ASSAY FOR CHAGAS' DISEASE AND REAGENTS FOR ITS USE

Abstract: An assay to confirm the presence of antibodies to (T. cruzi) in a test sample. The assay comprises detecting the presence of antibody to three (T. cruzi) antigens, Gp90, Gp60/50 and LPPG in a test sample. The presence of antibody in the test sample to at least two of three (T. cruzi) antibodies is indicative of a confirmed reactive sample. Also provided are diagnostic reagents for detection of (T. cruzi), a process for purifying GP60/50, a process for linking a protein and LPPG, and diagnostic test kits for use when assaying for antibodies to (T. cruzi).

Description: ASSAY FOR CHAGAS' DISEASE AND REAGENTS FOR ITS USE Background of the Invention This invention relates generally to the detection of parenterally transmitted diseases and their detection, and more particularly, relates to Trypanosoma cruzi which is the causative agent of Chagas' Disease, and assays for its detection in test samples. The protozoan parasite Trypanosoma cruzi is the causative agent responsible for a disease known as Chagas' Disease or as American Trypanosomiasis. The geographical range of this disease in the Americas is as far north as California and Maryland and as far south as regions in Argentina and Chile. It has been estimated that 90 million people are at risk, and that an additional 12 to 63 million individuals are infected with this parasite. See, for example, G. A. Schmunis, Transfusion 31:547- 557 (1991 ;: Anonymous. WHO Technical Report Series 1991 811:1-93 (19911: and S. Kingman, New Scientist 132:16-17 (1991). The first indigenous case of Chagas' Disease in North America was reported in 1955. N. C. Woody et al., JAMA 159:676- 677 (1955); R. J. Schiffler et al., JAMA 251:2983-84 (1984); J. D. Pearlman, A , I Med. 75:10571060 (1983); T. R. Navin. Am. J. Public Health 75:366-369 (1985); and Anonymous. Texas Health Bull. 159:111-13 (1955). Chagas 1 Disease is transmissible through blood products; the transmission of Chagas' Disease through blood transfusion in the United States has been recognized for some time. Two recent serological surveys in the Washington, D. C. area have indicated that several individuals residing in that area, originally from El Salvador and Nicaragua, had sera reactive for Chagas' Disease. L. V. Kirchhoff et al., JAMA 254:3058-3060 (1985); and L. V. Kirchhoff et al., Am. J. Med. 82:915-920 (1987). It has been estimated from this survey that there are up to 100,000 individuals living in the United States who are chronically infected with T. cruzi. A. Skolnick, JAMA 265:173 (1991). In a separate report, of 1027 consecutive blood donations in Los Angeles County during a three-month period which were screened by a complement fixation test for Chagas' Disease, there were ten initial reactives and one confirmed case. P. Kerndt et al., Transfusion 28:3 IS Abstract sl08 (1988) and P. Kerndt et al., Transfusion 31:814-818 (1991). Imported Chagas' Disease has been recognized in Europe as well, with a documented case of congenital T. cruzi infection reported in Sweden. P. O. Pehrson et al.. Scand. J. Infect. Pis. 13:307-308 (1981). Trypanosoma cruzi has one of the most complex life cycles of trypanosomes found in man. Trypomastigotes circulate in the blood of vertebrate hosts and are transmitted by blood-sucking triatomid insects. The disease also can be spread by blood transfusion, through intravenous drug use, by congenital transmission, by sexual activity, by organ transplant or through breast milk. See, for example, A. Skolnick, JAMA 262: 1433 (1989); P. Nickerson et al.. Ann. Intern. Med. 111:851- 853 (1989); Anonymous, Clinica 354:16 (1989); L. V. Kirchhoff. Ann. Intern. Med. 111:773-775 (1990); G. Bonfim et al.. ISBT/AABB Joint Congress. Abstract S445, 112 (Nov. 10-15,1989); P. Kerndt et al.. Transfusion 28:S108 (1988); I. H. Grant et al.. Ann. Intern. Med. 111:849-851(19891: M. Boxaca et al.. Int Conf. AIDS 6:437 (Abstract 3141) (1991); S. G. Sandier. Am. Red Cross Blood Services Letters 89:1- 10 (1989); A. L. Bittencourt. Am. J. Pis. Child 130:97-103 (1976); R. Hoff et al., Trans. R. Soc. Trop. Med. Hyg. 72:247-250 (1978); M. D. Gudino et al., in Emerging Global Patterns in Transfusion-Transmitted Infections. R G. Westphal et al., eds., Arlington, VA, American Association of Blood Banks, 65-86 (1990); I. G. Kagan et al.. Rev. Biol. Trop. 14:55-73 (1966); J. C. P. Dias et al.. Mem. Inst. Oswaldo Cruz 79:139-147 (1984); and J. H. Maguire et al., "American Trypanosomiasis" in Infectious Diseases P. D. Hoepricke et al., eds, Philadelphia: J. P. Lippincott, pp. 1257-1266 (1989). Diagnosis of the disease commonly is accomplished by identification of parasites in the blood, cerebrospinal fluid, fixed tissue or lymph node during periods of high fever; however, the organisms may be difficult to detect during the latent (or so-called indeterminant) phase, or during chronic stages of infection. In xenodiagnosis, the intestinal contents of insect vectors are examined for T. cruzi several weeks after these parasites feed on the blood of a suspected patient. However, this procedure is laborious and lacks sensitivity. E. L. Segura, "Xenodiagnosis" in Chagas' Disease Vectors. R R. Brenner et al., eds., 11:41-45, Boca Ratan, FL, CRC Press (1987) Several different serologic methodologies have been used to diagnose Chagas' Disease. These methodologies include indirect immunofluorescence, indirect hemagglutination, complement fixation and enzyme immunoassay. See, for example, F. Zicker et al., Bull. World Health Organ. 