Title of Invention

TETRA MALARIA STIX

Abstract

The present invention relates to Tetra Malaria strip (Fig 1) for analysis of the presence of all Plasmodium species of Malaria in a test sample, said strip comprising : a plurality of lines positioned at the intervals along the porous length of the said strip wherein the first line (Fig 3b) is impregnated with a monoclonal or polyclonal antibody against HRP-2, the second line (Fig 3c) is impregnated with a monoclonal or polyclonal antibody against falciparum specific pLDH, the third line (Fig 3d) is impregnated with a monoclonal or polyclonal antibody against vivax specific pLDH and the fourth line ( fig 3f) is impregnated with a monoclonal or polyclonal antibody against Pan specific pLDH and capable of immobilization against solvent transport by the sample, and a control line (fig 2& fig 3a) positioned upstream of the said line coated with goat antibody where by, said porous strip when dipped in to said chromatographic solvent, the relative mobility of the said test sample components containing complex facilitates migration of the said complex by capillary action and meeting the line of the corresponding immobilized antibody resulting in tapping of the said complex forming a pink purple band.

Full Text

FIELD OF THE INVENTION This invention relates to a Tetra Malaria Stix , a visual, rapid and sensitive immunoassay for the qualitative differential diagnos of P.falciparum and P.vivax from other two species P.malaria and P.ovale for testing of malaria. It is known that malaria is a serious; sometimes fatal, parasitic disease characterized by fever, chills, and anemia and is caused by a parasite that is transmitted from one human to another by the bite of infected Anopheles mosquitoes. The disease occurs in more than 90 countries world wide, and it is estimated that there are over 500 million clinical cases and 2.7 million malaria-caused deaths per year. It has been found that there are four kinds of parasites that can infect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. Conventionally, to detect whether the patient is infected by any of the above parasites a blood sample is taken from the patient and the disease is diagnosed microscopically by looking for the parasites in a drop of blood. The use of specific binding assays has been found to be of great value in a variety of clinical applications. Various biological fluids and tissue samples can be analyzed for a wide variety of components such as drugs, hormones, enzymes, proteins, antibodies, DNA and RNA fragments and other biological material. Specific binding assays include those assays wherein an analyte is measured which is a member of a specific binding pair consisting of a ligand and a receptor. The ligand and the receptor are related in that the receptor specifically binds to the ligand, being capable of distinguishing the ligand from other sample constituents having similar characteristics. Immunological assays depend on reactions between immunoglobulins (antibodies) which are capable of binding with specific antigenic determinants of various compounds and materials (antigens). Because the results of specific binding reactions are frequently not directly observable, various techniques have been devised for their indirect observation. Specific binding reactions may be observed by labelling of one of the members of the specific binding pair by well known techniques including radiolabelling and the use of chromophores, fluorophores and enzyme labels. Radiolabels, chromophores and fluorophores may be detected by use of radiation detectors, spectrophotometers or the naked eye. Where members of a specific binding pair are tagged with an enzyme label, their presence may be detected by the enzymatic activation of a reaction system wherein a compound such as a dyestuff, is activated to produce a detectable signal. Immunological assays are of three general types. In competitive binding assays, labelled reagents and unlabelied analyte compounds compete for binding sites on a binding material. After an incubation period, unbound materials are washed off and the amount of labelled reagent bound to the site is compared to reference amounts for a determination of the analyte concentration in the sample solution. A second type of immunological assay is known as a sandwich assay and generally involves contacting an analyte sample solution to a surface comprising a first binding material immunologically specific for that analyte. A second solution comprising a labelled binding material of the same type (antigen or antibody) as the first binding material is then added to the assay. The labelled binding material will bind to any analyte which is bound to the first binding material. The assay system is then subjected to a wash step to remove labelled binding material which failed to bind with the analyte and the amount of labelled material remaining is ordinarily proportional to the amount of bound analyte. A third type of immunological assay technique involves agglutination reaction techniques and is exemplified by well-known assays for blood antigens and serum types. Immunological cross-reactivity between antibodies within serum and antigens presented on red blood cell surfaces is indicated by the formation of a three dimensional cross-linked network of red blood cells and antibodies. The agglutination of the serum/red blood cell mixture results in the formation of a pellet which can be visible to the naked eye. These various assay procedures were originally performed according to liquid phase immunochemistry techniques wherein enzyme and radtolabelled reactions were carried out in liquid solution in apparatus such as microtiter plates. More recently, techniques and procedures have been adapted for carrying out "solid" phase assays wherein enzymatic and immunological reactions are carried out in solution on damp porous solid substrates. U.S. Pat. No. 4,168,146 to Grubb, et al., discloses the use of test strips for carrying out assertedly "solid phase" sandwich-type immunoassays. The strips are formed of bibulous carrier materials to which antibodies have been attached by adsorption, absorption or covalent bonding. Preferred test strip materials include cellulose fiber-containing materials such as filter paper, ion exchange paper and chromatographic paper. Also disclosed are uses of materials such as cellulose thin-layer chromatography discs, cellulose acetate discs, starch and three dimensional cross-linked materials such as Sephadex (Pharmacia Fine Chemicals, Uppsala Sweden). Immunoassays are carried out by wetting the test strips with measured amounts of an aqueous solution containing the suspected antigen. Antigen molecules within the test solution migrate by capillary action throughout the test strip, but because the bound antibodies retard the migration of the antigens for which they are specific, the extent of migration of the antigen molecules over a fixed time period is related as a function of the antigen concentration in the test solution. The antigen-containing areas of the diagnostic device are then indicated by the addition of labelled antibodies. European Patent Application No. 164,194 (published Dec. 11, 1985) discloses improvements on the methods of Zuk, et al. in that transported chromatographic materials have substantially the same rate of traversal along the longitudinal edge of the chromatographic strip as along the body of the strip. This allows the chromatographic transport front to remain substantially flat rather than concave. Of interest to the present patent application are two published patent applications. U.S. Pat. No. 4,452,901 discloses the use of porous nitrocellulose supports for immobilization of proteins. It also discloses that such nitrocellulose sheets may be utilized in immunoassay procedures if the residual binding capacities of the nitrocellulose sheets are saturated by blocking treatment with one or more types of proteins, different from those immobilized and not cross-reactive with any of the antibodies subsequently used in the assay. Of further interest to the background of the invention are the disclosures of Gordon, EPO Application 63,810, published Nov. 3, 1982, relating to devices for conducting immunological assays. The devices consist of a porous solid support containing a preselected array of delimited adsorption areas of antigens, antibodies or both, wherein residual adsorption sites on the substrate are saturated by protein blocking agents such as bovine serum albumin which do not cross-react with the antigens or antibodies employed in the assay. The porous supports are disclosed to have sufficient surface porosity to allow access by antibodies and surface affinity suitable for binding antigens. Such supports are disclosed to be selectable from a variety of natural and synthetic polymers and derivatives but are preferably nitrocellulose sheets 0.1 mm thick with pore size between about 0.15 .mu.m and about 15 .mu.m. Antigens or antibodies are applied to the porous solid support by direct contact followed by incubation with blocking agents. Assays for detection of unknown antigens or antibodies are then carried out through use of labelled antibodies which may also be anti immunoglobulin antibodies. Results of single or multiple assays are determined by detection of the labelled antibodies. In addition to the various specific binding assay procedures known in the prior art, there also are known numerous assay procedures involving the diffusive or chromatographic transport of assay reagents. Forgione, U.S. Pat. No. 3,875,014 discloses solid phase test indicators for the determination of concentrations of the enzyme aspartate aminotransferase (AST) in sera utilizing a pair of reactions. In the first reaction AST catalyzes the reaction of L-aspartic acid and alpha ketoglutaratic acid to form oxaloacetate. In the second reaction, oxaloacetate reacts with a diazonium salt to form a colored reaction product. The test indicator of Forgione, comprises a pair of bibulous materials, adhered to each other with an adhesive which is selectively permeable to oxaloacetic acid. The first material is impregnated with the substrates L-aspartic acid and alphaketoglutaric acid. The second material is impregnated with a dried diazonium salt dyestuff. The device is contacted with sera which, if it contains AST, catalyzes the reaction of the substrates to form oxaloacetic acid. Oxaloacetic acid then diffuses through the adhesive barrier to the second strip and activates a color reaction with the diazonium salt which may be compared against standards. Wieland and Determann, J. Chromatog., 28, 2-11 (1967) teaches relating to the use of Sephadex gels employed in a thin-layer chromatography format for separations of proteins. While Sephadex is not known as a conventional thin layer chromatography substrate and conventional thin layer chromatography is not practiced for the separation of proteins, highly cross-linked particles of Sephadex G-25 were used in ascending thin layer chromatography formats but a change in manufacture of Sephadex to bead form made ascending chromatography unworkable as the particles would not adhere and cohere satisfactorily. The use of large pore Sephadex types G-100 and G-200 in descending thin layer chromatography formats to the separation of proteins is also being taught. U.S. Pat. No. 4,435,504 discloses a test strip for analysis of analytes such as antigens, antibodies or polynucleotides employs a chromatographic medium and a solvent capable of transporting reagents and/or sample. Reagents are selected and disposed on the medium such that a labeled (first) reagent arrives at the detection (third) zone only after analyte is immobilized there and non-reactive sample componets have been transported beyond the detection zone. This sequential arrival is accomplished by the relative mobility of the reagent or sample; or by the site relationship of the zones. Preferably, the site relationship of the sample (second) zone and the label (first) zone is such that a plurality of pathways guide the sample and labeled reagent and any subsequent reagents to the detection zone in the recited order. US Patent no. 5827681 describes a test kit for the rapid detection and drug sensitivity of malaria is presented. The test kit of the present invention comprises a unique microscope/slide incubation chamber which permits rapid detection of malaria in wet blood samples using a regular transmitted light microscope to detect opaque hemozoin particles. Despite the great advances that have been made with respect to specific binding assay techniques in recent years, there still remain significant opportunities for improvement of these techniques. A particular limitation of current assay techniques is the requirement of numerous addition and wash steps. These steps, required to prevent undesired cross-reactions and remove excess reagents and interfering substances, complicate the procedure and effectively limit the type and level sophistication of analytical procedures that may be carried out. Elimination or reduction of the number of washing and addition steps which must be carried out by technical personnel will not only reduce time and expense of conducting assays and analyzing assay results, but will also reduce the difficulty of automating result analysis. For these reasons, new systems involving solid phase assay devices requiring a minimum number of addition and washing steps are highly desired. The disturbing rate at which malaria parasites are becoming resistant to antimalarial drugs emphasizes the need for a standardized test kit which can be readily used by medical and laboratory personnel to determine the parasite's susceptibility to various drugs. The test device should have substantially good shelf life and is also economical to produce with emphasis to give good results. The object of the present invention is to provide a visual, rapid and sensitive immunoassay device for the qualitative differential detection of P.falciparum and P.vivax for malaria. Accordingly the present invention relates to a diagnostic strip for analysis of an analyte in a test sample, said strip comprising: a plurality of lines positioned at the intervals along the porous length of the said strip wherein the first line is impregnated with a monoclonal or polyclonal antibody against HRP-2, the second line is impregnated with a monoclonal or polyclonal antibody against falciparum specific pLDH, the third line is impregnated with a monoclonal or polyclonal antibody against vivax specific pLDH and the fourth line is impregnated with a monoclonal or polyclonal antibody against Pan specific pLDH and capable of immobilization against solvent transport by the sample, and a control line positioned upstream of the said lines coated with goat anti mouse whereby, said porous strip half when dipped into said chromatographic solvent, the relative mobility of said test sample components containing complex facilitates migration of the said complex by capillary action and meeting the line of the corresponding immobilized antibody resulting in tapping of the said complex forming a pink purple band. SUMMARY OF THE INVENTION The present invention provides novel device for conducting specific binding assay procedure upon blood sample. This device requires a minimum of washing and is useful in carrying out qualitative assays for given sample. In use of test device according to the present invention, a monoclonal antibody is selectively immobilized at a site on a chromatographic strip and a sample is brought into contact with the immobilized antibody and unreacted materials are washed therefrom. Specifically, the devices according to the invention comprise a diagnostic test strip for the detection of presence of P.falciparum and P.vivaxfrom the other P.malaria and P.ovale in the given blood sample and assesses a drug sensitivity upto 99.8% and specificity upto 99.6% . The chromatographic strip has four lines. The lines is impregnated with a monoclonal or polyclonal antibodies and capable of immobilization against solvent transport by the sample. The control line positioned upstream of the second line coated with goat anti mouse. The invention will be described in short with reference to the accompanying drawings BRIEF DESCRIPTION OF THE ACCOMTETRAYING DRAWINGS Fig. 1 shows test device Fig. 2 shows front view plans of the device indicating the lines of antibodies. Fig. 3. Shows front view plans of the device showing the results of the practice of method of invention. DETAILED DESCRIPTION OF THE INVENTION The present relates to solid phase method for conducting specific binding assays upon sample fluid and more specially to the use of chromatographic techniques in conducting such assays. The device of the present invention utilize a chromatographic solvent to sequentially transport analyte containing sample material mixed with assay buffer i.e., colloidal gold conjugated to anti-HRP -2 antibody and anti-pan specific pLDH antibody which is capable of forming a complex with the sample material so as to trap the complex at the corresponding line of antibodies deposited upon the porous strip device forming a colour. The test uses monoclonal anti pf HRP2 antibody( test line Pf-), anti-Pf .specific pLDH antibody (test line Pf-2), monoclonal anti P.vivax specific pLDH antibody ((test line P.v.), monoclonal anti Pan specific pLDH antibody (( test line Pan) immobalised on the nitrocellulose strip. Chromatographic strip materials useful with the present invention include those materials having capillarity and the capacity for chromatographic solvent transport of non-immobilized reagents and reactive sample components by means of a selected chromatographic solvent. The device is essentially made of microporous materials made from nitrocellulose, by which term any nitric acid ester of cellulose is intended. Antibodies useful in conducting ten immunoassays of the present invention include those specifically reactive for the detection of presence of P. falciparum and P. vivax from the other P.malaria and P.ovale. Such antibody are preferably anti HRP-2 and anti pan specific pLDH antibody, which are essentially free of association with antibodies capable of binding with nonanalyte molecules. These antibodies are monoclonal or polyclonal. The antibodies are out sourced and concentration of these lines are 1 mg/ml to 5 mg/ml preferably 3mg/ml, 0.8 mg/ml to 5 mg/ml preferably 1 mg/ml, 2 mg/ml to 10 mg/ml preferably 3mg/ml and 0.5 mg/ml to 5 mg/ml preferably 3mg/ml for antibodies against HRP-2, falciparum specific pLDH, vivax specific pLDH and pan specific pLDH respectively. Fig. 1 shows the diagnostic test strip mainly consisting of a length of porous material and non porous material. The non porous material is shown as printed and is provided for holding the strip during use. The porous material is the chromatographic material preferably made of nitrocellulose and is impregnated with lines of antibodies (not shown) at intervals. Nitrocellulose membrane with pore size of 5µm to 20 µm is laminated on a support which could be made of plastic or cardboard. Fig, 2 shows front view plan of diagnostic strip for analysis of analyte In a sample fluid comprising of analyte and non analyte materials whereby the test device is impregnated with three lines. The first line is impregnated with a monoclonal or antibody and capable of immobilization against solvent transport by the sample. The second line, called the control line .positioned upstream of the second line is coated with goat anti-mouse. The concentration here is 0.05 mg/ml to 1 mg/ml preferably .1 mg/ml. If sample contains any of Plasmodium species, the colloidal gold conjugate on diagnostic test device complexes specific antibodies in the sample. This complex migrates through the device by capillary action. When the complex meets the line of the corresponding immobilized antibody, the complex is tapped forming a pink purple band which confirms a reactive test result. Absence of a coloured band in the test region indicates a negative test result. The gold conjugate used is prepared as follows: To an initial solution of .01% -0.5% w/v gold chloride a boiled sodium citrate solution 1-5% is added . The solution I rapidly stirred and boiled until a pink purple colour is developed. Tetra malarial antibodies are mixed in the buffer solution preferably HEPES, MOPS.TRIS or phosphate may be used. The molarity of buffer can range from 1mM to 50mM and pH 6.5 to 9.0. The antibody concentration preferably is 1 µg/ml to 10 µg/ml of gold colloid solution. The antibody solution is mixed with gold colloid for 1-2 hrs. Any non immune animal serum (mouse/goat/rabbit/sheep) is added o the above solution. The concentration of the serum could be 1 µg/ml to 10 µg/ml of gold colloid solution. The solution is mixed for 2 hr to over night. After this the solution is centrifuged and supernatant is discarded. The pellet is redissolved in a buffer containing stablizers like BSA, gelatin.50 to 500 OD / well preferred .200 OD well is added and dried at 37deg C for 2 hrs. Fig 3. shows the results of the practice of method of invention. As shown in Fig 3 (a) appearance of only one pink coloured line at control region indicates that the sample is non reactive for all Plasmodium species. As shown in Fig 3(b) iwo pink coloured line one each in Pan specific and control region indicates that the sample is reactive for P.malaria and P.ovale. Fig 3(c) appearance of two pink coloured line one each in Pan specific, Pv and control region indicates that the sample is reactive for P. vivax, P.malaria and P.ovale Fig 3(d), 3(e) and 3(f) appearance of four pink coloured line indicates that the sample is reactive for all Plasmodium species. A difference of intensity in colour may occur between the the test line and control line depending on the concentration of pLDH in the sample but does not effect the interpretation of the result. The Tetra Malaria Stix has been tested with positive and negative clinical samples tested by microscopic examination of whole blood . The results obtained are as follows (Table Removed) Precision Within un and between run precision hasve been determined by testing 10 relicates of three specimens : a negative . a low positive and a strong positive The CV(%) of negative . a low positive and strong positive values were within 10% of the time Numerous modifications and variations in practice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing descriptions of preferred embodiments thereof. Consequently, only such limitations should be placed on the invention as appear in the following claims. I claim, 1. A Tetra Malaria Strip for analysis of the presence of all Plasmodium species of Malaria in a test sample, said strip comprising: A porous length A non porous length, A plurality of lines positioned at the intervals along the porous length of the said strip where in the first line is impregnated with a monoclonal or polyclonal antibody against HRP-2, the second line is impregnated with a monoclonal or polyclonal antibody against falciparum specific pLDH, the third line is impregnated with monoclonal or polyclonal antibody against vivax specific pLDH and the fourth line is impregnated with a monoclonal or polyclonal antibody against Pan specific pLDH and capable of immobilization against solvent transport by the sample, and a control line positioned upstream of the said lines coated with goat anti bodies where by, said porous strip when dipped into said chromatographic solvent, the relative mobility of the said test sample components containing complex facilitates migration of the said complex by capillary action. 2. The Tetra Malaria strip as claimed in claim 1 where in the said porous length is made of thin layer chromatographic material. 3. The Tetra Malaria strip as claimed in claim 1 where in said thin layer chromatographic material is preferably nitrocellulosic material. 4. The Tetra Malaria Strip as claimed in claim 1 where in said complex is colloidal gold conjugate complex. 5. The Tetra Malaria stix as claimed in claim 1 wherein concentration of antibody in the first line is between 1-5 mg/ml preferably 3 mg/ml. 6. The Tetra Malaria stix as claimed in claim 1 wherein concentration of antibody in the second line is between .8-5 mg/ml preferably 1 mg/ml. 7. The Tetra Malaria stix as claimed in claim 1 wherein concentration of antibody in the third line is between 2-10 mg/ml preferably 5 mg/ml. 8. The Tetra Malaria stix as claimed in claim 1 wherein concentration of antibody in the fourth line is between .5-5 mg/ml preferably 3 mg/ml. 9. The Tetra Malaria stix such as hereinbefore described with reference to the accompanying drawings.

Documents:

75-DEL-2005-Abstract-(26-09-2008).pdf

75-del-2005-abstract.pdf

75-DEL-2005-Claims-(26-09-2008).pdf

75-del-2005-claims.pdf

75-DEL-2005-Correspondence-Others-(26-09-2008).pdf

75-del-2005-correspondence-others.pdf

75-del-2005-description (complete).pdf

75-del-2005-description (provisional).pdf

75-del-2005-drawings.pdf

75-del-2005-form-1.pdf

75-del-2005-form-13-(18-06-2007).pdf

75-del-2005-form-13-(29-10-2007).pdf

75-del-2005-form-13.pdf

75-del-2005-form-18.pdf

75-DEL-2005-Form-2-(26-09-2008).pdf

75-del-2005-form-2.pdf

75-DEL-2005-Form-26-(26-09-2008).pdf

75-del-2005-form-26.pdf

75-DEL-2005-Form-3-(26-09-2008).pdf

75-del-2005-form-3.pdf

75-del-2005-form-5.pdf

75-del-2005-form-9.pdf

75-del-2005-Petition-137-(18-01-2011).pdf

75-del-2005-petition-138.pdf

75-DEL-2005-Post Grant Opposition-(18-01-2011).pdf

75-del-2005-Post-Grant -(26-11-2010).pdf

75-del-2005-post-grant opposition u-s 25.pdf

75-DEL-2005-Post-Grant Opposition-(03-12-2010)-.pdf

75-DEL-2005-Post-Grant Opposition-(03-12-2010).pdf

75-DEL-2005-Post-Grant Opposition-(09-02-2011)-.pdf

75-DEL-2005-Post-Grant Opposition-(09-02-2011).pdf

75-del-2005-post-grant reply statement.pdf

75-del-2005-post-grant representation.pdf

75-DEL-2005-Post-Grant-Opposition-(04-01-2011).pdf