Title of Invention	DETECTION OF AUTOANTIBODIES REACTIVE WITH PANCREATIC ISLET CELL ANTIGENIC MOLECULES AND/OR INSULIN .
Abstract	1. A method of screening a sample of body fluid obtained from an animal subject for analyte autoantibodies reactive with one or more pancreatic islet cell antigenic molecules selected from the group consisting of GAD65, GAD67 and protein tyrosine phosphatase-like islet cell antigen (IA-2), or one or more variants, analogues, derivatives or fragments thereof, said method comprising: (a) providing one or more first sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from said group of pancreatic islet cell antigenic molecules, one or more variants, analogues, derivatives or fragments of said pancreatic islet cell antigenic molecules and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from pancreatic islet cell antigenic molecules, and said one or more variants, analogues, derivatives or fragments thereof; (b) providing one or more second sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from said group of pancreatic islet cell antigenic molecules, one or more variants, analogues, derivatives or fragments of said group of pancreatic islet cell antigenic molecules, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from pancreatic islet cell antigenic molecules, and said one or more variants, analogues, derivatives or fragments thereof; (c) contacting said antigenic molecules as provided by steps (a) and (b) simultaneously or successively with said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fluid can interact with said antigenic molecules so as to form one or more complexes comprising [antigenic molecule of said first source]-[analyte autoantibody]-[antigenic molecule of said second source], wherein said molecules of said first and second sources when present in said one or more complexes comprise, or are derived from, a common antigenic molecule, or wherein binding regions of said antigenic molecules of said first and second sources, for said analyte autoantibody, when present in said one or more complexes are present in, or are derived from, a common antigenic molecule; (d) prior to, or concurrent with, or subsequent to, step ( c ), providing immobilizing means whereby said antigenic molecule of said first source as present in a complex as formed in step (c ) is immobilized to a solid support prior to, or concurrent with, or subsequent to, step (c); (e) prior to, or concurrent with, or subsequent to, step ( c ), providing direct or indirect detectable labelling means whereby said antigenic molecule of said second source as present in a complex as formed in step ( c ) is provided with such direct or indirect detectable labelling means prior to, or concurrent with, or subsequent to, step (c); and (f) detecting the presence of complexes formed in (c ) immobilized according to (d) so as to provide an indication of analyte autoantibodies present in said sample.

Title of Invention

DETECTION OF AUTOANTIBODIES REACTIVE WITH PANCREATIC ISLET CELL ANTIGENIC MOLECULES AND/OR INSULIN .

Abstract

1. A method of screening a sample of body fluid obtained from an animal subject for analyte autoantibodies reactive with one or more pancreatic islet cell antigenic molecules selected from the group consisting of GAD65, GAD67 and protein tyrosine phosphatase-like islet cell antigen (IA-2), or one or more variants, analogues, derivatives or fragments thereof, said method comprising: (a) providing one or more first sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from said group of pancreatic islet cell antigenic molecules, one or more variants, analogues, derivatives or fragments of said pancreatic islet cell antigenic molecules and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from pancreatic islet cell antigenic molecules, and said one or more variants, analogues, derivatives or fragments thereof; (b) providing one or more second sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from said group of pancreatic islet cell antigenic molecules, one or more variants, analogues, derivatives or fragments of said group of pancreatic islet cell antigenic molecules, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from pancreatic islet cell antigenic molecules, and said one or more variants, analogues, derivatives or fragments thereof; (c) contacting said antigenic molecules as provided by steps (a) and (b) simultaneously or successively with said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fluid can interact with said antigenic molecules so as to form one or more complexes comprising [antigenic molecule of said first source]-[analyte autoantibody]-[antigenic molecule of said second source], wherein said molecules of said first and second sources when present in said one or more complexes comprise, or are derived from, a common antigenic molecule, or wherein binding regions of said antigenic molecules of said first and second sources, for said analyte autoantibody, when present in said one or more complexes are present in, or are derived from, a common antigenic molecule; (d) prior to, or concurrent with, or subsequent to, step ( c ), providing immobilizing means whereby said antigenic molecule of said first source as present in a complex as formed in step (c ) is immobilized to a solid support prior to, or concurrent with, or subsequent to, step (c); (e) prior to, or concurrent with, or subsequent to, step ( c ), providing direct or indirect detectable labelling means whereby said antigenic molecule of said second source as present in a complex as formed in step ( c ) is provided with such direct or indirect detectable labelling means prior to, or concurrent with, or subsequent to, step (c); and (f) detecting the presence of complexes formed in (c ) immobilized according to (d) so as to provide an indication of analyte autoantibodies present in said sample.

Full Text	The present invention relates to methods and kits useful in the detection of autoantibodies reactive with pancreatic islet cell antigenac molecules and / or insulin, for example methods and kits useful in me detection of autoantibodies indicative of the onset or presence of insulin dependent diabetes mellius (IDDM, type 1 diabetes). Type 1 diabetes is one of the more prevalent autoimmune diseases affecting man, with more man 2 million individuals in Europe and North America suffering from the disease (M A Atkinson, G S Eisenbarth. Type 1 diabetes: new perspectives oa disease pathogenesis and treatment. The Lancet, 2001 358: 221-229). Furthermore, the incidence of type 1 diabetes is increasing worldwide by 2.5% per year aad it is estimated mat the incidence of this disease will be 40% higher in 2010 that in 1998 (M A Atkinson, G S Eisenbarth. Type 1 diabetes: new perspectives on disease pathogenesis and treatment. The Lancet, 2001 358: 221-229). Autoantibodies to pancreatic β cell antigens are well recognised serological markers of type 1 diabetes and these include: autoantibodies reactive with islet eels in immunofluorescence tests (ICA), autoantibodies to ghitamic acid decarboxylase (GAD, in particular the 65 kDa isoform thereof,GAD65), autoaatibodies to protein tyrosine phosphatase-like islet cell antigen (which can be referred to as IA-2 or ICA512) and autoantibodies to insulin. GAD65 autoantibodies and IA-2 autoantibodies are components of ICA reactivity (M A Atkinson, G S Eisenbarth. Type 1 diabetes: new perspectives on disease pathogenesis and treatment The Lancet, 2001 358: 221-229). At least one of the three types of autoantibodies to GAD65, IA-2 and insulin ate present at diagnosis in sere from about 90% of patients with type 1 diabetes (M A Atkinson, G S Eisenbarm. Type 1 diabetes: new perspectives on disease pathogenesis and treatment. The Lancet, 2001 358: 221-229). Measurement of the above mentioned autoantibodies being serotogical matters of autoimmunity to pancreatic β cells can also be useful in monitoring patients involved in trials for prevention of diabetes (M A Atkinson, G S Eisenbarth. Type 1 diabetes: new perspectives on disease pathogenems and treatment. The Lancet, 2001 358: 221- 229). Currently, GAD65 autoantibodies are measured using imnamnopcerapitation assays (IPA) employing GAD65 radiaactfveiy labelled with 35S, 3H or 125I GAD65 labelled with 35S or 3H has been produced in an in vitro transcription/translation system (C E Grubin, T Daniels, B Toivola, M Landdin-Olsson, W A Hagopian, L Li, A E Karlsen, E Boel, B Michelsen, A Lemmark. A novel radioligand binding assay to determine diagnostic accuracy of isoform-specific giutamic acid decarboxylase antibodies in childhood IDDM. Diabetologia, 1994 37: 344-350; J S Patesm, K R Hejnaes, A Moody, A E Karlsen, M O Marshall, M H6ier-Madsen, E Bod, B K Michelaen, T Dyrberg. Detection of GADgs antibodies in diabetes and other autoimmune diseases using a simple radioligand assay. Diabetes, 1994 43: 459-467; sad A Falorni, E Ortqvist, B Persson, A Lcramark. Radioimmunoassays for gtutamic acid decarboxyiase (GAD65) and GAD65 autoantibodies using 35S or 3H recombinant human Ugands. Journal of Immunological Methods, 1995 186: 88-89). Native GAD65, for example rat or porcine GAD65 isolated from brain tissue (M J Rowley, I R Mackay, Q-Y Chen, W J Knowles, P Z Zimmet. Antibodies to glutamic acid decarboxyiase discriminate major types of diabetes meilitue Diabetes, 1992 41: 548:551; and M Ohta, H Obayasai, K Takahasbi, Y Kitagawa, K Nakano, S Matsuo, M Ohishi, N Itoh, K Hayashi, K Ohta. A simple solid-phase radioimunoassay for glutamic acid decarboxyiase (GAD) antibodies in patients wife diabetes mellitus. J Clin Biochem Nutr, 1996 20: 139-148) or recombinant humae GAD65, for example expressed in insect cells, yeast or mammalian cells (F Lufader, K-P Woltanski, L Mauch, H Haubruck, K-D Kohoert, I Rjasaoowski, D Michaelis, M Ziegler. Detection of autoantibodies to the 65-kD isoform of glutaante decarboxyiase by radioimmunoassay. European Journal of Endocrinolofy, 1994 130: 575-80; M Powell, L Prentice, T Asawa, R Kato, J Sawicka, H Tanaka, V Peteraen, A Munkley, S Morgan, B Rees Smith, J Furmaniak. Clinica Chimica Acta, 1996 256: 175-188; and T Matsuba, M Yano, No Abiru, H Talano, S Akazawa, S Nagataki, K Yasukawa. Expression of recombinant human ajhrtaouc acid decarboxylase (GAD) in myeloma cells and enzyme-linked irnrmmosotbent assay (EL1SA) for autotonbodies to GAD. J Biochem, 1997 121: 2O-24) have-been labelled with 125I and used in DP As. Recombinant fusion proteins of GAD65 and IA-2 have alao beea used in IP As for combined autoantibody testing (A Zavialov, M Ankelo, A Westcriuod-Karlsson, M Knip, J nioaen, A Hinkkanen Novel fusion proteins in the analysis of diabetes associated autoantibodies to GAD65 and IA-2. Journal of Immunological Methods, 2000 246: 91-96; and M Rickert, J Seissler, W Dangel, H Lorenz, W Richter. Fusion proteins for combined aoalysis of autoantibodies to the 65-kDa isoform of glutamic acid decarboxylase aad istet antigsn-2 in insulin depeadflBt diabetes mellitus. Clinical Chemistry, 2001 47(5):926-934). Currently available IP As based on 35S-GAD65 have, however, been technically demanding, difficult to standardise, relatively expensive and have not been adaptable to a kit format. Assays based on human recombinant 125I-GAD65 have been of equal sensitivity and specificity to assays bated on 35S-GAD65, aad have exhibited the added advantage of good reproductbility, technical simplicity, lower costs and have been easily adapted into a kit Unfortunately, however, assays based on 125I-GAD65 isolated from rat or porcine brain tissue have suffered from lower specificity due to contamination with GAD65 isoform. GAD65 autoantibodies have also been measured by differed types of ELISAs (A M Gronowski, E C C Wong, T R Wilhite, D L Martin, C H Smith, C A Parvin, M Landt. Detection of glutamic acid decarboxylase autoantibodies with the varelisa ELISA. Clinical Chemistry, 1995 41(10): 1532-1534; and H D Mehata, B S Void, S Minkin, E F Ullman. DELISA: sensitive nonisotopic assay for GAD65 autoantibodies, a key risk-assessment marker for insulin-dependent diabetes mettitus. Clinical Chemistry, 1996 42(2): 263-269). However, currently available tests to measure GAD65 autoantibodies by ELISA have poorer sensitivity and specificity than the above meotioaed IP As aad do not compare well with the IP As in proficiency studies (R S SchmidH, P G Cofanan, E Bonifacio, Participating Laboratories. Disease sensitivity and specificity of 52 assays for glutamic acid decarboxylase antibodies. The second international GADAb workshop. Diabetes, 1995 44: 636-640). The present invention, however, alleviates me problems hitherto associted with methods aad kits employed in the screening of autoaatibodies indicative of the onset or presence of type 1 diabetes and have general applicability in the detection of autoantibodies reactive with pancreatic islet cell antigenic molecules and / or insulin. In particular, the present invention employs methods and kits having unproved specificity and sensitivity when compared to prior art techniques as discussed above. There is provided by the present invention a method of screening a sample of body fluid obtained from an animal subject for analyte autoantibodies reactive with one or more antigenic molecules selected from pancreatic islet cell antigenic molecules and insulin, or one or more variants, analogues, derivatives or fragments thereof, said method comprising: (a) providing said sample of body fluid from said subject; (b) providing one or more first sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from pancreatic islet cell antigenic molecules, insulin, one or more variants, analogues, derivatives or fragments of said pancreatic islet cell antigenic molecules or insulin, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from pancreatic Met cell antigenic molecules, insulin and said one or more variants, analogues, derivatives or fragments thereof; (c) providing one or more second sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from pancreatic islet cell antigenic molecules, insulin, one or more variants, analogues, derivatives or fragments of said pancreatic islet cell antigenic molecules or insulin, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from pancreatic islet cell antigenic molecules, insulin and said one or more variants, analogues, derivatives or fragments thereof, (d) contacting said antigenic molecules as provided by steps (b) and (c) simultaneously or successively with-said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fhad can interact with said antigenic molecules so as to form one or more complexes comprising [antigenic molecule of said first souree]-[analyte autoantibody]- [antigenic molecule of said second source], wherein said antigenic molecules of said first aid second sources when present is said one or more complexes comprise, or ate derived from, a common antigeaic molecule, or wherein binding regions of said antigenic molecules of said first and second sources, for said analyte autoantibody, when present in said one or more complexes are present in, or are derived from, a common antigeaic molecule; (e) prior to, or concurrent with, or subsequent to, step (d), providing immobilising means whereby said antigenic moiecule of said first source as present in a complex as formed in step (d) is immobilised to a solid support prior to, or concurrent with, or subsequent to, step (d); (f) prior to, or concurrent with, or subsequent to, step (d), providing direct or indirect detectable labelling means whereby ami antigenic molecule of said second source as present in a complex as formed in step (d) is provided with such direct or indirect delectable labelling means prior to, or concurrent with, or subsequent to, step (d); and (g) detecting me presence of complexes formed in (d) immobilised according to (e) so as to provide an indication of analyte astoantibodies present in said sample. A method of screening for analyte autoantibodies according to the present invention is particularly advantageous in providing a method lor such autoantibody detection of high specificity and sensitivity. Hie one or more antigenic molecules, or one or mote variants, analogues, derivatives or fragments thereof, or fusion molecules thereof, that can react with analyte autoantibodies as required by the present invention can be selected from any pancreatic islet cell antigenic molecule aad insulin, and one or more variants, analogues, derivatives or fragments thereof; or fusion molecules thereof and in particular can suitably be selected from the group costisting pf glutamic acid decarboxylase (GAD, and in particular the 65KDa and 67KDa isoforms of gkttamic acid decarboxylase, GAD65 and GAD67), petein tyrosine phosphatase-like islet cell antigen (IA-2) and insulin, or one or more variants (such as, lor example, IA-2 beta), analogues, derivatives or fragments thereof, or fusion molecules comprising two or more of the above, with which analyte aotoantibodaes when present in said sample of body fluid can interact. In a particularly, preferred embodiment of die present invention, one or more of the above mentioned pancreatic islet cell antigenic molecules or insulin are employed, in particular the above mentioned pancreatic islet cell antigenic molecules, preferably one or more of GAD65, GAD67 or IA-2. Alternatively it may be preferred that one or more variants, analogues, derivatives or fragments of one or more of the above mentioned pancreatic islet cell antigenic molecules or insulin are employed, in particular one or more variants, analogues, derivatives or fragments of the above mentioned pancreatic islet cell antigenic molecules, preferably one or more variants, analogues, derivatives or fragments of one or more of GAD65, GAD67 or IA-2. In a further alternative preferred embodiment of the present invention, it may be prefered mat one or more fusion molecules are employed comprising two or more directly or indirectly fused antigenic molecules selected from the above mentioned pancreatic islet cell antigenic molecules, insulin and said one or more variants, analogues, derivatives or fragments thereof. It will be appreciated, therefore, that the terra GAD as used heron encompasses all isoforms of GAD, including in particular GAD65 or GAD67 and in certain embodiments of the present invention it may be preferred mat GAD65 is employed and in other embodiments of the present invention it may be preferred that GAD67 is employed. It will also be appreciated from the above that the present invention can include within its scope fusion molecules Ait can comprise GAD6$ or GAD67, or one or more fragments thereof (such as binding regions thereof). For example, fusion molecules for use within the scope of the present invention can comprise GAD65 directly or indirectly fused to GAD67, or one or more binding regions of GAD65 directly or indirectly fused to one or more binding regions or GAD67. Furthermore, it will also be appreciated that fusion molecules for use within the scope of the present invention can comprise IA-2, or one or more binding regions thereof, directly or indirectly fused to IA-2 beta, or one or more binding regions thereof. The present invention also allows for GAD65 or GAD67, or one or more variants, analogues, derivatives or fragments thereof, directly or indirectly fused to IA-2, or one or more variants, analogues, derivatives or fragments thereof. ID particular the one or more aniigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, that can react with such analyte autoratibodies as required by the present invention, can suitably be selected from the group consisting of glutamic acid decarboxyiase (in particular GAD65 or GAD67) and protein tyrosine phosphatase-like islet cell antigen (IA-2), or one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact. In a preferred aspect of the present invention, therefore, a method of screening substantially as hereinbefore described is for use in detecting analyte autoantibodies to GAD and / or IA-2, and the one or more antigenic molecules comprise GAD65 or GAD 67, or one or more variants, analogues, derivatives or fragments thereof, and / or IA-2, or one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact In the case where fusion antigenic molecules are employed in the present invention, suitable fusion molecules may comprise GAD65 or GAD67 or one or more variants, analogues, derivatives or fragments thereof, directly or indirectly fused to IA-2, or one or more variants, analogues, derivatives or fragments thereof; alternatively, suitable fusion molecules may comprise GAD65 directly or indirectly fused to GAD67, or one or more binding regions of GAD65 directly or indirectly fused to one or more binding regions or GAD67; alternatively suitable fusion molecules may comprise IA-2, or one or more binding regions thereof, directly or indirectly fused to IA-2 beta, or one or more binding regions thereof. Preferably a method according to the present invention comprises providing one or more first sources of antigenic molecules selected from GAD65, GAD67, IA-2, insulin, one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigeaic molecules selected from GAD65, GAD67, IA-2, insulin and said one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact, immobilised to a solid support according to step (e) of a method according to the present invention and one or more •second sources of antigenic molecules selected from GAD65, GAD67, IA-2, insulin, one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigeaic molecules selected from GAD65, GAD67, IA-2, insulin and said one or mare variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when patent in said sample of body fluid can interact, provided with labelling means according to step (f) of a method according to the present invention. More preferably, a method according to the present inventkn comprises providing one or more first sources of antigenic molecules selected from GAD65, GAD67, IA-2, one or more variants, analogues, derivatives or fragmeais thereof and fusion molecules comprising two or more directly or indirectly fused aatigenic molecules selected from GAD65, GAD67, IA-2, and said one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact (for example fusion molecules comprising GAD65, GAD67, or one or more variants, analogues, derivatives or fragments thereof directly or indirectly fused to IA-2 or one or more variants, aaslogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact), immobilised to a solid support aocndaig to step (e) of a method according to the present invention and a second source of antigenic molecules selected from GAD65, GAD67, IA-2, one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67, IA-2, and one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact (such as fusion molecules comprising GAD65 or GAD67, or one or more variants, analogues, derivatives or fragments thereof directly or indirectly fused to IA-2 or one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact), provided with labelling means according to step (f) of a method according to tbe present invention. It may be preferred that where a method according to the present invention is required to detect autoantibodies to GAD, the method comprises providing a first source-.of antigenic molecules consisting essentially of GAD antigenic moieaues selected from GAD65, OAD67, one or more variants, analogues, derivatives or fragments thereof with which analyte autoantibodies when present in said sample of body fluid can interact and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67 aad the one or more variants, analogues, derivatives or fragments thereof, (such as fusion molecules comprising GAD65 directly or indirectly fined to GAD67, or one or more binding regions of GAD65 directly or indirectly fused to one or more binding regions or GAD67, with which analyte autoantibodies when present in said sample of body fluid can interact), immobilised to a solid support according to step (e) of a method according to the present invention and a second source of antigenic molecules consisting essentially of GAD antigenic molecules selected from GAD65, GAD67, one or more variants, analogues, derivatives or fragments thereof with which analyte autoaotibodtes when present in said sample of body fluid can interact and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67 and one or more variants, analogues, derivatives or fragments thereof, wife which analyte autoantibodies when present in said sample of body fluid can interact (such as GAD65 directly or indirectly fused to GAD67, or one or more binding regions of GAD65 directly or indirectly fused to one or more binding regions or GAD67) provided with labelling means according to step (f) of a method according to the present invention. Alternatively, it may be preferred mat where a method according to the present invention is required to detect autoantibodies to IA-2, the method comprises providing a first source of antigenic molecules consisting essentially of IA- 2 antigenic molecules selected from IA-2, one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from IA-2 aad one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present m said sample of body fluid can interact (such as IA-2, or one or more binding regions thereof, directly or indirectly fused to IA-2 beta, or one or more binding regions (hereof) immobilised to a solid support according to step (e) of a method according to the present invention and a second source of antigenic molecules consisting essentially of IA-2 antigenic molecules selected from IA-2, one or more variants, analogues, derivatives or fragments thereof with which aoalyte autoantibodies when present in said sample of body fluid can interact aad fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from IA-2 and one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact (such as IA-2, or one or more binding regions thereof, directly or indirectly fused to IA-2 beta, or one or more binding regions thereof), provided with labelling means according to step (f) of a method according to the present invention. A still further alternative according to the present invention may be where a method according to the present invention is required to detect autoantibodies to both GAD and IA-2, where the method comprises providing (i) first and second sources of GAD antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact, and which GAD antigenic molecules are selected from GAD65, GAD67, one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67, IA-2, and said one or more variants, analogues, derivatives or fragments thereof and wherein at least one of said antigenic molecules of said fusion molecule comprises GAD65, GAD67, or one or more variants, analogues, derivatives or fragments thereof, wherein GAD antigenic molecules of said first source are immobilised to a solid support according to step (e) and GAD antigenic molecules of said second source are provided with labelling means according to step (f); and (ii) first and second sources of IA-2 antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which IA-2 antigenic molecules are selected from IA-2, one or more variants, analogues, derivatives or fragments thereof, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from IA-2, GAD65, GAD67 and said one or more variants, analogues, derivatives or fragments thereof and wherein at least one of said antigenic molecules of said fusion molecule comprises IA-2 or one or more variants, analogues, derivatives or fragments thereof, wherein IA-2 antigenic molecules of said first source are immobilised to a solid support according to step (e) and IA-2 antigenic molecules of said second source are provided with labelling means according to step (f). Accordingly, a preferred embodiment of the present invention may suitably comprise a method of screening a sample of body fluid obtained from an animal subject for analyte autoantibodies reactive with one or more antigenic molecules selected from pancreatic islet cell antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, said method comprising: (a) providing said sample of body fluid from said subject; (b) providing one or more first sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from GAD65, GAD67, IA-2, one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67, IA-2, and said one or more variants, analogues, derivatives or fragments thereof; (c) providing one or more second sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from GAD65, GAD67, IA-2, one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67, IA-2 and said one or more variants, analogues, derivatives or fragments thereof; (d) contacting said antigenic molecules as provided by steps (b) and (c) simultaneously or successively with said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fluid can interact with said antigenic molecules so as to form one or more complexes comprising [antigenic molecule of said first source]-[analyte autoantibody]- [antigenic molecule of said second source], wherein said antigenic molecules of said first and second sources when present in said one or more complexes comprise, or are derived from, a common antigenic molecule, or wherein binding regions of said antigenic molecules of said first and second sources, for said analyte autoantibody, when present in said one or more complexes are present in, or are derived from, a common antigenic molecule; (e) prior to, or concurrent with, or subsequent to, step (d), providing immobilising means whereby said antigenic molecule of said first source as present in a complex as formed in step (d) is immobilised to a solid support prior to, or concurrent with, or subsequent to, step (d); (f) prior to, or concurrent with, or subsequent to, step (d), providing direct or indirect detectable labelling means whereby said antigenic molecule of said second source as present in a complex as formed in step (d) is provided with such direct or indirect detectable labelling means prior to, or concurrent with, or subsequent to, step (d); and (g) detecting the presence of complexes formed in (d) immobilised according to (e) so as to provide an indication of analyte autoantibodies present in said sample. Alternatively, a preferred embodiment of the present invention may suitably comprise screening a sample of body fluid obtained from an animal subject for analyte autoantibodies reactive only with GAD, or one or more variants, analogues, derivatives or fragments thereof, said method comprising: (a) providing said sample of body fluid from said subject; (b) providing first and second sources of antigenic molecules consisting essentially of GAD antigenic molecules selected from GAD65, GAD67, one or more variants, analogues, derivatives or fragments thereof with which analyte autoantibodies when present in said sample of body fluid can interact and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67 and said one or more variants, analogues, derivatives or fragments thereof, wherein GAD antigenic molecules of said first source are immobilised to a solid support according to step (d) and GAD antigenic molecules of said second source are provided with labelling means according to step (e); (c) contacting said antigenic molecules as provided by step (b) simultaneously or successively with said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fluid can interact with said antigenic molecules so as to form one or more complexes comprising [GAD antigenic molecule of said first sourcej-[analyte autoantibodyH GAD antigenic molecule of said second source]; (d) prior to, or concurrent with, or subsequent to, step (c), providing immobilising means whereby said GAD antigenic molecule of said first source as present in a complex as formed in step (c) is immobilised to a solid support prior to, or concurrent with, or subsequent to, step (c); (e) prior to, or concurrent with, or subsequent to, step (c), providing direct or indirect detectable labelling means whereby said GAD antigenic molecule of said second source as present in a complex as formed in step (c) is provided with such direct or indirect detectable labelling means prior to, or concurrent with, or subsequent to, step (c); and (f) detecting the presence of complexes formed in (c) immobilised according to (d) so as to provide an indication of analyte autoantibodies present in said sample. Alternatively, a preferred embodiment of the present invention may suitably comprise screening a sample of body fluid obtained from an animal subject for analyte autoantibodies reactive with IA-2, or one or more variants, analogues, derivatives or fragments thereof, said method comprising: (a) providing said sample of body fluid from said subject; (b) providing first and second sources of antigenic molecules consisting essentially of IA-2 antigenic molecules selected from IA-2, one or more variants, analogues, derivatives or fragments thereof with which analyte autoantibodies when present in said sample of body fluid can interact and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from IA-2 and said one or more variants, analogues, derivatives or fragments thereof, wherein IA-2 antigenic molecules of said first source are immobilised to a solid support according to step (d) and IA-2 antigenic molecules of said second source are provided with labelling means according to step (e); (c) contacting said IA-2 antigenic molecules as provided by step (b) simultaneously or successively with said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fluid can interact with said IA-2 antigenic molecules so as to form one or more complexes comprising [IA-2 antigenic molecule of said first source]-[analyte autoantibody]-[IA-2 antigenic molecule of said second source]; (d) prior to, or concurrent with, or subsequent to, step (c), providing immobilising means whereby said IA-2 antigenic molecule of said first source as present in a complex as formed in step (c) is immobilised to a solid support prior to, or concurrent with, or subsequent to, step (c); (e) prior to, or concurrent with, or subsequent to, step (c), providing direct or indirect detectable labelling means whereby said IA-2 antigenic molecule of said second source as present in a complex as formed in step (c) is provided with such direct or indirect detectable labelling means prior to, or concurrent with, or subsequent to, step (c); and (f) detecting the presence of complexes formed in (c) immobilised according to (d) so as to provide an indication of analyte autoantibodies present in said sample. Alternatively, a preferred embodiment of the present invention may suitably comprise screening a sample of body fluid obtained from an animal subject for first and second analyte autoantibodies respectively reactive with GAD and IA-2, or one or more variants, analogues, derivatives or fragments thereof, said method comprising: (a) providing said sample of body fluid from said subject; (b) providing first and second sources of GAD antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact, and which GAD antigenic molecules are selected from GAD65, GAD67, one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67, IA-2, and said one or more variants, analogues, derivatives or fragments thereof and wherein at least one of said antigenic molecules of said fusion molecule comprises GAD65, GAD67, or one or more variants, analogues, derivatives or fragments thereof, wherein GAD antigenic molecules of said first source are immobilised to a solid support according to step (e) and GAD antigenic molecules of said second source are provided with labelling means according to step (f); (c) providing first and second sources of IA-2 antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which IA-2 antigenic molecules are selected from IA-2, one or more variants, analogues, derivatives or fragments thereof, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from IA-2, GAD65, GAD67 and said one or more variants, analogues, derivatives or fragments thereof and wherein at least one of said antigenic molecules of said fusion molecule comprises IA-2 or one or more variants, analogues, derivatives or fragments thereof, wherein IA-2 antigenic molecules of said first source are immobilised to a solid support according to step (e) and IA-2 antigenic molecules of said second source are provided with labelling means according to step (f); (d) contacting said antigenic molecules as provided by steps (b) and (c) simultaneously or successively with said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fluid can interact with said antigenic molecules so as to fonn one or more complexes comprising [GAD antigenic molecule of said first source]-[analyte autoantibody]-[GAD antigenic molecule of said second source] or [IA-2 antigenic molecule of said first source]-[analyte autoantibody]-[IA-2 arttigenic molecule of said second source]; (e) prior to, or concurrent with, or subsequent to, step (d), providing immobilising means whereby said GAD or IA-2 antigenic molecule of said first source as present in a complex as formed in step (d) is immobilised to a solid support prior to, or concurrent with, or subsequent to, step (d); (f) prior to, or concurrent with, or subsequent to, step (d), providing direct or indirect detectable labelling means whereby said GAD or IA-2 antigenic molecule of said second source as present in a complex as formed in step (d) is provided with such direct or indirect detectable labelling means prior to, or concurrent with, or subsequent to, step (d); and (g) detecting the presence of complexes formed in (d) immobilised according to (e) so as to provide an indication of analyte autoantibodies present in said sample. It may be preferred in a method according to the present invention that the antigenic molecules of the first and second sources when present in said one or more complexes substantially as hereinbefore described both comprise one or more common antigenic molecules. For example, antigenic molecules of said first and second sources when present in the one or more complexes can comprise GAD65 or GAD67; alternatively the antigenic molecules of the first and second sources when present in the one or more complexes can comprise IA-2. Alternatively, it may be preferred that one antigenic molecule, of either the first or second source, when present in the one or more complexes comprises an antigenic molecule selected from pancreatic islet cell antigenic molecules and insulin, and the other antigenic molecule when present in the one or more complexes comprises one or more variants, analogues, derivatives or fragments of the above mentioned one antigenic molecule with which analyte autoantibodies when present in said sample of body fluid can interact For example, where one of the antigenic molecules of the first or second source when present in the one or more complexes comprises GAD65 or GAD67, the other antigenic molecule when present in the one or more complexes can comprise one or more variants, analogues, derivatives or fragments of GAD65 or GAD67 with which analyte autoantibodies when present in said sample of body fluid can interact. A further example may be where one of the antigenic molecules of die first or second source when present in the one or more complexes comprises IA-2, and the other antigenic molecule when present in the one or more complexes can comprise one or more variants, analogues, derivatives or fragments of IA-2 with which analyte autoantibodies when present in said sample of body fluid can interact A still further alternative embodiment of the present invention may be where both antigenic molecules of the first: and second sources when present in the one or more complexes comprise a variant, analogue, derivative or fragment, which may be the same or different, of a common antigenic molecule, with which analyte autoantibodies when present in said sample of body fluid can interact. For example, both antigenic moleq\|ies of the first and second sources when present in the one or more complexes can comprise a variant, analogue, derivative or fragment, which may be the same or different, of GAD65 or GAD67, with which analyte autoantibodies when present in said sample of body fluid can interact. A further example may be where both antigenic molecules of the first and second sources when present in said one or more complexes comprise a variant, analogue, derivative or fragment, which may be the same or different, of IA-2, with which analyte autoantibodies when present in said sample of body fluid can interact. It will be appreciated from the above that where both antigenic molecules when present in the complexes formed according to a method of the present invention comprise variants, analogues, derivatives or fragments derived from a common antigenic molecule, such variants, analogues, derivative or fragments may be the same or different (for example variant 1 and variant 2, where variants 1 and 2 may respectively represent different variants of GAD65 or GAD67, or fragment 1 and fragment 2, where fragments 1 and 2 may respectively represent different fragments of GAD65 or GAD67, such as distinct, or possibly overlapping, epitopes of GAD65 or GAD67). In the case where fusion molecules are employed in a method according to the present invention, it will be appreciated that such fusion molecules can comprise -distinct antigenic molecules (for example GAD65 and IA-2) but that in a complex formed according to the present invention the binding regions of antigenic fusion molecules for an analyte autoantibody as respectively provided by the first and second sources will be present in, or will be derived from, a common antigenic molecule. Examples of complexes that may be formed employing one or more fusion molecules according to the present invention can include (i) [GAD-IA-2imm]-[analyte autoantibody]-[GAD- IA-2label] where in the case where the autoantibody comprises a GAD autoantibody this autoantibody interacts with binding regions of GAD present in [GAD-IA-2imn] and [GAD-IA-2label], or where the autoantibody comprises a IA-2 autoantibody this autoantibody interacts with binding regions of IA-2 present in [GAD-IA2imm] and [GAD-IA-2label], or (ii) [GAD(fragment)imm]-[analyte autoantibody]-[GAD-IA-2label] where a GAD analyte autoantibody interacts with binding regions of GAD present in [GAD(fragment)imm] and [GAD-IA-2iabd], or (iii) [IA-2(fogment)imm]-[analyte autoantibody]-[GAD-IA-2label] where a IA-2 analyte autoantibody interacts with binding regions of IA-2 present in [IA-2(fragment)imm] and [GAD-IA-2label], where GAD can represent GAD65 or GAD67. Suitably the detectable labelling means can be selected from the group consisting of enzymic labels, isotopic labels, chemiluminesceat labels, fluorescent labels, dyes and the like and typically can be selected from the group consisting of alkaline phosphatase, horseradish peroxidase, biotin or the like and in particular can comprise biotin. The monitoring can typically involve reaction of such detectable labelling means (when attached to the one or more antigenic molecules substantially as hereinbefore described, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof) with one or more substrates therefor (such as an avidin or streptavidin conjugate, for example, streptavidin horseradish peroxidase conjugate or the like), whereby the resulting conjugates can be detected suitably by measurement of optical density or the like. The detectable labelling means may be directly provided to the one or more antigenic molecules, or one or more one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, substantially as hereinbefore described. Additionally or alternatively the detectable labelling means may be indirectly provided to the one or more antigenic molecules, or one or-more one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, substantially as hereinbefore described, typically by providing the detectable labelling means to one or more antibodies or other binding agents which can bind with the one or more antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, substantially as hereinbefore described. A method of screening for autoantibodies according to the present invention typically comprises directly monitoring interaction of (i) such autoantibodies present in the sample of body fluid from the subject and (ii) one or more antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, substantially as hereinbefore described, as provided by the present invention, typically by employing non-competitive sandwich type assay techniques known in the art According to a preferred embodiment of the present invention antigenic molecules of one or more first sources, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, substantially as hereinbefore described, are immobilised to a solid support and antigenic molecules of one or more second sources, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, substantially as hereinbefore described, are provided with detectable labelling means, and wherein preferably the antigenic molecules of the one or more second sources are provided in solution phase as is conventional for use in known ELISA techniques. Preferably, in a method according to the present invention, antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, with which analyte autoantibodies when present in said sample of body fluid can interact, of one or more first sources are immobilised to a - solid support prior to contact with a sample of body fluid being screened. Such immobilised antigenic molecules of the one or more first sources, or more one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, with which analyte autoantibodies when present in said sample of body fluid can interact, are subsequently contacted with the sample of body fluid being screened either simultaneously or successively with contact of the sample of body fluid with antigenic molecules of one or more second sources, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, with which analyte autoantibodies when present in said sample of body fluid can interact. Particularly preferably, immobilised antigenic molecules of the one or more first sources, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, with which analyte autoantibodies when present in said sample of body fluid can interact, are contacted with the sample of body fluid being screened so as to form an intermediate complex comprising [antigenic molecule]-[analyte autoantibody] where the antigenic molecule is immobilised to a solid support and the thus formed immobilised intermediate complex is subsequently contacted with antigenic molecules of the one or more second sources, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, with which analyte autoantibodies when present in said sample of body fluid can interact, present in solution phase, so as to form the hitherto described complexes comprising [antigenic molecule of said first source]- [analyte autoantibody]-[antigenic molecule of said second source] immobilised to a solid support via the antigenic molecule of the first source. Accordingly, the present invention provides a method of screening a sample of body fluid obtained from an animal subject for analyte autoantibodies reactive with one or more antigenic molecules selected from pancreatic islet cell antigenic molecules and insulin, or one or more variants, analogues, derivatives or fragments thereof, said method comprising: (a) providing said sample of body fluid from said subject; (b) providing one or more first sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from pancreatic islet cell antigenic molecules, insulin, one or more variants, analogues, derivatives or fragments of said pancreatic islet cell antigenic molecules or insulin, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from pancreatic islet cell antigenic-molecules, insulin and said one or more variants, analogues, derivatives or fragments thereof, which antigenic molecules of said one or more first sources are immobilised to a solid support; (c) providing one or more second sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from pancreatic islet cell antigenic molecules, insulin, one or more variants, analogues, derivatives or fragments of said pancreatic islet cell antigenic molecules or insulin, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from pancreatic islet cell antigenic molecules, insulin and said one or more variants, analogues, derivatives or fragments thereof, which antigenic molecules of said one or more second sources are provided in solution phase; (d) contacting said antigenic molecules as provided by steps (b) and (c) simultaneously or successively with said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fluid can interact with said antigenic molecules so as to form one or more immobilised complexes comprising [antigenic molecule of said first source]-[analyte autoantibodyHantigenic molecule of said second source], wherein said antigenic molecules of said first and second sources when present in said one or more complexes comprise, or are derived from, a common antigenic molecule, or wherein binding regions of said antigenic molecules of said first and second sources, for said analyte autoantibody, when present in said one or more complexes are present in, or are derived from, a common antigenic molecule and wherein said complexes are immobilised to a solid support via said antigenic molecule of said first source; (e) prior to, or concurrent with, or subsequent to, step (d), providing direct or indirect detectable labelling means whereby said antigenic molecule of said second source as present in a complex as formed in step (d) is provided with such direct or indirect detectable labelling means prior to, or concurrent with, or subsequent to, step (d); and (f) detecting the presence of complexes formed in (d) so as to provide an indication of analyte autoantibodies present in said sample. The one or more antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, or fusion molecules thereof, that can react with analyte autoantibodies as required by the present invention can be selected from any pancreatic islet cell antigenic molecule and insulin, and one or more variants, analogues, derivatives or fragments thereof, or fusion molecules thereof and in particular can suitably be selected from the group consisting of glutamic acid decarboxylase (GAD, and in particular the 65KDa and 67KDa isoforms of glutamic acid decarboxylase, GAD65 and GAD67), protein tyrosine phosphatase-like islet cell antigen (IA-2) and insulin, or one or more variants (such as, for example, IA-2 beta), analogues, derivatives or fragments thereof, or fusion molecules comprising two or more of the above, with which analyte autoantibodies when present in said sample of body fluid can interact. In particular the one or more antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, that can react with such analyte autoantibodies as required by the present invention, can suitably be selected from the group consisting of glutamic acid decarboxylase (GAD, in particular GAD65 or GAD67) and protein tyrosine phosphatase-like islet cell antigen (IA-2), or one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact. Other contacting techniques are, however, possible, for example it is possible according to the present invention not to initially immobilise the antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, of the one or more first antigen sources, prior to a contacting step as described above, but to initially contact the solid support substantially as hereinbefore described with a binder for the antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, of the one or more first antigen sources and to subsequently contact the so treated solid support with the above described antigenic molecules of the one or more first and second sources, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, and the sample of body fluid being screened. In respect of the solid supports and conditions employed in the present invention, the supports and conditions do not generally fundamentally differ from conventional supports and conditions employed in known immunoassay techniques. A solid support for use according to the present invention can comprise an ELISA plate as currently employed in known ELISA techniques, or may employ any other suitable support for use in the present invention, such as tubes, particles, magnetic beads, nitrocellulose or the like. There is also provided by the present invention a kit for screening a sample of body fluid obtained from an animal subject for analyte autoantibodies reactive with one or more antigenic molecules selected from pancreatic islet cell antigenic molecules and insulin, or one or more variants, analogues, derivatives or fragments thereof, said kit comprising: (a) one or more first sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from pancreatic islet cell antigenic molecules, insulin, one or more variants, analogues, derivatives or fragments of said pancreatic islet cell antigenic molecules or insulin, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from pancreatic islet cell antigenic molecules, insulin and said one or more variants, analogues, derivatives or fragments thereof; (b) one or more second sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from pancreatic islet cell antigenic molecules, insulin, one or more variants, analogues, derivatives or fragments of said pancreatic islet cell antigenic molecules or insulin, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from pancreatic islet cell antigenic molecules, insulin and said one or more variants, analogues, derivatives or fragments thereof; (c) means for contacting said antigenic molecules as provided by (a) and (b) simultaneously or successively with said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fluid can interact with said antigenic molecules so as to form one or more complexes comprising [antigenic molecule of said first source]-[analyte autoantibody] - [antigenic molecule of said second source], wherein said antigenic molecules of said first and second sources when present in said one or more complexes comprise, or are derived from, a common antigenic molecule, or wherein binding regions of said antigenic molecules of said first and second sources, for said analyte autoantibody, when present in said one or more complexes are present in, or are derived from, a common antigenic molecule; (d) means for immobilising to a solid support, said antigenic molecule of said first source as present in a complex as defined in (c), prior to, or concurrent with, or subsequent to, contact of the antigenic molecule of said first source with the sample of body fluid being screened; (e) means for providing direct or indirect detectable labelling means to said antigenic molecule of said second source as present in a complex as defined in (c), prior to, or concurrent with, or subsequent to, contact of said antigenic molecule of said second source with said sample of body fluid being screened; and (f) means for detecting the presence of complexes as defined in (c) immobilised as defined in (d) so as to provide an indication of analyte autoantibodies present in said sample. A kit for screening for analyte autoantibodies according to the present invention is particularly advantageous in enabling such autoantibody detection of high specificity and sensitivity. The one or more antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof; or fusion molecules,thereof,, mat can react with analyte autoantibodies as required by the present invention can be selected from any pancreatic islet cell antigenic molecule and insulin, and one or more variants, analogues, derivatives or fragments thereof, or fusion molecules thereof and in particular can suitably be selected from the group consisting of glutamic acid decarboxylase (GAD, and in particular the 65KDa and 67KDa isoforms of glutamic acid decarboxylase, GAD65 and GAD67), protein tyrosine phosphatase-like islet cell antigen (IA-2) and insulin, or one or more variants (such as, for example, IA-2 beta), analogues, derivatives or fragments thereof, or fusion molecules comprising two or more of the above, with which analyte autoantibodies when present in said sample of body fluid can interact. In particular the one or more antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, that can react with such analyte autoantibodies as required by the present invention, can suitably be selected from the group consisting of glutamic acid decarboxylase (GAD, in particular GAD65 or GAD67) and protein tyrosine phosphatase-like islet cell antigen (IA-2), or one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact. Various embodiments of the antigenic molecules and suitable fusion molecules for use in a kit according to the present invention are substantially as hereinbefore described in greater detail with reference to a method according to the present invention. In a preferred aspect of the present invention, therefore, a kit for screening substantially as hereinbefore described is for use in detecting analyte autoantibodies to GAD65 or GAD67 and / or IA-2, or one or more variants, analogues, derivatives or fragments thereof, and the one or more antigenic molecules comprise GAD65 or GAD67, or one or more variants, analogues, derivatives or fragments thereof, and / or IA-2, or one or more variants, analogues, derivatives or fragments thereof. In the case where fusion antigenic molecules are employed in a kit according to the present invention, suitable fusion molecules may comprise two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67, IA-2 and said one or more variants, analogues, derivatives or fragments thereof substantially as hereinbefore described in greater detail with reference to a method according to the present invention. Preferably a kit according to the present invention comprises one or more first sources of antigenic molecules selected from GAD65, GAD67, IA-2, insulin, one or more variants, analogues, derivatives or fragments thereof, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67, IA-2, insulin and said one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact, immobilised to a solid support as defined in (d) and one or more second sources of antigenic molecules selected from GAD65, GAD67, IA-2, insulin, one or more variants, analogues, derivatives or fragments thereof, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67, IA-2, insulin and one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact, provided with labelling means as defined in (e). More preferably, a kit according to the present invention comprises one or more first sources of antigenic molecules selected from GAD65, GAD67, IA-2., one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67, IA-2 and said one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact, immobilised to a solid support as defined in (d) and one or more second sources of antigenic molecules selected from GAD65, GAD67, IA-2, one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67, IA-2 and said one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact, provided with labelling means as defined in (e). It may be preferred that a kit according to the present invention comprises a first source of antigenic molecules consisting essentially of GAD antigenic molecules selected from GAD65, GAD67, one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67 and said one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact, immobilised to a solid support as defined in (d), and a second source of antigenic molecules consisting essentially of GAD antigenic molecules selected from GAD65, GAD67, one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67 and said one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact, provided with labelling means as defined in (e). Alternatively, it may be preferred that a kit according to the present invention comprises a first source of antigenic molecules consisting essentially of IA-2 antigenic molecules selected from IA-2, one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from IA-2 and said one or more variants, analogues, derivatives or fragments thereof, immobilised to a solid support as defined in (d), and a second source of antigenic molecules consisting essentially of IA-2 antigenic molecules selected from IA-2, one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from IA-2 and said one or more variants, analogues, derivatives or fragments thereof, provided with labelling means as defined in (e). A still further alternative according to the present invention may be where a kit according to the present invention comprises (i) first and second sources of GAD antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact, and which GAD antigenic molecules are selected from GAD65, GAD67, one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67, IA-2 and said one or more variants, analogues, derivatives or fragments thereof and wherein at least one of said antigenic molecules of said fusion molecule comprises GAD65, GAD67, or one or more variants, analogues, derivatives or fragments thereof, wherein GAD antigenic molecules of said first source are immobilised to a solid support as defined in (d) and GAD antigenic molecules of said second source are provided with labelling means as defined in (e); and (ii) first and second sources of IA-2 antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which IA-2 antigenic molecules are selected from IA-2, one or more variants, analogues, derivatives or fragments thereof, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from IA-2, GAD65, GAD67 and said one or more variants, analogues, derivatives or fragments thereof and wherein at least one of said antigenic molecules of said fusion molecule comprises IA-2 or one or more variants, analogues, derivatives or fragments thereof, wherein IA-2 antigenic molecules of said first source are immobilised to a solid support according as defined in (d) and IA-2 antigenic molecules of said second, source are provided with labelling means as defined in (e). Accordingly, a preferred embodiment of the present invention may suitably comprise a kit for screening a sample of body fluid obtained from an animal subject for analyte autoantibodies reactive with one or more antigenic molecules selected from pancreatic islet cell antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, said kit comprising: (a) one or more first sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from GAD65, GAD67, IA-2, one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67, IA-2 and said one or more variants, analogues, derivatives or fragments thereof; (b) one or more second sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from GAD65, GAD67, IA-2, one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67, IA-2 and said one or more variants, analogues, derivatives or fragments thereof; (c) means for contacting said antigenic molecules as provided by (a) and (b) simultaneously or successively with said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fluid can interact with said antigenic molecules so as to form one or more complexes comprising [antigenic molecule of said first source]-[analyte autoantibody]- [antigenic molecule of said second source], wherein said antigenic molecules of said first and second sources when present in said one or more complexes comprise, or are derived from, a common antigenic molecule, or wherein binding regions of said antigenic molecules of said first and second sources, for said analyte autoantibody, when present in said one or more complexes are present in, or are derived from, a common antigenic molecule; (d) means for immobilising to a solid support said antigenic molecule of said first source as present in a complex as defined in (c), prior to, or concurrent with, or subsequent to, contact of said antigenic molecule of said first source with the sample of body fluid being screened; (e) means for providing direct or indirect detectable labelling means to said antigenic molecule of said second source as present in a complex as defined in (c), prior to, or concurrent with, or subsequent to, contact of said antigenic molecule of said second source with the sample of body fluid being screened; and (f) means for detecting the presence of complexes as defined in (c) immobilised as defined in (d) so as to provide an indication of analyte autoantibodies present in said sample. Alternatively, a preferred embodiment of the present invention may suitably comprise a kit for screening a sample of body fluid obtained from an animal subject for analyte autoantibodies reactive with GAD, or one or more variants, analogues, derivatives or fragments thereof, said kit comprising: (a) first and second sources of antigenic molecules consisting essentially of GAD antigenic molecules selected from GAD65, GAD67, one or more variants, analogues, derivatives or fragments thereof with which analyte autoantibodies when present in said sample of body fluid can interact and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67 and said one or more variants, analogues, derivatives or fragments Ihereof, wherein GAD antigenic molecules of said first source are immobilised to a solid support as defined in (c) and GAD antigenic molecules of said second source are provided with labelling means as defined in (d); (b) means for contacting said antigenic molecules as provided by (a) simultaneously or successively with said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fluid can interact with said antigenic molecules so as to form one or more complexes comprising [GAD antigenic molecule of said first source]-[analyte autoantibody]-[ GAD antigenic molecule of said second source]; (c) means for immobilising to a solid support said GAD antigenic molecule of said first source as present in a complex as defined in (b), prior to, or concurrent with, or subsequent to, contact of said GAD antigenic molecule of said first source with the sample of body fluid being screened; (d) means for providing direct or indirect detectable labelling means to said GAD antigenic molecule of said second source as present in a complex as defined in (b), prior to, or concurrent with, or subsequent to, contact of said GAD antigenic molecule of said second source with the sample of body fluid being screened; and (e) means for detecting the presence of complexes as defined in (b) immobilised as defined in (c) so as to provide an indication of analyte autoantibodies present in said sample. Alternatively, a preferred embodiment of the present invention may suitably comprise a kit for screening a sample of body fluid obtained from an animal subject for analyte autoantibodies reactive with IA-2, or one or more variants, analogues, derivatives or fragments thereof, said kit comprising: (a) first and second sources of antigenic molecules consisting essentially of IA-2 antigenic molecules selected from IA-2, one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from IA-2 and said one or more variants, analogues, derivatives or fragments thereof, wherein IA-2 antigenic molecules of said first source are immobilised to a solid support as defined in (c) and IA-2 antigenic molecules of said second source are provided with labelling means as defined in (d); (b) means for contacting said IA-2 antigenic molecules as provided by (a) simultaneously or successively with said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fluid can interact with said IA-2 antigenic molecules so as to form one or more complexes comprising [IA-2 antigenic molecule of said first source]-[analyte autoantibody]-[IA-2 antigenic molecule of said second source]; (c) means for immobilising to a solid support said IA-2 antigenic molecule of said first source as present in a complex as defined in (b), prior to, or concurrent with, or subsequent to, contact of said IA-2 antigenic molecule of said first source with the sample of body fluid being screened; (d) means for providing direct or indirect detectable labelling means to said IA-2 antigenic molecule of said second source as present in a complex as defined in (b), prior to, or concurrent with, or subsequent to, contact of said IA-2 antigenic molecule of said second source with the sample of body fluid being screened; and (e) means for detecting the presence of complexes as defined in (b) immobilised as defined in (c) so as to provide an indication of analyte autoantibodies present in said sample. Alternatively, a preferred embodiment of the present invention may suitably-comprise a kit for screening a sample of body fluid obtained from an animal subject for first and second analyte autoantibodies respectively reactive with GAD and IA-2, or one or more variants, analogues, derivatives or fragments thereof, said kit comprising: (a) first and second sources of GAD antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact, and which GAD antigenic molecules are selected from GAD65, GAD67, one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67, IA-2 and said one or more variants, analogues, derivatives or fragments thereof and wherein at least one of said antigenic molecules of said fusion molecule comprises GAD65, GAD67, or one or more variants, analogues, derivatives or fragments thereof, wherein GAD antigenic molecules of said first source are immobilised to a solid support as defined in (d) and GAD antigenic molecules of said second source are provided with labelling means as defined in (e); (b) first and second sources of IA-2 antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which IA-2 antigenic molecules are selected from IA-2, one or more variants, analogues, derivatives or fragments thereof, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from IA- 2, GAD65, GAD67 and said one or more variants, analogues, derivatives or fragments thereof and wherein at least one of said antigenic molecules of said fusion molecule comprises IA-2 or one or more variants, analogues, derivatives or fragments thereof, wherein IA-2 antigenic molecules of said first source are immobilised to a solid support according as defined in (d) and IA-2 antigenic molecules of said second source are provided with labelling means as defined in (e); (c) means for contacting said antigenic molecules as provided by (a) and (b) simultaneously or successively with said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fluid can interact with said antigenic molecules so as to form one or more complexes comprising [GAD antigenic molecule of said first source]-[analyte autoantibody]-[GAD antigenic molecule of said second source] or [IA-2 antigenic molecule of said first source]-[analyte autoantibody]-[IA-2 antigenic molecule of said second source]; (d) means for immobilising to a solid support said GAD or IA-2 antigenic molecule of said first source as present in a complex as defined in (c), prior to, or concurrent with, or subsequent to, contact of said GAD or IA-2 antigenic molecule of said first source with the sample of body fluid being screened; (e) means for providing direct or indirect detectable labelling means to said GAD or IA-2 antigenic molecule of said second source as present in a complex as defined in (c), prior to, or concurrent with, or subsequent to, contact of said GAD or IA-2 antigenic molecule of said second source with the sample of body fluid being screened; and (f) means for detecting the presence of complexes as defined in (c) immobilised as defined in (d) so as to provide an indication of analyte autoantibodies present in said sample. It may be preferred in a kit according to the present invention that the antigenic molecules of the first and second sources comprise one or more common antigenic molecules. For example, antigenic molecules of said first and second sources may comprise GAD65 or GAD67; alternatively antigenic molecules of the first and second sources may both comprise IA-2. Alternatively, it may be preferred that the antigenic molecules, of either the first or second source, are selected from pancreatic islet cell antigenic molecules and insulin, and the antigenic molecules of the other source comprises one or more variants, analogues, derivatives or fragments of the above mentioned antigenic molecules of either the first or second source, with which analyte autoantibodies when present in said sample of body fluid can interact For example, where the antigenic molecules of either the first or second source comprise GAD65 or GAD67, the antigenic molecules of the other source comprise one or more variants, analogues, derivatives, or fragments of GAD65 or GAD67 with which analyte autoantibodies when present in said sample of body fluid can interact. A further example may be where the antigenic molecules of either the first or second source comprise IA-2, and the antigenic molecules of the other source comprise one or more variants, analogues, derivatives or fragments of IA-2 with which analyte autoantibodies when present in said sample of body fluid can interact. A still further alternative embodiment of the present invention may be where both antigenic molecules of the first and second sources comprise a variant, analogue, derivative or fragment, which may be the same or different, of a common antigenic molecule, with which analyte autoantibodies when present in said sample of body fluid can interact. For example, antigenic molecules of both the first and second sources can comprise a variant, analogue, derivative or fragment, which may be the same or different, of GAD65 or GAD67, with which analyte autoantibodies when present in said sample of body fluid can interact. A further example may be where antigenic molecules of both the first and second sources comprise a variant, analogue, derivative or fragment, which may be the same or different, of IA-2, with which analyte autoantibodies when present in said sample of body fluid can interact. It will be appreciated from the above that where antigenic molecules of the first and second sources comprise variants, analogues, derivatives or fragments derived from a common antigenic molecule, such variants, analogues, derivative or fragments as provided by the respective sources, may be the same or different (for example variant 1 provided by the first source of antigenic molecules and variant 2 provided by the second source of antigenic molecules, where variants 1 and 2 may respectively represent different variants of GAD65 or GAD67, or fragment 1 provided by the first source of antigenic molecules and fragment 2 provided by the second source of antigenic molecules, where fragments 1 and 2 may respectively represent different fragments of GAD65 or GAD67, such as distinct, or possibly overlapping, epitopes of GAD65 or GAD67). Suitably the detectable labelling means can be selected from the group consisting of enzymic labels, isotopic labels, chemiluminescent labels, fluorescent labels, dyes and the like and typically can be selected from the group consisting of alkaline phosphatase, horseradish peroxidase, biotin-or the like and in particular can comprise biotin. Suitably such detectable labelling means (when attached to the one or more antigenic molecules substantially as hereinbefore described, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof) can be reacted with one or more substrates therefor (such as an avidin or streptavidin conjugate, for example, streptavidin horseradish peroxidase conjugate or the like), whereby the resulting conjugates can be detected suitably by measurement of optical density or the like. The detectable labelling means may be directly provided to the one or more antigenic molecules, or one or more one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, substantially as hereinbefore described. Additionally or alternatively the detectable labelling means may be indirectly provided to the one or more antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, substantially as hereinbefore described, typically by providing the detectable labelling means to one or more antibodies or other binding agents which can bind with the one or more antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, substantially as hereinbefore described. A kit for screening for autoantibodies according to the present invention typically further comprises means for directly monitoring interaction of (i) such autoantibodies present in the sample of body fluid from the subject and (ii) one or more antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, substantially as hereinbefore described, as provided by the present invention, typically by employing non-competitive sandwich type assay techniques known in the art. According to a preferred embodiment of the present invention antigenic molecules of one or more first sources, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, substantially as hereinbefore described, are immobilised to a solid support and antigenic molecules of one or more second sources, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion- molecules thereof, substantially as hereinbefore described, are provided with detectable labelling means, and wherein preferably the antigenic molecules of the one or more second sources are provided in solution phase as is conventional for use in known ELIS A techniques. Preferably, a kit according to the present invention comprises immobilising means whereby antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, with which analyte autoantibodies when present in said sample of body fluid can interact, of one or more first sources are immobilised to a solid support prior to contact with a sample of body fluid being screened. Suitably a kit according to the present invention comprises contacting means whereby such immobilised antigenic molecules of the one or more first sources, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, with which analyte autoantibodies when present in said sample of body fluid can interact, can be contacted with the sample of body fluid being screened either simultaneously or successively with contact of the sample of body fluid with antigenic molecules of one or more second sources, or more one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, with which analyte autoantibodies when present in said sample of body fluid can interact. Particularly preferably, contacting means are provided whereby immobilised antigenic molecules of the one or more first sources, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, with which analyte autoantibodies when present in said sample of body fluid can interact, are contacted with the sample of body fluid being screened so as to form an intermediate complex comprising [antigenic molecule] - [analyte autoantibody] where the antigenic molecule is immobilised to a solid support and the thus formed immobilised intermediate complex is subsequently contacted with molecules of the one or more second sources, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, with which analyte autoantibodies when present in said sample of body fluid can interact, present in solution phase, so as to form the hitherto described complex comprising [antigenic molecule of said first source]-[analyte autoantibody]-[antigenic molecule of said second source] immobilised to a solid support via the antigenic molecule of said first source. Accordingly, the present invention provides a kit for screening a sample of body fluid obtained from an animal subject for analyte autoantibodies reactive with one or more antigenic molecules selected from pancreatic islet cell antigenic molecules and insulin, or one or more variants, analogues, derivatives or fragments thereof, said kit comprising: (a) one or more first sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from pancreatic islet cell antigenic molecules, insulin, one or more variants, analogues, derivatives or fragments of said pancreatic islet cell antigenic molecules or insulin, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from pancreatic islet cell antigenic molecules, insulin and said one or more variants, analogues, derivatives or fragments thereof, wherein said antigenic molecules of said one or more first sources are immobilised to a solid support; (b) one or more second sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from pancreatic islet cell antigenic molecules, insulin, one or more variants, analogues, derivatives or fragments of said pancreatic islet cell antigenic molecules or insulin, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from pancreatic islet cell antigenic molecules, insulin and said one or more variants, analogues, derivatives or fragments thereof, which antigenic molecules of said one or more second sources are provided in solution phase and are provided with labelling means as defined in (d); (c) means for contacting said antigenic molecules as provided by (a) and (b) simultaneously or successively with said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fluid can interact with said antigenic molecules so as to form one or more immobilised complexes comprising [antigenic molecule of said first source]-[analyte autoantibody]-[antigenic molecule of said second source], wherein said antigenic molecules of said first and second sources when present in said one or more complexes comprise, or are derived from, a common antigenic molecule, or wherein binding regions of said antigenic molecules of said first and second sources, for said analyte autoantibody, when present in said one or more complexes are present in, or are derived from, a common antigenic molecule, which immobilised complexes are immobilised to a solid support via said antigenic molecule of said first source present in said complex; (d) means for providing direct or indirect detectable labelling means to said antigenic molecule of said second source as present in a complex as defined in (c), prior to, or concurrent with, or subsequent to, contact of said antigenic molecule of said second source with said sample of body fluid being screened; and (e) means for detecting the presence of complexes as defined in (c) so as to provide an indication of analyte autoantibodies present in said sample. Other contacting means may, however, be present in a kit according to the present invention, for example it is possible according to the present invention not to provide means for initially immobilising the antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, of the one or more first antigen sources, prior to a contacting step as described above, but to provide means to initially contact the solid support substantially as hereinbefore described with a binder for the antigenic molecules, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, of the one or more first antigen sources and for the kit to comprise means for subsequently contacting the so treated solid support with the above described antigenic molecules or the one or more first and second sources, or one or more variants, analogues, derivatives or fragments thereof, or one or more fusion molecules thereof, and the sample of body fluid being screened. In respect of the solid supports and conditions employed in a kit according to the present invention, the supports and conditions do not generally fundamentally differ from conventional supports and conditions employed in known immunoassay techniques. A solid support for use in a kit according to the present invention can comprise an ELISA plate as currently employed in known ELISA techniques, or may employ any other suitable support for use in the present invention, such as tubes, particles, magnetic beads, nitrocellulose or the like. Detection of analyte autoantibodies reactive with one or more antigenic molecules selected from pancreatic islet cell antigenic molecules and insulin according to a method provided by the present invention, or employing a kit provided by the present invention, can be useful in the diagnosis of a number of disease states with which the presence of such analyte autoantibodies is associated. In particular, the present invention can be employed in screening a sample of body fluid from an animal subject suspected of suffering from, susceptible to or having any of the following disease states - type 1 diabetes mellitus and / or stiff man syndrome, type 2 diabetes mellitus, one or more autoimmune thyroid diseases, celiac disease, one or more connective tissue diseases, adrenal autoimmunity, or a combination of two or more different autoimmune diseases and the present invention further provides a method of diagnosing any of the above disease states in an animal subject. It will be appreciated from the foregoing description that the present invention provides assay methods and kits for detecting, in a sample of body fluid obtained from an animal subject, analyte autoantibodies reactive with one or more antigenic molecules selected from pancreatic islet cell antigenic molecules and insulin and indicative of the onset or presence of the above disease states. The detection of such autoantibodies in the sample of body fluid (or at least the level of such autoantibodies in the sample) is indicative of the likely onset or presence of such disease states in the subject from which the sample is obtained and can, therefore, enable the diagnosis of the likely onset or presence of such disease states. There is, therefore, further provided by the present invention a method of diagnosing the likely onset or presence of disease states, associated with the presence of autoantibodies reactive with one or more antigenic molecules selected from pancreatic islet cell antigenic molecules and insulin, in an animal subject (in particular a human subject); suspected of having or being susceptible to these disease states, the method . comprising detecting autoantibodies reactive with one or more antigenic molecules selected from pancreatic islet cell antigenic molecules and insulin, and-being indicative of the likely onset or presence of these disease states in a sample of body fluid from the subject substantially as hereinbefore described, and whereby the detected autoantibodies can provide a diagnosis of the likely onset or presence of these disease states. There is, therefore, still further provided by the present invention a method of delaying or preventing the onset of disease states, associated with the presence of autoantibodies reactive with one or more antigenic molecules selected from pancreatic islet cell antigenic molecules and insulin, in an animal subject (in particular a human subject), or treating an animal subject (in particular a human subject) having or recovering from such disease states, which method comprises initially detecting autoantibodies reactive with one or more antigenic molecules selected from pancreatic islet cell antigenic molecules and insulin, and being indicative of the onset or presence of such disease states, in a sample of body fluid obtained from the subject substantially as hereinbefore described, thereby providing a diagnosis of the likely onset or presence of such disease states in the subject, and administering to the subject a therapeutically effective amount of at least one therapeutic agent effective in delaying onset, obviating, preventing and/or treating these disease states. There is, therefore, still further provided by the present invention a method of assessing the effectiveness of delaying or preventing the onset of disease states, associated with the presence of autoantibodies reactive with one or more antigenic molecules selected from pancreatic islet cell antigenic molecules and insulin, in an animal subject (in particular a human subject), or assessing the effectiveness of treating an animal subject (in particular a human subject) having or recovering from such disease states, which method comprises administering to the subject a therapeutically effective amount of at least one therapeutic agent effective in delaying onset, obviating, preventing and/or treating such disease states associated with the presence of autoantibodies reactive with one or more antigenic molecules selected from pancreatic islet cell antigenic molecules and insulin, and detecting autoantibodies indicative of the onset or presence of such disease states in a sample of body fluid obtained from the subject, so as to provide an indication of the presence of said autoantibodies in said sample, thereby providing an indication of the effectiveness of treating the subject, or of delaying or preventing the onset of such disease states in the subject. There is still further provided by the present invention, in combination, a kit substantially as hereinbefore described, together with a therapeutically effective amount of at least one therapeutic agent effective in the treatment of disease states associated with the presence of autoantibodies reactive with one or more antigenic molecules selected from pancreatic islet cell antigenic molecules and insulin. A sample of body fluid being screened by the present invention will typically comprise blood samples or other fluid blood fractions, such as in particular serum samples or plasma samples, but the sample may in principle be another biological fluid, such as saliva or urine or solubilised tissue extracts. The term "antigen" or "antigenic molecules" as used herein denotes a compound with which antibodies as described herein can interact and which is capable of binding to an antibody to form specific antibody-antigen complexes. The antigen or antigenic molecules may be natural or synthetic and modifications thereto are preferably such as to not detrimentally affect the binding properties thereof to a specific antibody. As indicated above, the present invention covers the use of "variants", "analogs", "derivatives" and "fragments" of antigenic molecules as described herein and the terms "variants", "analogs", "derivatives" and "fragments" as used herein can be characterised as polypeptides which retain essentially the same biological function or activity as naturally occurring antigenic molecules and in particular in respect of the binding properties thereof for a specific antibody. Suitably, variants, analogs, derivatives and fragments, and variants, analogs and derivatives of the fragments as described herein, have a primary structural conformation of amino acids in which several or a few (such as 5 to 10, I to 5 or 1 to 3) amino acid residues of the naturally occurring antigenic molecules are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions are deletions which do not alter or substantially alter the biological activity or function of the naturally occurring antigenic molecules. Conservative substitutions can be preferred as hereinafter described in greater detail. More particularly, variants, analogs or derivatives of antigenic molecules suitable for use according to the present invention may be ones in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), or ones in which one or more of the amino acid resides includes a substituent group or the like. Such variants, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein. Most typically, variants, analogs or derivatives are those that vary from a reference (such as naturally occurring antigenic molecules as referred to herein) by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids A, V, L and I; among the hydroxyl residues S and T; among the acidic residues D and E; among the amide residues N and Q; among the basic residues K and R; and among the aromatic residues F and Y. More particularly, the term "fragment" as used herein denotes a polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of naturally occurring amino acids as referred to herein, and variants or derivatives thereof and such fragments may be "free standing", i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region. In the context of the present invention, it will be appreciated that particularly preferred fragments for use according to the present invention may be one or more epitopes of antigenic molecules as referred to herein and as described above such epitope fragments may be used in "free standing" form or may be used within a larger polypeptide, such as a scaffold polypeptide, of which they form a part or region. The use of such epitope fragments in substantially isolated or free standing form according to the present invention can be advantageous in terms of enhanced stability of such substantially isolated or free standing epitope fragments when compared with the use of a full length antigen and / or enhanced specificity associated, with the use of such substantially isolated or free standing epitope fragments compared with the use of a full length antigen. The present invention will now be further illustrated by the following example, which does not limit the scope of the invention in any way. Example: Preparation of GAD65 coated ELISA plates Human recombinant GAD65 of >95% purity as assessed by SDS-PAGE (M Powell, L Prentice, T Asawa, R Kato, J Sawicka, H Tanaka, V Petersen, A Munkley, S Morgan, B Rees Smith, J Furmaniak. Clinica Chimica Acta, 1996 256: 175-188) was diluted to 150ug/L in a coating buffer comprising 1.59g/L Na2CO3, 2.94g/L NaHCO3, 0.1g/L NaN3, 0.01g/L Phenol Red, and 5mg/L BSA (pH 9.2) and 150uL of the resulting solution was added to 96-well ELISA plates (available under the trade mark Nunc F8 MaxiSorp). The plates were then incubated overnight at 4°C, the contents of the wells aspirated and the wells washed three times with a high salt buffer (HSB) comprising 10g/L BSA, lg/L NaN3, 11.69g/L NaCl, 18.17g/L Tris, and 10mL/L Polyoxyethylene-Sorbitan Monolaurate (available under the trade mark Tween 20 (pH 8.3). A post-coat buffer comprising 3g/L BSA, 9g/L NaCl, 20g/L sucrose and 0.2g/L NaN3 was added to the wells (250uL per well) and incubated for 30 minutes at room temperature. The post-coat buffer was then aspirated, the plates dried under vacuum and stored in sealed bags with silica gel at 4°C until use. Labelling of GAD65 with bio tin Human recombinant GAD65 in PBS (1.15g/L Na2HPO4, 0.2g/L KH2PO4, 0.2g/L KCl, 8g/L NaCl) was reacted with a commercial biotinylation reagent (Sulfo-NHS-LC-LC- Biotin available under the trade mark EZ-Link from Perbio Science), at a molar ratio of 1 part GAD65 to 24 parts biotinylation reagent for 30 minutes at room temperature. The reaction was stopped by dialysis against PBS at 4°C and the resulting material was stored at -70°C in small aliquots. The resulting GAD65 labelled with bio tin according to the above techniques (GAD65- Bi) was diluted to 3mg/L in 20g/L BSA, filtered through a 0.22m filter, aliquotted at 1mL per vial, freeze dried and then stored at -20°C. To use, 7.5mL of HSB was added to the vial, giving a final concentration of GAD65-Bi of 400g/L. Preparation of calibrators for GAD65 autoannbody assay IgG preparations of three human monoclonal autoantibodies to GAD65 (M J Powell, N Hayakawa, M Masuda, J Sanders, M Evans, LDKE Premawardhana, J Furmaniak, B Rees Smith. Isolation and characterization of three human monoclonal antibodies to glutamic acid decarboxylase (GAD65) from a patient without clinical diabetes. Diabetes/Metabolism Research and Reviews, 2001 17 (SI): 021) were diluted in HSB to give an absorbance at OD450nm from 4.0 to 0.1. These monoclonal antibodies are known to be representative of GAD65 autoantibodies found in sera from patients with type 1 diabetes (M J Powell, N Hayakawa, M Masuda, J Sanders, M Evans, LDKE Premawardhana, J Furmaniak, B Rees Smith. Isolation and characterization of three human monoclonal antibodies to glutamic acid decarboxylase (GAD65) from a patient without clinical diabetes. Diabetes/Metabolism Research and Reviews, 2001 17 (S1): 021). GAD65 Autoantibodv Detection Plates coated with GAD65 prepared according to the above techniques were brought to room temperature and 25L of undiluted serum sample obtained from test donors, or calibrators prepared according to the above techniques, were added to the plate wells in duplicate and incubated for 1 hour at room temperature with shaking at 200rpm. The samples were then aspirated, the wells washed three times with buffer (8.7g/L NaCl, 2.4g/L Tris, 0.5mL/L Tween 20, pH 7.6), followed by addition of lOOuL of GAD65-Bi diluted in HSB (400g/L) prepared according to the above techniques and incubation for 1 hour at room temperature, with shaking at 200rpm. After the incubation the contents of the wells were aspirated and wells washed three times with buffer (8.7g/L NaCl, 2.4g/L Tris, 0.5mL/L Tween 20, pH 7.6). Thereafter, lOOuL of a streptavidin horseradish peroxidase conjugate diluted to 1g/mL was added and incubated for 20 minutes at room temperature, with shaking at 200rpm. After aspiration, the wells were washed three times with buffer (8.7g/L NaCl, 2.4g/L Tris, 0.5mL/L Tween 20, pH 7.6) and once with water.. lOOuL of tetramethyl benzidine was then added and incubated for 20 minutes in the dark, followed by addition of 50uL of 0.5 mol/L H2SO4. The absorbance of the wells was then measured in an ELISA plate reader at 450nm or in the case of samples giving an OD 450nm>4.0, absorbance was also measured at 405nm. The above procedures were applied to IA-2 autoantibodies and also to the combined detection of GAD65 and IA-2 autoantibodies (in particular, involving the preparation of IA-2 labelled with biotin, IA-2-Bi). Reference preparations of IA-2 autoantibodies for generation of a calibration curve were prepared from polyclonal IA-2 autoantibodies. Results A typical calibration curve for GAD65 obtained at OD 450nm is shown in Figure la. Seven calibrators of arbitrarily assigned GAD65 autoantibody ELISA units from 0 ELISA units/mL to 512 ELISA units/mL were included. OD 450nm values for the calibrators were 0.033, 0.168, 0.310, 1.020, 3.187, >4.0 and >4.0 for calibrators 0, 1.0, 2.0, 8.0, 32, 128 and 512 respectively. OD 405nm values were 0.008, 0.048, 0.090, 0.290, 0.929, 1.385 and 1.620 for calibrators 0, 1.0, 2.0, 8.0, 32, 128 and 512 respectively as shown in Figure 1b. Consequently, samples that gave OD 450nm values >4.0 could be measured within the calibration curve range at OD 405nm as shown in Figure lb. The mean ±SD absorbance at 450nm for n=25 sera from healthy blood donors was 0.04 ± 0.015. Results of GAD65 autoantibody measurement according to the present invention are shown in Tables 1 and 2. Results of GAD65 autoantibodies in sera from n=18 patients with type 1 diabetes mellitus and n=14 patients with Graves' disease are shown in Table 1. All 18 diabetic sera were positive for GAD65 autoantibodies when ELISA based techniques according to the present invention were employed (levels >1.0 ELISA units/mL), however only 13 of these patients were positive in radioimmunoassay (RIA). The 5 samples which were negative in RIA showed low levels of GAD65 autoantibodies in ELISA based techniques according to the present invention with OD 450nm between 0.117 to 0.980 (1.3 to 10.7 ELISA units/mL). In the case of samples with low levels in RIA (1.0 to 7.5 RIA units/mL), the OD 450nm values in ELISA based techniques according to the present invention were between 0.290 and 1.060 (12.0 to 105.8 ELISA units/mL). In the case of samples with higher levels of GAD65 autoantibodies in RIA (26.8 to 118.8 RIA units/mL), OD 450nm in ELISA based techniques according to the present invention were very high>4.0(>128 ELISA units/mL) and, therefore, the relationship between the GAD65 autoantibody levels in the sample and the calibration curve was calculated after measurement at 405nm. Preincubation of test sera with non-labelled GAD65 (at final concentration from 0.0005 to 0.01g/mL) prior to performing the ELISA resulted in a dose dependent reduction of OD 450nm values. These experiments confirmed the specificity of the GAD65 autoantibody ELISA based techniques according to the present invention. In the case of n=14 sera from patients with Graves' disease, OD 450nm values for all samples were below 0.05 ( to the present invention); all these samples were negative for GAD65 autoantibodies in RIA ( Comparison of the lowest detection limit for GAD65 autoantibodies in ELISA based techniques according to the present invention and RIA is shown in Table 2. In the RIA dilutions of WHO reference preparation for islet cell autoantibodies [NIBSC 97/550] from 250 to 31.25 WHO units/mL, detectable levels of GAD65 autoantibodies were seen. However, in the ELISA based techniques according to the present invention, GAD65 autoantibodies were detectable in the case of WHO standard dilutions from 250 to 3.91 WHO units/mL. Further, an example of the dilution profile of a serum sample Z (from a patient with high levels of GAD65 autoantibodies) is shown in Table 2. In the case of RIA, GAD65 autoantibodies were detectable in the sample Z in the range from 1/8 to 1/8192 dilution. In the case of ELISA based techniques according to the present invention, GAD65 autoantibodies were detectable in the range from 1/8 to 1/32768 dilution. Consequently, the end point dilution analyses of WHO standard and patient's serum indicated that a 4-fold to 8-fold greater dilutions were detectable in ELISA based techniques according to the present invention compared to RIA. An example of results of IA-2 autoantibody measurement by ELISA based techniques according to the present invention is shown in Table 3. Typical OD 450nm values for the set of IA-2 autoantibody calibrators (0, 1:0, 2.0, 4.0, 8.0, 16, 32, 64, 128 ELISA units/mL) were from 0.054 to >4.0 and OD405 values were from 0.008 to 1.874. Sera from n=10 patients with type 1 diabetes mellitus who had IA-2 autoantibody levels in RIA from 2.0 to 38.9 RIA units/mL were all positive in ELISA based techniques according to the present invention. These samples gave OD 450nm values between 0.209 to >4.0 (2.6 to >32 ELISA units/mL). WHO standard was detectable in IA-2 autoantibody RIA at 250 WHO units/mL and 125 WHO units/mL however, further dilutions (62.5 WHO units/mL and 31.25 WHO units/mL) were detectable in ELISA based techniques according to the present invention. OD 450nm values in the case of n=10 sera from healthy blood donors (all negative for IA-2 autoantibodies in RIA) were between 0.030 and 0.071 ( Results of a combination assay for GAD65 autoantibodies and IA-2 autoantibodies are shown in Tables 4 and 5. Table 4 shows results of combined measurement of GAD65 autoantibodies and IA-2 autoantibodies in ELISA based techniques according to the present invention, when IA-2-Bi or GAD65-Bi or IA-2-Bi plus GAD65-Bi were used in the assay. In the case of IA-2 autoantibody calibrators, no signal in the ELISA based techniques according to the present invention was detected when GAE>65-Bi was used, whereas dose dependent increase of OD 450nm was observed when IA-2-Bi or IA-2- Bi plus GAL>65-Bi were used. The OD 450nm signal with IA-2-Bi alone was essentially the same as signal with IA-2-Bi plus GAD65-Bi. In the case of GAD65 autoantibody calibrators, no signal was detected when IA-2-Bi was used in the assay while dose dependent response of OD 450nm was observed with GAD65-Bi and IA-2- Bi plus GAD65-BL The OD 450nm signals in the case of GAD65-Bi alone and IA-2- Bi plus GAD65-Bi plus GAD65-Bi were comparable. Measurement of both autoantibodies in different dilutions of serum sample Z showed a good agreement with the results of IA-2 autoantibodies or GAD65 autoantibodies alone. Serum from an individual healthy blood donor or healthy blood donor pool serum gave very low OD 450nm values from 0.006 to 0.046 irrespective of which biotinylated antigen was used. This experiment shows that in the combined assay to measure IA-2 autoantibodies and GAD65 autoantibodies, the results are specific and reflect the presence of the two autoantibodies in the test sample. More detailed results of the combined measurement of IA-2 autoantibodies and GAD65 autoantibodies are shown in Table 5. Sera from patients with Type 1 diabetes were tested in the assay. IA-2 autoantibodies and GAD65 autoantibodies wore assessed in these sera by RIA; sera 1-3 were positive for both IA-2 autoantibodies and GAD65 autoantibodies, sera 4-6 were negative for GAD65 autoantibodies but positive for IA-2 autoantibodies, sera 7-9 were positive for GAD65 autoantibodies but negative for IA-2 autoantibodies and sera 10-12 were negative for both IA-2 or GAD65 autoantibodies. As shown in Table 5, in the combined assay one or the other or bom autoantibodies were detected in serum samples. Furthermore, in the case of serum 6 that had borderline levels of IA-2 autoantibodies by RIA (0.9 RIA units/mL), the signal in the combined ELISA based techniques according to the present invention was clearly positive. Additional examples of results with ELISA for GAD65 autoantibodies, IA-2 autoantibodies and combined G AD65 and IA-2 autoantibodies according to the present invention for different patient subgroups are shown in Tables 6, 7 and 8 respectively. Conclusions The above results show that the non-radioactive based techniques according to the present invention allow measurement of GAD65 autoantibodies in test samples. The non-radioactive based techniques according to the present invention can also be employed to measure IA-2 autoantibodies separately or simultaneously. The measurements are specific and they are in agreement with the results of GAD65 autoantibodies and IA-2 autoantibodies measurements by established radioactive reference methods (M Powell, L Prentice, T Asawa, R Kato, J Sawicka, H Tanaka, V Petersen, A Munkley, S Morgan, B Rees Smith, J Furmaniak. Clinica Chimica Acta, 1996 256: 175-188; and M Masuda, M Powell, S Chen, C Beer, P Fichna, B Rees Smith, J Furmaniak. Autoantibodies to IA-2 in insulin-dependent diabetes mellitus. Measurements with a new immunoprecipitation assay. Clinica Chimica Acta, 2000 291: 53-66). Furthermore, measurements of GAD65 autoantibodies and IA-2 autoantibodies by non-radioactive based techniques according to the present invention are at least as sensitive compared to results by RIA (Tables 1, 2 and 3). The techniques according to the present invention provide a non-radioactive assay to measure GAD65 autoantibodies or IA-2 autoantibodies with a high sensitivity that is convenient to use for diagnostic and screening purposes. A combination assay for GAD65 and IA-2 autoantibodies according to the present invention can be of particular benefit for use in large population screening programmes. Footnote: GAD Ab levels above 1 RIA unit/mL were considered positive in RIA. * WHO units/mL. Values of samples positive in GAD65 Ab RIA were 33, 100 and 2500 units/mL. The same samples were negative, negative and >500 units/mL in the ELISA respectively. 3 Range of values: 5.2 - greater than 500 units/mL. 4 Range of values: negative - 3800 units/mL. 5 Value of a sample positive in GAD65 Ab ELISA was 40 units/mL. The same sample was negative in the RIA. 6 Values of samples positive in GAD65 Ab ELISA were 306 and 500 units/mL. The same samples were 354 units/mL and 1700 units/mL in the RIA, respectively. 7 Values of samples positive in GAD65 Ab RIA were 78, 354 and 1700 units/mL. The same samples were negative, 306 and 500 units/mL in the ELISA, respectively. 8 Value of a sample positive in GAD65 Ab ELISA was 24 units/ml; this sample was negative in the RIA. 9 The same sample was positive in GAD65 Ab ELISA and RIA with values of 15 units/mL and 30 units/mL, respectively. 1 Values of samples positive in IA-2 Ab ELIS A were 44 units/mL and 179 units/mL (units/mL are WHO 97/550). Adsorption experiments indicated the presence of specific IA-2 Ab in these samples. The same samples were negative in the RIA. 2 Values of IA-2 Ab positive samples in RIA were 150 units/mL and 28S units/mL. The same samples were negative in the ELIS A. 3 Range of values: 132 - >4000 units/mL. 4 Range of values: 178- 4508 units/mL. 5 Value of IA-2 Ab positive sample was 101 units/mL. This sample was negative in the RIA. 1 Value of GAD65 Ab for samples positive in ELISA ranged from 7.3 - >500 units/mL (units/mL are WHO 97/550). 2 Values of IA-2 Ab for samples positive in ELISA ranged from 34-3613 units/mL (units/mL are WHO 97/550). 3 All combined ELISA results were expressed as index calculated as follows: Oik™ n^ of sample ; OE^sonm of pool of healthy blood donor sera values of 2.0 or greater are positive 18 sera were positive for GAD65 Ab and negative for IA-2 Ab; all 18 were positive in the combined ELISA. 4 sera were positive for IA-2 Ab and negative for GAD6s Ab; all 4 were positive in the combined ELISA 10 sera were positive for GAD65 Ab and for IA-2 Ab; all 10 were positive in the combined ELISA 1 serum was negative for GAD65 Ab and for IA-2 Ab but positive in the combined ELISA 2 sera were negative in all 3 assays. 4 GAD65 Ab levels were 10 units/mL and IA-2 Ab were negative in the one positive sample. 5 GAD65 Ab were negative and IA-2 Ab levels were 76 units/mL in the one positive sample. 6 The positive samples were the same as described in 4 and 5 (index value 7.8 and 4.8 respectively). GAD65 Ab ELISA, IA-2 Ab ELISA and combined GAD65 Ab + IA-2 Ab ELISA results in different patient groups. 7 GAD65 Ab levels were 9 and >500 units/mL but IA-2 Ab were negative in the two positive samples. The positive samples were the same as described in 7. The index values for these samples were: 3.6 and 55.0 respectively. GAD65 Ab levels were 15 units/mL but IA-2 Ab were negative. The positive sample was the same as described in 9. The index value was 6.0. WE CLAIM: 1. A method of screening a sample of body fluid obtained from an animal subject for analyte autoantibodies reactive with one or more pancreatic islet cell antigenic molecules selected from the group consisting of GAD65, GAD67 and protein tyrosine phosphatase-like islet cell antigen (IA-2), or one or more variants, analogues, derivatives or fragments thereof, said method comprising: (a) providing one or more first sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from said group of pancreatic islet cell antigenic molecules, one or more variants, analogues, derivatives or fragments of said pancreatic islet cell antigenic molecules and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from pancreatic islet cell antigenic molecules, and said one or more variants, analogues, derivatives or fragments thereof; (b) providing one or more second sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from said group of pancreatic islet cell antigenic molecules, one or more variants, analogues, derivatives or fragments of said group of pancreatic islet cell antigenic molecules, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from pancreatic islet cell antigenic molecules, and said one or more variants, analogues, derivatives or fragments thereof; (c) contacting said antigenic molecules as provided by steps (a) and (b) simultaneously or successively with said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fluid can interact with said antigenic molecules so as to form one or more complexes comprising [antigenic molecule of said first source]-[analyte autoantibody]-[antigenic molecule of said second source], wherein said molecules of said first and second sources when present in said one or more complexes comprise, or are derived from, a common antigenic molecule, or wherein binding regions of said antigenic molecules of said first and second sources, for said analyte autoantibody, when present in said one or more complexes are present in, or are derived from, a common antigenic molecule; (d) prior to, or concurrent with, or subsequent to, step ( c ), providing immobilizing means whereby said antigenic molecule of said first source as present in a complex as formed in step (c ) is immobilized to a solid support prior to, or concurrent with, or subsequent to, step (c); (e) prior to, or concurrent with, or subsequent to, step ( c ), providing direct or indirect detectable labelling means whereby said antigenic molecule of said second source as present in a complex as formed in step ( c ) is provided with such direct or indirect detectable labelling means prior to, or concurrent with, or subsequent to, step (c); and (f) detecting the presence of complexes formed in (c ) immobilized according to (d) so as to provide an indication of analyte autoantibodies present in said sample. 2. A method as claimed in any of claim 1, which comprises providing (i) first and second sources of GAD antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact, and which GAD antigenic molecules are selected from GAD 65, GAD67, one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67, protein tyrosine phosphatase-like islet cell antigen (IA-2) and said one of more variants, analogues, derivatives or fragments thereof and wherein at least one of said antigenic molecules of said fusion molecule comprises GAD65, GAD67, or one or more variants, analogues, derivatives or fragments thereof, wherein GAD antigenic molecules of said first source are immobilized to a solid support according to step (d) and GAD antigenic molecules of said second source are provided with labelling means according to step (e); and (ii) first and second sources of protein tyrosine phosphatase-like islet cell antigen (IA-2), antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which protein tyrosine phosphatase-like islet cell antigen (IA-2) and antigenic molecules are selected from protein tyrosine phosphate-like islet cell antigen (IA-2), one or more variants, analogues, derivatives or fragments thereof, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from protein tyrosine phosphatase-like islet cell antigen (IA-2), GAD65, GAD67 and said one or more variants, analogues, derivatives or fragments thereof and wherein at least one of said antigenic molecules of said fusion molecules comprises protein tyrosine phosphatase-like islet cell antigen (IA-2) or one or more variants, analogues, derivatives or fragments thereof, wherein protein tyrosine phosphatase-like islet cell antigen (IA-2), antigenic molecules of said first source are immobilised to a solid support according to step (d) and protein tyrosine phosphatase-like islet cell antigen (IA-2) antigenic molecules of said second source are provided with labelling means according to step (e). 3. A method as claimed in any of claims 1 to 2, wherein the antigenic molecules of the first and second sources when present in said one or more complexes comprise one or more common antigenic molecules. 4. A method as claimed in claim 1, as dependent on any of claims 1,2 or 3, wherein one of the antigenic molecules of the first or second source when present in the one or more complexes comprises GAD65, or GAD67, the other antigenic molecule when present in the one or more complexes comprises one or more variants, analogues, derivatives or fragments of GAD65, GAD67, with which analyte autoantibodies when present in said sample of body fluid can interact. 5. A method as claimed in claim 1, as dependent on any of claims 1,2, wherein one of the antigenic molecules of the first or second source when present in the one or more complexes comprises protein tyrosine phosphatase-like islet cell antigen (IA-2) and the other antigenic molecule when present in the one or more complexes comprises one or more variants, analogues, derivatives or fragments of protein tyrosine phosphatase-like islet cell antigen with which analyte autoantibodies when present in said sample of body fluid can interact. 6. A method as claimed in any of claims 1 to 3, wherein both antigenic molecules of the first and second sources when present in the one or more complexes comprise variant, analogue, derivative or fragment, which may be the same or different, of a common antigenic molecule, with which analyte autoantibodies when present in said sample of body fluid can interact. 7. A method as claimed in claim 6, as dependent on any of claims 1 or 2, wherein both antigenic molecules of the first and second sources when present in the one or more complexes comprise a variant, derivative or fragment, which may be the same or different, of GAD65 or GAD67, with which analyte autoantibodies when present in said sample of body fluid can interact. 8. A method as claimed in claim 6, as dependent on any of claims 1, 2 wherein both antigenic molecules of the first and second sources when present in said one or more complexes comprise a variant, analogue, derivative or fragment, which may be the same or different, of protein tyrosine phosphatase-like islet cell antigen (IA-2), with which analyte autoantibodies when present in said sample of body fluid can interact. 9. A method as claimed in any of claims 1 to 8, wherein said antigenic molecules of said one of more first sources are immobilized to a solid support prior to contact with a sample of body fluid being screened. 10. A method as claimed in claim 9, wherein said immobilized antigenic molecules of said one or more first sources are subsequently contacted with the sample of body fluid being screened either simultaneously or successively with contact of the sample of body fluid with said antigenic molecules of said one or more second sources. 11. A method as claimed in claim 10, wherein said immobilized antigenic molecules of said one or more first sources are subsequently contacted with the sample of body fluid being screened so as to form an intermediate complex comprising [antigenic molecule]-[analyte autoantibody] where the antigenic molecule is immobilized to a solid support and the thus immobilized intermediate complex is subsequently contacted with said antigenic molecules of said one or more second sources, present in solution phase, so as to form said complexes comprising [antigenic molecule of said first source]-[analyte autoantibody]-]antigenic molecule of said second source] immobilized to a solid support via the antigenic molecule of the first source. 12. A kit for screening a sample of body fluid obtained from an animal subject for analyte autoantibodies reactive with one or more pancreatic islet cell antigenic molecules selected from the group consisting of GAD65, GAD67 and protein tyrosine phosphatase-like islet cell antigen (IA-2), one or more variants, analogues, derivatives or fragments thereof, said kit comprising: (a) one or more first sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from said group of pancreatic islet cell antigenic molecules, one or more variants, analogues, derivatives or fragments of said pancreatic islet cell antigenic molecules, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from said pancreatic islet cell antigenic molecules, and said one or more variants, analogues, derivatives or fragments thereof; (b) one or more second sources of antigenic molecules with which analtye autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from said pancreatic islet cell antigenic molecules, or one or more variants, analogues, derivatives or fragments of said pancreatic islet cell antigenic molecules, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from said pancreatic islet cell antigenic molecules, and said one or more variants, analogues, derivatives or fragments thereof. (c) means for contacting said antigenic molecules as provided by (a) and (b) simultaneously or successively with said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fluid can interact with said antigenic molecules so as to form one or more complexes comprising [antigenic molecule of said first source]-[analyte autoantibody]- [antigenic molecule of said second source], wherein said antigenic molecules of said first and second sources when present in said one or more complexes comprise, or are derived from, a common antigenic molecule, or wherein binding regions of said antigenic molecules of said first and second sources, for said analtye autoantibody, when present in said one or more complexes are present in, or are derived from, a common antigenic molecule; (d) means for immobilizing to a solid support, said antigenic molecule of said first source as present in a complex as defined in ( c ), prior to, or concurrent with, or subsequent to, contact of the antigenic molecule of said first source with the sample of body fluid being screened; (e) means for providing direct or indirect detectable labelling means to said antigenic molecule of said second source as present in a complex as defined in ( c ), prior to , or concurrent with, or subsequent to, contact of said antigenic molecule of said second source with said sample of body fluid being screened; and (f) means for detecting the presence of complexes as defined in ( c) immobilized as defined in (d) so as to provide an indication of analyte autoantibodies in said sample. 13. A kit as claimed in claim 17, which comprises a first source of antigenic molecules consisting of GAD antigenic molecules selected from GAD65, GAD67, one or more variants, analogues, derivatives or fragments thereof with which analyte autoantibodies when present in said sample of body fluid can interact and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67, and said one or more variants, analogues, derivatives or fragments thereof, immobilized to a solid support as defined in (d), and a second source of antigenic molecules consisting essentially of GAD antigenic molecules selected from GAD65, GAD67, one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67 and said one or more variants, analogues, derivatives or fragments thereof, provided with labelling means as defined in (e). 14. A kit as claimed in claim 17, which comprises a first source of antigenic molecules consisting essentially of protein tyrosine phosphatase-like islet cell antigen (IA-2), antigenic molecules selected from protein tyrosine phosphatase-like islet cell antigen (IA-2), one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from protein tyrosine phosphatase-like islet cell antigen (IA-2) and said one or more variants, analogues, derivatives or fragments thereof, immobilized to a solid support as defined in (d), and a second source of antigenic molecules consisting essentially of protein tyrosine phosphatase-like islet cell antigen (IA-2), selected from protein tyrosine phosphatase-like islet cell antigen (IA-2), one or more variants, analogues, derivatives or fragments thereof, with which analyte autoantibodies when present in said sample of body fluid can interact and fusion molecules selected from protein tyrosine phosphatase-like islet cell antigen (IA-2) and said one or more variants, analogues, derivatives or fragments thereof, provided with labelling means as defined in (e). 15. A kit as claimed in claim 17, which comprises (i) first and second sources of GAD antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact, and which GAD antigenic molecules are selected from GAD65, GAD67, one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly of indirectly fused antigenic (IA-2) molecules selected from GAD65, GAD67, protein tyrosine phosphatase-like islet cell antigen (IA-2) and said one or more variants, analogues, derivatives or fragments thereof and wherein at least one of said antigenic molecules of said fusion molecules comprises GAD65, GAD67, or one or more variants, analogues, derivatives or fragments thereof, wherein GAD antigenic molecules of said first source are immobilized to a solid support as defined in (d) and GAD antigenic molecules of said second source are provided with labelling means as defined in (e); and (ii) first and second sources of protein tyrosine phosphates-like islet cell antigen (IA-2), antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which protein tyrosine phosphatase-like islet cell antigen (IA-2) antigenic molecules are selected from protein tyrosine phosphatase-like islet cell antigen (IA-2), one or more variants, analogues, derivatives or fragments thereof, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from protein tyrosine phosphatase-like islet cell antigen, GAD65, GAD67 and said one or more variants, analogues, derivatives of fragments thereof and wherein at least one of said antigenic molecules of said fusion molecule comprises protein tyrosine phosphatase-like islet cell antigen (IA-2) or one or more variants, analogues, derivatives or fragments thereof, wherein protein tyrosine phosphatase-like islet cell antigen (IA-2) antigenic molecules of said first source are immobilized to a solid support according as defined in (d) and protein tyrosine phosphatase like islet cell antigen (IA-2) antigenic molecules of said second source are provided with labelling means as defined in (e). 16. A kit as claimed in any of claims 12 to 15, wherein the antigenic molecules of the first and second sources comprise one or more common antigenic molecules. 17. A kit as claimed in claim 16, as dependent on any of claims 12 to 13 and 15, wherein said antigenic molecules of said first and second sources comprise GAD65 or GAD67. 18. A kit as claimed in claim 16, as dependent on any of claims 12,14 and 15, wherein said antigenic molecules of the first and second sources comprise protein tyrosine phosphatase-like islet cell antigen (IA-2). 19. A kit as claimed in any of claims 12 to 15, wherein antigenic molecules, of either the first or second source, are selected from said pancreatic islet cell antigenic molecules, and the antigenic molecules of the other source comprises one or more variants, analogues, derivatives or fragments of said antigenic molecules of either the first or second source, with which analyte autoantibodies when present in said sample of body fluid can interact. 20. A kit as claimed in claim 19, as dependent on any of claims 12 to 13 and 15, wherein antigenic molecules of either the first or second source comprise GAD65 or GAD67, and the antigenic molecules of the other source comprise one or more variants, analogues, derivatives or fragments of GAD65 or GAD67, with which analyte autoantibodies when present in said sample of body fluid can interact. 21. A kit as claimed in claim 19, as dependent on any of claims 12,14 and 15, wherein antigenic molecules of either the first or second source comprise protein tyrosine phosphatase-like islet cell antigen (IA-2), and the antigenic molecules of the other source comprise one or more variants, analogues, derivatives or fragments of protein tyrosine phosphatase-like islet cell antigen (IA-2) with which analyte autoanitbodies when present in said sample of body fluid can interact. 22. A kit as claimed in any of claims 12 to 16, wherein both antigenic molecules of the first and second sources comprise a variant, analogue, derivative or fragment, which may be the same or different, of a common antigenic molecule, with which analyte autoantibodies when present in said sample of body fluid can interact. 23. A kit as claimed in claim 22, as dependent of any of claims 12 to 13 and 15, wherein antigenic molecules of both the first and second sources comprise a variant, analogue, derivative or fragment, which may be the same or different, of GAD65 or GAD67, with which analyte autoantibodies when present in said sample of body fluid can interact. 24. A kit as claimed in claim 22, as dependent on any of claims 12,14 and 15, wherein antigenic molecules of both the first and second sources comprise a variant, analogue, derivative or fragment, which may be the same or different, of protein tyrosine phosphatase-like islet cell antigen, with which analyte autoantibodies when present in said sample of body fluid can interact. 25. A kit as claimed in any of claims 12 to 24, which comprises immobilising means whereby antigenic molecules of said one or more first sources are immobilized to a solid support prior to contact with a sample of body fluid being screened. 26. A kit as claimed in claim 25, which comprises contacting means whereby such immobilized antigenic molecules of said one or more first sources are contacted with the sample of body fluid being screened either simultaneously or successively with contact of the sample of body fluid with antigenic molecules of said one or more second sources. 27. A kit as claimed in claim 26, wherein contacting means are provided whereby immobilized antigenic molecules of said one or more first sources are contacted with the sample of body fluid being screened so as to form as intermediate complex comprising [antigenic molecule]- [analyte autoantibody] where the antigenic molecule is immobilized to a solid support and the thus formed immobilized intermediate complex is subsequently contacted with antigenic molecules of said one ore more second source present in solution phase, so as to form said one or more complexes comprising [antigenic molecule of said first source]-[analyte autoantibody]-[antigenic molecule of said second source] immobilized to a solid support via the antigenic molecule of said first source. 28. A method as claimed in any of claims 1 to 11, or a kit as claimed in any of claims 12 to 27, wherein the said sample of body fluid is from an animal subject suspected of suffering from, susceptible to or having one or more of the following disease states, type 1 diabetes mellitus and/or stiff man syndrome, type 2 diabetes mellitus one or more autoimmune thyroid diseases, celiac disease, or more connective tissue diseases, adrenal autoimmunity, or a combination of two or more different autoimmune diseases. 29. A kit as claimed in any of claims 12 to 27, together with a therapeutically effective amount of at least one therapeutic agent effective in the treatment of one more disease states associated with the presence of autoantibodies reactive with one more antigenic molecules selected from said pancreatic islet cell antigenic molecules in an animal subject suspected of suffering from, susceptible to or having type 1 diabetes mellitus and/or stiff man syndrome. 30. A kit as claimed in any of claims 12 to 27, together with a therapeutically effective amount of at least one therapeutic agent effective in the treatment of one or more disease states associated with the present of autoantibodies reactive with one or more antigenic molecules selected from said pancreatic islet cell antigenic molecules in an animal subject suspected of suffering from, susceptible to or having type 2 diabetes mellitus, one or more autoimmune thyroid diseases, celiac disease, one or more connective tissue diseases, adrenal autoimmunity, or a combination of two or more different autoimmune diseases.+++++ 1. A method of screening a sample of body fluid obtained from an animal subject for analyte autoantibodies reactive with one or more pancreatic islet cell antigenic molecules selected from the group consisting of GAD65, GAD67 and protein tyrosine phosphatase-like islet cell antigen (IA-2), or one or more variants, analogues, derivatives or fragments thereof, said method comprising: (a) providing one or more first sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from said group of pancreatic islet cell antigenic molecules, one or more variants, analogues, derivatives or fragments of said pancreatic islet cell antigenic molecules and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from pancreatic islet cell antigenic molecules, and said one or more variants, analogues, derivatives or fragments thereof; (b) providing one or more second sources of antigenic molecules with which analyte autoantibodies when present in said sample of body fluid can interact and which antigenic molecules are selected from said group of pancreatic islet cell antigenic molecules, one or more variants, analogues, derivatives or fragments of said group of pancreatic islet cell antigenic molecules, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from pancreatic islet cell antigenic molecules, and said one or more variants, analogues, derivatives or fragments thereof; (c) contacting said antigenic molecules as provided by steps (a) and (b) simultaneously or successively with said sample of body fluid being screened, whereby analyte autoantibodies when present in said sample of body fluid can interact with said antigenic molecules so as to form one or more complexes comprising [antigenic molecule of said first source]-[analyte autoantibody]-[antigenic molecule of said second source], wherein said molecules of said first and second sources when present in said one or more complexes comprise, or are derived from, a common antigenic molecule, or wherein binding regions of said antigenic molecules of said first and second sources, for said analyte autoantibody, when present in said one or more complexes are present in, or are derived from, a common antigenic molecule; (d) prior to, or concurrent with, or subsequent to, step ( c ), providing immobilizing means whereby said antigenic molecule of said first source as present in a complex as formed in step (c ) is immobilized to a solid support prior to, or concurrent with, or subsequent to, step (c); (e) prior to, or concurrent with, or subsequent to, step ( c ), providing direct or indirect detectable labelling means whereby said antigenic molecule of said second source as present in a complex as formed in step ( c ) is provided with such direct or indirect detectable labelling means prior to, or concurrent with, or subsequent to, step (c); and (f) detecting the presence of complexes formed in (c ) immobilized according to (d) so as to provide an indication of analyte autoantibodies present in said sample.

Full Text

The present invention relates to methods and kits useful in the detection of
autoantibodies reactive with pancreatic islet cell antigenac molecules and / or insulin,
for example methods and kits useful in me detection of autoantibodies indicative of
the onset or presence of insulin dependent diabetes mellius (IDDM, type 1 diabetes).
Type 1 diabetes is one of the more prevalent autoimmune diseases affecting man, with
more man 2 million individuals in Europe and North America suffering from the
disease (M A Atkinson, G S Eisenbarth. Type 1 diabetes: new perspectives oa disease
pathogenesis and treatment. The Lancet, 2001 358: 221-229). Furthermore, the
incidence of type 1 diabetes is increasing worldwide by 2.5% per year aad it is
estimated mat the incidence of this disease will be 40% higher in 2010 that in 1998
(M A Atkinson, G S Eisenbarth. Type 1 diabetes: new perspectives on disease
pathogenesis and treatment. The Lancet, 2001 358: 221-229).
Autoantibodies to pancreatic β cell antigens are well recognised serological markers
of type 1 diabetes and these include: autoantibodies reactive with islet eels in
immunofluorescence tests (ICA), autoantibodies to ghitamic acid decarboxylase
(GAD, in particular the 65 kDa isoform thereof,GAD65), autoaatibodies to protein
tyrosine phosphatase-like islet cell antigen (which can be referred to as IA-2 or
ICA512) and autoantibodies to insulin. GAD65 autoantibodies and IA-2
autoantibodies are components of ICA reactivity (M A Atkinson, G S Eisenbarth.
Type 1 diabetes: new perspectives on disease pathogenesis and treatment The
Lancet, 2001 358: 221-229). At least one of the three types of autoantibodies to
GAD65, IA-2 and insulin ate present at diagnosis in sere from about 90% of patients
with type 1 diabetes (M A Atkinson, G S Eisenbarm. Type 1 diabetes: new
perspectives on disease pathogenesis and treatment. The Lancet, 2001 358: 221-229).
Measurement of the above mentioned autoantibodies being serotogical matters of
autoimmunity to pancreatic β cells can also be useful in monitoring patients involved
in trials for prevention of diabetes (M A Atkinson, G S Eisenbarth. Type 1 diabetes:

new perspectives on disease pathogenems and treatment. The Lancet, 2001 358: 221-
229).
Currently, GAD65 autoantibodies are measured using imnamnopcerapitation assays
(IPA) employing GAD65 radiaactfveiy labelled with 35S, 3H or 125I GAD65 labelled
with 35S or 3H has been produced in an in vitro transcription/translation system (C E
Grubin, T Daniels, B Toivola, M Landdin-Olsson, W A Hagopian, L Li, A E Karlsen,
E Boel, B Michelsen, A Lemmark. A novel radioligand binding assay to determine
diagnostic accuracy of isoform-specific giutamic acid decarboxylase antibodies in
childhood IDDM. Diabetologia, 1994 37: 344-350; J S Patesm, K R Hejnaes, A
Moody, A E Karlsen, M O Marshall, M H6ier-Madsen, E Bod, B K Michelaen, T
Dyrberg. Detection of GADgs antibodies in diabetes and other autoimmune diseases
using a simple radioligand assay. Diabetes, 1994 43: 459-467; sad A Falorni, E
Ortqvist, B Persson, A Lcramark. Radioimmunoassays for gtutamic acid
decarboxyiase (GAD65) and GAD65 autoantibodies using 35S or 3H recombinant
human Ugands. Journal of Immunological Methods, 1995 186: 88-89). Native
GAD65, for example rat or porcine GAD65 isolated from brain tissue (M J Rowley, I R
Mackay, Q-Y Chen, W J Knowles, P Z Zimmet. Antibodies to glutamic acid
decarboxyiase discriminate major types of diabetes meilitue Diabetes, 1992 41:
548:551; and M Ohta, H Obayasai, K Takahasbi, Y Kitagawa, K Nakano, S Matsuo,
M Ohishi, N Itoh, K Hayashi, K Ohta. A simple solid-phase radioimunoassay for
glutamic acid decarboxyiase (GAD) antibodies in patients wife diabetes mellitus. J
Clin Biochem Nutr, 1996 20: 139-148) or recombinant humae GAD65, for example
expressed in insect cells, yeast or mammalian cells (F Lufader, K-P Woltanski, L
Mauch, H Haubruck, K-D Kohoert, I Rjasaoowski, D Michaelis, M Ziegler.
Detection of autoantibodies to the 65-kD isoform of glutaante decarboxyiase by
radioimmunoassay. European Journal of Endocrinolofy, 1994 130: 575-80; M
Powell, L Prentice, T Asawa, R Kato, J Sawicka, H Tanaka, V Peteraen, A Munkley,
S Morgan, B Rees Smith, J Furmaniak. Clinica Chimica Acta, 1996 256: 175-188;
and T Matsuba, M Yano, No Abiru, H Talano, S Akazawa, S Nagataki, K Yasukawa.
Expression of recombinant human ajhrtaouc acid decarboxylase (GAD) in myeloma
cells and enzyme-linked irnrmmosotbent assay (EL1SA) for autotonbodies to GAD. J
Biochem, 1997 121: 2O-24) have-been labelled with 125I and used in DP As.
Recombinant fusion proteins of GAD65 and IA-2 have alao beea used in IP As for

combined autoantibody testing (A Zavialov, M Ankelo, A Westcriuod-Karlsson, M
Knip, J nioaen, A Hinkkanen Novel fusion proteins in the analysis of diabetes
associated autoantibodies to GAD65 and IA-2. Journal of Immunological Methods,
2000 246: 91-96; and M Rickert, J Seissler, W Dangel, H Lorenz, W Richter. Fusion
proteins for combined aoalysis of autoantibodies to the 65-kDa isoform of glutamic
acid decarboxylase aad istet antigsn-2 in insulin depeadflBt diabetes mellitus. Clinical
Chemistry, 2001 47(5):926-934).
Currently available IP As based on 35S-GAD65 have, however, been technically
demanding, difficult to standardise, relatively expensive and have not been adaptable
to a kit format. Assays based on human recombinant 125I-GAD65 have been of equal
sensitivity and specificity to assays bated on 35S-GAD65, aad have exhibited the
added advantage of good reproductbility, technical simplicity, lower costs and have
been easily adapted into a kit Unfortunately, however, assays based on 125I-GAD65
isolated from rat or porcine brain tissue have suffered from lower specificity due to
contamination with GAD65 isoform.
GAD65 autoantibodies have also been measured by differed types of ELISAs (A M
Gronowski, E C C Wong, T R Wilhite, D L Martin, C H Smith, C A Parvin, M Landt.
Detection of glutamic acid decarboxylase autoantibodies with the varelisa ELISA.
Clinical Chemistry, 1995 41(10): 1532-1534; and H D Mehata, B S Void, S Minkin, E
F Ullman. DELISA: sensitive nonisotopic assay for GAD65 autoantibodies, a key
risk-assessment marker for insulin-dependent diabetes mettitus. Clinical Chemistry,
1996 42(2): 263-269).
However, currently available tests to measure GAD65 autoantibodies by ELISA have
poorer sensitivity and specificity than the above meotioaed IP As aad do not compare
well with the IP As in proficiency studies (R S SchmidH, P G Cofanan, E Bonifacio,
Participating Laboratories. Disease sensitivity and specificity of 52 assays for
glutamic acid decarboxylase antibodies. The second international GADAb workshop.
Diabetes, 1995 44: 636-640).
The present invention, however, alleviates me problems hitherto associted with
methods aad kits employed in the screening of autoaatibodies indicative of the onset

or presence of type 1 diabetes and have general applicability in the detection of
autoantibodies reactive with pancreatic islet cell antigenic molecules and / or insulin.
In particular, the present invention employs methods and kits having unproved
specificity and sensitivity when compared to prior art techniques as discussed above.
There is provided by the present invention a method of screening a sample of body
fluid obtained from an animal subject for analyte autoantibodies reactive with one or
more antigenic molecules selected from pancreatic islet cell antigenic molecules and
insulin, or one or more variants, analogues, derivatives or fragments thereof, said
method comprising:
(a) providing said sample of body fluid from said subject;
(b) providing one or more first sources of antigenic molecules with which
analyte autoantibodies when present in said sample of body fluid can interact
and which antigenic molecules are selected from pancreatic islet cell antigenic
molecules, insulin, one or more variants, analogues, derivatives or fragments
of said pancreatic islet cell antigenic molecules or insulin, and fusion
molecules comprising two or more directly or indirectly fused antigenic
molecules selected from pancreatic Met cell antigenic molecules, insulin and
said one or more variants, analogues, derivatives or fragments thereof;
(c) providing one or more second sources of antigenic molecules with which
analyte autoantibodies when present in said sample of body fluid can interact
and which antigenic molecules are selected from pancreatic islet cell antigenic
molecules, insulin, one or more variants, analogues, derivatives or fragments
of said pancreatic islet cell antigenic molecules or insulin, and fusion
molecules comprising two or more directly or indirectly fused antigenic
molecules selected from pancreatic islet cell antigenic molecules, insulin and
said one or more variants, analogues, derivatives or fragments thereof,
(d) contacting said antigenic molecules as provided by steps (b) and (c)
simultaneously or successively with-said sample of body fluid being screened,
whereby analyte autoantibodies when present in said sample of body fhad can

interact with said antigenic molecules so as to form one or more complexes
comprising [antigenic molecule of said first souree]-[analyte autoantibody]-
[antigenic molecule of said second source], wherein said antigenic molecules
of said first aid second sources when present is said one or more complexes
comprise, or ate derived from, a common antigeaic molecule, or wherein
binding regions of said antigenic molecules of said first and second sources,
for said analyte autoantibody, when present in said one or more complexes are
present in, or are derived from, a common antigeaic molecule;
(e) prior to, or concurrent with, or subsequent to, step (d), providing
immobilising means whereby said antigenic moiecule of said first source as
present in a complex as formed in step (d) is immobilised to a solid support
prior to, or concurrent with, or subsequent to, step (d);
(f) prior to, or concurrent with, or subsequent to, step (d), providing direct or
indirect detectable labelling means whereby ami antigenic molecule of said
second source as present in a complex as formed in step (d) is provided with
such direct or indirect delectable labelling means prior to, or concurrent with,
or subsequent to, step (d); and
(g) detecting me presence of complexes formed in (d) immobilised according
to (e) so as to provide an indication of analyte astoantibodies present in said
sample.
A method of screening for analyte autoantibodies according to the present invention is
particularly advantageous in providing a method lor such autoantibody detection of
high specificity and sensitivity.
Hie one or more antigenic molecules, or one or mote variants, analogues, derivatives
or fragments thereof, or fusion molecules thereof, that can react with analyte
autoantibodies as required by the present invention can be selected from any
pancreatic islet cell antigenic molecule aad insulin, and one or more variants,
analogues, derivatives or fragments thereof; or fusion molecules thereof and in
particular can suitably be selected from the group costisting pf glutamic acid

decarboxylase (GAD, and in particular the 65KDa and 67KDa isoforms of gkttamic
acid decarboxylase, GAD65 and GAD67), petein tyrosine phosphatase-like islet cell
antigen (IA-2) and insulin, or one or more variants (such as, lor example, IA-2 beta),
analogues, derivatives or fragments thereof, or fusion molecules comprising two or
more of the above, with which analyte aotoantibodaes when present in said sample of
body fluid can interact. In a particularly, preferred embodiment of die present
invention, one or more of the above mentioned pancreatic islet cell antigenic
molecules or insulin are employed, in particular the above mentioned pancreatic islet
cell antigenic molecules, preferably one or more of GAD65, GAD67 or IA-2.
Alternatively it may be preferred that one or more variants, analogues, derivatives or
fragments of one or more of the above mentioned pancreatic islet cell antigenic
molecules or insulin are employed, in particular one or more variants, analogues,
derivatives or fragments of the above mentioned pancreatic islet cell antigenic
molecules, preferably one or more variants, analogues, derivatives or fragments of
one or more of GAD65, GAD67 or IA-2. In a further alternative preferred embodiment
of the present invention, it may be prefered mat one or more fusion molecules are
employed comprising two or more directly or indirectly fused antigenic molecules
selected from the above mentioned pancreatic islet cell antigenic molecules, insulin
and said one or more variants, analogues, derivatives or fragments thereof.
It will be appreciated, therefore, that the terra GAD as used heron encompasses all
isoforms of GAD, including in particular GAD65 or GAD67 and in certain
embodiments of the present invention it may be preferred mat GAD65 is employed
and in other embodiments of the present invention it may be preferred that GAD67 is
employed. It will also be appreciated from the above that the present invention can
include within its scope fusion molecules Ait can comprise GAD6$ or GAD67, or one
or more fragments thereof (such as binding regions thereof). For example, fusion
molecules for use within the scope of the present invention can comprise GAD65
directly or indirectly fused to GAD67, or one or more binding regions of GAD65
directly or indirectly fused to one or more binding regions or GAD67. Furthermore, it
will also be appreciated that fusion molecules for use within the scope of the present
invention can comprise IA-2, or one or more binding regions thereof, directly or
indirectly fused to IA-2 beta, or one or more binding regions thereof. The present
invention also allows for GAD65 or GAD67, or one or more variants, analogues,

derivatives or fragments thereof, directly or indirectly fused to IA-2, or one or more
variants, analogues, derivatives or fragments thereof.
ID particular the one or more aniigenic molecules, or one or more variants, analogues,
derivatives or fragments thereof, that can react with such analyte autoratibodies as
required by the present invention, can suitably be selected from the group consisting
of glutamic acid decarboxyiase (in particular GAD65 or GAD67) and protein tyrosine
phosphatase-like islet cell antigen (IA-2), or one or more variants, analogues,
derivatives or fragments thereof, with which analyte autoantibodies when present in
said sample of body fluid can interact.
In a preferred aspect of the present invention, therefore, a method of screening
substantially as hereinbefore described is for use in detecting analyte autoantibodies
to GAD and / or IA-2, and the one or more antigenic molecules comprise GAD65 or
GAD 67, or one or more variants, analogues, derivatives or fragments thereof, and / or
IA-2, or one or more variants, analogues, derivatives or fragments thereof, with which
analyte autoantibodies when present in said sample of body fluid can interact In the
case where fusion antigenic molecules are employed in the present invention, suitable
fusion molecules may comprise GAD65 or GAD67 or one or more variants, analogues,
derivatives or fragments thereof, directly or indirectly fused to IA-2, or one or more
variants, analogues, derivatives or fragments thereof; alternatively, suitable fusion
molecules may comprise GAD65 directly or indirectly fused to GAD67, or one or more
binding regions of GAD65 directly or indirectly fused to one or more binding regions
or GAD67; alternatively suitable fusion molecules may comprise IA-2, or one or more
binding regions thereof, directly or indirectly fused to IA-2 beta, or one or more
binding regions thereof.
Preferably a method according to the present invention comprises providing one or
more first sources of antigenic molecules selected from GAD65, GAD67, IA-2, insulin,
one or more variants, analogues, derivatives or fragments thereof and fusion
molecules comprising two or more directly or indirectly fused antigeaic molecules
selected from GAD65, GAD67, IA-2, insulin and said one or more variants, analogues,
derivatives or fragments thereof, with which analyte autoantibodies when present in
said sample of body fluid can interact, immobilised to a solid support according to

step (e) of a method according to the present invention and one or more •second
sources of antigenic molecules selected from GAD65, GAD67, IA-2, insulin, one or
more variants, analogues, derivatives or fragments thereof and fusion molecules
comprising two or more directly or indirectly fused antigeaic molecules selected from
GAD65, GAD67, IA-2, insulin and said one or mare variants, analogues, derivatives or
fragments thereof, with which analyte autoantibodies when patent in said sample of
body fluid can interact, provided with labelling means according to step (f) of a
method according to the present invention.
More preferably, a method according to the present inventkn comprises providing
one or more first sources of antigenic molecules selected from GAD65, GAD67, IA-2,
one or more variants, analogues, derivatives or fragmeais thereof and fusion
molecules comprising two or more directly or indirectly fused aatigenic molecules
selected from GAD65, GAD67, IA-2, and said one or more variants, analogues,
derivatives or fragments thereof, with which analyte autoantibodies when present in
said sample of body fluid can interact (for example fusion molecules comprising
GAD65, GAD67, or one or more variants, analogues, derivatives or fragments thereof
directly or indirectly fused to IA-2 or one or more variants, aaslogues, derivatives or
fragments thereof, with which analyte autoantibodies when present in said sample of
body fluid can interact), immobilised to a solid support aocndaig to step (e) of a
method according to the present invention and a second source of antigenic molecules
selected from GAD65, GAD67, IA-2, one or more variants, analogues, derivatives or
fragments thereof and fusion molecules comprising two or more directly or indirectly
fused antigenic molecules selected from GAD65, GAD67, IA-2, and one or more
variants, analogues, derivatives or fragments thereof, with which analyte
autoantibodies when present in said sample of body fluid can interact (such as fusion
molecules comprising GAD65 or GAD67, or one or more variants, analogues,
derivatives or fragments thereof directly or indirectly fused to IA-2 or one or more
variants, analogues, derivatives or fragments thereof, with which analyte
autoantibodies when present in said sample of body fluid can interact), provided with
labelling means according to step (f) of a method according to tbe present invention.
It may be preferred that where a method according to the present invention is required
to detect autoantibodies to GAD, the method comprises providing a first source-.of
antigenic molecules consisting essentially of GAD antigenic moieaues selected from

GAD65, OAD67, one or more variants, analogues, derivatives or fragments thereof
with which analyte autoantibodies when present in said sample of body fluid can
interact and fusion molecules comprising two or more directly or indirectly fused
antigenic molecules selected from GAD65, GAD67 aad the one or more variants,
analogues, derivatives or fragments thereof, (such as fusion molecules comprising
GAD65 directly or indirectly fined to GAD67, or one or more binding regions of
GAD65 directly or indirectly fused to one or more binding regions or GAD67, with
which analyte autoantibodies when present in said sample of body fluid can interact),
immobilised to a solid support according to step (e) of a method according to the
present invention and a second source of antigenic molecules consisting essentially of
GAD antigenic molecules selected from GAD65, GAD67, one or more variants,
analogues, derivatives or fragments thereof with which analyte autoaotibodtes when
present in said sample of body fluid can interact and fusion molecules comprising two
or more directly or indirectly fused antigenic molecules selected from GAD65, GAD67
and one or more variants, analogues, derivatives or fragments thereof, wife which
analyte autoantibodies when present in said sample of body fluid can interact (such as
GAD65 directly or indirectly fused to GAD67, or one or more binding regions of
GAD65 directly or indirectly fused to one or more binding regions or GAD67)
provided with labelling means according to step (f) of a method according to the
present invention. Alternatively, it may be preferred mat where a method according
to the present invention is required to detect autoantibodies to IA-2, the method
comprises providing a first source of antigenic molecules consisting essentially of IA-
2 antigenic molecules selected from IA-2, one or more variants, analogues,
derivatives or fragments thereof, with which analyte autoantibodies when present in
said sample of body fluid can interact and fusion molecules comprising two or more
directly or indirectly fused antigenic molecules selected from IA-2 aad one or more
variants, analogues, derivatives or fragments thereof, with which analyte
autoantibodies when present m said sample of body fluid can interact (such as IA-2,
or one or more binding regions thereof, directly or indirectly fused to IA-2 beta, or
one or more binding regions (hereof) immobilised to a solid support according to step
(e) of a method according to the present invention and a second source of antigenic
molecules consisting essentially of IA-2 antigenic molecules selected from IA-2, one
or more variants, analogues, derivatives or fragments thereof with which aoalyte
autoantibodies when present in said sample of body fluid can interact aad fusion

molecules comprising two or more directly or indirectly fused antigenic molecules
selected from IA-2 and one or more variants, analogues, derivatives or fragments
thereof, with which analyte autoantibodies when present in said sample of body fluid
can interact (such as IA-2, or one or more binding regions thereof, directly or
indirectly fused to IA-2 beta, or one or more binding regions thereof), provided with
labelling means according to step (f) of a method according to the present invention.
A still further alternative according to the present invention may be where a method
according to the present invention is required to detect autoantibodies to both GAD
and IA-2, where the method comprises providing (i) first and second sources of GAD
antigenic molecules with which analyte autoantibodies when present in said sample of
body fluid can interact, and which GAD antigenic molecules are selected from
GAD65, GAD67, one or more variants, analogues, derivatives or fragments thereof and
fusion molecules comprising two or more directly or indirectly fused antigenic
molecules selected from GAD65, GAD67, IA-2, and said one or more variants,
analogues, derivatives or fragments thereof and wherein at least one of said antigenic
molecules of said fusion molecule comprises GAD65, GAD67, or one or more variants,
analogues, derivatives or fragments thereof, wherein GAD antigenic molecules of said
first source are immobilised to a solid support according to step (e) and GAD
antigenic molecules of said second source are provided with labelling means
according to step (f); and (ii) first and second sources of IA-2 antigenic molecules
with which analyte autoantibodies when present in said sample of body fluid can
interact and which IA-2 antigenic molecules are selected from IA-2, one or more
variants, analogues, derivatives or fragments thereof, and fusion molecules
comprising two or more directly or indirectly fused antigenic molecules selected from
IA-2, GAD65, GAD67 and said one or more variants, analogues, derivatives or
fragments thereof and wherein at least one of said antigenic molecules of said fusion
molecule comprises IA-2 or one or more variants, analogues, derivatives or fragments
thereof, wherein IA-2 antigenic molecules of said first source are immobilised to a
solid support according to step (e) and IA-2 antigenic molecules of said second source
are provided with labelling means according to step (f).
Accordingly, a preferred embodiment of the present invention may suitably comprise
a method of screening a sample of body fluid obtained from an animal subject for
analyte autoantibodies reactive with one or more antigenic molecules selected from

pancreatic islet cell antigenic molecules, or one or more variants, analogues,
derivatives or fragments thereof, said method comprising:
(a) providing said sample of body fluid from said subject;
(b) providing one or more first sources of antigenic molecules with which
analyte autoantibodies when present in said sample of body fluid can interact
and which antigenic molecules are selected from GAD65, GAD67, IA-2, one or
more variants, analogues, derivatives or fragments thereof and fusion
molecules comprising two or more directly or indirectly fused antigenic
molecules selected from GAD65, GAD67, IA-2, and said one or more variants,
analogues, derivatives or fragments thereof;
(c) providing one or more second sources of antigenic molecules with which
analyte autoantibodies when present in said sample of body fluid can interact
and which antigenic molecules are selected from GAD65, GAD67, IA-2, one or
more variants, analogues, derivatives or fragments thereof and fusion
molecules comprising two or more directly or indirectly fused antigenic
molecules selected from GAD65, GAD67, IA-2 and said one or more variants,
analogues, derivatives or fragments thereof;
(d) contacting said antigenic molecules as provided by steps (b) and (c)
simultaneously or successively with said sample of body fluid being screened,
whereby analyte autoantibodies when present in said sample of body fluid can
interact with said antigenic molecules so as to form one or more complexes
comprising [antigenic molecule of said first source]-[analyte autoantibody]-
[antigenic molecule of said second source], wherein said antigenic molecules
of said first and second sources when present in said one or more complexes
comprise, or are derived from, a common antigenic molecule, or wherein
binding regions of said antigenic molecules of said first and second sources,
for said analyte autoantibody, when present in said one or more complexes are
present in, or are derived from, a common antigenic molecule;

(e) prior to, or concurrent with, or subsequent to, step (d), providing
immobilising means whereby said antigenic molecule of said first source as
present in a complex as formed in step (d) is immobilised to a solid support
prior to, or concurrent with, or subsequent to, step (d);
(f) prior to, or concurrent with, or subsequent to, step (d), providing direct or
indirect detectable labelling means whereby said antigenic molecule of said
second source as present in a complex as formed in step (d) is provided with
such direct or indirect detectable labelling means prior to, or concurrent with,
or subsequent to, step (d); and
(g) detecting the presence of complexes formed in (d) immobilised according
to (e) so as to provide an indication of analyte autoantibodies present in said
sample.
Alternatively, a preferred embodiment of the present invention may suitably comprise
screening a sample of body fluid obtained from an animal subject for analyte
autoantibodies reactive only with GAD, or one or more variants, analogues,
derivatives or fragments thereof, said method comprising:
(a) providing said sample of body fluid from said subject;
(b) providing first and second sources of antigenic molecules consisting
essentially of GAD antigenic molecules selected from GAD65, GAD67, one or
more variants, analogues, derivatives or fragments thereof with which analyte
autoantibodies when present in said sample of body fluid can interact and
fusion molecules comprising two or more directly or indirectly fused antigenic
molecules selected from GAD65, GAD67 and said one or more variants,
analogues, derivatives or fragments thereof, wherein GAD antigenic
molecules of said first source are immobilised to a solid support according to
step (d) and GAD antigenic molecules of said second source are provided with
labelling means according to step (e);

(c) contacting said antigenic molecules as provided by step (b) simultaneously
or successively with said sample of body fluid being screened, whereby
analyte autoantibodies when present in said sample of body fluid can interact
with said antigenic molecules so as to form one or more complexes
comprising [GAD antigenic molecule of said first sourcej-[analyte
autoantibodyH GAD antigenic molecule of said second source];
(d) prior to, or concurrent with, or subsequent to, step (c), providing
immobilising means whereby said GAD antigenic molecule of said first source
as present in a complex as formed in step (c) is immobilised to a solid support
prior to, or concurrent with, or subsequent to, step (c);
(e) prior to, or concurrent with, or subsequent to, step (c), providing direct or
indirect detectable labelling means whereby said GAD antigenic molecule of
said second source as present in a complex as formed in step (c) is provided
with such direct or indirect detectable labelling means prior to, or concurrent
with, or subsequent to, step (c); and
(f) detecting the presence of complexes formed in (c) immobilised according
to (d) so as to provide an indication of analyte autoantibodies present in said
sample.
Alternatively, a preferred embodiment of the present invention may suitably comprise
screening a sample of body fluid obtained from an animal subject for analyte
autoantibodies reactive with IA-2, or one or more variants, analogues, derivatives or
fragments thereof, said method comprising:
(a) providing said sample of body fluid from said subject;
(b) providing first and second sources of antigenic molecules consisting
essentially of IA-2 antigenic molecules selected from IA-2, one or more
variants, analogues, derivatives or fragments thereof with which analyte
autoantibodies when present in said sample of body fluid can interact and
fusion molecules comprising two or more directly or indirectly fused antigenic

molecules selected from IA-2 and said one or more variants, analogues,
derivatives or fragments thereof, wherein IA-2 antigenic molecules of said
first source are immobilised to a solid support according to step (d) and IA-2
antigenic molecules of said second source are provided with labelling means
according to step (e);
(c) contacting said IA-2 antigenic molecules as provided by step (b)
simultaneously or successively with said sample of body fluid being screened,
whereby analyte autoantibodies when present in said sample of body fluid can
interact with said IA-2 antigenic molecules so as to form one or more
complexes comprising [IA-2 antigenic molecule of said first source]-[analyte
autoantibody]-[IA-2 antigenic molecule of said second source];
(d) prior to, or concurrent with, or subsequent to, step (c), providing
immobilising means whereby said IA-2 antigenic molecule of said first source
as present in a complex as formed in step (c) is immobilised to a solid support
prior to, or concurrent with, or subsequent to, step (c);

(e) prior to, or concurrent with, or subsequent to, step (c), providing direct or
indirect detectable labelling means whereby said IA-2 antigenic molecule of
said second source as present in a complex as formed in step (c) is provided
with such direct or indirect detectable labelling means prior to, or concurrent
with, or subsequent to, step (c); and
(f) detecting the presence of complexes formed in (c) immobilised according
to (d) so as to provide an indication of analyte autoantibodies present in said
sample.
Alternatively, a preferred embodiment of the present invention may suitably comprise
screening a sample of body fluid obtained from an animal subject for first and second
analyte autoantibodies respectively reactive with GAD and IA-2, or one or more
variants, analogues, derivatives or fragments thereof, said method comprising:
(a) providing said sample of body fluid from said subject;

(b) providing first and second sources of GAD antigenic molecules with which
analyte autoantibodies when present in said sample of body fluid can interact,
and which GAD antigenic molecules are selected from GAD65, GAD67, one or
more variants, analogues, derivatives or fragments thereof and fusion
molecules comprising two or more directly or indirectly fused antigenic
molecules selected from GAD65, GAD67, IA-2, and said one or more variants,
analogues, derivatives or fragments thereof and wherein at least one of said
antigenic molecules of said fusion molecule comprises GAD65, GAD67, or one
or more variants, analogues, derivatives or fragments thereof, wherein GAD
antigenic molecules of said first source are immobilised to a solid support
according to step (e) and GAD antigenic molecules of said second source are
provided with labelling means according to step (f);
(c) providing first and second sources of IA-2 antigenic molecules with which
analyte autoantibodies when present in said sample of body fluid can interact
and which IA-2 antigenic molecules are selected from IA-2, one or more
variants, analogues, derivatives or fragments thereof, and fusion molecules
comprising two or more directly or indirectly fused antigenic molecules
selected from IA-2, GAD65, GAD67 and said one or more variants, analogues,
derivatives or fragments thereof and wherein at least one of said antigenic
molecules of said fusion molecule comprises IA-2 or one or more variants,
analogues, derivatives or fragments thereof, wherein IA-2 antigenic molecules
of said first source are immobilised to a solid support according to step (e) and
IA-2 antigenic molecules of said second source are provided with labelling
means according to step (f);
(d) contacting said antigenic molecules as provided by steps (b) and (c)
simultaneously or successively with said sample of body fluid being screened,
whereby analyte autoantibodies when present in said sample of body fluid can
interact with said antigenic molecules so as to fonn one or more complexes
comprising [GAD antigenic molecule of said first source]-[analyte
autoantibody]-[GAD antigenic molecule of said second source] or [IA-2

antigenic molecule of said first source]-[analyte autoantibody]-[IA-2 arttigenic
molecule of said second source];
(e) prior to, or concurrent with, or subsequent to, step (d), providing
immobilising means whereby said GAD or IA-2 antigenic molecule of said
first source as present in a complex as formed in step (d) is immobilised to a
solid support prior to, or concurrent with, or subsequent to, step (d);
(f) prior to, or concurrent with, or subsequent to, step (d), providing direct or
indirect detectable labelling means whereby said GAD or IA-2 antigenic
molecule of said second source as present in a complex as formed in step (d) is
provided with such direct or indirect detectable labelling means prior to, or
concurrent with, or subsequent to, step (d); and
(g) detecting the presence of complexes formed in (d) immobilised according
to (e) so as to provide an indication of analyte autoantibodies present in said
sample.
It may be preferred in a method according to the present invention that the antigenic
molecules of the first and second sources when present in said one or more complexes
substantially as hereinbefore described both comprise one or more common antigenic
molecules. For example, antigenic molecules of said first and second sources when
present in the one or more complexes can comprise GAD65 or GAD67; alternatively
the antigenic molecules of the first and second sources when present in the one or
more complexes can comprise IA-2.
Alternatively, it may be preferred that one antigenic molecule, of either the first or
second source, when present in the one or more complexes comprises an antigenic
molecule selected from pancreatic islet cell antigenic molecules and insulin, and the
other antigenic molecule when present in the one or more complexes comprises one
or more variants, analogues, derivatives or fragments of the above mentioned one
antigenic molecule with which analyte autoantibodies when present in said sample of
body fluid can interact For example, where one of the antigenic molecules of the
first or second source when present in the one or more complexes comprises GAD65

or GAD67, the other antigenic molecule when present in the one or more complexes
can comprise one or more variants, analogues, derivatives or fragments of GAD65 or
GAD67 with which analyte autoantibodies when present in said sample of body fluid
can interact. A further example may be where one of the antigenic molecules of die
first or second source when present in the one or more complexes comprises IA-2,
and the other antigenic molecule when present in the one or more complexes can
comprise one or more variants, analogues, derivatives or fragments of IA-2 with
which analyte autoantibodies when present in said sample of body fluid can interact
A still further alternative embodiment of the present invention may be where both
antigenic molecules of the first: and second sources when present in the one or more
complexes comprise a variant, analogue, derivative or fragment, which may be the
same or different, of a common antigenic molecule, with which analyte
autoantibodies when present in said sample of body fluid can interact. For example,
both antigenic moleq|ies of the first and second sources when present in the one or
more complexes can comprise a variant, analogue, derivative or fragment, which may
be the same or different, of GAD65 or GAD67, with which analyte autoantibodies
when present in said sample of body fluid can interact. A further example may be
where both antigenic molecules of the first and second sources when present in said
one or more complexes comprise a variant, analogue, derivative or fragment, which
may be the same or different, of IA-2, with which analyte autoantibodies when
present in said sample of body fluid can interact. It will be appreciated from the
above that where both antigenic molecules when present in the complexes formed
according to a method of the present invention comprise variants, analogues,
derivatives or fragments derived from a common antigenic molecule, such variants,
analogues, derivative or fragments may be the same or different (for example variant
1 and variant 2, where variants 1 and 2 may respectively represent different variants
of GAD65 or GAD67, or fragment 1 and fragment 2, where fragments 1 and 2 may
respectively represent different fragments of GAD65 or GAD67, such as distinct, or
possibly overlapping, epitopes of GAD65 or GAD67).
In the case where fusion molecules are employed in a method according to the present
invention, it will be appreciated that such fusion molecules can comprise -distinct
antigenic molecules (for example GAD65 and IA-2) but that in a complex formed

according to the present invention the binding regions of antigenic fusion molecules
for an analyte autoantibody as respectively provided by the first and second sources
will be present in, or will be derived from, a common antigenic molecule. Examples
of complexes that may be formed employing one or more fusion molecules according
to the present invention can include (i) [GAD-IA-2imm]-[analyte autoantibody]-[GAD-
IA-2label] where in the case where the autoantibody comprises a GAD autoantibody
this autoantibody interacts with binding regions of GAD present in [GAD-IA-2imn]
and [GAD-IA-2label], or where the autoantibody comprises a IA-2 autoantibody this
autoantibody interacts with binding regions of IA-2 present in [GAD-IA2imm] and
[GAD-IA-2label], or (ii) [GAD(fragment)imm]-[analyte autoantibody]-[GAD-IA-2label]
where a GAD analyte autoantibody interacts with binding regions of GAD present in
[GAD(fragment)imm] and [GAD-IA-2iabd], or (iii) [IA-2(fogment)imm]-[analyte
autoantibody]-[GAD-IA-2label] where a IA-2 analyte autoantibody interacts with
binding regions of IA-2 present in [IA-2(fragment)imm] and [GAD-IA-2label], where
GAD can represent GAD65 or GAD67.
Suitably the detectable labelling means can be selected from the group consisting of
enzymic labels, isotopic labels, chemiluminesceat labels, fluorescent labels, dyes and
the like and typically can be selected from the group consisting of alkaline
phosphatase, horseradish peroxidase, biotin or the like and in particular can comprise
biotin. The monitoring can typically involve reaction of such detectable labelling
means (when attached to the one or more antigenic molecules substantially as
hereinbefore described, or one or more variants, analogues, derivatives or fragments
thereof, or one or more fusion molecules thereof) with one or more substrates therefor
(such as an avidin or streptavidin conjugate, for example, streptavidin horseradish
peroxidase conjugate or the like), whereby the resulting conjugates can be detected
suitably by measurement of optical density or the like.
The detectable labelling means may be directly provided to the one or more antigenic
molecules, or one or more one or more variants, analogues, derivatives or fragments
thereof, or one or more fusion molecules thereof, substantially as hereinbefore
described. Additionally or alternatively the detectable labelling means may be
indirectly provided to the one or more antigenic molecules, or one or-more one or
more variants, analogues, derivatives or fragments thereof, or one or more fusion

molecules thereof, substantially as hereinbefore described, typically by providing the
detectable labelling means to one or more antibodies or other binding agents which
can bind with the one or more antigenic molecules, or one or more variants,
analogues, derivatives or fragments thereof, or one or more fusion molecules thereof,
substantially as hereinbefore described.
A method of screening for autoantibodies according to the present invention typically
comprises directly monitoring interaction of (i) such autoantibodies present in the
sample of body fluid from the subject and (ii) one or more antigenic molecules, or one
or more variants, analogues, derivatives or fragments thereof, or one or more fusion
molecules thereof, substantially as hereinbefore described, as provided by the present
invention, typically by employing non-competitive sandwich type assay techniques
known in the art
According to a preferred embodiment of the present invention antigenic molecules of
one or more first sources, or one or more variants, analogues, derivatives or fragments
thereof, or one or more fusion molecules thereof, substantially as hereinbefore
described, are immobilised to a solid support and antigenic molecules of one or more
second sources, or one or more variants, analogues, derivatives or fragments thereof,
or one or more fusion molecules thereof, substantially as hereinbefore described, are
provided with detectable labelling means, and wherein preferably the antigenic
molecules of the one or more second sources are provided in solution phase as is
conventional for use in known ELISA techniques.
Preferably, in a method according to the present invention, antigenic molecules, or
one or more variants, analogues, derivatives or fragments thereof, or one or more
fusion molecules thereof, with which analyte autoantibodies when present in said
sample of body fluid can interact, of one or more first sources are immobilised to a -
solid support prior to contact with a sample of body fluid being screened. Such
immobilised antigenic molecules of the one or more first sources, or more one or
more variants, analogues, derivatives or fragments thereof, or one or more fusion
molecules thereof, with which analyte autoantibodies when present in said sample of
body fluid can interact, are subsequently contacted with the sample of body fluid
being screened either simultaneously or successively with contact of the sample of

body fluid with antigenic molecules of one or more second sources, or one or more
variants, analogues, derivatives or fragments thereof, or one or more fusion molecules
thereof, with which analyte autoantibodies when present in said sample of body fluid
can interact. Particularly preferably, immobilised antigenic molecules of the one or
more first sources, or one or more variants, analogues, derivatives or fragments
thereof, or one or more fusion molecules thereof, with which analyte autoantibodies
when present in said sample of body fluid can interact, are contacted with the sample
of body fluid being screened so as to form an intermediate complex comprising
[antigenic molecule]-[analyte autoantibody] where the antigenic molecule is
immobilised to a solid support and the thus formed immobilised intermediate complex
is subsequently contacted with antigenic molecules of the one or more second
sources, or one or more variants, analogues, derivatives or fragments thereof, or one
or more fusion molecules thereof, with which analyte autoantibodies when present in
said sample of body fluid can interact, present in solution phase, so as to form the
hitherto described complexes comprising [antigenic molecule of said first source]-
[analyte autoantibody]-[antigenic molecule of said second source] immobilised to a
solid support via the antigenic molecule of the first source.
Accordingly, the present invention provides a method of screening a sample of body
fluid obtained from an animal subject for analyte autoantibodies reactive with one or
more antigenic molecules selected from pancreatic islet cell antigenic molecules and
insulin, or one or more variants, analogues, derivatives or fragments thereof, said
method comprising:
(a) providing said sample of body fluid from said subject;
(b) providing one or more first sources of antigenic molecules with which
analyte autoantibodies when present in said sample of body fluid can interact
and which antigenic molecules are selected from pancreatic islet cell antigenic
molecules, insulin, one or more variants, analogues, derivatives or fragments
of said pancreatic islet cell antigenic molecules or insulin, and fusion
molecules comprising two or more directly or indirectly fused antigenic
molecules selected from pancreatic islet cell antigenic-molecules, insulin and
said one or more variants, analogues, derivatives or fragments thereof, which

antigenic molecules of said one or more first sources are immobilised to a
solid support;
(c) providing one or more second sources of antigenic molecules with which
analyte autoantibodies when present in said sample of body fluid can interact
and which antigenic molecules are selected from pancreatic islet cell antigenic
molecules, insulin, one or more variants, analogues, derivatives or fragments
of said pancreatic islet cell antigenic molecules or insulin, and fusion
molecules comprising two or more directly or indirectly fused antigenic
molecules selected from pancreatic islet cell antigenic molecules, insulin and
said one or more variants, analogues, derivatives or fragments thereof, which
antigenic molecules of said one or more second sources are provided in
solution phase;
(d) contacting said antigenic molecules as provided by steps (b) and (c)
simultaneously or successively with said sample of body fluid being screened,
whereby analyte autoantibodies when present in said sample of body fluid can
interact with said antigenic molecules so as to form one or more immobilised
complexes comprising [antigenic molecule of said first source]-[analyte
autoantibodyHantigenic molecule of said second source], wherein said
antigenic molecules of said first and second sources when present in said one
or more complexes comprise, or are derived from, a common antigenic
molecule, or wherein binding regions of said antigenic molecules of said first
and second sources, for said analyte autoantibody, when present in said one or
more complexes are present in, or are derived from, a common antigenic
molecule and wherein said complexes are immobilised to a solid support via
said antigenic molecule of said first source;
(e) prior to, or concurrent with, or subsequent to, step (d), providing direct or
indirect detectable labelling means whereby said antigenic molecule of said
second source as present in a complex as formed in step (d) is provided with
such direct or indirect detectable labelling means prior to, or concurrent with,
or subsequent to, step (d); and

(f) detecting the presence of complexes formed in (d) so as to provide an
indication of analyte autoantibodies present in said sample.
The one or more antigenic molecules, or one or more variants, analogues, derivatives
or fragments thereof, or fusion molecules thereof, that can react with analyte
autoantibodies as required by the present invention can be selected from any
pancreatic islet cell antigenic molecule and insulin, and one or more variants,
analogues, derivatives or fragments thereof, or fusion molecules thereof and in
particular can suitably be selected from the group consisting of glutamic acid
decarboxylase (GAD, and in particular the 65KDa and 67KDa isoforms of glutamic
acid decarboxylase, GAD65 and GAD67), protein tyrosine phosphatase-like islet cell
antigen (IA-2) and insulin, or one or more variants (such as, for example, IA-2 beta),
analogues, derivatives or fragments thereof, or fusion molecules comprising two or
more of the above, with which analyte autoantibodies when present in said sample of
body fluid can interact. In particular the one or more antigenic molecules, or one or
more variants, analogues, derivatives or fragments thereof, that can react with such
analyte autoantibodies as required by the present invention, can suitably be selected
from the group consisting of glutamic acid decarboxylase (GAD, in particular GAD65
or GAD67) and protein tyrosine phosphatase-like islet cell antigen (IA-2), or one or
more variants, analogues, derivatives or fragments thereof, with which analyte
autoantibodies when present in said sample of body fluid can interact.
Other contacting techniques are, however, possible, for example it is possible
according to the present invention not to initially immobilise the antigenic molecules,
or one or more variants, analogues, derivatives or fragments thereof, or one or more
fusion molecules thereof, of the one or more first antigen sources, prior to a
contacting step as described above, but to initially contact the solid support
substantially as hereinbefore described with a binder for the antigenic molecules, or
one or more variants, analogues, derivatives or fragments thereof, or one or more
fusion molecules thereof, of the one or more first antigen sources and to subsequently
contact the so treated solid support with the above described antigenic molecules of
the one or more first and second sources, or one or more variants, analogues,
derivatives or fragments thereof, or one or more fusion molecules thereof, and the
sample of body fluid being screened.

In respect of the solid supports and conditions employed in the present invention, the
supports and conditions do not generally fundamentally differ from conventional
supports and conditions employed in known immunoassay techniques. A solid
support for use according to the present invention can comprise an ELISA plate as
currently employed in known ELISA techniques, or may employ any other suitable
support for use in the present invention, such as tubes, particles, magnetic beads,
nitrocellulose or the like.
There is also provided by the present invention a kit for screening a sample of body
fluid obtained from an animal subject for analyte autoantibodies reactive with one or
more antigenic molecules selected from pancreatic islet cell antigenic molecules and
insulin, or one or more variants, analogues, derivatives or fragments thereof, said kit
comprising:
(a) one or more first sources of antigenic molecules with which analyte
autoantibodies when present in said sample of body fluid can interact and
which antigenic molecules are selected from pancreatic islet cell antigenic
molecules, insulin, one or more variants, analogues, derivatives or fragments
of said pancreatic islet cell antigenic molecules or insulin, and fusion
molecules comprising two or more directly or indirectly fused antigenic
molecules selected from pancreatic islet cell antigenic molecules, insulin and
said one or more variants, analogues, derivatives or fragments thereof;
(b) one or more second sources of antigenic molecules with which analyte
autoantibodies when present in said sample of body fluid can interact and
which antigenic molecules are selected from pancreatic islet cell antigenic
molecules, insulin, one or more variants, analogues, derivatives or fragments
of said pancreatic islet cell antigenic molecules or insulin, and fusion
molecules comprising two or more directly or indirectly fused antigenic
molecules selected from pancreatic islet cell antigenic molecules, insulin and
said one or more variants, analogues, derivatives or fragments thereof;

(c) means for contacting said antigenic molecules as provided by (a) and (b)
simultaneously or successively with said sample of body fluid being screened,
whereby analyte autoantibodies when present in said sample of body fluid can
interact with said antigenic molecules so as to form one or more complexes
comprising [antigenic molecule of said first source]-[analyte autoantibody] -
[antigenic molecule of said second source], wherein said antigenic molecules
of said first and second sources when present in said one or more complexes
comprise, or are derived from, a common antigenic molecule, or wherein
binding regions of said antigenic molecules of said first and second sources,
for said analyte autoantibody, when present in said one or more complexes are
present in, or are derived from, a common antigenic molecule;
(d) means for immobilising to a solid support, said antigenic molecule of said
first source as present in a complex as defined in (c), prior to, or concurrent
with, or subsequent to, contact of the antigenic molecule of said first source
with the sample of body fluid being screened;
(e) means for providing direct or indirect detectable labelling means to said
antigenic molecule of said second source as present in a complex as defined in
(c), prior to, or concurrent with, or subsequent to, contact of said antigenic
molecule of said second source with said sample of body fluid being screened;
and
(f) means for detecting the presence of complexes as defined in (c)
immobilised as defined in (d) so as to provide an indication of analyte
autoantibodies present in said sample.
A kit for screening for analyte autoantibodies according to the present invention is
particularly advantageous in enabling such autoantibody detection of high specificity
and sensitivity.
The one or more antigenic molecules, or one or more variants, analogues, derivatives
or fragments thereof; or fusion molecules,thereof,, mat can react with analyte
autoantibodies as required by the present invention can be selected from any

pancreatic islet cell antigenic molecule and insulin, and one or more variants,
analogues, derivatives or fragments thereof, or fusion molecules thereof and in
particular can suitably be selected from the group consisting of glutamic acid
decarboxylase (GAD, and in particular the 65KDa and 67KDa isoforms of glutamic
acid decarboxylase, GAD65 and GAD67), protein tyrosine phosphatase-like islet cell
antigen (IA-2) and insulin, or one or more variants (such as, for example, IA-2 beta),
analogues, derivatives or fragments thereof, or fusion molecules comprising two or
more of the above, with which analyte autoantibodies when present in said sample of
body fluid can interact. In particular the one or more antigenic molecules, or one or
more variants, analogues, derivatives or fragments thereof, that can react with such
analyte autoantibodies as required by the present invention, can suitably be selected
from the group consisting of glutamic acid decarboxylase (GAD, in particular GAD65
or GAD67) and protein tyrosine phosphatase-like islet cell antigen (IA-2), or one or
more variants, analogues, derivatives or fragments thereof, with which analyte
autoantibodies when present in said sample of body fluid can interact. Various
embodiments of the antigenic molecules and suitable fusion molecules for use in a kit
according to the present invention are substantially as hereinbefore described in
greater detail with reference to a method according to the present invention.
In a preferred aspect of the present invention, therefore, a kit for screening
substantially as hereinbefore described is for use in detecting analyte autoantibodies
to GAD65 or GAD67 and / or IA-2, or one or more variants, analogues, derivatives or
fragments thereof, and the one or more antigenic molecules comprise GAD65 or
GAD67, or one or more variants, analogues, derivatives or fragments thereof, and / or
IA-2, or one or more variants, analogues, derivatives or fragments thereof. In the case
where fusion antigenic molecules are employed in a kit according to the present
invention, suitable fusion molecules may comprise two or more directly or indirectly
fused antigenic molecules selected from GAD65, GAD67, IA-2 and said one or more
variants, analogues, derivatives or fragments thereof substantially as hereinbefore
described in greater detail with reference to a method according to the present
invention.
Preferably a kit according to the present invention comprises one or more first sources
of antigenic molecules selected from GAD65, GAD67, IA-2, insulin, one or more

variants, analogues, derivatives or fragments thereof, and fusion molecules
comprising two or more directly or indirectly fused antigenic molecules selected from
GAD65, GAD67, IA-2, insulin and said one or more variants, analogues, derivatives or
fragments thereof, with which analyte autoantibodies when present in said sample of
body fluid can interact, immobilised to a solid support as defined in (d) and one or
more second sources of antigenic molecules selected from GAD65, GAD67, IA-2,
insulin, one or more variants, analogues, derivatives or fragments thereof, and fusion
molecules comprising two or more directly or indirectly fused antigenic molecules
selected from GAD65, GAD67, IA-2, insulin and one or more variants, analogues,
derivatives or fragments thereof, with which analyte autoantibodies when present in
said sample of body fluid can interact, provided with labelling means as defined in
(e).
More preferably, a kit according to the present invention comprises one or more first
sources of antigenic molecules selected from GAD65, GAD67, IA-2., one or more
variants, analogues, derivatives or fragments thereof and fusion molecules comprising
two or more directly or indirectly fused antigenic molecules selected from GAD65,
GAD67, IA-2 and said one or more variants, analogues, derivatives or fragments
thereof, with which analyte autoantibodies when present in said sample of body fluid
can interact, immobilised to a solid support as defined in (d) and one or more second
sources of antigenic molecules selected from GAD65, GAD67, IA-2, one or more
variants, analogues, derivatives or fragments thereof and fusion molecules comprising
two or more directly or indirectly fused antigenic molecules selected from GAD65,
GAD67, IA-2 and said one or more variants, analogues, derivatives or fragments
thereof, with which analyte autoantibodies when present in said sample of body fluid
can interact, provided with labelling means as defined in (e). It may be preferred that
a kit according to the present invention comprises a first source of antigenic
molecules consisting essentially of GAD antigenic molecules selected from GAD65,
GAD67, one or more variants, analogues, derivatives or fragments thereof and fusion
molecules comprising two or more directly or indirectly fused antigenic molecules
selected from GAD65, GAD67 and said one or more variants, analogues, derivatives or
fragments thereof, with which analyte autoantibodies when present in said sample of
body fluid can interact, immobilised to a solid support as defined in (d), and a second
source of antigenic molecules consisting essentially of GAD antigenic molecules

selected from GAD65, GAD67, one or more variants, analogues, derivatives or
fragments thereof and fusion molecules comprising two or more directly or indirectly
fused antigenic molecules selected from GAD65, GAD67 and said one or more
variants, analogues, derivatives or fragments thereof, with which analyte
autoantibodies when present in said sample of body fluid can interact, provided with
labelling means as defined in (e). Alternatively, it may be preferred that a kit
according to the present invention comprises a first source of antigenic molecules
consisting essentially of IA-2 antigenic molecules selected from IA-2, one or more
variants, analogues, derivatives or fragments thereof, with which analyte
autoantibodies when present in said sample of body fluid can interact and fusion
molecules comprising two or more directly or indirectly fused antigenic molecules
selected from IA-2 and said one or more variants, analogues, derivatives or fragments
thereof, immobilised to a solid support as defined in (d), and a second source of
antigenic molecules consisting essentially of IA-2 antigenic molecules selected from
IA-2, one or more variants, analogues, derivatives or fragments thereof, with which
analyte autoantibodies when present in said sample of body fluid can interact and
fusion molecules comprising two or more directly or indirectly fused antigenic
molecules selected from IA-2 and said one or more variants, analogues, derivatives or
fragments thereof, provided with labelling means as defined in (e). A still further
alternative according to the present invention may be where a kit according to the
present invention comprises (i) first and second sources of GAD antigenic molecules
with which analyte autoantibodies when present in said sample of body fluid can
interact, and which GAD antigenic molecules are selected from GAD65, GAD67, one
or more variants, analogues, derivatives or fragments thereof and fusion molecules
comprising two or more directly or indirectly fused antigenic molecules selected from
GAD65, GAD67, IA-2 and said one or more variants, analogues, derivatives or
fragments thereof and wherein at least one of said antigenic molecules of said fusion
molecule comprises GAD65, GAD67, or one or more variants, analogues, derivatives
or fragments thereof, wherein GAD antigenic molecules of said first source are
immobilised to a solid support as defined in (d) and GAD antigenic molecules of said
second source are provided with labelling means as defined in (e); and (ii) first and
second sources of IA-2 antigenic molecules with which analyte autoantibodies when
present in said sample of body fluid can interact and which IA-2 antigenic molecules
are selected from IA-2, one or more variants, analogues, derivatives or fragments

thereof, and fusion molecules comprising two or more directly or indirectly fused
antigenic molecules selected from IA-2, GAD65, GAD67 and said one or more
variants, analogues, derivatives or fragments thereof and wherein at least one of said
antigenic molecules of said fusion molecule comprises IA-2 or one or more variants,
analogues, derivatives or fragments thereof, wherein IA-2 antigenic molecules of said
first source are immobilised to a solid support according as defined in (d) and IA-2
antigenic molecules of said second, source are provided with labelling means as
defined in (e).
Accordingly, a preferred embodiment of the present invention may suitably comprise
a kit for screening a sample of body fluid obtained from an animal subject for analyte
autoantibodies reactive with one or more antigenic molecules selected from pancreatic
islet cell antigenic molecules, or one or more variants, analogues, derivatives or
fragments thereof, said kit comprising:
(a) one or more first sources of antigenic molecules with which analyte
autoantibodies when present in said sample of body fluid can interact and
which antigenic molecules are selected from GAD65, GAD67, IA-2, one or
more variants, analogues, derivatives or fragments thereof and fusion
molecules comprising two or more directly or indirectly fused antigenic
molecules selected from GAD65, GAD67, IA-2 and said one or more variants,
analogues, derivatives or fragments thereof;
(b) one or more second sources of antigenic molecules with which analyte
autoantibodies when present in said sample of body fluid can interact and
which antigenic molecules are selected from GAD65, GAD67, IA-2, one or
more variants, analogues, derivatives or fragments thereof and fusion
molecules comprising two or more directly or indirectly fused antigenic
molecules selected from GAD65, GAD67, IA-2 and said one or more variants,
analogues, derivatives or fragments thereof;
(c) means for contacting said antigenic molecules as provided by (a) and (b)
simultaneously or successively with said sample of body fluid being screened,
whereby analyte autoantibodies when present in said sample of body fluid can

interact with said antigenic molecules so as to form one or more complexes
comprising [antigenic molecule of said first source]-[analyte autoantibody]-
[antigenic molecule of said second source], wherein said antigenic molecules
of said first and second sources when present in said one or more complexes
comprise, or are derived from, a common antigenic molecule, or wherein
binding regions of said antigenic molecules of said first and second sources,
for said analyte autoantibody, when present in said one or more complexes are
present in, or are derived from, a common antigenic molecule;
(d) means for immobilising to a solid support said antigenic molecule of said
first source as present in a complex as defined in (c), prior to, or concurrent
with, or subsequent to, contact of said antigenic molecule of said first source
with the sample of body fluid being screened;
(e) means for providing direct or indirect detectable labelling means to said
antigenic molecule of said second source as present in a complex as defined in
(c), prior to, or concurrent with, or subsequent to, contact of said antigenic
molecule of said second source with the sample of body fluid being screened;
and
(f) means for detecting the presence of complexes as defined in (c)
immobilised as defined in (d) so as to provide an indication of analyte
autoantibodies present in said sample.
Alternatively, a preferred embodiment of the present invention may suitably comprise
a kit for screening a sample of body fluid obtained from an animal subject for analyte
autoantibodies reactive with GAD, or one or more variants, analogues, derivatives or
fragments thereof, said kit comprising:
(a) first and second sources of antigenic molecules consisting essentially of
GAD antigenic molecules selected from GAD65, GAD67, one or more variants,
analogues, derivatives or fragments thereof with which analyte autoantibodies
when present in said sample of body fluid can interact and fusion molecules
comprising two or more directly or indirectly fused antigenic molecules

selected from GAD65, GAD67 and said one or more variants, analogues,
derivatives or fragments Ihereof, wherein GAD antigenic molecules of said
first source are immobilised to a solid support as defined in (c) and GAD
antigenic molecules of said second source are provided with labelling means
as defined in (d);
(b) means for contacting said antigenic molecules as provided by (a)
simultaneously or successively with said sample of body fluid being screened,
whereby analyte autoantibodies when present in said sample of body fluid can
interact with said antigenic molecules so as to form one or more complexes
comprising [GAD antigenic molecule of said first source]-[analyte
autoantibody]-[ GAD antigenic molecule of said second source];
(c) means for immobilising to a solid support said GAD antigenic molecule of
said first source as present in a complex as defined in (b), prior to, or
concurrent with, or subsequent to, contact of said GAD antigenic molecule of
said first source with the sample of body fluid being screened;
(d) means for providing direct or indirect detectable labelling means to said
GAD antigenic molecule of said second source as present in a complex as
defined in (b), prior to, or concurrent with, or subsequent to, contact of said
GAD antigenic molecule of said second source with the sample of body fluid
being screened; and
(e) means for detecting the presence of complexes as defined in (b)
immobilised as defined in (c) so as to provide an indication of analyte
autoantibodies present in said sample.
Alternatively, a preferred embodiment of the present invention may suitably comprise
a kit for screening a sample of body fluid obtained from an animal subject for analyte
autoantibodies reactive with IA-2, or one or more variants, analogues, derivatives or
fragments thereof, said kit comprising:

(a) first and second sources of antigenic molecules consisting essentially of
IA-2 antigenic molecules selected from IA-2, one or more variants, analogues,
derivatives or fragments thereof, with which analyte autoantibodies when
present in said sample of body fluid can interact and fusion molecules
comprising two or more directly or indirectly fused antigenic molecules
selected from IA-2 and said one or more variants, analogues, derivatives or
fragments thereof, wherein IA-2 antigenic molecules of said first source are
immobilised to a solid support as defined in (c) and IA-2 antigenic molecules
of said second source are provided with labelling means as defined in (d);
(b) means for contacting said IA-2 antigenic molecules as provided by (a)
simultaneously or successively with said sample of body fluid being screened,
whereby analyte autoantibodies when present in said sample of body fluid can
interact with said IA-2 antigenic molecules so as to form one or more
complexes comprising [IA-2 antigenic molecule of said first source]-[analyte
autoantibody]-[IA-2 antigenic molecule of said second source];

(c) means for immobilising to a solid support said IA-2 antigenic molecule of
said first source as present in a complex as defined in (b), prior to, or
concurrent with, or subsequent to, contact of said IA-2 antigenic molecule of
said first source with the sample of body fluid being screened;
(d) means for providing direct or indirect detectable labelling means to said
IA-2 antigenic molecule of said second source as present in a complex as
defined in (b), prior to, or concurrent with, or subsequent to, contact of said
IA-2 antigenic molecule of said second source with the sample of body fluid
being screened; and
(e) means for detecting the presence of complexes as defined in (b)
immobilised as defined in (c) so as to provide an indication of analyte
autoantibodies present in said sample.
Alternatively, a preferred embodiment of the present invention may suitably-comprise
a kit for screening a sample of body fluid obtained from an animal subject for first and

second analyte autoantibodies respectively reactive with GAD and IA-2, or one or
more variants, analogues, derivatives or fragments thereof, said kit comprising:
(a) first and second sources of GAD antigenic molecules with which analyte
autoantibodies when present in said sample of body fluid can interact, and
which GAD antigenic molecules are selected from GAD65, GAD67, one or
more variants, analogues, derivatives or fragments thereof and fusion
molecules comprising two or more directly or indirectly fused antigenic
molecules selected from GAD65, GAD67, IA-2 and said one or more variants,
analogues, derivatives or fragments thereof and wherein at least one of said
antigenic molecules of said fusion molecule comprises GAD65, GAD67, or one
or more variants, analogues, derivatives or fragments thereof, wherein GAD
antigenic molecules of said first source are immobilised to a solid support as
defined in (d) and GAD antigenic molecules of said second source are
provided with labelling means as defined in (e);
(b) first and second sources of IA-2 antigenic molecules with which analyte
autoantibodies when present in said sample of body fluid can interact and
which IA-2 antigenic molecules are selected from IA-2, one or more variants,
analogues, derivatives or fragments thereof, and fusion molecules comprising
two or more directly or indirectly fused antigenic molecules selected from IA-
2, GAD65, GAD67 and said one or more variants, analogues, derivatives or
fragments thereof and wherein at least one of said antigenic molecules of said
fusion molecule comprises IA-2 or one or more variants, analogues,
derivatives or fragments thereof, wherein IA-2 antigenic molecules of said
first source are immobilised to a solid support according as defined in (d) and
IA-2 antigenic molecules of said second source are provided with labelling
means as defined in (e);
(c) means for contacting said antigenic molecules as provided by (a) and (b)
simultaneously or successively with said sample of body fluid being screened,
whereby analyte autoantibodies when present in said sample of body fluid can
interact with said antigenic molecules so as to form one or more complexes
comprising [GAD antigenic molecule of said first source]-[analyte

autoantibody]-[GAD antigenic molecule of said second source] or [IA-2
antigenic molecule of said first source]-[analyte autoantibody]-[IA-2 antigenic
molecule of said second source];
(d) means for immobilising to a solid support said GAD or IA-2 antigenic
molecule of said first source as present in a complex as defined in (c), prior to,
or concurrent with, or subsequent to, contact of said GAD or IA-2 antigenic
molecule of said first source with the sample of body fluid being screened;
(e) means for providing direct or indirect detectable labelling means to said
GAD or IA-2 antigenic molecule of said second source as present in a
complex as defined in (c), prior to, or concurrent with, or subsequent to,
contact of said GAD or IA-2 antigenic molecule of said second source with
the sample of body fluid being screened; and
(f) means for detecting the presence of complexes as defined in (c)
immobilised as defined in (d) so as to provide an indication of analyte
autoantibodies present in said sample.
It may be preferred in a kit according to the present invention that the antigenic
molecules of the first and second sources comprise one or more common antigenic
molecules. For example, antigenic molecules of said first and second sources may
comprise GAD65 or GAD67; alternatively antigenic molecules of the first and second
sources may both comprise IA-2.
Alternatively, it may be preferred that the antigenic molecules, of either the first or
second source, are selected from pancreatic islet cell antigenic molecules and insulin,
and the antigenic molecules of the other source comprises one or more variants,
analogues, derivatives or fragments of the above mentioned antigenic molecules of
either the first or second source, with which analyte autoantibodies when present in
said sample of body fluid can interact For example, where the antigenic molecules of
either the first or second source comprise GAD65 or GAD67, the antigenic molecules
of the other source comprise one or more variants, analogues, derivatives, or fragments
of GAD65 or GAD67 with which analyte autoantibodies when present in said sample

of body fluid can interact. A further example may be where the antigenic molecules
of either the first or second source comprise IA-2, and the antigenic molecules of the
other source comprise one or more variants, analogues, derivatives or fragments of
IA-2 with which analyte autoantibodies when present in said sample of body fluid can
interact.
A still further alternative embodiment of the present invention may be where both
antigenic molecules of the first and second sources comprise a variant, analogue,
derivative or fragment, which may be the same or different, of a common antigenic
molecule, with which analyte autoantibodies when present in said sample of body
fluid can interact. For example, antigenic molecules of both the first and second
sources can comprise a variant, analogue, derivative or fragment, which may be the
same or different, of GAD65 or GAD67, with which analyte autoantibodies when
present in said sample of body fluid can interact. A further example may be where
antigenic molecules of both the first and second sources comprise a variant, analogue,
derivative or fragment, which may be the same or different, of IA-2, with which
analyte autoantibodies when present in said sample of body fluid can interact. It will
be appreciated from the above that where antigenic molecules of the first and second
sources comprise variants, analogues, derivatives or fragments derived from a
common antigenic molecule, such variants, analogues, derivative or fragments as
provided by the respective sources, may be the same or different (for example variant
1 provided by the first source of antigenic molecules and variant 2 provided by the
second source of antigenic molecules, where variants 1 and 2 may respectively
represent different variants of GAD65 or GAD67, or fragment 1 provided by the first
source of antigenic molecules and fragment 2 provided by the second source of
antigenic molecules, where fragments 1 and 2 may respectively represent different
fragments of GAD65 or GAD67, such as distinct, or possibly overlapping, epitopes of
GAD65 or GAD67).
Suitably the detectable labelling means can be selected from the group consisting of
enzymic labels, isotopic labels, chemiluminescent labels, fluorescent labels, dyes and
the like and typically can be selected from the group consisting of alkaline
phosphatase, horseradish peroxidase, biotin-or the like and in particular can comprise
biotin. Suitably such detectable labelling means (when attached to the one or more

antigenic molecules substantially as hereinbefore described, or one or more variants,
analogues, derivatives or fragments thereof, or one or more fusion molecules thereof)
can be reacted with one or more substrates therefor (such as an avidin or streptavidin
conjugate, for example, streptavidin horseradish peroxidase conjugate or the like),
whereby the resulting conjugates can be detected suitably by measurement of optical
density or the like.
The detectable labelling means may be directly provided to the one or more antigenic
molecules, or one or more one or more variants, analogues, derivatives or fragments
thereof, or one or more fusion molecules thereof, substantially as hereinbefore
described. Additionally or alternatively the detectable labelling means may be
indirectly provided to the one or more antigenic molecules, or one or more variants,
analogues, derivatives or fragments thereof, or one or more fusion molecules thereof,
substantially as hereinbefore described, typically by providing the detectable labelling
means to one or more antibodies or other binding agents which can bind with the one
or more antigenic molecules, or one or more variants, analogues, derivatives or
fragments thereof, or one or more fusion molecules thereof, substantially as
hereinbefore described.
A kit for screening for autoantibodies according to the present invention typically
further comprises means for directly monitoring interaction of (i) such autoantibodies
present in the sample of body fluid from the subject and (ii) one or more antigenic
molecules, or one or more variants, analogues, derivatives or fragments thereof, or
one or more fusion molecules thereof, substantially as hereinbefore described, as
provided by the present invention, typically by employing non-competitive sandwich
type assay techniques known in the art.
According to a preferred embodiment of the present invention antigenic molecules of
one or more first sources, or one or more variants, analogues, derivatives or fragments
thereof, or one or more fusion molecules thereof, substantially as hereinbefore
described, are immobilised to a solid support and antigenic molecules of one or more
second sources, or one or more variants, analogues, derivatives or fragments thereof,
or one or more fusion- molecules thereof, substantially as hereinbefore described, are
provided with detectable labelling means, and wherein preferably the antigenic

molecules of the one or more second sources are provided in solution phase as is
conventional for use in known ELIS A techniques.
Preferably, a kit according to the present invention comprises immobilising means
whereby antigenic molecules, or one or more variants, analogues, derivatives or
fragments thereof, or one or more fusion molecules thereof, with which analyte
autoantibodies when present in said sample of body fluid can interact, of one or more
first sources are immobilised to a solid support prior to contact with a sample of body
fluid being screened. Suitably a kit according to the present invention comprises
contacting means whereby such immobilised antigenic molecules of the one or more
first sources, or one or more variants, analogues, derivatives or fragments thereof, or
one or more fusion molecules thereof, with which analyte autoantibodies when
present in said sample of body fluid can interact, can be contacted with the sample of
body fluid being screened either simultaneously or successively with contact of the
sample of body fluid with antigenic molecules of one or more second sources, or
more one or more variants, analogues, derivatives or fragments thereof, or one or
more fusion molecules thereof, with which analyte autoantibodies when present in
said sample of body fluid can interact. Particularly preferably, contacting means are
provided whereby immobilised antigenic molecules of the one or more first sources,
or one or more variants, analogues, derivatives or fragments thereof, or one or more
fusion molecules thereof, with which analyte autoantibodies when present in said
sample of body fluid can interact, are contacted with the sample of body fluid being
screened so as to form an intermediate complex comprising [antigenic molecule] -
[analyte autoantibody] where the antigenic molecule is immobilised to a solid support
and the thus formed immobilised intermediate complex is subsequently contacted
with molecules of the one or more second sources, or one or more variants, analogues,
derivatives or fragments thereof, or one or more fusion molecules thereof, with which
analyte autoantibodies when present in said sample of body fluid can interact, present
in solution phase, so as to form the hitherto described complex comprising [antigenic
molecule of said first source]-[analyte autoantibody]-[antigenic molecule of said
second source] immobilised to a solid support via the antigenic molecule of said first
source.

Accordingly, the present invention provides a kit for screening a sample of body fluid
obtained from an animal subject for analyte autoantibodies reactive with one or more
antigenic molecules selected from pancreatic islet cell antigenic molecules and
insulin, or one or more variants, analogues, derivatives or fragments thereof, said kit
comprising:
(a) one or more first sources of antigenic molecules with which analyte
autoantibodies when present in said sample of body fluid can interact and
which antigenic molecules are selected from pancreatic islet cell antigenic
molecules, insulin, one or more variants, analogues, derivatives or fragments
of said pancreatic islet cell antigenic molecules or insulin, and fusion
molecules comprising two or more directly or indirectly fused antigenic
molecules selected from pancreatic islet cell antigenic molecules, insulin and
said one or more variants, analogues, derivatives or fragments thereof,
wherein said antigenic molecules of said one or more first sources are
immobilised to a solid support;
(b) one or more second sources of antigenic molecules with which analyte
autoantibodies when present in said sample of body fluid can interact and
which antigenic molecules are selected from pancreatic islet cell antigenic
molecules, insulin, one or more variants, analogues, derivatives or fragments
of said pancreatic islet cell antigenic molecules or insulin, and fusion
molecules comprising two or more directly or indirectly fused antigenic
molecules selected from pancreatic islet cell antigenic molecules, insulin and
said one or more variants, analogues, derivatives or fragments thereof, which
antigenic molecules of said one or more second sources are provided in
solution phase and are provided with labelling means as defined in (d);
(c) means for contacting said antigenic molecules as provided by (a) and (b)
simultaneously or successively with said sample of body fluid being screened,
whereby analyte autoantibodies when present in said sample of body fluid can
interact with said antigenic molecules so as to form one or more immobilised
complexes comprising [antigenic molecule of said first source]-[analyte
autoantibody]-[antigenic molecule of said second source], wherein said

antigenic molecules of said first and second sources when present in said one
or more complexes comprise, or are derived from, a common antigenic
molecule, or wherein binding regions of said antigenic molecules of said first
and second sources, for said analyte autoantibody, when present in said one or
more complexes are present in, or are derived from, a common antigenic
molecule, which immobilised complexes are immobilised to a solid support
via said antigenic molecule of said first source present in said complex;
(d) means for providing direct or indirect detectable labelling means to said
antigenic molecule of said second source as present in a complex as defined in
(c), prior to, or concurrent with, or subsequent to, contact of said antigenic
molecule of said second source with said sample of body fluid being screened;
and
(e) means for detecting the presence of complexes as defined in (c) so as to
provide an indication of analyte autoantibodies present in said sample.
Other contacting means may, however, be present in a kit according to the present
invention, for example it is possible according to the present invention not to provide
means for initially immobilising the antigenic molecules, or one or more variants,
analogues, derivatives or fragments thereof, or one or more fusion molecules thereof,
of the one or more first antigen sources, prior to a contacting step as described above,
but to provide means to initially contact the solid support substantially as hereinbefore
described with a binder for the antigenic molecules, or one or more variants,
analogues, derivatives or fragments thereof, or one or more fusion molecules thereof,
of the one or more first antigen sources and for the kit to comprise means for
subsequently contacting the so treated solid support with the above described
antigenic molecules or the one or more first and second sources, or one or more
variants, analogues, derivatives or fragments thereof, or one or more fusion molecules
thereof, and the sample of body fluid being screened.
In respect of the solid supports and conditions employed in a kit according to the
present invention, the supports and conditions do not generally fundamentally differ
from conventional supports and conditions employed in known immunoassay

techniques. A solid support for use in a kit according to the present invention can
comprise an ELISA plate as currently employed in known ELISA techniques, or may
employ any other suitable support for use in the present invention, such as tubes,
particles, magnetic beads, nitrocellulose or the like.
Detection of analyte autoantibodies reactive with one or more antigenic molecules
selected from pancreatic islet cell antigenic molecules and insulin according to a
method provided by the present invention, or employing a kit provided by the present
invention, can be useful in the diagnosis of a number of disease states with which the
presence of such analyte autoantibodies is associated. In particular, the present
invention can be employed in screening a sample of body fluid from an animal subject
suspected of suffering from, susceptible to or having any of the following disease
states - type 1 diabetes mellitus and / or stiff man syndrome, type 2 diabetes mellitus,
one or more autoimmune thyroid diseases, celiac disease, one or more connective
tissue diseases, adrenal autoimmunity, or a combination of two or more different
autoimmune diseases and the present invention further provides a method of
diagnosing any of the above disease states in an animal subject.
It will be appreciated from the foregoing description that the present invention
provides assay methods and kits for detecting, in a sample of body fluid obtained
from an animal subject, analyte autoantibodies reactive with one or more antigenic
molecules selected from pancreatic islet cell antigenic molecules and insulin and
indicative of the onset or presence of the above disease states. The detection of such
autoantibodies in the sample of body fluid (or at least the level of such autoantibodies
in the sample) is indicative of the likely onset or presence of such disease states in the
subject from which the sample is obtained and can, therefore, enable the diagnosis of
the likely onset or presence of such disease states.
There is, therefore, further provided by the present invention a method of diagnosing
the likely onset or presence of disease states, associated with the presence of
autoantibodies reactive with one or more antigenic molecules selected from pancreatic
islet cell antigenic molecules and insulin, in an animal subject (in particular a human
subject); suspected of having or being susceptible to these disease states, the method .
comprising detecting autoantibodies reactive with one or more antigenic molecules

selected from pancreatic islet cell antigenic molecules and insulin, and-being
indicative of the likely onset or presence of these disease states in a sample of body
fluid from the subject substantially as hereinbefore described, and whereby the
detected autoantibodies can provide a diagnosis of the likely onset or presence of
these disease states.
There is, therefore, still further provided by the present invention a method of
delaying or preventing the onset of disease states, associated with the presence of
autoantibodies reactive with one or more antigenic molecules selected from pancreatic
islet cell antigenic molecules and insulin, in an animal subject (in particular a human
subject), or treating an animal subject (in particular a human subject) having or
recovering from such disease states, which method comprises initially detecting
autoantibodies reactive with one or more antigenic molecules selected from pancreatic
islet cell antigenic molecules and insulin, and being indicative of the onset or presence
of such disease states, in a sample of body fluid obtained from the subject
substantially as hereinbefore described, thereby providing a diagnosis of the likely
onset or presence of such disease states in the subject, and administering to the subject
a therapeutically effective amount of at least one therapeutic agent effective in
delaying onset, obviating, preventing and/or treating these disease states.
There is, therefore, still further provided by the present invention a method of
assessing the effectiveness of delaying or preventing the onset of disease states,
associated with the presence of autoantibodies reactive with one or more antigenic
molecules selected from pancreatic islet cell antigenic molecules and insulin, in an
animal subject (in particular a human subject), or assessing the effectiveness of
treating an animal subject (in particular a human subject) having or recovering from
such disease states, which method comprises administering to the subject a
therapeutically effective amount of at least one therapeutic agent effective in delaying
onset, obviating, preventing and/or treating such disease states associated with the
presence of autoantibodies reactive with one or more antigenic molecules selected
from pancreatic islet cell antigenic molecules and insulin, and detecting
autoantibodies indicative of the onset or presence of such disease states in a sample of
body fluid obtained from the subject, so as to provide an indication of the presence of
said autoantibodies in said sample, thereby providing an indication of the

effectiveness of treating the subject, or of delaying or preventing the onset of such
disease states in the subject.
There is still further provided by the present invention, in combination, a kit
substantially as hereinbefore described, together with a therapeutically effective
amount of at least one therapeutic agent effective in the treatment of disease states
associated with the presence of autoantibodies reactive with one or more antigenic
molecules selected from pancreatic islet cell antigenic molecules and insulin.
A sample of body fluid being screened by the present invention will typically
comprise blood samples or other fluid blood fractions, such as in particular serum
samples or plasma samples, but the sample may in principle be another biological
fluid, such as saliva or urine or solubilised tissue extracts.
The term "antigen" or "antigenic molecules" as used herein denotes a compound with
which antibodies as described herein can interact and which is capable of binding to
an antibody to form specific antibody-antigen complexes. The antigen or antigenic
molecules may be natural or synthetic and modifications thereto are preferably such
as to not detrimentally affect the binding properties thereof to a specific antibody.
As indicated above, the present invention covers the use of "variants", "analogs",
"derivatives" and "fragments" of antigenic molecules as described herein and the
terms "variants", "analogs", "derivatives" and "fragments" as used herein can be
characterised as polypeptides which retain essentially the same biological function or
activity as naturally occurring antigenic molecules and in particular in respect of the
binding properties thereof for a specific antibody. Suitably, variants, analogs,
derivatives and fragments, and variants, analogs and derivatives of the fragments as
described herein, have a primary structural conformation of amino acids in which
several or a few (such as 5 to 10, I to 5 or 1 to 3) amino acid residues of the naturally
occurring antigenic molecules are substituted, deleted or added, in any combination.
Especially preferred among these are silent substitutions, additions are deletions
which do not alter or substantially alter the biological activity or function of the
naturally occurring antigenic molecules. Conservative substitutions can be preferred
as hereinafter described in greater detail.

More particularly, variants, analogs or derivatives of antigenic molecules suitable for
use according to the present invention may be ones in which one or more of the amino
acid residues are substituted with a conserved or non-conserved amino acid residue
(preferably a conserved amino acid residue), or ones in which one or more of the
amino acid resides includes a substituent group or the like. Such variants, derivatives
and analogs are deemed to be within the scope of those skilled in the art from the
teachings herein.
Most typically, variants, analogs or derivatives are those that vary from a reference
(such as naturally occurring antigenic molecules as referred to herein) by conservative
amino acid substitutions. Such substitutions are those that substitute a given amino
acid in a polypeptide by another amino acid of like characteristics. Typically seen as
conservative substitutions are the replacements, one for another, among the aliphatic
amino acids A, V, L and I; among the hydroxyl residues S and T; among the acidic
residues D and E; among the amide residues N and Q; among the basic residues K and
R; and among the aromatic residues F and Y.
More particularly, the term "fragment" as used herein denotes a polypeptide having
an amino acid sequence that entirely is the same as part but not all of the amino acid
sequence of naturally occurring amino acids as referred to herein, and variants or
derivatives thereof and such fragments may be "free standing", i.e. not part of or
fused to other amino acids or polypeptides, or they may be comprised within a larger
polypeptide of which they form a part or region. In the context of the present
invention, it will be appreciated that particularly preferred fragments for use
according to the present invention may be one or more epitopes of antigenic
molecules as referred to herein and as described above such epitope fragments may be
used in "free standing" form or may be used within a larger polypeptide, such as a
scaffold polypeptide, of which they form a part or region. The use of such epitope
fragments in substantially isolated or free standing form according to the present
invention can be advantageous in terms of enhanced stability of such substantially
isolated or free standing epitope fragments when compared with the use of a full
length antigen and / or enhanced specificity associated, with the use of such

substantially isolated or free standing epitope fragments compared with the use of a
full length antigen.
The present invention will now be further illustrated by the following example, which
does not limit the scope of the invention in any way.
Example:
Preparation of GAD65 coated ELISA plates
Human recombinant GAD65 of >95% purity as assessed by SDS-PAGE (M Powell, L
Prentice, T Asawa, R Kato, J Sawicka, H Tanaka, V Petersen, A Munkley, S Morgan,
B Rees Smith, J Furmaniak. Clinica Chimica Acta, 1996 256: 175-188) was diluted
to 150ug/L in a coating buffer comprising 1.59g/L Na2CO3, 2.94g/L NaHCO3, 0.1g/L
NaN3, 0.01g/L Phenol Red, and 5mg/L BSA (pH 9.2) and 150uL of the resulting
solution was added to 96-well ELISA plates (available under the trade mark Nunc F8
MaxiSorp). The plates were then incubated overnight at 4°C, the contents of the wells
aspirated and the wells washed three times with a high salt buffer (HSB) comprising
10g/L BSA, lg/L NaN3, 11.69g/L NaCl, 18.17g/L Tris, and 10mL/L
Polyoxyethylene-Sorbitan Monolaurate (available under the trade mark Tween 20 (pH
8.3). A post-coat buffer comprising 3g/L BSA, 9g/L NaCl, 20g/L sucrose and 0.2g/L
NaN3 was added to the wells (250uL per well) and incubated for 30 minutes at room
temperature. The post-coat buffer was then aspirated, the plates dried under vacuum
and stored in sealed bags with silica gel at 4°C until use.
Labelling of GAD65 with bio tin
Human recombinant GAD65 in PBS (1.15g/L Na2HPO4, 0.2g/L KH2PO4, 0.2g/L KCl,
8g/L NaCl) was reacted with a commercial biotinylation reagent (Sulfo-NHS-LC-LC-
Biotin available under the trade mark EZ-Link from Perbio Science), at a molar ratio
of 1 part GAD65 to 24 parts biotinylation reagent for 30 minutes at room temperature.
The reaction was stopped by dialysis against PBS at 4°C and the resulting material
was stored at -70°C in small aliquots.
The resulting GAD65 labelled with bio tin according to the above techniques (GAD65-
Bi) was diluted to 3mg/L in 20g/L BSA, filtered through a 0.22m filter, aliquotted at

1mL per vial, freeze dried and then stored at -20°C. To use, 7.5mL of HSB was
added to the vial, giving a final concentration of GAD65-Bi of 400g/L.
Preparation of calibrators for GAD65 autoannbody assay
IgG preparations of three human monoclonal autoantibodies to GAD65 (M J Powell,
N Hayakawa, M Masuda, J Sanders, M Evans, LDKE Premawardhana, J Furmaniak,
B Rees Smith. Isolation and characterization of three human monoclonal antibodies
to glutamic acid decarboxylase (GAD65) from a patient without clinical diabetes.
Diabetes/Metabolism Research and Reviews, 2001 17 (SI): 021) were diluted in HSB
to give an absorbance at OD450nm from 4.0 to 0.1. These monoclonal antibodies are
known to be representative of GAD65 autoantibodies found in sera from patients with
type 1 diabetes (M J Powell, N Hayakawa, M Masuda, J Sanders, M Evans, LDKE
Premawardhana, J Furmaniak, B Rees Smith. Isolation and characterization of three
human monoclonal antibodies to glutamic acid decarboxylase (GAD65) from a patient
without clinical diabetes. Diabetes/Metabolism Research and Reviews, 2001 17 (S1):
021).
GAD65 Autoantibodv Detection
Plates coated with GAD65 prepared according to the above techniques were brought to
room temperature and 25L of undiluted serum sample obtained from test donors, or
calibrators prepared according to the above techniques, were added to the plate wells
in duplicate and incubated for 1 hour at room temperature with shaking at 200rpm.
The samples were then aspirated, the wells washed three times with buffer (8.7g/L
NaCl, 2.4g/L Tris, 0.5mL/L Tween 20, pH 7.6), followed by addition of lOOuL of
GAD65-Bi diluted in HSB (400g/L) prepared according to the above techniques and
incubation for 1 hour at room temperature, with shaking at 200rpm. After the
incubation the contents of the wells were aspirated and wells washed three times with
buffer (8.7g/L NaCl, 2.4g/L Tris, 0.5mL/L Tween 20, pH 7.6). Thereafter, lOOuL of
a streptavidin horseradish peroxidase conjugate diluted to 1g/mL was added and
incubated for 20 minutes at room temperature, with shaking at 200rpm. After
aspiration, the wells were washed three times with buffer (8.7g/L NaCl, 2.4g/L Tris,
0.5mL/L Tween 20, pH 7.6) and once with water.. lOOuL of tetramethyl benzidine
was then added and incubated for 20 minutes in the dark, followed by addition of

50uL of 0.5 mol/L H2SO4. The absorbance of the wells was then measured in an
ELISA plate reader at 450nm or in the case of samples giving an OD 450nm>4.0,
absorbance was also measured at 405nm.
The above procedures were applied to IA-2 autoantibodies and also to the combined
detection of GAD65 and IA-2 autoantibodies (in particular, involving the preparation
of IA-2 labelled with biotin, IA-2-Bi). Reference preparations of IA-2 autoantibodies
for generation of a calibration curve were prepared from polyclonal IA-2
autoantibodies.
Results
A typical calibration curve for GAD65 obtained at OD 450nm is shown in Figure la.
Seven calibrators of arbitrarily assigned GAD65 autoantibody ELISA units from 0
ELISA units/mL to 512 ELISA units/mL were included. OD 450nm values for the
calibrators were 0.033, 0.168, 0.310, 1.020, 3.187, >4.0 and >4.0 for calibrators 0,
1.0, 2.0, 8.0, 32, 128 and 512 respectively. OD 405nm values were 0.008, 0.048,
0.090, 0.290, 0.929, 1.385 and 1.620 for calibrators 0, 1.0, 2.0, 8.0, 32, 128 and 512
respectively as shown in Figure 1b. Consequently, samples that gave OD 450nm
values >4.0 could be measured within the calibration curve range at OD 405nm as
shown in Figure lb. The mean ±SD absorbance at 450nm for n=25 sera from healthy
blood donors was 0.04 ± 0.015.
Results of GAD65 autoantibody measurement according to the present invention are
shown in Tables 1 and 2. Results of GAD65 autoantibodies in sera from n=18 patients
with type 1 diabetes mellitus and n=14 patients with Graves' disease are shown in
Table 1. All 18 diabetic sera were positive for GAD65 autoantibodies when ELISA
based techniques according to the present invention were employed (levels >1.0
ELISA units/mL), however only 13 of these patients were positive in
radioimmunoassay (RIA). The 5 samples which were negative in RIA showed low
levels of GAD65 autoantibodies in ELISA based techniques according to the present
invention with OD 450nm between 0.117 to 0.980 (1.3 to 10.7 ELISA units/mL). In
the case of samples with low levels in RIA (1.0 to 7.5 RIA units/mL), the OD 450nm
values in ELISA based techniques according to the present invention were between
0.290 and 1.060 (12.0 to 105.8 ELISA units/mL). In the case of samples with higher

levels of GAD65 autoantibodies in RIA (26.8 to 118.8 RIA units/mL), OD 450nm in
ELISA based techniques according to the present invention were very high>4.0(>128
ELISA units/mL) and, therefore, the relationship between the GAD65 autoantibody
levels in the sample and the calibration curve was calculated after measurement at
405nm. Preincubation of test sera with non-labelled GAD65 (at final concentration
from 0.0005 to 0.01g/mL) prior to performing the ELISA resulted in a dose
dependent reduction of OD 450nm values. These experiments confirmed the
specificity of the GAD65 autoantibody ELISA based techniques according to the
present invention.
In the case of n=14 sera from patients with Graves' disease, OD 450nm values for all
samples were below 0.05 ( to the present invention); all these samples were negative for GAD65 autoantibodies in
RIA ( Comparison of the lowest detection limit for GAD65 autoantibodies in ELISA based
techniques according to the present invention and RIA is shown in Table 2. In the
RIA dilutions of WHO reference preparation for islet cell autoantibodies [NIBSC
97/550] from 250 to 31.25 WHO units/mL, detectable levels of GAD65 autoantibodies
were seen. However, in the ELISA based techniques according to the present
invention, GAD65 autoantibodies were detectable in the case of WHO standard
dilutions from 250 to 3.91 WHO units/mL. Further, an example of the dilution profile
of a serum sample Z (from a patient with high levels of GAD65 autoantibodies) is
shown in Table 2. In the case of RIA, GAD65 autoantibodies were detectable in the
sample Z in the range from 1/8 to 1/8192 dilution. In the case of ELISA based
techniques according to the present invention, GAD65 autoantibodies were detectable
in the range from 1/8 to 1/32768 dilution. Consequently, the end point dilution
analyses of WHO standard and patient's serum indicated that a 4-fold to 8-fold
greater dilutions were detectable in ELISA based techniques according to the present
invention compared to RIA.
An example of results of IA-2 autoantibody measurement by ELISA based techniques
according to the present invention is shown in Table 3. Typical OD 450nm values for
the set of IA-2 autoantibody calibrators (0, 1:0, 2.0, 4.0, 8.0, 16, 32, 64, 128 ELISA

units/mL) were from 0.054 to >4.0 and OD405 values were from 0.008 to 1.874. Sera
from n=10 patients with type 1 diabetes mellitus who had IA-2 autoantibody levels in
RIA from 2.0 to 38.9 RIA units/mL were all positive in ELISA based techniques
according to the present invention. These samples gave OD 450nm values between
0.209 to >4.0 (2.6 to >32 ELISA units/mL). WHO standard was detectable in IA-2
autoantibody RIA at 250 WHO units/mL and 125 WHO units/mL however, further
dilutions (62.5 WHO units/mL and 31.25 WHO units/mL) were detectable in ELISA
based techniques according to the present invention. OD 450nm values in the case of
n=10 sera from healthy blood donors (all negative for IA-2 autoantibodies in RIA)
were between 0.030 and 0.071 ( Results of a combination assay for GAD65 autoantibodies and IA-2 autoantibodies are
shown in Tables 4 and 5. Table 4 shows results of combined measurement of GAD65
autoantibodies and IA-2 autoantibodies in ELISA based techniques according to the
present invention, when IA-2-Bi or GAD65-Bi or IA-2-Bi plus GAD65-Bi were used in
the assay. In the case of IA-2 autoantibody calibrators, no signal in the ELISA based
techniques according to the present invention was detected when GAE>65-Bi was used,
whereas dose dependent increase of OD 450nm was observed when IA-2-Bi or IA-2-
Bi plus GAL>65-Bi were used. The OD 450nm signal with IA-2-Bi alone was
essentially the same as signal with IA-2-Bi plus GAD65-Bi. In the case of GAD65
autoantibody calibrators, no signal was detected when IA-2-Bi was used in the assay
while dose dependent response of OD 450nm was observed with GAD65-Bi and IA-2-
Bi plus GAD65-BL The OD 450nm signals in the case of GAD65-Bi alone and IA-2-
Bi plus GAD65-Bi plus GAD65-Bi were comparable. Measurement of both
autoantibodies in different dilutions of serum sample Z showed a good agreement
with the results of IA-2 autoantibodies or GAD65 autoantibodies alone. Serum from
an individual healthy blood donor or healthy blood donor pool serum gave very low
OD 450nm values from 0.006 to 0.046 irrespective of which biotinylated antigen was
used. This experiment shows that in the combined assay to measure IA-2
autoantibodies and GAD65 autoantibodies, the results are specific and reflect the
presence of the two autoantibodies in the test sample.
More detailed results of the combined measurement of IA-2 autoantibodies and
GAD65 autoantibodies are shown in Table 5. Sera from patients with Type 1 diabetes

were tested in the assay. IA-2 autoantibodies and GAD65 autoantibodies wore
assessed in these sera by RIA; sera 1-3 were positive for both IA-2 autoantibodies and
GAD65 autoantibodies, sera 4-6 were negative for GAD65 autoantibodies but positive
for IA-2 autoantibodies, sera 7-9 were positive for GAD65 autoantibodies but negative
for IA-2 autoantibodies and sera 10-12 were negative for both IA-2 or GAD65
autoantibodies. As shown in Table 5, in the combined assay one or the other or bom
autoantibodies were detected in serum samples. Furthermore, in the case of serum 6
that had borderline levels of IA-2 autoantibodies by RIA (0.9 RIA units/mL), the
signal in the combined ELISA based techniques according to the present invention
was clearly positive.
Additional examples of results with ELISA for GAD65 autoantibodies, IA-2
autoantibodies and combined G AD65 and IA-2 autoantibodies according to the present
invention for different patient subgroups are shown in Tables 6, 7 and 8 respectively.
Conclusions
The above results show that the non-radioactive based techniques according to the
present invention allow measurement of GAD65 autoantibodies in test samples. The
non-radioactive based techniques according to the present invention can also be
employed to measure IA-2 autoantibodies separately or simultaneously. The
measurements are specific and they are in agreement with the results of GAD65
autoantibodies and IA-2 autoantibodies measurements by established radioactive
reference methods (M Powell, L Prentice, T Asawa, R Kato, J Sawicka, H Tanaka, V
Petersen, A Munkley, S Morgan, B Rees Smith, J Furmaniak. Clinica Chimica Acta,
1996 256: 175-188; and M Masuda, M Powell, S Chen, C Beer, P Fichna, B Rees
Smith, J Furmaniak. Autoantibodies to IA-2 in insulin-dependent diabetes mellitus.
Measurements with a new immunoprecipitation assay. Clinica Chimica Acta, 2000
291: 53-66). Furthermore, measurements of GAD65 autoantibodies and IA-2
autoantibodies by non-radioactive based techniques according to the present invention
are at least as sensitive compared to results by RIA (Tables 1, 2 and 3).
The techniques according to the present invention provide a non-radioactive assay to
measure GAD65 autoantibodies or IA-2 autoantibodies with a high sensitivity that is
convenient to use for diagnostic and screening purposes. A combination assay for

GAD65 and IA-2 autoantibodies according to the present invention can be of particular
benefit for use in large population screening programmes.

Footnote: GAD Ab levels above 1 RIA unit/mL were considered positive in RIA. *
WHO units/mL.

Values of samples positive in GAD65 Ab RIA were 33, 100 and 2500
units/mL. The same samples were negative, negative and >500 units/mL in the
ELISA respectively.
3 Range of values: 5.2 - greater than 500 units/mL.
4 Range of values: negative - 3800 units/mL.
5 Value of a sample positive in GAD65 Ab ELISA was 40 units/mL. The same
sample was negative in the RIA.
6 Values of samples positive in GAD65 Ab ELISA were 306 and 500 units/mL.
The same samples were 354 units/mL and 1700 units/mL in the RIA, respectively.
7 Values of samples positive in GAD65 Ab RIA were 78, 354 and 1700
units/mL. The same samples were negative, 306 and 500 units/mL in the ELISA,
respectively.
8 Value of a sample positive in GAD65 Ab ELISA was 24 units/ml; this sample
was negative in the RIA.
9 The same sample was positive in GAD65 Ab ELISA and RIA with values of
15 units/mL and 30 units/mL, respectively.

1 Values of samples positive in IA-2 Ab ELIS A were 44 units/mL and 179
units/mL (units/mL are WHO 97/550). Adsorption experiments indicated the
presence of specific IA-2 Ab in these samples. The same samples were negative in
the RIA.
2 Values of IA-2 Ab positive samples in RIA were 150 units/mL and 28S
units/mL. The same samples were negative in the ELIS A.
3 Range of values: 132 - >4000 units/mL.

4 Range of values: 178- 4508 units/mL.
5 Value of IA-2 Ab positive sample was 101 units/mL. This sample was
negative in the RIA.

1 Value of GAD65 Ab for samples positive in ELISA ranged from 7.3 - >500
units/mL (units/mL are WHO 97/550).
2 Values of IA-2 Ab for samples positive in ELISA ranged from 34-3613
units/mL (units/mL are WHO 97/550).
3 All combined ELISA results were expressed as index calculated as follows:
Oik™ n^ of sample ;
OE^sonm of pool of healthy blood donor sera
values of 2.0 or greater are positive

18 sera were positive for GAD65 Ab and negative for IA-2 Ab; all 18 were positive in
the combined ELISA.
4 sera were positive for IA-2 Ab and negative for GAD6s Ab; all 4 were positive in
the combined ELISA
10 sera were positive for GAD65 Ab and for IA-2 Ab; all 10 were positive in the
combined ELISA
1 serum was negative for GAD65 Ab and for IA-2 Ab but positive in the combined
ELISA
2 sera were negative in all 3 assays.

4 GAD65 Ab levels were 10 units/mL and IA-2 Ab were negative in the one
positive sample.
5 GAD65 Ab were negative and IA-2 Ab levels were 76 units/mL in the one
positive sample.
6 The positive samples were the same as described in 4 and 5 (index value 7.8
and 4.8 respectively).
GAD65 Ab ELISA, IA-2 Ab ELISA and combined GAD65 Ab + IA-2 Ab ELISA
results in different patient groups.
7 GAD65 Ab levels were 9 and >500 units/mL but IA-2 Ab were negative in the
two positive samples.
The positive samples were the same as described in 7. The index values for
these samples were: 3.6 and 55.0 respectively.
GAD65 Ab levels were 15 units/mL but IA-2 Ab were negative.
The positive sample was the same as described in 9. The index value was 6.0.

WE CLAIM:
1. A method of screening a sample of body fluid obtained from an animal
subject for analyte autoantibodies reactive with one or more pancreatic
islet cell antigenic molecules selected from the group consisting of
GAD65, GAD67 and protein tyrosine phosphatase-like islet cell antigen
(IA-2), or one or more variants, analogues, derivatives or fragments
thereof, said method comprising:
(a) providing one or more first sources of antigenic molecules with
which analyte autoantibodies when present in said sample of body
fluid can interact and which antigenic molecules are selected from
said group of pancreatic islet cell antigenic molecules, one or more
variants, analogues, derivatives or fragments of said pancreatic islet
cell antigenic molecules and fusion molecules comprising two or
more directly or indirectly fused antigenic molecules selected from
pancreatic islet cell antigenic molecules, and said one or more
variants, analogues, derivatives or fragments thereof;
(b) providing one or more second sources of antigenic molecules with
which analyte autoantibodies when present in said sample of body
fluid can interact and which antigenic molecules are selected from
said group of pancreatic islet cell antigenic molecules, one or more
variants, analogues, derivatives or fragments of said group of
pancreatic islet cell antigenic molecules, and fusion molecules
comprising two or more directly or indirectly fused antigenic
molecules selected from pancreatic islet cell antigenic molecules,
and said one or more variants, analogues, derivatives or fragments
thereof;

(c) contacting said antigenic molecules as provided by steps (a) and (b)
simultaneously or successively with said sample of body fluid
being screened, whereby analyte autoantibodies when present in
said sample of body fluid can interact with said antigenic molecules
so as to form one or more complexes comprising [antigenic
molecule of said first source]-[analyte autoantibody]-[antigenic
molecule of said second source], wherein said molecules of said
first and second sources when present in said one or more
complexes comprise, or are derived from, a common antigenic
molecule, or wherein binding regions of said antigenic molecules of
said first and second sources, for said analyte autoantibody, when
present in said one or more complexes are present in, or are
derived from, a common antigenic molecule;
(d) prior to, or concurrent with, or subsequent to, step ( c ), providing
immobilizing means whereby said antigenic molecule of said first
source as present in a complex as formed in step (c ) is
immobilized to a solid support prior to, or concurrent with, or
subsequent to, step (c);
(e) prior to, or concurrent with, or subsequent to, step ( c ), providing
direct or indirect detectable labelling means whereby said antigenic
molecule of said second source as present in a complex as formed
in step ( c ) is provided with such direct or indirect detectable
labelling means prior to, or concurrent with, or subsequent to, step
(c); and

(f) detecting the presence of complexes formed in (c ) immobilized
according to (d) so as to provide an indication of analyte
autoantibodies present in said sample.
2. A method as claimed in any of claim 1, which comprises providing (i)
first and second sources of GAD antigenic molecules with which
analyte autoantibodies when present in said sample of body fluid can
interact, and which GAD antigenic molecules are selected from GAD
65, GAD67, one or more variants, analogues, derivatives or fragments
thereof and fusion molecules comprising two or more directly or
indirectly fused antigenic molecules selected from GAD65, GAD67,
protein tyrosine phosphatase-like islet cell antigen (IA-2) and said one
of more variants, analogues, derivatives or fragments thereof and
wherein at least one of said antigenic molecules of said fusion
molecule comprises GAD65, GAD67, or one or more variants,
analogues, derivatives or fragments thereof, wherein GAD antigenic
molecules of said first source are immobilized to a solid support
according to step (d) and GAD antigenic molecules of said second
source are provided with labelling means according to step (e); and (ii)
first and second sources of protein tyrosine phosphatase-like islet cell
antigen (IA-2), antigenic molecules with which analyte autoantibodies
when present in said sample of body fluid can interact and which
protein tyrosine phosphatase-like islet cell antigen (IA-2) and antigenic
molecules are selected from protein tyrosine phosphate-like islet cell
antigen (IA-2), one or more variants, analogues, derivatives or
fragments thereof, and fusion molecules comprising two or more
directly or indirectly fused antigenic molecules selected from protein
tyrosine phosphatase-like islet cell antigen (IA-2), GAD65, GAD67 and

said one or more variants, analogues, derivatives or fragments thereof
and wherein at least one of said antigenic molecules of said fusion
molecules comprises protein tyrosine phosphatase-like islet cell
antigen (IA-2) or one or more variants, analogues, derivatives or
fragments thereof, wherein protein tyrosine phosphatase-like islet cell
antigen (IA-2), antigenic molecules of said first source are immobilised
to a solid support according to step (d) and protein tyrosine
phosphatase-like islet cell antigen (IA-2) antigenic molecules of said
second source are provided with labelling means according to step (e).
3. A method as claimed in any of claims 1 to 2, wherein the antigenic
molecules of the first and second sources when present in said one or
more complexes comprise one or more common antigenic molecules.
4. A method as claimed in claim 1, as dependent on any of claims 1,2 or
3, wherein one of the antigenic molecules of the first or second source
when present in the one or more complexes comprises GAD65, or
GAD67, the other antigenic molecule when present in the one or more
complexes comprises one or more variants, analogues, derivatives or
fragments of GAD65, GAD67, with which analyte autoantibodies when
present in said sample of body fluid can interact.
5. A method as claimed in claim 1, as dependent on any of claims 1,2,
wherein one of the antigenic molecules of the first or second source
when present in the one or more complexes comprises protein tyrosine
phosphatase-like islet cell antigen (IA-2) and the other antigenic
molecule when present in the one or more complexes comprises one or
more variants, analogues, derivatives or fragments of protein tyrosine

phosphatase-like islet cell antigen with which analyte autoantibodies
when present in said sample of body fluid can interact.
6. A method as claimed in any of claims 1 to 3, wherein both antigenic
molecules of the first and second sources when present in the one or
more complexes comprise variant, analogue, derivative or fragment,
which may be the same or different, of a common antigenic molecule,
with which analyte autoantibodies when present in said sample of
body fluid can interact.
7. A method as claimed in claim 6, as dependent on any of claims 1 or 2,
wherein both antigenic molecules of the first and second sources when
present in the one or more complexes comprise a variant, derivative or
fragment, which may be the same or different, of GAD65 or GAD67,
with which analyte autoantibodies when present in said sample of
body fluid can interact.
8. A method as claimed in claim 6, as dependent on any of claims 1, 2
wherein both antigenic molecules of the first and second sources when
present in said one or more complexes comprise a variant, analogue,
derivative or fragment, which may be the same or different, of protein
tyrosine phosphatase-like islet cell antigen (IA-2), with which analyte
autoantibodies when present in said sample of body fluid can interact.
9. A method as claimed in any of claims 1 to 8, wherein said antigenic
molecules of said one of more first sources are immobilized to a solid
support prior to contact with a sample of body fluid being screened.

10. A method as claimed in claim 9, wherein said immobilized antigenic
molecules of said one or more first sources are subsequently contacted
with the sample of body fluid being screened either simultaneously or
successively with contact of the sample of body fluid with said
antigenic molecules of said one or more second sources.
11. A method as claimed in claim 10, wherein said immobilized antigenic
molecules of said one or more first sources are subsequently contacted
with the sample of body fluid being screened so as to form an
intermediate complex comprising [antigenic molecule]-[analyte
autoantibody] where the antigenic molecule is immobilized to a solid
support and the thus immobilized intermediate complex is
subsequently contacted with said antigenic molecules of said one or
more second sources, present in solution phase, so as to form said
complexes comprising [antigenic molecule of said first source]-[analyte
autoantibody]-]antigenic molecule of said second source] immobilized
to a solid support via the antigenic molecule of the first source.
12. A kit for screening a sample of body fluid obtained from an animal
subject for analyte autoantibodies reactive with one or more pancreatic
islet cell antigenic molecules selected from the group consisting of
GAD65, GAD67 and protein tyrosine phosphatase-like islet cell antigen
(IA-2), one or more variants, analogues, derivatives or fragments
thereof, said kit comprising:
(a) one or more first sources of antigenic molecules with which analyte
autoantibodies when present in said sample of body fluid can
interact and which antigenic molecules are selected from said
group of pancreatic islet cell antigenic molecules, one or more

variants, analogues, derivatives or fragments of said pancreatic islet
cell antigenic molecules, and fusion molecules comprising two or
more directly or indirectly fused antigenic molecules selected from
said pancreatic islet cell antigenic molecules, and said one or more
variants, analogues, derivatives or fragments thereof;
(b) one or more second sources of antigenic molecules with which
analtye autoantibodies when present in said sample of body fluid
can interact and which antigenic molecules are selected from said
pancreatic islet cell antigenic molecules, or one or more variants,
analogues, derivatives or fragments of said pancreatic islet cell
antigenic molecules, and fusion molecules comprising two or more
directly or indirectly fused antigenic molecules selected from said
pancreatic islet cell antigenic molecules, and said one or more
variants, analogues, derivatives or fragments thereof.
(c) means for contacting said antigenic molecules as provided by (a)
and (b) simultaneously or successively with said sample of body
fluid being screened, whereby analyte autoantibodies when present
in said sample of body fluid can interact with said antigenic
molecules so as to form one or more complexes comprising
[antigenic molecule of said first source]-[analyte autoantibody]-
[antigenic molecule of said second source], wherein said antigenic
molecules of said first and second sources when present in said one
or more complexes comprise, or are derived from, a common
antigenic molecule, or wherein binding regions of said antigenic
molecules of said first and second sources, for said analtye
autoantibody, when present in said one or more complexes are
present in, or are derived from, a common antigenic molecule;

(d) means for immobilizing to a solid support, said antigenic molecule
of said first source as present in a complex as defined in ( c ), prior
to, or concurrent with, or subsequent to, contact of the antigenic
molecule of said first source with the sample of body fluid being
screened;
(e) means for providing direct or indirect detectable labelling means to
said antigenic molecule of said second source as present in a
complex as defined in ( c ), prior to , or concurrent with, or
subsequent to, contact of said antigenic molecule of said second
source with said sample of body fluid being screened; and
(f) means for detecting the presence of complexes as defined in ( c)
immobilized as defined in (d) so as to provide an indication of
analyte autoantibodies in said sample.
13. A kit as claimed in claim 17, which comprises a first source of antigenic
molecules consisting of GAD antigenic molecules selected from
GAD65, GAD67, one or more variants, analogues, derivatives or
fragments thereof with which analyte autoantibodies when present in
said sample of body fluid can interact and fusion molecules
comprising two or more directly or indirectly fused antigenic
molecules selected from GAD65, GAD67, and said one or more variants,
analogues, derivatives or fragments thereof, immobilized to a solid
support as defined in (d), and a second source of antigenic molecules
consisting essentially of GAD antigenic molecules selected from
GAD65, GAD67, one or more variants, analogues, derivatives or
fragments thereof, with which analyte autoantibodies when present in

said sample of body fluid can interact and fusion molecules
comprising two or more directly or indirectly fused antigenic
molecules selected from GAD65, GAD67 and said one or more variants,
analogues, derivatives or fragments thereof, provided with labelling
means as defined in (e).
14. A kit as claimed in claim 17, which comprises a first source of antigenic
molecules consisting essentially of protein tyrosine phosphatase-like
islet cell antigen (IA-2), antigenic molecules selected from protein
tyrosine phosphatase-like islet cell antigen (IA-2), one or more
variants, analogues, derivatives or fragments thereof, with which
analyte autoantibodies when present in said sample of body fluid can
interact, and fusion molecules comprising two or more directly or
indirectly fused antigenic molecules selected from protein tyrosine
phosphatase-like islet cell antigen (IA-2) and said one or more variants,
analogues, derivatives or fragments thereof, immobilized to a solid
support as defined in (d), and a second source of antigenic molecules
consisting essentially of protein tyrosine phosphatase-like islet cell
antigen (IA-2), selected from protein tyrosine phosphatase-like islet
cell antigen (IA-2), one or more variants, analogues, derivatives or
fragments thereof, with which analyte autoantibodies when present in
said sample of body fluid can interact and fusion molecules selected
from protein tyrosine phosphatase-like islet cell antigen (IA-2) and
said one or more variants, analogues, derivatives or fragments thereof,
provided with labelling means as defined in (e).
15. A kit as claimed in claim 17, which comprises (i) first and second
sources of GAD antigenic molecules with which analyte
autoantibodies when present in said sample of body fluid can interact,

and which GAD antigenic molecules are selected from GAD65, GAD67,
one or more variants, analogues, derivatives or fragments thereof and
fusion molecules comprising two or more variants, analogues,
derivatives or fragments thereof and fusion molecules comprising two
or more directly of indirectly fused antigenic (IA-2) molecules selected
from GAD65, GAD67, protein tyrosine phosphatase-like islet cell
antigen (IA-2) and said one or more variants, analogues, derivatives or
fragments thereof and wherein at least one of said antigenic molecules
of said fusion molecules comprises GAD65, GAD67, or one or more
variants, analogues, derivatives or fragments thereof, wherein GAD
antigenic molecules of said first source are immobilized to a solid
support as defined in (d) and GAD antigenic molecules of said second
source are provided with labelling means as defined in (e); and (ii) first
and second sources of protein tyrosine phosphates-like islet cell
antigen (IA-2), antigenic molecules with which analyte autoantibodies
when present in said sample of body fluid can interact and which
protein tyrosine phosphatase-like islet cell antigen (IA-2) antigenic
molecules are selected from protein tyrosine phosphatase-like islet cell
antigen (IA-2), one or more variants, analogues, derivatives or
fragments thereof, and fusion molecules comprising two or more
directly or indirectly fused antigenic molecules selected from protein
tyrosine phosphatase-like islet cell antigen, GAD65, GAD67 and said
one or more variants, analogues, derivatives of fragments thereof and
wherein at least one of said antigenic molecules of said fusion
molecule comprises protein tyrosine phosphatase-like islet cell antigen
(IA-2) or one or more variants, analogues, derivatives or fragments
thereof, wherein protein tyrosine phosphatase-like islet cell antigen
(IA-2) antigenic molecules of said first source are immobilized to a
solid support according as defined in (d) and protein tyrosine

phosphatase like islet cell antigen (IA-2) antigenic molecules of said
second source are provided with labelling means as defined in (e).
16. A kit as claimed in any of claims 12 to 15, wherein the antigenic
molecules of the first and second sources comprise one or more
common antigenic molecules.
17. A kit as claimed in claim 16, as dependent on any of claims 12 to 13
and 15, wherein said antigenic molecules of said first and second
sources comprise GAD65 or GAD67.
18. A kit as claimed in claim 16, as dependent on any of claims 12,14 and
15, wherein said antigenic molecules of the first and second sources
comprise protein tyrosine phosphatase-like islet cell antigen (IA-2).
19. A kit as claimed in any of claims 12 to 15, wherein antigenic molecules,
of either the first or second source, are selected from said pancreatic
islet cell antigenic molecules, and the antigenic molecules of the other
source comprises one or more variants, analogues, derivatives or
fragments of said antigenic molecules of either the first or second
source, with which analyte autoantibodies when present in said
sample of body fluid can interact.
20. A kit as claimed in claim 19, as dependent on any of claims 12 to 13
and 15, wherein antigenic molecules of either the first or second source
comprise GAD65 or GAD67, and the antigenic molecules of the other
source comprise one or more variants, analogues, derivatives or
fragments of GAD65 or GAD67, with which analyte autoantibodies
when present in said sample of body fluid can interact.

21. A kit as claimed in claim 19, as dependent on any of claims 12,14 and
15, wherein antigenic molecules of either the first or second source
comprise protein tyrosine phosphatase-like islet cell antigen (IA-2),
and the antigenic molecules of the other source comprise one or more
variants, analogues, derivatives or fragments of protein tyrosine
phosphatase-like islet cell antigen (IA-2) with which analyte
autoanitbodies when present in said sample of body fluid can interact.
22. A kit as claimed in any of claims 12 to 16, wherein both antigenic
molecules of the first and second sources comprise a variant, analogue,
derivative or fragment, which may be the same or different, of a
common antigenic molecule, with which analyte autoantibodies when
present in said sample of body fluid can interact.
23. A kit as claimed in claim 22, as dependent of any of claims 12 to 13 and
15, wherein antigenic molecules of both the first and second sources
comprise a variant, analogue, derivative or fragment, which may be
the same or different, of GAD65 or GAD67, with which analyte
autoantibodies when present in said sample of body fluid can interact.
24. A kit as claimed in claim 22, as dependent on any of claims 12,14 and
15, wherein antigenic molecules of both the first and second sources
comprise a variant, analogue, derivative or fragment, which may be
the same or different, of protein tyrosine phosphatase-like islet cell
antigen, with which analyte autoantibodies when present in said
sample of body fluid can interact.

25. A kit as claimed in any of claims 12 to 24, which comprises
immobilising means whereby antigenic molecules of said one or more
first sources are immobilized to a solid support prior to contact with a
sample of body fluid being screened.
26. A kit as claimed in claim 25, which comprises contacting means
whereby such immobilized antigenic molecules of said one or more
first sources are contacted with the sample of body fluid being
screened either simultaneously or successively with contact of the
sample of body fluid with antigenic molecules of said one or more
second sources.
27. A kit as claimed in claim 26, wherein contacting means are provided
whereby immobilized antigenic molecules of said one or more first
sources are contacted with the sample of body fluid being screened so
as to form as intermediate complex comprising [antigenic molecule]-
[analyte autoantibody] where the antigenic molecule is immobilized to
a solid support and the thus formed immobilized intermediate
complex is subsequently contacted with antigenic molecules of said
one ore more second source present in solution phase, so as to form
said one or more complexes comprising [antigenic molecule of said
first source]-[analyte autoantibody]-[antigenic molecule of said second
source] immobilized to a solid support via the antigenic molecule of
said first source.
28. A method as claimed in any of claims 1 to 11, or a kit as claimed in any
of claims 12 to 27, wherein the said sample of body fluid is from an
animal subject suspected of suffering from, susceptible to or having
one or more of the following disease states, type 1 diabetes mellitus

and/or stiff man syndrome, type 2 diabetes mellitus one or more
autoimmune thyroid diseases, celiac disease, or more connective tissue
diseases, adrenal autoimmunity, or a combination of two or more
different autoimmune diseases.
29. A kit as claimed in any of claims 12 to 27, together with a
therapeutically effective amount of at least one therapeutic agent
effective in the treatment of one more disease states associated with the
presence of autoantibodies reactive with one more antigenic molecules
selected from said pancreatic islet cell antigenic molecules in an animal
subject suspected of suffering from, susceptible to or having type 1
diabetes mellitus and/or stiff man syndrome.
30. A kit as claimed in any of claims 12 to 27, together with a
therapeutically effective amount of at least one therapeutic agent
effective in the treatment of one or more disease states associated with
the present of autoantibodies reactive with one or more antigenic
molecules selected from said pancreatic islet cell antigenic molecules in
an animal subject suspected of suffering from, susceptible to or having
type 2 diabetes mellitus, one or more autoimmune thyroid diseases,
celiac disease, one or more connective tissue diseases, adrenal
autoimmunity, or a combination of two or more different autoimmune
diseases.+++++

1. A method of screening a sample of body fluid obtained from an animal
subject for analyte autoantibodies reactive with one or more pancreatic
islet cell antigenic molecules selected from the group consisting of
GAD65, GAD67 and protein tyrosine phosphatase-like islet cell antigen
(IA-2), or one or more variants, analogues, derivatives or fragments
thereof, said method comprising:
(a) providing one or more first sources of antigenic molecules with
which analyte autoantibodies when present in said sample of body
fluid can interact and which antigenic molecules are selected from
said group of pancreatic islet cell antigenic molecules, one or more
variants, analogues, derivatives or fragments of said pancreatic islet
cell antigenic molecules and fusion molecules comprising two or
more directly or indirectly fused antigenic molecules selected from
pancreatic islet cell antigenic molecules, and said one or more
variants, analogues, derivatives or fragments thereof;
(b) providing one or more second sources of antigenic molecules with
which analyte autoantibodies when present in said sample of body
fluid can interact and which antigenic molecules are selected from
said group of pancreatic islet cell antigenic molecules, one or more
variants, analogues, derivatives or fragments of said group of
pancreatic islet cell antigenic molecules, and fusion molecules
comprising two or more directly or indirectly fused antigenic
molecules selected from pancreatic islet cell antigenic molecules,
and said one or more variants, analogues, derivatives or fragments
thereof;

(c) contacting said antigenic molecules as provided by steps (a) and (b)
simultaneously or successively with said sample of body fluid
being screened, whereby analyte autoantibodies when present in
said sample of body fluid can interact with said antigenic molecules
so as to form one or more complexes comprising [antigenic
molecule of said first source]-[analyte autoantibody]-[antigenic
molecule of said second source], wherein said molecules of said
first and second sources when present in said one or more
complexes comprise, or are derived from, a common antigenic
molecule, or wherein binding regions of said antigenic molecules of
said first and second sources, for said analyte autoantibody, when
present in said one or more complexes are present in, or are
derived from, a common antigenic molecule;
(d) prior to, or concurrent with, or subsequent to, step ( c ), providing
immobilizing means whereby said antigenic molecule of said first
source as present in a complex as formed in step (c ) is
immobilized to a solid support prior to, or concurrent with, or
subsequent to, step (c);
(e) prior to, or concurrent with, or subsequent to, step ( c ), providing
direct or indirect detectable labelling means whereby said antigenic
molecule of said second source as present in a complex as formed
in step ( c ) is provided with such direct or indirect detectable
labelling means prior to, or concurrent with, or subsequent to, step
(c); and

(f) detecting the presence of complexes formed in (c ) immobilized
according to (d) so as to provide an indication of analyte
autoantibodies present in said sample.

Documents:

692-KOLNP-2004-FORM-27.pdf

692-kolnp-2004-granted-abstract.pdf

692-kolnp-2004-granted-claims.pdf

692-kolnp-2004-granted-correspondence.pdf

692-kolnp-2004-granted-description (complete).pdf

692-kolnp-2004-granted-drawings.pdf

692-kolnp-2004-granted-examination report.pdf

692-kolnp-2004-granted-form 1.pdf

692-kolnp-2004-granted-form 18.pdf

692-kolnp-2004-granted-form 2.pdf

692-kolnp-2004-granted-form 26.pdf

692-kolnp-2004-granted-form 3.pdf

692-kolnp-2004-granted-form 5.pdf

692-kolnp-2004-granted-reply to examination report.pdf

692-kolnp-2004-granted-specification.pdf

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Patent Number

226484

Indian Patent Application Number

692/KOLNP/2004

PG Journal Number

51/2008

Publication Date

19-Dec-2008

Grant Date

17-Dec-2008

Date of Filing

25-May-2004

Name of Patentee

RSR LIMITED

Applicant Address

AVENUE PARK, PENTWYN, CARDIFF CF23 8HE

Inventors:

#	Inventor's Name	Inventor's Address
1	SMITH, BERNARD REES	RICHMOND HOUSE DRUIDSTONE ROAD OLD ST MELLONS CARDIFF CF3 6XD
2	FURMANIAK, JADWIGA	35 HAVENWOOD DRIVE HUNTERS GREEN THORNHILL CARDIFF CF4 9HX
3	POWELL, MICHAEL	141 PENCISELY ROAD CANTON CARDIFF CF5 1DN

PCT International Classification Number

G01N 33/564

PCT International Application Number

PCT/GB02/05285

PCT International Filing date

2002-11-26

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	0128583.2	2001-11-28	U.K.