68:465-471 (1990); ME Carmargo, Rev. Inst. Med. Trop. Sao Paulo 8:227-234 (1977); ME Carmargo et al., Bull. Pan Am. Health Organ 19:233-244 (1985); A.A. Pan et al., J. Infect. Pis. (165:585-588 [1992]); A. A. Pan et al., Am. J. Trop. Med. Hvg. 45:120 Abstract 66 (1991); A, F. Ferreira et al.. Rev. Inst. Med. Trop. San Paulo 33:123-128 (1991). Specific antibody responses to the parasite are detectable after infection, and the titers typically remain high for life. P. M. Israelski et al., Am. J. Trop. Med. Hyg. 39:445-455 (1988); R. Lelchuck et al.. Clin. Exp. Immunol. 6:547-555 (1970); N. H. Vattuone et al.. Am. J. Trop. Med. Hvg. 76:45-47(1973). The natural transmission of Chagas Pisease in humans occurs when skin or mucosa come in contact with feces of infected bugs. The signs and symptoms are so mild that the recent infection usually is not associated with T. cruzi infection. Even without treatment, the majority of individuals recover from the acute stages of disease. After a latent period of years or decades, a percentage of individuals (20-40%) develo the cardiac or gastrointestinal symptoms that characterize chronic Chagas' Pisease. Persistence of parasitemia in asymptomatic individuals and survival of the parasite in banked blood and blood components increases the dangers of transmission of Chagas' Disease by blood transmission; infected blood used for transfusion in blood banks poses a significant public health problem. A rapid and reliable assay with standardize components for screening and confirming blood donors for Chagas 1 Disease thus would be extremely useful in preventing transfusion of the disease by blood banks. Summary Of The Invention The present invention provides a method which can be utilized as a confirmatory test for the presence of antibodies to T. cruzi in a test sample. The method comprises (a) determining the presence of a first T. cruzi antibody in a test sample, which comprises (i) contacting a first aliquot of a test sample with a first T. cruzi antigen attached to a solid support to form a mixture and incubating same to form first antigen/antibody complexes, (ii) contacting said first antigen/antibody complexes with an indicator reagent for a time and under conditions sufficient to form first antigen/antibody /indicator reagent complexes, and (iϋ) detecting the presence of the first T. cruzi antibody by measuring the signal generated; (b) determining the presence of a second T. cruzi antibody in a test sample, which comprises (i) contacting a secon aliquot of a test sample with a second T. cruzi antigen attached to a solid support to form a mixture and incubating same to form second antigen/antibody complexes, (ϋ) contacting said second antigen antibody complexes with an indicator reagent for a time and under conditions sufficient to form second antigen/antibody /indicator reagent complexes, and (iii) detecting the presence of the second T. cruzi antibody by measuring the signal generated; and (c) determining the presence of a third T. cruzi antibody in a test sample, which comprises (i) contacting a third aliquot of a test sample with a third T. cruzi antigen attached to a solid support to form a mixture and incubating same to form third antigen antibody complexes, (ϋ) contacting said third antigen antibody complexes with an indicator reagent for a time and under conditions sufficient to form third antigen antibody /indicator reagent complexes and (iϋ) detecting the presence of the third T. cruzi antibody by measuring the signal generate The presence of at least two specific antibodies confirms the presence of T. cruzi antibody in the test sample. T. cruzi antigens for use in the assay include Gp90, Gp 60/50 and LPPG. The antigens are used with the proviso that the antigen used as the first antigen is not used for the second antigen in step (b) or the third antigen in step (c), the antigen used as the second antigen is not used for the first antigen in step (a) the third antigen in step (c), and the antigen used as the third antigen is not used for t first antigen in step (a) or the second antigen in step (b). The indicator reagent used i the assay comprises a label which is selected from the group consisting of a chromogen, a catalyst, a luminescent compound, a chemiluminescent compound, a radioactive element, and a direct visual label. The present invention also provides diagnostic reagents for use in a method f detection of L cruzi antibody. These reagents comprise Gp90, Gp60/50 and LPPG. Also provided are diagnostic test kits for confirmatory tests which include Gp 90, Gp60/50 and LPPG. In addition, processes for purifying the Gp60/50 and the LPPG antigens are provided. The process for purifying the Gp60/50 antigen of T. cruzi. comprises (a) isolating the membrane of the epimastigote stage of T. cruzi by dounce homogenization; (b) extracting the Gp60/50 antigen; (c) applying the resultant extract of step (b) to a Galanthus nivalis lectin affinity column and eluting with a carbohydrate; and (d) purifying the eluate with an affinity column comprising a monoclonal antibody specific for Gp60/50. The extraction step preferably is performed with a non-ionic detergent Further, the present invention provides a process for linking an antigenic glycoϋpid of T. cruzi to a protein carrier, which comprises (a) obtaining the glycoUpi hpophosphonopeptidoglycan (LPPG) from the epimastogote stage of T. cruzi: (b) unking LPPG of step (a) with a protein using ethyldimethyl-amino-propyl- carbodiimide (ED AC) by (i) contacting LPPG with ED AC to form a resultant mixtu and incubating said mixture for a time and under conditions sufficient to activate the mixture, (ii) incubating the activated mixture with the protein for a time and under conditions sufficient to link the LPPG with the protein; (c) purifying the mixture; an (d) eluting the LPPG from the mixture. The protein preferred to be linked is bovine serum albumin (BSA). Further, it is preferred that the purification of step (c) is performed by passing the mixture over blue dextran Sepharose. Brief Description of the Drawings FIGURE 1A is a bar graph wherein the number of sera from a negative population reactive with T. cruzi Gp90 (90kD) is shown along the vertical axis while the Signal to Negative ratio (S/Neg) is shown along the horizontal axis of the graph, wherein the number of sera tested was 289. FIGURE IB is a bar graph wherein the number of sera from a negative population reactive with T. cruzi Gp60/50 is shown along the vertical axis while the Signal to Negative ratio (S/Neg) is shown along the horizontal axis of the graph, wherein the number of sera tested was 289. FIGURE IC is a bar graph wherein the number of sera from a negative population reactive with T. cruzi LPPG is shown along the vertical axis whϋe the Signal to Negative ratio (S/Neg) is shown along the horizontal axis of the graph, wherein the number of sera tested was 289. FIGURES 2 A through 2D show the results of the assay of the invention graphed as a function of Optical Density (OD) versus the dilution factor of the (previously positive) tested sera, wherein a solid circle indicates gp90 bead, an open circle indicates gp60/50 bead, and an open triangle indicates LPPG-BSA bead. FIGURE 2A is a graph of sample C, obtained from San Antonio, Texas; FIGURE 2B is a graph of sample b, obtained from Los Angeles, CA; FIGURE 2C is a graph of a sample from Brazϋ; FIGURE D is a Chagas' Disease EIA Latin American Positive Control (Abbott Laboratories, Abbott Park, IL). FIGURE 3 is a scheme for confirmation of seropositivity for T. cruzi. Detaϋed Description of the Invention An assay for the detection ofT.cruzi antibody analyte in a test sample is provided. The assay preferably is performed as an immunoassay, although the present invention is not limited to immunoreactive assays. Any assay utilizing specific binding members can be performed. A "specific binding member," as used herein, is a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. Therefore, in addition to antigen and antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding member, for example, an analyte-analog. Immunoreactive specific binding members include antigens, antigen fragments; antibodies and antibod fragments, both monoclonal and polyclonal; and complexes thereof, including those formed by recombinant DNA methods. "Analyte," as used herein, is the substance to be detected which may be present in the test sample. The analyte can be any substance for which there exists a naturally occurring specific binding member (such as, an antibody), or for which a specific binding member can be prepared. Thus, an analyte is a substance that can bind to one or more specific binding members in an assay. "Analyte" also includes any antigenic substances, haptens, antibodies, and combinations thereof. As a member of a specific binding pair, the analyte can be detected by means of naturally occurring specific binding partners (pairs) such as the use of intrinsic factor protein in the capture and/or indicator reagents for the determination of vitamin B 12, or the use of a lectin in the capture and/or indicator reagents for the determination of a carbohydrate. The analyte can include a protein, a peptide, an amino acid, a hormone, a steroid, a vitamin, a drug including those administered for therapeutic purposes as well as those administered for ϋϋcit purposes, a bacterium, a virus, and metaboϋtes of or antibodies to any of the above substances. The test sample can be a mammalian biological fluid such as whole blood or whole blood components including red blood cells, white blood cells including lymphocyte or lymphocyte subset preparations, platelets, serum and plasma; ascites; saliva; stools; cerebrospinal fluid; urine; sputum; trachael aspirates and other constituents of the body which may contain or be suspected of containing the analyte(s) of interest The test sample also can be a culture fluid supernatant, or a suspension of cultured cells. Mammals or others whose body fluids can be assayed for T.cruzi antibody analyte according to the present invention include humans and primates, as well as other mammals who are suspected of containing these analytes of interest. The indicator reagent comprises a label conjugated to a specific binding member of each analyte. Each indicator reagent produces a detectable signal at a level relative to the amount of analyte, if any, present in the test sample. In a preferred embodiment each indicator reagent, whϋe comprising a specific binding member of a different analyte, is conjugated to the same signal generating compound (label), whic is capable of generating a detectable signal. In general, the indicator reagent is detected or measured after it is captured on the solid phase material. In the present invention, the total signal generated by the indicator reagent(s) indicates the presence of one or more of the analytes in the test sample. It is contemplated that different signal generating compounds can be utiϋzed in the practice of the present invention. Thus, for example, different fluorescent compounds could be utilized as the signal generating compounds, one for each indicator reagent and detection ,.could be determined by reading at different wavelengths. Or, a short-Uved chemϋuminescent compound such as an acridinium or phenanthridium compound and a long-Uved chemϋuminescent compound such as a dioxetane can be utiϋzed to generate signals at different times for different analytes. Methods which detail the use of two or more chemϋuminescent compounds which are capable of generating signals at different times are the subject matter of International Pubϋcation No. WO92/12255 (U.S. Seria No. 636,038). Acridinium and phenanthridinium compounds are described in E. P.publication no.O 273 115 (U.S. Serial No. 07/271,763). In addition to being either an antigen or an antibody member of a specific binding pair, the specific binding member of the indicator reagent can be a member of any specific binding pair, including either biotin or avidin, a carbohydrate or a lectin, complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor or an enzyme, an enzyme inhibitor or an enzyme, and the like. An immunoreactive specific binding member can be an antibody, an antigen, or an antibody/antigen complex that is capable of binding either to the analyte as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancϋlary specific binding member as in an indirect assay. If an antibody is used, it can be a monoclonal antibody, a polyclonal antibody, an antibody fragment, a recombinant antibody, a mixture thereof, or a mixture of an antibody and other specific binding members. The detaϋs of the preparation of such antibodies and their suitabϋity for us as specific binding members are weϋ known to those in the art The signal generating compound (label) of the indicator reagent is capable of generating a measurable signal detectable by external means. The various signal generating compounds Oabels) contemplated include chromagens; catalysts such as enzymes for example, horseradish peroxidase, alkaϋne phosphatase, and B- galactosidase; luminescent compounds such as fluorescein and rhodamine; chemiluminescent compounds such as acridinium compounds, phenanthridinium compounds and dioxetane compounds; radioactive elements; and direct visual labels. The selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances. A variety of different indicator reagents can be formed by varying either the label or the specific binding member. The capture reagents of the present invention comprise a specific binding member for each of the analytes of interest which are attached to at least one solid phase and which are unlabeled. Although the capture reagent is specific for the analyte as in a sandwich assay, it can be specific for an indicator reagent or analyte in a competitive assay, or for an anciUary specific binding member, which itself is specific for the analyte, as in an indirect assay. The capture reagent can be directly or indirectly bound to a soϋd phase material before the performance of the assay or during the performance of the assay, thereby enabϋng the separation of immobiϋzed complexes from the test sample. This attachment can be achieved, for example, by coating the specific binding member onto the soϋd phases by absorption or covalent coupling. Coating methods, and other known means of attachment are known to those in the art. The specific binding member of the capture reagent can be any molecule capable of specificaUy binding with another molecule. The specific binding member of the capture reagent can be an immunoreactive compound such as an antibody, antigen, or antibody/antigen complex. If an antibody is used, it can be a monoclonal antibody, a polyclonal antibody, an antibody fragment a recombinant antibody, a mixture thereof, or a mixture of an antibody and other specific binding members. The "soϋd phase" is not critical and can be selected by one skiϋed in the art. Thus, latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of wells of reaction trays, glass or siϋcon chips and tanned sheep red blood cells are all suitable examples. Suitable methods for immobiϋzing capture reagents on solid phases include ionic, hydrophobic, covalent interactions and the like. A "soϋd phase", as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The soϋd phase can be chosen for its intrinsic abiϋty to attract and immobilize the capture reagent Alternatively, the soϋd phase can retain an additional receptor which has the abiϋty to attract and immobiϋze the capture reagent. The additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent As yet another alternative, the receptor molecule can be any specific binding member which is immobϋized upon (attached to) the soϋd phase and which has the abiϋty to immobiϋze the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a soϋd phase material before the performance of the assay or during the performance of the assay. The solid phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or sϋicon surface of a test tube, microtiter wel sheet, bead, microparticle, chip, and other configurations known to those of ordinar skϋl in the art. It is contemplated and within the scope of the invention that the soϋd phase also can comprise any suitable porous material with sufficient porosity to allow acces by detection antibodies and a suitable surface affinity to bind antigens. Microporous structures are generaϋy preferred, but materials with gel structure in the hydrated stat may be used as well. Such useful soϋd supports include: natural polymeric carbohydrates and their syntheticaUy modified, crossUnked or substituted derivatives such as agar, agarose, cross-ϋnked alginic acid, substituted and cross-ϋnked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulo esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins a derivatives, including cross-Unked or modified gelatins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers which may be prepared with suitably porous structures, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and othe polymers, such as polyurethanes or polyepoxides; porous inorganic materials such a sulfates or carbonates of alkaline earth metals and magnesium, including barium sulfate, calcium sulfate, calcium carbonate, siϋcates of alkali and all aϋne earth metal aluminum and magnesium; and aluminum or siϋcon oxides or hydrates, such as clay alumina, talc, kaolin, zeoϋte, silica gel, or glass (these materials may be used as filte with the above polymeric materials); and mixtures or copolymers of the above classe such as graft copolymers obtained by initiaϋzing polymerization of synthetic polyme on a pre-existing natural polymer. AU of these materials may be used in suitable shapes, such as films, sheets, or plates, or they may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or fabrics. The porous structure of nitrocellulose has excellent absorption and adsorptio quaϋties for a wide variety of reagents including monoclonal antibodies. Nylon also possesses similar characteristics and also is suitable. It is contemplated that such porous soϋd supports described hereinabove are preferably in the form of sheets of thickness from about 0.01 to 0.5 mm, preferably about 0.1 mm. The pore size may vary within wide limits, and is preferably from about 0.025 to 15 microns, especially from about 0.15 to 15 microns. The surfaces such supports may be activated by chemical processes which cause covalent Unkage o the antigen or antibody to the support. The irreversible binding of the antigen or antibody is obtained, however, in general, by adsorption on the porous material by poorly understood hydrophobic forces. Preferred solid phase materials for flow-through assay devices include filter paper such as a porous fiberglass material or other fiber matrix materials. The thickness of such material is not critical and wϋl be a matter of choice, largely based upon the properties of the sample or analyte being assayed, such as the fluidity of the test sample. To change or enhance the intrinsic charge of the soϋd phase, a charged substance can be coated directly to the material or onto microparticles which then are retained by a solid phase support material. Alternatively, microparticles can serve as the soϋd phase, by being retained in a column or being suspended in the mixture of soluble reagents and test sample, or the particles themselves can be retained and immobUized by a solid phase support material. By "retained and immobiