Title of Invention	NON-COMPETITIVE IMMUNOASSAY FOR SMALL ANALYTES
Abstract	The invention is directed to a non-competitive immunoas- say for small analytes, wherein the analyte is reacted with 5 two binding partners. The first binding partner binds to the analyte to form a complex between the first binding part- ner and the analyte. and the second binding partner binds to the complex formed by the first binding partner and the analyte. The resulting complex formed between the ana- 10 lyte and the binding partners is detected. The binding part- ners are proteins, such as antibodies including antibody fragments. The invention further relates to reagent pairs and test kits useful in the assays, as well as to the use of 15 Novel reagents and means for their preparation are also provided.

Title of Invention

NON-COMPETITIVE IMMUNOASSAY FOR SMALL ANALYTES

Abstract

The invention is directed to a non-competitive immunoas- say for small analytes, wherein the analyte is reacted with 5 two binding partners. The first binding partner binds to the analyte to form a complex between the first binding part- ner and the analyte. and the second binding partner binds to the complex formed by the first binding partner and the analyte. The resulting complex formed between the ana- 10 lyte and the binding partners is detected. The binding part- ners are proteins, such as antibodies including antibody fragments. The invention further relates to reagent pairs and test kits useful in the assays, as well as to the use of 15 Novel reagents and means for their preparation are also provided.

Full Text	1 Non-competitive immunoassay for small analytes Field or the Invention The present invention relates to immunoassays and especially to 5 non-competitive immunoassays for small analyies. The present invention fur- ther relates to reagent pairs and test-kits useful in the assays, as well as to the use of the reagent pairs and to a process for their preparation. Novel reagents and means for their preparation are also provided. 10 Technical Background of the Invention In a competitive immunoassay, external reagent that competes with the analyte has to be added, which is not the case in the non-competitive as- say format. The method of choice for the detection of analytes by immuno- chemistry is nowadays a non-competitive immunoassay, where two antibodies 15 bind to two different epitopes of the analyte creating a so-called sandwich-type assay. Such an assay is well suited for high molecular weight analytes and it provides improved speed, sensitivity, and specificity, which are needed in modern immunoassays. However, it has been a difficult task to develop non- competitive assays for small analytes, because low molecular weight mo- 20 lecules are not large enough for binding simultaneously to more than one anti- body independently. Therefore, despite many fundamental problems with re- spect to specificity and sensitivity, the competitive immunoassay format has been almost exclusively used for the detection of small analytes. However, there are few publications where the development of a 25 non-competitive immunoassay for a small analyte has been reported. In these papers, a secondary anti-immune complex (anti-IC) antibody, which binds primary anti-analyte antibody that is combined with the analyte but which does not bind the primary antibody or the analyte alone, has been developed (Ull- man et al., 1993; Self et al., 1994; Towbin et al., 1995). Ullman ef al., 1993, 30 describe an antibody that recognizes an immune complex of an antibody to tetrahydrocannabinol (THC). The anti-IC antibody was obtained by using an affinity labelled anti-THC antibody as immunogen and selecting an anti-IC anti- body the binding of which was enhanced by the presence of 9THC. Self et al., 1994, used the same principle in preparing anti-IC antibodies for determin- 35 ing digoxin. Towbin et al., 1995, report a sandwich immunoassay for the hapten angiotensin II, wherein the immunisation involves tolerization with un- 2 complexed primary antibody prior to immunisation with the anti-immune com- plex to obtain the anti-IC antibodies. The anti-IC antibodies used in non-com- petitive immunoassays for small analytes have so far been conventional poly- clonal or monoclonal antibodies obtained by immunisation, and the assays de- 5 scribed here include labelling of the primary antibody and immobilisation of the secondary or vice versa. So-called 'idiometric' non-competitive immunoassay for small ana- lytes has been developed by Mares et al., 1995. They used two types of anti- idiotypic antibodies, which recognize different epitopes within the hypervari- 10 able region of the oeastradiol specifc primary antibody. The first anti-idiotypic antibody (betatype) possesses the capacity of competing with the analyte for an epitope at the binding site of the primary antibody. The second anti-idiotype (alphatype) recognizes an epitope within the variable region of the primary an- tibody and the binding is not sensitive to the presence of the analyte. The al- 15 phatype is, however, sterically hindered from binding to the primary antibody in the presence of the betatype. These three types of antibodies permit the de- velopment of a non-competitive assay for small analytes. So-called open sandwich immunoassays have been developed for the detection of haptens (Suzuki et al., 2000; Suzuki et al., 1999; Yokozeki et 20 al, 2002). They are non-competitive assays based on a phenomenon accord- ing to which the association of separated VH and VL chains in some antibodies is strongly favoured in the presence of antigen (Ueda et al., 1996), Despite the significant benefits of the non-competitive immunoas- say format, only few examples of that kind of assays for small analytes have 25 been reported. The reason for that is most probably the difficulty of producing secondary (anti-immuno complex, or anti-idiotypic) antibodies by immunising animals. For example anti-hapten monoclonal antibodies, which have been developed by hybridoma technology (Kohler and Milstein, 1975), are self-anti- gens for mice and raising immunoresponse against them is difficult 30 (Maruyama et al., 2002; Ullman et al., 1993; Kobayashi et al., 2000), A further problem is that the immune complex used for immunisation tends to break down before the response to the immuno complex is obtained (Ullman et al., 1993; Kobayashi et al.,2000). The present invention now provides a non-competitive immunoas- 35 say protocol for small analytes, which circumvents the immunisation of anim- als with the immune complex, which has been so far the most challenging task 3 when anti-IC antibodies have been developed. The invention also facilitates a homogenous immunoassay, which further improves the speed, sensitivity and simplicity of the assay. 5 Summary of the Invention The difficulties associated with raising anti-lC antibodies for use in Immunoassays for small analytes can now be avoided by providing the neces- sary anti-IC antibodies from a display recombinant binding partner library in- stead of from immunised animals. A phage display antibody library may be 10 constructed, which contains a vast number of clones, from which Those coding the desired binding partners, such as antibody fragments, can be enriched and selected through sequential panning. This protocol opens new possibilities for developing rapid, reliable and simple immunoassays for small analytes in a cost-effective and feasible way. 15 Consequently, one object of the present invention is a non-compet- itive immunoassay for a small analyte comprising reacting a sample containing said analyte with a reagent pair comprising a first binding partner that binds to said analyte, and a second binding partner that binds to the complex of said analyte and said first binding partner. The immunoassay is characterized in 20 that the second binding partner is obtained from a display recombinant binding partner library by selecting a binding partner that binds to said complex of the analyte and first binding partner, and determining the binding of the second binding partner thus indicating the presence of the analyte in the sample. Another object of the invention is a reagent pair for a non-competit- 25 ive immunoassay for a small analyte, comprising a first binding partner that binds to said analyte, and a second binding partner that binds to the complex of said analyte and said first binding partner, characterized in that the second binding partner is obtained from a display recombinant binding partner library by selecting a binding partner that binds to said complex of the analyte and 30 first binding partner. Still another object of the present invention is a test kit for a non- competitive immunoassay for a small analyte, said kit comprising a reagent pair comprising a first binding partner that binds to said analyte, and a second binding partner that binds to the complex of said analyte and said first binding 35 partner, characterized in mat the second binding partner is obtained from a 4 display recombinant binding partner library by selecting a binding partner that binds to said complex of the analyte and first binding partner. The invention Is also directed to the use of a reagent pair compris- ing a first binding partner that binds to an analyte, and a second binding part- 5 ner that binds to the complex of said analyte and said first binding partner, in a non-competitive immunoassay for a small analyte, whereby the second bind- ing partner is obtained from a display recombinant binding partner library by selecting a binding partner that binds to said complex of the analyte and first binding partner. 10 A still further object of the invention is a process for preparing a re- agent pair for a non-competitive immunoassay for a small analyte comprising providing a first binding partner that binds to said analyte, and a second bind- ing partner that binds to the complex of said analyte and said first binding part- ner, characterized in that the second binding partner is obtained from a display 15 recombinant binding partner library by selecting a binding partner that binds to said complex of the analyte and first binding partner. The invention also provides novel recombinant binding proteins, characterized in that they comprise the ligand-binding portion of M1 Fab com- prising SEQ ID NO 1 and SEQ ID NO 2; M2 Fab comprising SEQ ID NO 3 and 20 SEQ ID NO 4: or K11 scFv comprising SEQ ID NO 5. DNA encoding the novel binding proteins as well as host cells ex- pressing them are also included in the invention. Advantagous embodiments of the invention are set forth in the de- pendent claims. 25 Other objects, details and advantages of the present invention will become apparent from the following drawings, detailed description and ex- amples. Brief Description of the Drawings Figure 1. A competitive ELISA using M1 Fab. Urine+ is urine spiked 30 with 1 g/ml of morphine. Figure 2. A non-competitive time resolved fluorescent immunoassay (TR-FIA) with the Eu labelled anti-M1 +morphine immune complex scFv frag- ment K11. a) Cross-reactivity and sensitivity of the assay, b) Sensitivity of the assay with S1 urine control dilutions as samples. 35 Figure 3. A homogenous TR-FRET immunoassay of morphine. 5 5 Figure 4. Analysis of cross-reactivity of M1 anti-morphine Fab-frag- ment with codeine, heroin, noscapine and papaverine by a competitive ELISA. Figure 5. A comparative TR-FRET based homogeneous immunoas- say for morphine. ' 5 Detailed Description of the Invention The reagent pair for the non-competitive immunoassay of the inven- tion comprises a first binding partner and a second binding partner. The first binding partner binds to the analyte to form a complex between the first bind- ing partner and the analyte. The second binding partner binds to the complex 10 formed by the first binding partner and the analyte. The binding partners are usually proteins such as antibodies including antibody fragments that have the desired binding properties. An antibody is an immunoglobuiin molecule and it can belong to any of classes IgG, IgM, IgE, IgA or IgD; lgG and IgM being the most frequently used. Preferably the binding partners are antibody fragments 15 comprising the Iigand-binding site, such as Fab, or scFv fragments. The frag- ment known as the Fab fragment (fragment antigen binding) consists of the variable and constant domain of an immunoglobulin light chain covalently at- tached by a disulfide bridge to the variable and first constant domain of an im- munoglobulin heavy chain. Fv (variable domain) means the variable regions of 20 the immunoglobulin molecule that are responsible for the ligand binding. ScFv (single chain Fv) means a molecule wherein the variable domains of the heavy and light chain of an antibody are linked by a peptide to form a single poly- peptide chain synthesized from a single mRNA molecule. The variable regions of an immunoglobulin heavy chain and light chain are together responsible for 25 the ligand binding. Ligand is the substance to which the binding partner binds, in connection with antibodies it is an antigen or a hapten. The first binding partner may be a conventional polyclonal or mono- clonal antibody or fragment thereof, but preferably it is a recombinant one, as is the second binding partner. When the first binding partner has been selec- 30 ted for, it is complexed with its ligand and this complex is used to select for the second binding partner from a recombinant library. The first binding partner without the ligand is used as contra selection. The second binding partner should only recognise complexes, not free first binding partner nor free antigen to any significant extent. 35 The recombinant binding partner library is conveniently an expres- sion library, which is typically a display library. The general principle of the dis- 6 play recombinant binding partner libraries is that they present the binding part- ner as a fusion protein on the surface, which may be the surface of a microbial cell such as a yeast or bacterial cell, or a phage. The display recombinant binding partner library can also be a display library, where stable complexes of 5 nascent protein and mRNA are produced in an in vitro expression system. Phage display libraries are the most frequently used. Antibody phage display technology and its applications are described e.g. in Hoogenboom et al., 1998. A phage display antibody library may be constructed by cloning im- mnunoglobulin domains coding cDNAs into an appropriate phage display vec- 10 tor. DNA encoding for millions of variants of antibody fragments is batch- cloned into the vector as part of the phage coat protein. Large libraries con- taining millions of antibody fragments with different specificities can be ob- tained by transforming the vectors in bacteria. Cultivation of the bacteria leads to the expression of phages displaying antibody fragments on their surface. 15 The gene for the displayed antibody is carried in the phage genome, thus link- ing genotype with phenotype. The physical linkage between the displayed pro- tein and its DNA allows screening of vast numbers of variants of the protein, each linked to its corresponding DNA, by a simple in vitro selection procedure called panning. In its simplest form, panning is carried out by incubating the 20 pool of phage-displayed variants with the ligand of interest that has been im- mobilized on a carrier, washing away unbound phage, and eluting specifically bound phage by disrupting the binding to the ligand. The eluted phage is then amplified in vivo. The process is repeated several times, resulting in stepwise enrichment of the phage pool in favour of the tightest binding sequences. After 25 about 3 to 6 rounds of selection and amplification, the best clones are se- quenced and transformed into a host cell for further expression. The host cell may be a eucaryotic or procaryotic cell e.g. a yeast, animal, plant or insect cell or bacterial cell. It may even be a hybridoma cell, which after transformation produces a recombinant monoclonal antibody. The recombinant binding part- 30 ner or at least part of it may also be produced synthetically. The concept to use recombinant antibody libraries makes the non- competitive sandwich assay for small analytes feasible. The sandwich can be detected by all the standard immunoassays. Usually one partner is immobil- ized on a carrier, such as a microtiter well or a bead. A sandwich is formed in 35 the presence of analyte and the other binding partner. The sandwich may be detected e.g. by using secondary antibodies or by labelling at least one of the 7 binding partners. The label can be any conventional label, such as a radioact- ive label, an enzyme, or a fluorescent compound. The assay can be e.g. ELISA or FIA. A great advantage of the reagent pair of the present invention is 5 that it enables a homogenous non-competitive immunoassay, i.e. an immun- oassay that is carried out in solution. The avoidance of immobilising and wash- ing steps makes the assay extremely simple. Such a test is also suitable for testing on-site i.e. in places elsewhere than the laboratory. A preferred homogenous immunoassay is one based on fluores- 10 cence resonance energy transfer (FRET), for review see Szöllösi et al., 1998. In FRET, energy from a molecular fluorophore (donor) is excited to a high-en- ergy state and transferred to another fluorophore (acceptor) via intermolecular dipole-dlpole coupling. This is possible only if the distance between the donor and fhe acceptor is short (10-100 A) and the fluorescence spectrum of the 15 donor and the absorption spectrum of the acceptor partially overlap. The en- ergy transfer is then detected as a change in fluorescence. Often time-re- solved fluorescence is utilized (Hemmilä et al., 1988). FRET is applied to the present invention by labelling the two binding partners, which preferably are antibody fragments, with fluorophores that form 20 a FRET donor-acceptor pair. When the binding partners and the analyte are small the fluorophores come into very close proximity, and a measurable FRET signal is obtained. The invention provides a convenient and rapid analytical tool for low molecular weight analytes, such as therapeutic and abused drugs, steroids, 25 hormones, metabolites, and environmental pollutants and toxins. A common feature of these small analytes is that they are too small for conventional sandwich assays where two antibodies recognizing different epitopes of the antigen are used. The molecular weight of these small analytes are normally less than 5000, but the limits are not absolute. 30 The immunoassay may be employed in all kinds of investigations, such as in detecting environmental hazards, toxic compounds in food and feed, chemicals indicative of ongoing processes e.g. of microbial processes in buildings, metabolic, processes of living organisms, and in clinical tests, drug monitoring and pharmacological research. The assays are extremely suitable 35 for detecting drugs of abuse, such as opiates (e.g. morphine), amphetamines, cannabinoids (e.g. tetrahydrocannabinol (THC)), barbiturates, benzo- 8 diazepines, cocaine, LSD, methadone, methaqualone, phencyclidine, pro- poxyphene, tricyclic antidepressants. The homogenous assay provides an ex- cellent and convenient tool for on-site tests e.g. to be used by the police in raiding drivers etc. The sample to be analysed for e.g. drugs and abused 5 drugs may be any body fluid sample, such as blood, serum, urine or saliva. The reagent pair of the invention may be included in a test-kit. This test-kit may further comprise any other reagents needed for the assay, such as reaction solutions, buffers, washing solutions and detecting means, such as labels and optionally a fluorometer. Preferably the test-kit comprises multiple 10 reagent pairs physically separated from each other, e.g. many in the form of a microarray, whereby e.g. many different drugs of abuse may be tested simul- taneously from a single saliva sample. In a special embodiment of the invention, a reagent pair for detect- ing morphine is produced and employed in a homogenous immunoassay for 15 said substance. The first binding partner is a Fab fragment obtained from a phage display antibody library produced from cDNA from a mouse immunized with morphine conjugated to an immunocarrier BSA. Morphine specific anti- body phages were enriched by selecting those binding to morphine conjugated BSA and sorting out those binding to BSA alone. After several panning rounds 20 two high binding clones are sequenced and expressed. The expressed Fab fragments were named M1 Fab and M2 Fab. The second binding partner is obtained from a naive scFv antibody fragment phage display library by selecting antibodies that bind to a complex of morphine and M1 Fab. First the phages are preincubated to bound M1 Fab 25 to sort out those binding to M1 Fab as such. The unbound phages are separ- ated and incubated with a mixture of morphine and immobilised M1 Fab to se- lect the phages that bind to the immunocomplex formed between the immobil- ized M1 Fab and morphine. Unbound phages are washed away, and then those bound to the complex are eluted. The background is monitored by 30 checking the binding to M1 Fab in the absence of morphine. After several pan- ning rounds a number of clones are picked up, sequenced and expressed res- ulting in scFv fragment K11. A fluorescence-based immunoassay is performed using M1 Fab la- belled with europium as a first binding partner, and scFv fragment K11 labelled 35 with Cy5 as a second binding partner. The binding partners are incubated with saliva or urine samples containing morphine and then fluorescence is meas- 9 ured after a predetermined time. The assay is completely homogeneous and the signal is readable in about 5 min. The sensitivity for both urine and saliva is clearly higher than that demanded by the authorities in the case of morphine, our model analyte. The performance of the test is such that the re- 5 agents are in the well of a microtiter plate and dilution series of either saliva or urine is added. In a preferred mode for e.g. police field use, the reagents are in dry form in a vessel. Saliva is added, which dissolves the reagents and the result can be read without further processes. The novel recombinant binding proteins provided by the invention 10 comprise any ligand-binding portion of M1 Fab, M2 Fab or K11 scFv. By "lig- and-binding portion" is meant that part of the molecule that is responsible for the binding. Minor variations or modifications of the sequences set forth in the description and claims are still within the scope of the invention provided that they do not affect the binding activity of the proteins. 15 The invention is illustrated by the following non-limiting example. It should be understood, however, that the embodiments given in the description above and in the examples are for illustrative purposes only, and that various changes and modifications are possible within the scope of the invention. Example 1 20 Development of an anti-morphine antibody immunisation of mice Four six-week-old female Balb/c mice were immunised in three- week intervals with morphine conjugated BSA (Fitzgerald) in Freund's ad- juvant. Serum samples were tested after second booster and the mouse 25 showing the best response against the antigen in direct ELISA was selected to be the source of an antibody phage display library. Construction of the antibody phage display library All basic recombinant DNA methods were performed essentially as described (Sambrook et al. 1990). The mouse with the highest antibody re- 30 sponse to morphine-BSA conjugate was sacrificed and the total RNA was isol- ated from the spleen cells using the RNagents Total RNA Isolation System (Promega Co., Wl, USA). The mRNA pool of the total RNA was isolated with the Oligotex mRNA Kit (OIAGEN Inc., Germany). The cDNA was synthesised from the mRNA with oligo-dT priming. Genes encoding antibody Fab frag- 35 ments were amplified with PCR using antibody kappa light chain and heavy 10 10 chain variable region and constant region specific primers. Antibody light chain PCR products were pooled and digested with Nhel and AscI restriction en- zymes, purified by preparative agarose gel in combination with the QIAquick Gel Extraction Kit (OlAGEN Inc.. Germany). The agarose gel purified antibody 5 light chain DNA was ligated into the Fab phagemid vector phagemid9 derived from pComb3 (Barbas et al., 1991) and transformed into the E.coli XL1-Blue strain (Stratagene) by electroporation. Plasmid DNA was isolated with the OlA- GEN Plasmid Midi Kit (Q1AGEN Inc., Germany) from the overnight culture. PCR products encoding the Fd region (variable and first constant region) of 10 the heavy chain were pooled and digested with Sfil and Notl restriction en- zymes, purified by preparative agarose gel isolation and ligated to the phagemid vector containing the light chain DNA. The phagemid vector encod- ing both the heavy and light chain of the Fab fragment was transformed into the E.coli TOP10F1 bacteria (Invitrogen Inc., CA, USA) by electroporation. 15 Transformed bacteria were incubated over night at +37 C on a shaker and plasmid DNA was isolated with the QIAGEN Plasmid Midi Kit. The diversity of the antibody library was ensured by sequencing partial VL-or VH-gene regions of individual clones. A phage display antibody library was made as follows: 4 g of anti- 20 body library plasmid DNA was transformed into E.coli TOP1 0F1 by electropora- tion in two parallel transformations. After transformations, cells were suspen- ded into 2,8 ml of SOC medium and incubated for 1 h at +35 C on a shaker. 7 ml Of prewarmed (+37 C) SB medium. 20 g/ml of carbenicillin, and 10 g/ml of tetracycline was added. After 1 -h incubation at +35 C on a shaker, 30 g/ml 25 of carbenicillin was added and the incubation was continued for 1 h after which 1 ml ( 1011 pfu) of helper phage VCS-M13 (Stratagene) was added and the phages were let to infect the bacteria for 20 min at +35 C with a slow shaking. The parallel transformations were joined together and 80 ml of prewarmed (+37 C) SB medium with 50 g/ml carbenicillin and 10 g/ml of tetracycline 30 was added. After 2-h incubation at +35 C on a shaker, 70 g/ml of kanamycin was added and the incubation was continued over night. Cells were centrifuged for 15 min at 4000g at +4 C. 20 ml of 20% PEG, 2.5 M NaCl (PEG/NaCI) was added ID the supernatant and it was incub- ated for 30 min on ice. PEG precipitated phages were centrifuged for 20 min 35 at 13000g at +4 C. The pellet was suspended in 2 ml of PBS and 1 ml was transferred into two eppendorf tubes. After centrifugation for 5 min at +4 C, 11 phages were precipitated by adding 200 \| of PEG/NaCl to the supernatant. The solution was mixed and centrifuged for 5 min at +4 C. The pellet was sus- pended in 1 ml of PBS orPBS+1% BSA. 1 \| of 20% Na-azide was added as a preservative. The phage display antibody library was stored at +4 C. 5 Selection of the anti-morphine library Morphine specific antibodies were selected from the phage display antibody library by the following selection procedure: A microtiter well was coated over night at +4 C with 1 g of morphine conjugated BSA (morphine- BSA; Fitzgerald Industries International, Inc., MA, USA) in 100 \| of 100 mM 10 Na-bicarbonate buffer. pH 9.8. The well was washed two times with PBS and blocked with 1 % BSA in PBS for 1 h at +37 C. 100 \| of the phage library was incubated in the well for 1 h at RT with shaking, after which the unbound phages were removed and the well was washed 22 times with PBS. Bound phages were eluted with 100 \| of 100 mM HCI (pH 2.2) for 15 min. Eluted 15 phages were removed from the well and neutralised with 1 M Tris. 3 ml of fresh E.coli XL1-Blue cells (OD600 1) grown in SB supplemented with 10 g/ml of tetracycline was infected with eluted phages at +35 C for 15 min. 7 ml of prewarmed SB (+37 C) with 20 g/ml of carbenicillin and 10 g/ml of tetracyc- line was added and the culture was incubated for 1 h at +35 C on a shaker. 30 20 g/ml of carbenicillin was added and incubation was continued for 1 h. 1 ml of helper phage VCS-M13 was added to the culture and incubated at +35 C for 15 min with a slow shaking. The culture was diluted with 90 ml of prewarmed (+37 C) SB with 50 g/ml carbenicillin and 10 g/ml tetracycline. After 2 h in- cubation at +35 C on a shaker, 70 g/ml of kanamycin was added and the in- 25 cubation was continued over night. The purification of the amplified phages was performed by PEG precipitation as described above. The purified phages were used in further enrichment rounds. After four selection rounds, morphine specific antibody phages had been enriched over 1000 times when compared to the background. The background binding 30 of the phages was monitored in parallel in each selection round by incubating the phage library in a BSA coated microtiter well. The well was washed and the phages were eluted as in the morphine-BSA coated well. The enrichment of the morphine specific phages was monitored by comparing the amount of the eluted phages from the morphine-BSA coated well to the amount of eluted 35 phages from the background well. Characterisation of individual clones 12 After the fourth panning round, the phagemid DNA was isolated with the QIAGEN Plasmid Midi Kit. The plasmid was digested with Nhel and Notl restriction enzymes to isolate the Fab gene fragment. The agarose gel isolated DNA was ligated to the expression vector pKKtac. The ligation reac- 5 tion was transformed into the E.coli XL1-Blue cells. Individual clones were picked and miniprep DNA was extracted with the QIAprep Spin Miniprep Kit (QIAGEN Inc., Germany). According to sequen- cing of the minipreps, two different Fab clones were found. These clones were named M1 and M2. The amino acid sequences of the M1 Fab fragment were 10 SEQ ID NO 1 (light chain) and SEQ ID NO 2 (heavy chain). Amino acids no. 3 to 108 represent the variable region and no. 109 to 215 the constant region of the M1 light chain (SEQ ID NO 1). Amino acids no 4 to 123 represent the vari- able region and no. 124 to 226 the constant region of the M1 heavy chain (SEQ ID NO 2). The amino acid sequences of M2 Fab fragment were SEQ ID 15 NO 3 (light chain) and SEQ ID NO 4 (heavy chain). Amino acids no 3 to 108 represent the variable region and no. 109 to 215 the constant region of the M2 light chain (SEQ ID NO 3). Amino acids no. 4 to 123 represent the variable re- gion and no. 124 to 226 the constant region of the M2 heavy chain (SEQ ID NO 4). The rest of the amino acids derive from the cloning technique, and 20 some of the C-terminal amino acids facilitate the isolation and purification of the protein. Small-scale (3 ml) Fab expression cultures were also made. Peri- plasmic fraction of the cells was isolated by freezing and thawing the cells for three times in PBS. The binding of individual Fab clones to morphine was tested by 25 ELISA in morphine-BSA coated microtiter wells: Microtiter wells were coated with 200 ng of morphine-BSA in 0.1 M Na-bicarbonate buffer, pH 9.8 for over night at +4 C. Control wells were filled only with the buffer. After coating, wells were washed three times with PBS and blocked with 0.5% BSA in PBS (BSA/PBS) for 1 h at RT. Wells were washed three times with PBS and 1:10 30 dilution of the periplasmic fractions was added into wells in 100 \| of BSA/PBS. After 1-h incubation at RT on a shaker, wells were washed three times with PBS and 1:2000 diluted alkaline phosphatase conjugated anti- mouse Fab specific antibody (Sigma, A-1293) was added in 100 \| of BSA/PBS. The wells were incubated for 1 h at RT on a shaker and washed 35 three times with PBS. 100 \| of alkaline phosphatase substrate solution (2 mg/ml of p-nitrophenylphosphate di-Na-salt in diethanolamine-MgCI-buffer) 13 was added to the wells and absorbance was measured. According to the pre- liminary results, the M1 Fab showed better performance in the assay and was therefore chosen for the continuation. Fermentation and purification 5 The anti-morphine Fab fragment M1 in the expression vector pKKtac was transformed in the E.coli expression strain RV308. The Fab was expressed by fed-batch fermentation in a Bio-Flow IV fermenter (New Brun- swick) and purified by the Sepharose SP ion-exchange and protein G chroma- tography (Pharmacia). 10 Performance of the primary antibody in a competitive immunoassay The performance of the M1 anti-morphine Fab fragment was tested in a competitive ELISA assay using the commercial urine toxicology controls S1 and S3 (Bio-Rad). The S1 urine toxicology control contains drugs and drug metabolites (including morphine) at concentrations 20-25% below immunoas- 15 say cut-off levels as recommended by the U.S. Substance Abuse and Mental Health Services Administration (SAMSHA) and other agencies. The S3 urine toxicology control contains drugs and drug metabolites at concentrations ap- proximately three times immunoassay cut-off levels. Microtiter wells were coated over night at +4 C with 500 ng of morphine-BSA in 100 \| of Na-bicar- 20 bonate buffer, pH 9.8. Wells were washed three times with PBS and blocked with BSA/PBS for 1 h at RT. After three washes with PBS, S1, S3 (Bio-Rad), positive control, or negative control urine dilutions were added to the wells in 100 \| of BSA/PBS spiked with 1 ng of M1 Fab. Wells were incubated for 2 h at RT on a shaker and washed three times with PBS. 1:2000 diluted alkaline 25 phosphatase conjugated anti-Fab antibody (Sigma, A-1293) was added to the wells in 100 \| of BSA/PBS and incubated for 1 h at RT on a shaker. Wells were washed three times with PBS, 100 \| of alkaline phosphatase substrate solution was added and A405 was measured. Results are shown in Figure 1. A positive result could be achieved with 1:64 dilution of the sample. 30 Development of an anti-immune complex antibody specific to the im- mune complex of M1 Fab and morphine Selection of the immune complex specific antibody from a naive hu- man scFv phaae display library M1 Fab fragment was biotinylated with ImmunoPure Sulfo-NHS-LC- 35 Biotin Kit (Pierce). Biotinylated antibody was purified and buffer was changed to PBS with Econo-Pac 10DG Columns (Bio-Rad, CA, USA). 200 \| of a naïve 14 human scFv phage display library (Kappa or Lambda light chain) in BSA/PBS was preincubated with 10 \| of streptavidin coated magnetic beads (Dynal, M- 280) and 0.5 g of biotinylated M1 Fab for over night at +4 C. The naive hu- man scFv phage display library was constructed from pooled lymphocytes of 5 50 healthy individuals. The size of the library was estimated to be 1x108 clones. The naive human scFv phage display library contains the IgM specific VH-genes combined either with the kappa or lambda specific VL-genes. Un- bound phages were separated from the beads and 100 \| of them was incub- ated with 100 ng of morphine, 500 ng of biotinylated M1 Fab, and 5 \| of strep- 10 tavidin coated magnetic beads for 1 h at RT on a shaker. The background for the selection procedure was implemented in a similar way but omitting the morphine from the binding reaction. Magnetic beads were washed five times with 0.5 ml of PBS and bound phages were eluted with 100 \| of HCI (pH 2.2) for 30 min. The eluted phages were neutralised with 1M Tris and E.coli XL1- 15 Blue cells were infected. Ceils were grown and phages were purified as de- scribed previously. After five panning rounds enrichment was seen with the scFv library having the kappa light chains, when the amount of eluted phages was compared to the amount of eluted phages from the background control well. The enrichment of specific binders to the immune complex, formed by 20 M1 Fab and morphine, was also clearly seen in a phage ELISA when the eluted phage pools from the selection rounds were tested. Characterisation of individual clones Individual phage clones were picked and they were sequenced. All of the clones had the same sequence (SEQ ID NO 5). The amino acid se- 25 quence of anti-M1+morphine immune complex scFv fragment was named K11 scFv. Amino acids no- 3 to 120 represent the heavy chain variable region, no. 140 to 246 represent the light chain variable region, and no. 121 to 139 repre- sent the linker of K11 scFv (SEQ ID NO 5). The rest of the amino acids derive from the cloning technique, and some of the C-terminal amino acids facilitate 30 the isolation and purification of the protein. Expression and purification The gene encoding the scFv fragment K 11 was inserted into the expression vector pKKtac and transformed into the E.coli RV308 strain. Cells were inoculated into 20 ml of LB with 100 g/ml ampicillin and were incubated 35 over night at +37 C on a shaker. From the overnight culture a 10 ml inoculate was added into two erlenmeyer bottles containing 500 ml of LB with 100 g/ml 15 15 of ampicillin. Cells were incubated at +37 C on a shaker until the OD600 was 1 after which 0.5 ml of 1M IPTG and 100 g/ml of ampicillin were added into the culture and the incubation was continued over night at +30 C on a shaker. Both the supernatant of the culture medium and the periplasmic fraction of the 5 cells were used for the purification of the K11 scFv. Cells were centrifuged at 4000g for 15 min and the supernatant was poured into a clean flask. The cells were resuspended in 20 ml of PBS. The periplasmic fraction of the cells was isolated by freezing (-70 C) and thawing (+37 C) the cells for three times. After centrifugation at 12000g for 30 min, the clear periplasmic supernatant was 10 taken. The culture medium and the periplasmic fraction were treated with 2 mg/ml of DNasel to remove residual chromosomal DNA for two hours at +37 C. Since the expressed K11 scFv has a 6xHis-tag in its C-terminus, it could be purified by immobilised metal affinity chromatography (IMAC). The K11 scFv was purified by expanded bed chromatography using STREAMLINE 15 Chelating (Amershampharmacia biotech) as a matrix and copper as the metal chelate. The purity of K11 scFv was checked with SDS-PAGE. Development of a non-competitive immunoassay for morphine Labelling of the anti-immune complex antibody K11 scFv with europium 20 The purified anti-M1 and morphine immune complex scFv fragment K11 was labelled with europium chelate by the DELFIA Eu-Labelling Kit (Wal- lac) as described by the manufacturer. Buffer of the labelled K11 scFv was changed to 50 mM Tris pH 7.8, 0.9% NaCI. Labelling yield was 0.4 Eu/scFv, A non-competitive immunoassay for morphine 25 The sensitivity and cross-reactivity of K11 scFv was studied by a non-competitive immunoassay. Transparent Streptawell (Roche) streptavidin coated microtiter wells were washed three times with PBS. 500 ng of biot- inylated anti-morphine M1 Fab was added into the wells in 100 \| of BSA/PBS and the wells were incubated for 30 min at RT on a shaker. After three PBS 30 washes, sample dilutions spiked with morphine, amphetamine, tetrahydrocan- nabinol (THC) or S1 urine control were added into the wells in 50 \| of BSA/PBS. 1:100 diluted europium labelled K11 scFv was added into the wells in 50 \| of BSA/PBS and the incubation was continued for 1 h. Wells were washed three times with PBS and 100 \| of Enhancement solution (Wallac) 35 was added into the wells. After 15-min shaking at RT, fluorescence was detec- ted by VictorV fluorometer (Wallac). Results are shown in Figure 2. There is a 16 significant difference in affinity between the binding of K11 scFv to the primary antibody fragment M1 and to the M1 and morphine immune complex. This al- lows the detection of 1 ng/ml of morphine in a sample. No cross-reactivity with amphetamine or THC was detected. 5 A homogenous time resolved fluorescent resonance energy transfer (TR-FRET) The anti-morphine Fab fragment M1 was labelled with europium by the LANCE Eu-W1024 ITC chelate Kit (Wattac). The buffer of me labelled M1 was changed to 50 mM Tris, pH 7.8, 0.9% NaCl. The labelling yield was 1.1 10 Eu/Fab. The anti-M1 +morphine immune complex scFv fragment K11 scFv was labelled with Cy5 by the FluoroLink-Ab Cy5 labelling kit (Amershampharmacia biotech). The labelling yield was 2 Cy5/scFv. Saliva was spiked with various dilutions of morphine and these samples were filtered through cotton wool before adding into the black mi- 15 crotiter wells (Nunc). 1 g of europium labelled M1 Fab and 1 g of Cy5 la- belled K11 scFv was added into the wells in 50 \| of BSA/PBS. TR-FRET was read after 5, 15, 30, or 60 min incubation at RT by VictorV fluorometer (Wal- lac). Results are shown in Figure 3. After five minutes incubation. 1 ng/ml of morphine in saliva was detected. 20 While one strategy for providing reagents and assays of the inven- tion has been described, numerous variations and modifications will become apparent to the person skilled in the art. Example 2 Cross-reactivity of the M1 Fab-fragment with codeine, heroin, noscapine 25 and papaverine Cross-reactivity of M1 anti-morphine Fab-fragment with codeine. heroin, noscapine and papaverine was tested in a competitive ELISA. Mi- crotiter plate wells were coated o/n at +4°C with 500 ng of morphine-BSA in 100 \| of Na-bicarbonate buffer, pH 9.8. The wells were washed three times 30 with PBS and blocked with 0.5% BSA/PBS for 1 h at RT and washed again three times with PBS. Two parallel 100 \| samples containing morphine, codeine, heroin, noscapine or papaverine (39, 78. 156, 313, 625, 1250, 2500 or 5000 ng/ml) In PBS with 5 ng of purified M1 Fab were added into the wells and incubated for 30 min at RT on a shaker and washed three times with PBS. 35 1:2000 diluted alkaline phosphatase conjugated anti-Fab antibody (Sigma, A- 17 1293) was added to the wells in 100 \| of 0.5% BSA/PBS and incubated for 30 min at RT on a shaker. Wells were washed three limes with PBS, 100 \| of al- kaline phosphatase substrate solution was added and A405 was measured. The results are shown in Figure 4. According to the competitive ELISA result M1 5 antibody has high cross-reactivity to codeine and heroin, which are structurally very similar with morphine, A homogenous time resolved fluorescent resonance energy transfer (TR- FRET) imunoassay for morphine The anti-morphine Fab fragment M1 was labelled with europium by 10 LANCE Eu-W1024 ITC chelate Kit (Wallac). The buffer of the labelled M1 was changed to 50 mM Tris, pH 7.8, 0.9% NaCI. The labelling yield was 1.1 Eu/Fab. The anti-M1+morphine immune complex scFv fragment K11 was la- belled with Cy5 by FluoroLink-Ab Cy5 labelling kit (Amersham Pharmacia Bi- otech), The labelling yield was 2 Cy5/scFv. 15 Saliva was spiked with a high concentration (10 µg/ml) of the follow- ing drugs: morphine, heroin, codeine, papaverine and noscapine. The samples were filtered through cotton wool before adding into black microtiter wells (Nunc). 1 g of europium labelled M1 Fab and 1 g of Cy5 labelled K11 scFv was added into the wells in 50 \| of BSA/PBS. As the control the background 20 fluorescence from the M1 and K11 antibody pair without any added drug was measured. TR-FRET was read after 5 min incubation at RT by VictorV fluoro- meter (Wallac). The results are shown in Figure 5. Saliva sample spiked with morphine is giving a high fluorescent value, whereas the other drugs are giving fluorescent values that are similar to 25 the background fluorescence detected from the control (labelled M1 and K11 scFv fragments without any added drug). K11 scFv binds the immune complex formed between M1 and mophine with extremely high specificity. K11 is able to discriminate completely M1 and morphine immune complex from the im- mune complexes between M1 and heroin or M1 and codeine, which gave 30 fluorescent values corresponding to the background level. 18 References Barbas, C.F. Ill, Kang, A.S., Lerner, R.A., Benkovic, S.J. (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. 5 Prac. Nati. Acad. Sci. USA 88:7978-7982. Hernmilä, I., Malminen, O., Mikola, H., Lövgren, T. (1988) Homogeneous time- resolved fluoroimmunoassay of thyroxin in serum, Clin. Chem. 34:2320- 2322. Hoogenboom H.R., de Bruïne, A.P., Hufton, S.E., Hoet, R.M., Arends. J.-W., 10 and Roovers. R.C. (1998) Review article: Antibody phage display tech- nology and its applications, Immunotechnology 4,1-20. Kobayashi, N., Oiwa, H., Kubota, K., Sakoda, S., Goto, J, (2000) Monoclonal antibodies generated against an affinity-labeled immune complex of an anti-bile acid metabolite antibody: an approach to noncompetitive hap- 5 ten immunoassays based on anti-idiotype or anti-metatype antibodies. J Immunol Methods, 245, 95-108. Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256, 495-7. Mares. A., De Boever, J., Osher, J.T Quiroga, S., Barnard. G. and Kohen, F. 20 (1995) A direct non-competitive idiometric enzyme immunoassay for serum oestradiol. J Immunol Methods, 181, 83-90. Maruyama, H., Speriagh, M., Zaloudik, J., Liang, S., Mizuki, K., Molthoff, C. and Herlyn, D. (2002) Immunization procedures for anti-idiotypic anti- body induction in mice and rats. J Immunol Methods, 264,121. 25 Sambrook, J., Fritsch, E.F., and Maniatis. T. (1990) Molecular Cloning: A laboratory Manual, 2nd Ed., Cold Spring Horbor Laboratory Press, Cold Spring Harbor, NY. Self, C.H., Dessi, J.L. and Winger, L.A. (1994) High-performance assays of small molecules: enhanced sensitivity, rapidity, and convenience 30 demonstrated with a noncompetitive immunometric anti-immune com- plex assay system for digoxin. Clin Chem, 40, 2035-41. Suzuki, C, Ueda, H., Mahoney, W. and Nagamune, T. (2000) Open sandwich enzyme-linked immunosorbent assay for the quantitation of small haptens. Anal Biochem, 286, 238-46. 19 Suzuki, C, Ueda. H., Tsumoto, K., Mahoney, W.C., Kumagai, I. and Nagamune, T. (1999) Open sandwich ELISA with V(H)-/V(L)-alkaline phosphatase fusion proteins. J Immunol Methods, 224, 171-84. Szöllösi, J., Damjanovich, S., Matyus, L. (1998) Application of Fluorescence 5 Resonance Energy Transfer in the Clinical Laboratory: Routine and Re- search. Communications in Clinical Cytometry, 34:159-179. Towbin, H., Motz, J., Oroszlan. P. and Zingel. O. (1995) Sandwich immunoas- say for the hapten angiotensin lI. A novel assay principle based on anti- bodies against immune complexes. J Immunol Methods, 181, 167-76. 10 Ueda. H., Tsumoto. K . Kubota. K . Suzuki, E., Nagamune, T., Nishimura, H., Schueier, P.A., Winter, G., Kumagai, I. and Mohoney, W.C. (1996) Open sandwich ELISA: a novel immunoassay based on the interchain interaction of antibody variable region. Nat Biotechnol, 14,1714-8. Ullman, E.F., Milburn, G., Jelesko, J., Radika, K.. Pirio, M., Kempe, T. and 15 Skold, C. (1993) Anti-immune complex antibodies enhance affinity and specificity of primary antibodies. Proc Natl Acad Sci USA, 90, 1184-9. Yokozeki, T., Ueda, H., Arai, R., Mahoney, W. and Nagamune, T. (2002) A ho- mogeneous noncompetitive immunoassay for the detection of small haptens. Anal Chem, 74. 2500-4. 20 20 Claims 1. Non-competitive immunoassay for a small analyte comprising re- acting a sample containing said analyte with a reagent pair comprising a first binding partner that binds to said analyte. and a second binding partner that 5 binds to the complex of said analyte and said first binding partner, wherein said second binding partner is obtained from a display recombinant binding partner library by selecting a binding partner that binds to said complex of the analyte and first binding partner, and determining the binding of the second binding partner, thus indicating the presence of the analyte in the sample. 10 2. The assay of claim 1, wherein the first and second binding part- ners are selected from antibody fragments Fab and scFv. 3. The assay of claim 1 or 2, which assay is a homogeneous assay. 4. The assay of claim 3, which assay is based on fluorescence res- onance energy transfer (FRET). 15 5. The assay of any of the preceding claims, wherein the snalyte is a drug of abuse. 6. The assay of claim 5, wherein the analyte is morphine, tetrahy- drocannabinol (THG) or amphetamine. 7. Reagent pair for a non-competitive immunoassay for a small ana- 20 lyte, comprising a first binding partner that binds to said analyte, and a second binding partner that binds to the complex of said analyte and said first binding partner, wherein said second binding partner is obtained from a display recom- binant binding partner library by selecting a binding partner that binds to said complex of the analyte and first binding partner. 25 8. Test Kit for a non-competitive immunoassay for a small analyte, said kit comprising a reagent pair comprising a first binding partner that binds to said analyte, and a second binding partner that binds to the complex of said analyte and said first binding partner, wherein said second binding partner is obtained from a display recombinant binding partner library by selecting a 30 binding partner that binds to said complex of the analyte and first binding part- ner. 9. The test kit of claim 8, wherein the first and second binding part- ners are selected from antibody fragments Fab and scFv. 10. The test kit of claim 8 or 9, comprising reagents for a homogen- 35 eous assay. 21 11. The test kit of claim 10, comprising reagents for a fluorescence resonance energy transfer (FRET) based assay. 12. The test kit of any of claims 8 to 11, comprising reagents for as- saying a drug of abuse. 5 13. The test kit of claim 12, comprising multiple reagent pairs for as- saying multiple drugs of abuse. 14. The test kit of any of claims 8 to 13, comprising reagents for as- saying morphine, tetrahydrocannabinol (THC) or amphetamine. 15. The test kit of claim 14. comprising one or more reagents from 10 the group consisting of the ligand-binding portion of M1 Fab comprising SEQ ID NO 1 and SEQ ID NO 2; M2 Fab comprising SEQ ID NO 3 and SEQ ID NO 4; and K11 scFv comprising SEQ ID NO 5. 16. The test kit of claim 15, wherein said ligand binding portion is formed by amino acids no. 3 to 108 of SEQ ID NO 1 and amino acids no. 4 to 15 123 of SEQ ID NO 2; or of amino acids no. 3 to 108 of SEQ ID NO 3 and of amino acids no. 4 to 123 of SEQ ID NO 4; or of amino acids no. 3 to 120 and no. 140 to 246 of SEQ ID NO 5. 17. Use of a reagent pair comprising a first binding partner that binds to an analyte, and a second binding partner that binds to the complex of 20 said analyte and said first binding partner, in a non-competitive immunoassay for a small analyte, whereby the second binding partner is obtained from a dis- play recombinant binding partner library by selecting a binding partner that binds to said complex of the analyte and first binding partner. 18. Process for preparing a reagent pair for a non-competitive im- 25 munoassay for a small analyte, comprising providing a first binding partner that binds to said analyte, and a second binding partner that binds to the com- plex of said analyte and said first binding partner, wherein said second binding partner is obtained from a display recombinant binding partner library by se- lecting a binding partner that binds to said complex of the analyte and first 30 binding partner. 19. The process of claim 18, wherein recombinant antibody frag- ments are prepared from a phage display libraty. 20. The process of claim 18 or 19, wherein the first binding partner is also obtained from a display recombinant binding partner library. 22 21. Recombinant binding protein, comprising the ligand-binding por- tion of M1 Fab comprising SEQ ID NO 1 and SEQ ID NO 2; M2 Fab compris- ing SEQ ID NO 3 and SEQ ID NO 4; or K11 scFv comprising SEQ ID NO 5. 22. The recombinant binding protein of claim 21, wherein said lig- 5 and binding portion of said protein is formed by amino acids no. 3 to 108 of SEQ ID NO1 and amino acids no. 4 to 123 of SEQ ID NO 2; or of amino acids no. 3 to 108 of SEQ ID NO 3 and of amino acids no. 4 to 123 of SEQ ID NO 4; or of amino acids no. 3 to 120 and no. 140 to 246 of SEQ ID NO 5. 23. The recombinant binding protein of claim 21, which protein has 10 the amino acid sequence SEQ ID NO 1 and SEQ ID NO 2; SEQ ID NO 3 and SEQ ID NO 4; or SEQ ID NO 5. 24. DNA, which encodes a recombinant binding protein of claim 21, 22 or 23. 25. Host cell, which expresses a recombinant binding protein of 15 claim 21, 22 or 23. The invention is directed to a non-competitive immunoas- say for small analytes, wherein the analyte is reacted with 5 two binding partners. The first binding partner binds to the analyte to form a complex between the first binding part- ner and the analyte. and the second binding partner binds to the complex formed by the first binding partner and the analyte. The resulting complex formed between the ana- 10 lyte and the binding partners is detected. The binding part- ners are proteins, such as antibodies including antibody fragments. The invention further relates to reagent pairs and test kits useful in the assays, as well as to the use of 15 Novel reagents and means for their preparation are also provided.

Full Text

1
Non-competitive immunoassay for small analytes
Field or the Invention
The present invention relates to immunoassays and especially to
5 non-competitive immunoassays for small analyies. The present invention fur-
ther relates to reagent pairs and test-kits useful in the assays, as well as to the
use of the reagent pairs and to a process for their preparation. Novel reagents
and means for their preparation are also provided.
10 Technical Background of the Invention
In a competitive immunoassay, external reagent that competes with
the analyte has to be added, which is not the case in the non-competitive as-
say format. The method of choice for the detection of analytes by immuno-
chemistry is nowadays a non-competitive immunoassay, where two antibodies
15 bind to two different epitopes of the analyte creating a so-called sandwich-type
assay. Such an assay is well suited for high molecular weight analytes and it
provides improved speed, sensitivity, and specificity, which are needed in
modern immunoassays. However, it has been a difficult task to develop non-
competitive assays for small analytes, because low molecular weight mo-
20 lecules are not large enough for binding simultaneously to more than one anti-
body independently. Therefore, despite many fundamental problems with re-
spect to specificity and sensitivity, the competitive immunoassay format has
been almost exclusively used for the detection of small analytes.
However, there are few publications where the development of a
25 non-competitive immunoassay for a small analyte has been reported. In these
papers, a secondary anti-immune complex (anti-IC) antibody, which binds
primary anti-analyte antibody that is combined with the analyte but which does
not bind the primary antibody or the analyte alone, has been developed (Ull-
man et al., 1993; Self et al., 1994; Towbin et al., 1995). Ullman ef al., 1993,
30 describe an antibody that recognizes an immune complex of an antibody to
tetrahydrocannabinol (THC). The anti-IC antibody was obtained by using an
affinity labelled anti-THC antibody as immunogen and selecting an anti-IC anti-
body the binding of which was enhanced by the presence of 9THC. Self et
al., 1994, used the same principle in preparing anti-IC antibodies for determin-
35 ing digoxin. Towbin et al., 1995, report a sandwich immunoassay for the
hapten angiotensin II, wherein the immunisation involves tolerization with un-

2
complexed primary antibody prior to immunisation with the anti-immune com-
plex to obtain the anti-IC antibodies. The anti-IC antibodies used in non-com-
petitive immunoassays for small analytes have so far been conventional poly-
clonal or monoclonal antibodies obtained by immunisation, and the assays de-
5 scribed here include labelling of the primary antibody and immobilisation of the
secondary or vice versa.
So-called 'idiometric' non-competitive immunoassay for small ana-
lytes has been developed by Mares et al., 1995. They used two types of anti-
idiotypic antibodies, which recognize different epitopes within the hypervari-
10 able region of the oeastradiol specifc primary antibody. The first anti-idiotypic
antibody (betatype) possesses the capacity of competing with the analyte for
an epitope at the binding site of the primary antibody. The second anti-idiotype
(alphatype) recognizes an epitope within the variable region of the primary an-
tibody and the binding is not sensitive to the presence of the analyte. The al-
15 phatype is, however, sterically hindered from binding to the primary antibody in
the presence of the betatype. These three types of antibodies permit the de-
velopment of a non-competitive assay for small analytes.
So-called open sandwich immunoassays have been developed for
the detection of haptens (Suzuki et al., 2000; Suzuki et al., 1999; Yokozeki et
20 al, 2002). They are non-competitive assays based on a phenomenon accord-
ing to which the association of separated VH and VL chains in some antibodies
is strongly favoured in the presence of antigen (Ueda et al., 1996),
Despite the significant benefits of the non-competitive immunoas-
say format, only few examples of that kind of assays for small analytes have
25 been reported. The reason for that is most probably the difficulty of producing
secondary (anti-immuno complex, or anti-idiotypic) antibodies by immunising
animals. For example anti-hapten monoclonal antibodies, which have been
developed by hybridoma technology (Kohler and Milstein, 1975), are self-anti-
gens for mice and raising immunoresponse against them is difficult
30 (Maruyama et al., 2002; Ullman et al., 1993; Kobayashi et al., 2000), A further
problem is that the immune complex used for immunisation tends to break
down before the response to the immuno complex is obtained (Ullman et al.,
1993; Kobayashi et al.,2000).
The present invention now provides a non-competitive immunoas-
35 say protocol for small analytes, which circumvents the immunisation of anim-
als with the immune complex, which has been so far the most challenging task

3
when anti-IC antibodies have been developed. The invention also facilitates a
homogenous immunoassay, which further improves the speed, sensitivity and
simplicity of the assay.
5 Summary of the Invention
The difficulties associated with raising anti-lC antibodies for use in
Immunoassays for small analytes can now be avoided by providing the neces-
sary anti-IC antibodies from a display recombinant binding partner library in-
stead of from immunised animals. A phage display antibody library may be
10 constructed, which contains a vast number of clones, from which Those coding
the desired binding partners, such as antibody fragments, can be enriched and
selected through sequential panning. This protocol opens new possibilities for
developing rapid, reliable and simple immunoassays for small analytes in a
cost-effective and feasible way.
15 Consequently, one object of the present invention is a non-compet-
itive immunoassay for a small analyte comprising reacting a sample containing
said analyte with a reagent pair comprising a first binding partner that binds to
said analyte, and a second binding partner that binds to the complex of said
analyte and said first binding partner. The immunoassay is characterized in
20 that the second binding partner is obtained from a display recombinant binding
partner library by selecting a binding partner that binds to said complex of the
analyte and first binding partner, and determining the binding of the second
binding partner thus indicating the presence of the analyte in the sample.
Another object of the invention is a reagent pair for a non-competit-
25 ive immunoassay for a small analyte, comprising a first binding partner that
binds to said analyte, and a second binding partner that binds to the complex
of said analyte and said first binding partner, characterized in that the second
binding partner is obtained from a display recombinant binding partner library
by selecting a binding partner that binds to said complex of the analyte and
30 first binding partner.
Still another object of the present invention is a test kit for a non-
competitive immunoassay for a small analyte, said kit comprising a reagent
pair comprising a first binding partner that binds to said analyte, and a second
binding partner that binds to the complex of said analyte and said first binding
35 partner, characterized in mat the second binding partner is obtained from a

4
display recombinant binding partner library by selecting a binding partner that
binds to said complex of the analyte and first binding partner.
The invention Is also directed to the use of a reagent pair compris-
ing a first binding partner that binds to an analyte, and a second binding part-
5 ner that binds to the complex of said analyte and said first binding partner, in a
non-competitive immunoassay for a small analyte, whereby the second bind-
ing partner is obtained from a display recombinant binding partner library by
selecting a binding partner that binds to said complex of the analyte and first
binding partner.
10 A still further object of the invention is a process for preparing a re-
agent pair for a non-competitive immunoassay for a small analyte comprising
providing a first binding partner that binds to said analyte, and a second bind-
ing partner that binds to the complex of said analyte and said first binding part-
ner, characterized in that the second binding partner is obtained from a display
15 recombinant binding partner library by selecting a binding partner that binds to
said complex of the analyte and first binding partner.
The invention also provides novel recombinant binding proteins,
characterized in that they comprise the ligand-binding portion of M1 Fab com-
prising SEQ ID NO 1 and SEQ ID NO 2; M2 Fab comprising SEQ ID NO 3 and
20 SEQ ID NO 4: or K11 scFv comprising SEQ ID NO 5.
DNA encoding the novel binding proteins as well as host cells ex-
pressing them are also included in the invention.
Advantagous embodiments of the invention are set forth in the de-
pendent claims.
25 Other objects, details and advantages of the present invention will
become apparent from the following drawings, detailed description and ex-
amples.
Brief Description of the Drawings
Figure 1. A competitive ELISA using M1 Fab. Urine+ is urine spiked
30 with 1 g/ml of morphine.
Figure 2. A non-competitive time resolved fluorescent immunoassay
(TR-FIA) with the Eu labelled anti-M1 +morphine immune complex scFv frag-
ment K11. a) Cross-reactivity and sensitivity of the assay, b) Sensitivity of the
assay with S1 urine control dilutions as samples.
35 Figure 3. A homogenous TR-FRET immunoassay of morphine.

5 5
Figure 4. Analysis of cross-reactivity of M1 anti-morphine Fab-frag-
ment with codeine, heroin, noscapine and papaverine by a competitive ELISA.
Figure 5. A comparative TR-FRET based homogeneous immunoas-
say for morphine. '
5 Detailed Description of the Invention
The reagent pair for the non-competitive immunoassay of the inven-
tion comprises a first binding partner and a second binding partner. The first
binding partner binds to the analyte to form a complex between the first bind-
ing partner and the analyte. The second binding partner binds to the complex
10 formed by the first binding partner and the analyte. The binding partners are
usually proteins such as antibodies including antibody fragments that have the
desired binding properties. An antibody is an immunoglobuiin molecule and it
can belong to any of classes IgG, IgM, IgE, IgA or IgD; lgG and IgM being the
most frequently used. Preferably the binding partners are antibody fragments
15 comprising the Iigand-binding site, such as Fab, or scFv fragments. The frag-
ment known as the Fab fragment (fragment antigen binding) consists of the
variable and constant domain of an immunoglobulin light chain covalently at-
tached by a disulfide bridge to the variable and first constant domain of an im-
munoglobulin heavy chain. Fv (variable domain) means the variable regions of
20 the immunoglobulin molecule that are responsible for the ligand binding. ScFv
(single chain Fv) means a molecule wherein the variable domains of the heavy
and light chain of an antibody are linked by a peptide to form a single poly-
peptide chain synthesized from a single mRNA molecule. The variable regions
of an immunoglobulin heavy chain and light chain are together responsible for
25 the ligand binding. Ligand is the substance to which the binding partner binds,
in connection with antibodies it is an antigen or a hapten.
The first binding partner may be a conventional polyclonal or mono-
clonal antibody or fragment thereof, but preferably it is a recombinant one, as
is the second binding partner. When the first binding partner has been selec-
30 ted for, it is complexed with its ligand and this complex is used to select for the
second binding partner from a recombinant library. The first binding partner
without the ligand is used as contra selection. The second binding partner
should only recognise complexes, not free first binding partner nor free antigen
to any significant extent.
35 The recombinant binding partner library is conveniently an expres-
sion library, which is typically a display library. The general principle of the dis-

6
play recombinant binding partner libraries is that they present the binding part-
ner as a fusion protein on the surface, which may be the surface of a microbial
cell such as a yeast or bacterial cell, or a phage. The display recombinant
binding partner library can also be a display library, where stable complexes of
5 nascent protein and mRNA are produced in an in vitro expression system.
Phage display libraries are the most frequently used. Antibody phage display
technology and its applications are described e.g. in Hoogenboom et al., 1998.
A phage display antibody library may be constructed by cloning im-
mnunoglobulin domains coding cDNAs into an appropriate phage display vec-
10 tor. DNA encoding for millions of variants of antibody fragments is batch-
cloned into the vector as part of the phage coat protein. Large libraries con-
taining millions of antibody fragments with different specificities can be ob-
tained by transforming the vectors in bacteria. Cultivation of the bacteria leads
to the expression of phages displaying antibody fragments on their surface.
15 The gene for the displayed antibody is carried in the phage genome, thus link-
ing genotype with phenotype. The physical linkage between the displayed pro-
tein and its DNA allows screening of vast numbers of variants of the protein,
each linked to its corresponding DNA, by a simple in vitro selection procedure
called panning. In its simplest form, panning is carried out by incubating the
20 pool of phage-displayed variants with the ligand of interest that has been im-
mobilized on a carrier, washing away unbound phage, and eluting specifically
bound phage by disrupting the binding to the ligand. The eluted phage is then
amplified in vivo. The process is repeated several times, resulting in stepwise
enrichment of the phage pool in favour of the tightest binding sequences. After
25 about 3 to 6 rounds of selection and amplification, the best clones are se-
quenced and transformed into a host cell for further expression. The host cell
may be a eucaryotic or procaryotic cell e.g. a yeast, animal, plant or insect cell
or bacterial cell. It may even be a hybridoma cell, which after transformation
produces a recombinant monoclonal antibody. The recombinant binding part-
30 ner or at least part of it may also be produced synthetically.
The concept to use recombinant antibody libraries makes the non-
competitive sandwich assay for small analytes feasible. The sandwich can be
detected by all the standard immunoassays. Usually one partner is immobil-
ized on a carrier, such as a microtiter well or a bead. A sandwich is formed in
35 the presence of analyte and the other binding partner. The sandwich may be
detected e.g. by using secondary antibodies or by labelling at least one of the

7
binding partners. The label can be any conventional label, such as a radioact-
ive label, an enzyme, or a fluorescent compound. The assay can be e.g.
ELISA or FIA.
A great advantage of the reagent pair of the present invention is
5 that it enables a homogenous non-competitive immunoassay, i.e. an immun-
oassay that is carried out in solution. The avoidance of immobilising and wash-
ing steps makes the assay extremely simple. Such a test is also suitable for
testing on-site i.e. in places elsewhere than the laboratory.
A preferred homogenous immunoassay is one based on fluores-
10 cence resonance energy transfer (FRET), for review see Szöllösi et al., 1998.
In FRET, energy from a molecular fluorophore (donor) is excited to a high-en-
ergy state and transferred to another fluorophore (acceptor) via intermolecular
dipole-dlpole coupling. This is possible only if the distance between the donor
and fhe acceptor is short (10-100 A) and the fluorescence spectrum of the
15 donor and the absorption spectrum of the acceptor partially overlap. The en-
ergy transfer is then detected as a change in fluorescence. Often time-re-
solved fluorescence is utilized (Hemmilä et al., 1988).
FRET is applied to the present invention by labelling the two binding
partners, which preferably are antibody fragments, with fluorophores that form
20 a FRET donor-acceptor pair. When the binding partners and the analyte are
small the fluorophores come into very close proximity, and a measurable
FRET signal is obtained.
The invention provides a convenient and rapid analytical tool for low
molecular weight analytes, such as therapeutic and abused drugs, steroids,
25 hormones, metabolites, and environmental pollutants and toxins. A common
feature of these small analytes is that they are too small for conventional
sandwich assays where two antibodies recognizing different epitopes of the
antigen are used. The molecular weight of these small analytes are normally
less than 5000, but the limits are not absolute.
30 The immunoassay may be employed in all kinds of investigations,
such as in detecting environmental hazards, toxic compounds in food and
feed, chemicals indicative of ongoing processes e.g. of microbial processes in
buildings, metabolic, processes of living organisms, and in clinical tests, drug
monitoring and pharmacological research. The assays are extremely suitable
35 for detecting drugs of abuse, such as opiates (e.g. morphine), amphetamines,
cannabinoids (e.g. tetrahydrocannabinol (THC)), barbiturates, benzo-

8
diazepines, cocaine, LSD, methadone, methaqualone, phencyclidine, pro-
poxyphene, tricyclic antidepressants. The homogenous assay provides an ex-
cellent and convenient tool for on-site tests e.g. to be used by the police in
raiding drivers etc. The sample to be analysed for e.g. drugs and abused
5 drugs may be any body fluid sample, such as blood, serum, urine or saliva.
The reagent pair of the invention may be included in a test-kit. This
test-kit may further comprise any other reagents needed for the assay, such
as reaction solutions, buffers, washing solutions and detecting means, such as
labels and optionally a fluorometer. Preferably the test-kit comprises multiple
10 reagent pairs physically separated from each other, e.g. many in the form of a
microarray, whereby e.g. many different drugs of abuse may be tested simul-
taneously from a single saliva sample.
In a special embodiment of the invention, a reagent pair for detect-
ing morphine is produced and employed in a homogenous immunoassay for
15 said substance. The first binding partner is a Fab fragment obtained from a
phage display antibody library produced from cDNA from a mouse immunized
with morphine conjugated to an immunocarrier BSA. Morphine specific anti-
body phages were enriched by selecting those binding to morphine conjugated
BSA and sorting out those binding to BSA alone. After several panning rounds
20 two high binding clones are sequenced and expressed. The expressed Fab
fragments were named M1 Fab and M2 Fab.
The second binding partner is obtained from a naive scFv antibody
fragment phage display library by selecting antibodies that bind to a complex
of morphine and M1 Fab. First the phages are preincubated to bound M1 Fab
25 to sort out those binding to M1 Fab as such. The unbound phages are separ-
ated and incubated with a mixture of morphine and immobilised M1 Fab to se-
lect the phages that bind to the immunocomplex formed between the immobil-
ized M1 Fab and morphine. Unbound phages are washed away, and then
those bound to the complex are eluted. The background is monitored by
30 checking the binding to M1 Fab in the absence of morphine. After several pan-
ning rounds a number of clones are picked up, sequenced and expressed res-
ulting in scFv fragment K11.
A fluorescence-based immunoassay is performed using M1 Fab la-
belled with europium as a first binding partner, and scFv fragment K11 labelled
35 with Cy5 as a second binding partner. The binding partners are incubated with
saliva or urine samples containing morphine and then fluorescence is meas-

9
ured after a predetermined time. The assay is completely homogeneous and
the signal is readable in about 5 min. The sensitivity for both urine and saliva
is clearly higher than that demanded by the authorities in the case of
morphine, our model analyte. The performance of the test is such that the re-
5 agents are in the well of a microtiter plate and dilution series of either saliva or
urine is added. In a preferred mode for e.g. police field use, the reagents are
in dry form in a vessel. Saliva is added, which dissolves the reagents and the
result can be read without further processes.
The novel recombinant binding proteins provided by the invention
10 comprise any ligand-binding portion of M1 Fab, M2 Fab or K11 scFv. By "lig-
and-binding portion" is meant that part of the molecule that is responsible for
the binding. Minor variations or modifications of the sequences set forth in the
description and claims are still within the scope of the invention provided that
they do not affect the binding activity of the proteins.
15 The invention is illustrated by the following non-limiting example. It
should be understood, however, that the embodiments given in the description
above and in the examples are for illustrative purposes only, and that various
changes and modifications are possible within the scope of the invention.
Example 1
20 Development of an anti-morphine antibody
immunisation of mice
Four six-week-old female Balb/c mice were immunised in three-
week intervals with morphine conjugated BSA (Fitzgerald) in Freund's ad-
juvant. Serum samples were tested after second booster and the mouse
25 showing the best response against the antigen in direct ELISA was selected to
be the source of an antibody phage display library.
Construction of the antibody phage display library
All basic recombinant DNA methods were performed essentially as
described (Sambrook et al. 1990). The mouse with the highest antibody re-
30 sponse to morphine-BSA conjugate was sacrificed and the total RNA was isol-
ated from the spleen cells using the RNagents Total RNA Isolation System
(Promega Co., Wl, USA). The mRNA pool of the total RNA was isolated with
the Oligotex mRNA Kit (OIAGEN Inc., Germany). The cDNA was synthesised
from the mRNA with oligo-dT priming. Genes encoding antibody Fab frag-
35 ments were amplified with PCR using antibody kappa light chain and heavy

10 10
chain variable region and constant region specific primers. Antibody light chain
PCR products were pooled and digested with Nhel and AscI restriction en-
zymes, purified by preparative agarose gel in combination with the QIAquick
Gel Extraction Kit (OlAGEN Inc.. Germany). The agarose gel purified antibody
5 light chain DNA was ligated into the Fab phagemid vector phagemid9 derived
from pComb3 (Barbas et al., 1991) and transformed into the E.coli XL1-Blue
strain (Stratagene) by electroporation. Plasmid DNA was isolated with the OlA-
GEN Plasmid Midi Kit (Q1AGEN Inc., Germany) from the overnight culture.
PCR products encoding the Fd region (variable and first constant region) of
10 the heavy chain were pooled and digested with Sfil and Notl restriction en-
zymes, purified by preparative agarose gel isolation and ligated to the
phagemid vector containing the light chain DNA. The phagemid vector encod-
ing both the heavy and light chain of the Fab fragment was transformed into
the E.coli TOP10F1 bacteria (Invitrogen Inc., CA, USA) by electroporation.
15 Transformed bacteria were incubated over night at +37 C on a shaker and
plasmid DNA was isolated with the QIAGEN Plasmid Midi Kit. The diversity of
the antibody library was ensured by sequencing partial VL-or VH-gene regions
of individual clones.
A phage display antibody library was made as follows: 4 g of anti-
20 body library plasmid DNA was transformed into E.coli TOP1 0F1 by electropora-
tion in two parallel transformations. After transformations, cells were suspen-
ded into 2,8 ml of SOC medium and incubated for 1 h at +35 C on a shaker. 7
ml Of prewarmed (+37 C) SB medium. 20 g/ml of carbenicillin, and 10 g/ml
of tetracycline was added. After 1 -h incubation at +35 C on a shaker, 30 g/ml
25 of carbenicillin was added and the incubation was continued for 1 h after which
1 ml ( 1011 pfu) of helper phage VCS-M13 (Stratagene) was added and the
phages were let to infect the bacteria for 20 min at +35 C with a slow shaking.
The parallel transformations were joined together and 80 ml of prewarmed
(+37 C) SB medium with 50 g/ml carbenicillin and 10 g/ml of tetracycline
30 was added. After 2-h incubation at +35 C on a shaker, 70 g/ml of kanamycin
was added and the incubation was continued over night.
Cells were centrifuged for 15 min at 4000g at +4 C. 20 ml of 20%
PEG, 2.5 M NaCl (PEG/NaCI) was added ID the supernatant and it was incub-
ated for 30 min on ice. PEG precipitated phages were centrifuged for 20 min
35 at 13000g at +4 C. The pellet was suspended in 2 ml of PBS and 1 ml was
transferred into two eppendorf tubes. After centrifugation for 5 min at +4 C,

11
phages were precipitated by adding 200 | of PEG/NaCl to the supernatant.
The solution was mixed and centrifuged for 5 min at +4 C. The pellet was sus-
pended in 1 ml of PBS orPBS+1% BSA. 1 | of 20% Na-azide was added as
a preservative. The phage display antibody library was stored at +4 C.
5 Selection of the anti-morphine library
Morphine specific antibodies were selected from the phage display
antibody library by the following selection procedure: A microtiter well was
coated over night at +4 C with 1 g of morphine conjugated BSA (morphine-
BSA; Fitzgerald Industries International, Inc., MA, USA) in 100 | of 100 mM
10 Na-bicarbonate buffer. pH 9.8. The well was washed two times with PBS and
blocked with 1 % BSA in PBS for 1 h at +37 C. 100 | of the phage library was
incubated in the well for 1 h at RT with shaking, after which the unbound
phages were removed and the well was washed 22 times with PBS. Bound
phages were eluted with 100 | of 100 mM HCI (pH 2.2) for 15 min. Eluted
15 phages were removed from the well and neutralised with 1 M Tris. 3 ml of
fresh E.coli XL1-Blue cells (OD600 1) grown in SB supplemented with 10 g/ml
of tetracycline was infected with eluted phages at +35 C for 15 min. 7 ml of
prewarmed SB (+37 C) with 20 g/ml of carbenicillin and 10 g/ml of tetracyc-
line was added and the culture was incubated for 1 h at +35 C on a shaker. 30
20 g/ml of carbenicillin was added and incubation was continued for 1 h. 1 ml of
helper phage VCS-M13 was added to the culture and incubated at +35 C for
15 min with a slow shaking. The culture was diluted with 90 ml of prewarmed
(+37 C) SB with 50 g/ml carbenicillin and 10 g/ml tetracycline. After 2 h in-
cubation at +35 C on a shaker, 70 g/ml of kanamycin was added and the in-
25 cubation was continued over night. The purification of the amplified phages
was performed by PEG precipitation as described above.
The purified phages were used in further enrichment rounds. After
four selection rounds, morphine specific antibody phages had been enriched
over 1000 times when compared to the background. The background binding
30 of the phages was monitored in parallel in each selection round by incubating
the phage library in a BSA coated microtiter well. The well was washed and
the phages were eluted as in the morphine-BSA coated well. The enrichment
of the morphine specific phages was monitored by comparing the amount of
the eluted phages from the morphine-BSA coated well to the amount of eluted
35 phages from the background well.
Characterisation of individual clones

12
After the fourth panning round, the phagemid DNA was isolated
with the QIAGEN Plasmid Midi Kit. The plasmid was digested with Nhel and
Notl restriction enzymes to isolate the Fab gene fragment. The agarose gel
isolated DNA was ligated to the expression vector pKKtac. The ligation reac-
5 tion was transformed into the E.coli XL1-Blue cells.
Individual clones were picked and miniprep DNA was extracted with
the QIAprep Spin Miniprep Kit (QIAGEN Inc., Germany). According to sequen-
cing of the minipreps, two different Fab clones were found. These clones were
named M1 and M2. The amino acid sequences of the M1 Fab fragment were
10 SEQ ID NO 1 (light chain) and SEQ ID NO 2 (heavy chain). Amino acids no. 3
to 108 represent the variable region and no. 109 to 215 the constant region of
the M1 light chain (SEQ ID NO 1). Amino acids no 4 to 123 represent the vari-
able region and no. 124 to 226 the constant region of the M1 heavy chain
(SEQ ID NO 2). The amino acid sequences of M2 Fab fragment were SEQ ID
15 NO 3 (light chain) and SEQ ID NO 4 (heavy chain). Amino acids no 3 to 108
represent the variable region and no. 109 to 215 the constant region of the M2
light chain (SEQ ID NO 3). Amino acids no. 4 to 123 represent the variable re-
gion and no. 124 to 226 the constant region of the M2 heavy chain (SEQ ID
NO 4). The rest of the amino acids derive from the cloning technique, and
20 some of the C-terminal amino acids facilitate the isolation and purification of
the protein. Small-scale (3 ml) Fab expression cultures were also made. Peri-
plasmic fraction of the cells was isolated by freezing and thawing the cells for
three times in PBS.
The binding of individual Fab clones to morphine was tested by
25 ELISA in morphine-BSA coated microtiter wells: Microtiter wells were coated
with 200 ng of morphine-BSA in 0.1 M Na-bicarbonate buffer, pH 9.8 for over
night at +4 C. Control wells were filled only with the buffer. After coating, wells
were washed three times with PBS and blocked with 0.5% BSA in PBS
(BSA/PBS) for 1 h at RT. Wells were washed three times with PBS and 1:10
30 dilution of the periplasmic fractions was added into wells in 100 | of
BSA/PBS. After 1-h incubation at RT on a shaker, wells were washed three
times with PBS and 1:2000 diluted alkaline phosphatase conjugated anti-
mouse Fab specific antibody (Sigma, A-1293) was added in 100 | of
BSA/PBS. The wells were incubated for 1 h at RT on a shaker and washed
35 three times with PBS. 100 | of alkaline phosphatase substrate solution (2
mg/ml of p-nitrophenylphosphate di-Na-salt in diethanolamine-MgCI-buffer)

13
was added to the wells and absorbance was measured. According to the pre-
liminary results, the M1 Fab showed better performance in the assay and was
therefore chosen for the continuation.
Fermentation and purification
5 The anti-morphine Fab fragment M1 in the expression vector
pKKtac was transformed in the E.coli expression strain RV308. The Fab was
expressed by fed-batch fermentation in a Bio-Flow IV fermenter (New Brun-
swick) and purified by the Sepharose SP ion-exchange and protein G chroma-
tography (Pharmacia).
10 Performance of the primary antibody in a competitive immunoassay
The performance of the M1 anti-morphine Fab fragment was tested
in a competitive ELISA assay using the commercial urine toxicology controls
S1 and S3 (Bio-Rad). The S1 urine toxicology control contains drugs and drug
metabolites (including morphine) at concentrations 20-25% below immunoas-
15 say cut-off levels as recommended by the U.S. Substance Abuse and Mental
Health Services Administration (SAMSHA) and other agencies. The S3 urine
toxicology control contains drugs and drug metabolites at concentrations ap-
proximately three times immunoassay cut-off levels. Microtiter wells were
coated over night at +4 C with 500 ng of morphine-BSA in 100 | of Na-bicar-
20 bonate buffer, pH 9.8. Wells were washed three times with PBS and blocked
with BSA/PBS for 1 h at RT. After three washes with PBS, S1, S3 (Bio-Rad),
positive control, or negative control urine dilutions were added to the wells in
100 | of BSA/PBS spiked with 1 ng of M1 Fab. Wells were incubated for 2 h
at RT on a shaker and washed three times with PBS. 1:2000 diluted alkaline
25 phosphatase conjugated anti-Fab antibody (Sigma, A-1293) was added to the
wells in 100 | of BSA/PBS and incubated for 1 h at RT on a shaker. Wells
were washed three times with PBS, 100 | of alkaline phosphatase substrate
solution was added and A405 was measured. Results are shown in Figure 1. A
positive result could be achieved with 1:64 dilution of the sample.
30 Development of an anti-immune complex antibody specific to the im-
mune complex of M1 Fab and morphine
Selection of the immune complex specific antibody from a naive hu-
man scFv phaae display library
M1 Fab fragment was biotinylated with ImmunoPure Sulfo-NHS-LC-
35 Biotin Kit (Pierce). Biotinylated antibody was purified and buffer was changed
to PBS with Econo-Pac 10DG Columns (Bio-Rad, CA, USA). 200 | of a naïve

14
human scFv phage display library (Kappa or Lambda light chain) in BSA/PBS
was preincubated with 10 | of streptavidin coated magnetic beads (Dynal, M-
280) and 0.5 g of biotinylated M1 Fab for over night at +4 C. The naive hu-
man scFv phage display library was constructed from pooled lymphocytes of
5 50 healthy individuals. The size of the library was estimated to be 1x108
clones. The naive human scFv phage display library contains the IgM specific
VH-genes combined either with the kappa or lambda specific VL-genes. Un-
bound phages were separated from the beads and 100 | of them was incub-
ated with 100 ng of morphine, 500 ng of biotinylated M1 Fab, and 5 | of strep-
10 tavidin coated magnetic beads for 1 h at RT on a shaker. The background for
the selection procedure was implemented in a similar way but omitting the
morphine from the binding reaction. Magnetic beads were washed five times
with 0.5 ml of PBS and bound phages were eluted with 100 | of HCI (pH 2.2)
for 30 min. The eluted phages were neutralised with 1M Tris and E.coli XL1-
15 Blue cells were infected. Ceils were grown and phages were purified as de-
scribed previously. After five panning rounds enrichment was seen with the
scFv library having the kappa light chains, when the amount of eluted phages
was compared to the amount of eluted phages from the background control
well. The enrichment of specific binders to the immune complex, formed by
20 M1 Fab and morphine, was also clearly seen in a phage ELISA when the
eluted phage pools from the selection rounds were tested.
Characterisation of individual clones
Individual phage clones were picked and they were sequenced. All
of the clones had the same sequence (SEQ ID NO 5). The amino acid se-
25 quence of anti-M1+morphine immune complex scFv fragment was named K11
scFv. Amino acids no- 3 to 120 represent the heavy chain variable region, no.
140 to 246 represent the light chain variable region, and no. 121 to 139 repre-
sent the linker of K11 scFv (SEQ ID NO 5). The rest of the amino acids derive
from the cloning technique, and some of the C-terminal amino acids facilitate
30 the isolation and purification of the protein.
Expression and purification
The gene encoding the scFv fragment K 11 was inserted into the
expression vector pKKtac and transformed into the E.coli RV308 strain. Cells
were inoculated into 20 ml of LB with 100 g/ml ampicillin and were incubated
35 over night at +37 C on a shaker. From the overnight culture a 10 ml inoculate
was added into two erlenmeyer bottles containing 500 ml of LB with 100 g/ml

15 15
of ampicillin. Cells were incubated at +37 C on a shaker until the OD600 was 1
after which 0.5 ml of 1M IPTG and 100 g/ml of ampicillin were added into the
culture and the incubation was continued over night at +30 C on a shaker.
Both the supernatant of the culture medium and the periplasmic fraction of the
5 cells were used for the purification of the K11 scFv. Cells were centrifuged at
4000g for 15 min and the supernatant was poured into a clean flask. The cells
were resuspended in 20 ml of PBS. The periplasmic fraction of the cells was
isolated by freezing (-70 C) and thawing (+37 C) the cells for three times. After
centrifugation at 12000g for 30 min, the clear periplasmic supernatant was
10 taken. The culture medium and the periplasmic fraction were treated with 2
mg/ml of DNasel to remove residual chromosomal DNA for two hours at
+37 C. Since the expressed K11 scFv has a 6xHis-tag in its C-terminus, it
could be purified by immobilised metal affinity chromatography (IMAC). The
K11 scFv was purified by expanded bed chromatography using STREAMLINE
15 Chelating (Amershampharmacia biotech) as a matrix and copper as the metal
chelate. The purity of K11 scFv was checked with SDS-PAGE.
Development of a non-competitive immunoassay for morphine
Labelling of the anti-immune complex antibody K11 scFv with
europium
20 The purified anti-M1 and morphine immune complex scFv fragment
K11 was labelled with europium chelate by the DELFIA Eu-Labelling Kit (Wal-
lac) as described by the manufacturer. Buffer of the labelled K11 scFv was
changed to 50 mM Tris pH 7.8, 0.9% NaCI. Labelling yield was 0.4 Eu/scFv,
A non-competitive immunoassay for morphine
25 The sensitivity and cross-reactivity of K11 scFv was studied by a
non-competitive immunoassay. Transparent Streptawell (Roche) streptavidin
coated microtiter wells were washed three times with PBS. 500 ng of biot-
inylated anti-morphine M1 Fab was added into the wells in 100 | of BSA/PBS
and the wells were incubated for 30 min at RT on a shaker. After three PBS
30 washes, sample dilutions spiked with morphine, amphetamine, tetrahydrocan-
nabinol (THC) or S1 urine control were added into the wells in 50 | of
BSA/PBS. 1:100 diluted europium labelled K11 scFv was added into the wells
in 50 | of BSA/PBS and the incubation was continued for 1 h. Wells were
washed three times with PBS and 100 | of Enhancement solution (Wallac)
35 was added into the wells. After 15-min shaking at RT, fluorescence was detec-
ted by VictorV fluorometer (Wallac). Results are shown in Figure 2. There is a

16
significant difference in affinity between the binding of K11 scFv to the primary
antibody fragment M1 and to the M1 and morphine immune complex. This al-
lows the detection of 1 ng/ml of morphine in a sample. No cross-reactivity with
amphetamine or THC was detected.
5 A homogenous time resolved fluorescent resonance energy transfer
(TR-FRET)
The anti-morphine Fab fragment M1 was labelled with europium by
the LANCE Eu-W1024 ITC chelate Kit (Wattac). The buffer of me labelled M1
was changed to 50 mM Tris, pH 7.8, 0.9% NaCl. The labelling yield was 1.1
10 Eu/Fab. The anti-M1 +morphine immune complex scFv fragment K11 scFv was
labelled with Cy5 by the FluoroLink-Ab Cy5 labelling kit (Amershampharmacia
biotech). The labelling yield was 2 Cy5/scFv.
Saliva was spiked with various dilutions of morphine and these
samples were filtered through cotton wool before adding into the black mi-
15 crotiter wells (Nunc). 1 g of europium labelled M1 Fab and 1 g of Cy5 la-
belled K11 scFv was added into the wells in 50 | of BSA/PBS. TR-FRET was
read after 5, 15, 30, or 60 min incubation at RT by VictorV fluorometer (Wal-
lac). Results are shown in Figure 3. After five minutes incubation. 1 ng/ml of
morphine in saliva was detected.
20 While one strategy for providing reagents and assays of the inven-
tion has been described, numerous variations and modifications will become
apparent to the person skilled in the art.
Example 2
Cross-reactivity of the M1 Fab-fragment with codeine, heroin, noscapine
25 and papaverine
Cross-reactivity of M1 anti-morphine Fab-fragment with codeine.
heroin, noscapine and papaverine was tested in a competitive ELISA. Mi-
crotiter plate wells were coated o/n at +4°C with 500 ng of morphine-BSA in
100 | of Na-bicarbonate buffer, pH 9.8. The wells were washed three times
30 with PBS and blocked with 0.5% BSA/PBS for 1 h at RT and washed again
three times with PBS. Two parallel 100 | samples containing morphine,
codeine, heroin, noscapine or papaverine (39, 78. 156, 313, 625, 1250, 2500
or 5000 ng/ml) In PBS with 5 ng of purified M1 Fab were added into the wells
and incubated for 30 min at RT on a shaker and washed three times with PBS.
35 1:2000 diluted alkaline phosphatase conjugated anti-Fab antibody (Sigma, A-

17
1293) was added to the wells in 100 | of 0.5% BSA/PBS and incubated for 30
min at RT on a shaker. Wells were washed three limes with PBS, 100 | of al-
kaline phosphatase substrate solution was added and A405 was measured. The
results are shown in Figure 4. According to the competitive ELISA result M1
5 antibody has high cross-reactivity to codeine and heroin, which are structurally
very similar with morphine,
A homogenous time resolved fluorescent resonance energy transfer (TR-
FRET) imunoassay for morphine
The anti-morphine Fab fragment M1 was labelled with europium by
10 LANCE Eu-W1024 ITC chelate Kit (Wallac). The buffer of the labelled M1 was
changed to 50 mM Tris, pH 7.8, 0.9% NaCI. The labelling yield was 1.1
Eu/Fab. The anti-M1+morphine immune complex scFv fragment K11 was la-
belled with Cy5 by FluoroLink-Ab Cy5 labelling kit (Amersham Pharmacia Bi-
otech), The labelling yield was 2 Cy5/scFv.
15 Saliva was spiked with a high concentration (10 µg/ml) of the follow-
ing drugs: morphine, heroin, codeine, papaverine and noscapine. The samples
were filtered through cotton wool before adding into black microtiter wells
(Nunc). 1 g of europium labelled M1 Fab and 1 g of Cy5 labelled K11 scFv
was added into the wells in 50 | of BSA/PBS. As the control the background
20 fluorescence from the M1 and K11 antibody pair without any added drug was
measured. TR-FRET was read after 5 min incubation at RT by VictorV fluoro-
meter (Wallac). The results are shown in Figure 5.
Saliva sample spiked with morphine is giving a high fluorescent
value, whereas the other drugs are giving fluorescent values that are similar to
25 the background fluorescence detected from the control (labelled M1 and K11
scFv fragments without any added drug). K11 scFv binds the immune complex
formed between M1 and mophine with extremely high specificity. K11 is able
to discriminate completely M1 and morphine immune complex from the im-
mune complexes between M1 and heroin or M1 and codeine, which gave
30 fluorescent values corresponding to the background level.

18
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20 20
Claims
1. Non-competitive immunoassay for a small analyte comprising re-
acting a sample containing said analyte with a reagent pair comprising a first
binding partner that binds to said analyte. and a second binding partner that
5 binds to the complex of said analyte and said first binding partner, wherein
said second binding partner is obtained from a display recombinant binding
partner library by selecting a binding partner that binds to said complex of the
analyte and first binding partner, and determining the binding of the second
binding partner, thus indicating the presence of the analyte in the sample.
10 2. The assay of claim 1, wherein the first and second binding part-
ners are selected from antibody fragments Fab and scFv.
3. The assay of claim 1 or 2, which assay is a homogeneous assay.
4. The assay of claim 3, which assay is based on fluorescence res-
onance energy transfer (FRET).
15 5. The assay of any of the preceding claims, wherein the snalyte is
a drug of abuse.
6. The assay of claim 5, wherein the analyte is morphine, tetrahy-
drocannabinol (THG) or amphetamine.
7. Reagent pair for a non-competitive immunoassay for a small ana-
20 lyte, comprising a first binding partner that binds to said analyte, and a second
binding partner that binds to the complex of said analyte and said first binding
partner, wherein said second binding partner is obtained from a display recom-
binant binding partner library by selecting a binding partner that binds to said
complex of the analyte and first binding partner.
25 8. Test Kit for a non-competitive immunoassay for a small analyte,
said kit comprising a reagent pair comprising a first binding partner that binds
to said analyte, and a second binding partner that binds to the complex of said
analyte and said first binding partner, wherein said second binding partner is
obtained from a display recombinant binding partner library by selecting a
30 binding partner that binds to said complex of the analyte and first binding part-
ner.
9. The test kit of claim 8, wherein the first and second binding part-
ners are selected from antibody fragments Fab and scFv.
10. The test kit of claim 8 or 9, comprising reagents for a homogen-
35 eous assay.

21
11. The test kit of claim 10, comprising reagents for a fluorescence
resonance energy transfer (FRET) based assay.
12. The test kit of any of claims 8 to 11, comprising reagents for as-
saying a drug of abuse.
5 13. The test kit of claim 12, comprising multiple reagent pairs for as-
saying multiple drugs of abuse.
14. The test kit of any of claims 8 to 13, comprising reagents for as-
saying morphine, tetrahydrocannabinol (THC) or amphetamine.
15. The test kit of claim 14. comprising one or more reagents from
10 the group consisting of the ligand-binding portion of M1 Fab comprising SEQ
ID NO 1 and SEQ ID NO 2; M2 Fab comprising SEQ ID NO 3 and SEQ ID
NO 4; and K11 scFv comprising SEQ ID NO 5.
16. The test kit of claim 15, wherein said ligand binding portion is
formed by amino acids no. 3 to 108 of SEQ ID NO 1 and amino acids no. 4 to
15 123 of SEQ ID NO 2; or of amino acids no. 3 to 108 of SEQ ID NO 3 and of
amino acids no. 4 to 123 of SEQ ID NO 4; or of amino acids no. 3 to 120 and
no. 140 to 246 of SEQ ID NO 5.
17. Use of a reagent pair comprising a first binding partner that
binds to an analyte, and a second binding partner that binds to the complex of
20 said analyte and said first binding partner, in a non-competitive immunoassay
for a small analyte, whereby the second binding partner is obtained from a dis-
play recombinant binding partner library by selecting a binding partner that
binds to said complex of the analyte and first binding partner.
18. Process for preparing a reagent pair for a non-competitive im-
25 munoassay for a small analyte, comprising providing a first binding partner
that binds to said analyte, and a second binding partner that binds to the com-
plex of said analyte and said first binding partner, wherein said second binding
partner is obtained from a display recombinant binding partner library by se-
lecting a binding partner that binds to said complex of the analyte and first
30 binding partner.
19. The process of claim 18, wherein recombinant antibody frag-
ments are prepared from a phage display libraty.
20. The process of claim 18 or 19, wherein the first binding partner
is also obtained from a display recombinant binding partner library.

22
21. Recombinant binding protein, comprising the ligand-binding por-
tion of M1 Fab comprising SEQ ID NO 1 and SEQ ID NO 2; M2 Fab compris-
ing SEQ ID NO 3 and SEQ ID NO 4; or K11 scFv comprising SEQ ID NO 5.
22. The recombinant binding protein of claim 21, wherein said lig-
5 and binding portion of said protein is formed by amino acids no. 3 to 108 of
SEQ ID NO1 and amino acids no. 4 to 123 of SEQ ID NO 2; or of amino acids
no. 3 to 108 of SEQ ID NO 3 and of amino acids no. 4 to 123 of SEQ ID NO 4;
or of amino acids no. 3 to 120 and no. 140 to 246 of SEQ ID NO 5.
23. The recombinant binding protein of claim 21, which protein has
10 the amino acid sequence SEQ ID NO 1 and SEQ ID NO 2; SEQ ID NO 3 and
SEQ ID NO 4; or SEQ ID NO 5.
24. DNA, which encodes a recombinant binding protein of claim 21,
22 or 23.
25. Host cell, which expresses a recombinant binding protein of
15 claim 21, 22 or 23.

The invention is directed to a non-competitive immunoas-
say for small analytes, wherein the analyte is reacted with
5 two binding partners. The first binding partner binds to the
analyte to form a complex between the first binding part-
ner and the analyte. and the second binding partner binds
to the complex formed by the first binding partner and the
analyte. The resulting complex formed between the ana-
10 lyte and the binding partners is detected. The binding part-
ners are proteins, such as antibodies including antibody
fragments. The invention further relates to reagent pairs
and test kits useful in the assays, as well as to the use of
15 Novel reagents and means for their preparation are also
provided.

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Patent Number

216841

Indian Patent Application Number

00870/KOLNP/2005

PG Journal Number

12/2008

Publication Date

21-Mar-2008

Grant Date

19-Mar-2008

Date of Filing

12-May-2005

Name of Patentee

VALTION TEKNILLINEN TUTKIMUSKESKUS

Applicant Address

VUORIMIEHENTIE 5, FI-02150 ESPOO, FINLAND.

Inventors:

#	Inventor's Name	Inventor's Address
1	HÖYHTYÄ MATTI	CALONIUKSENKATU 9 C 54 FI-00100 HELSINKI, FINLAND.
2	TAKKINEN KRISTIINA	HALTILANTIE 12 AS 1 FI-02200 ESPOO, FINLAND.
3	PULLI TIMO	MYLLYPELLONPOLKU 3B 28 FI-00650 HELSINKI, FINLAND.
4	SÖDERLUND HANS	SALONKITIE 19 FI-02940 ESPOO, FINLAND.

PCT International Classification Number

C07K16/44

PCT International Application Number

PCT/FI2003/000875

PCT International Filing date

2003-11-17

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	20022048	2002-11-18	Finland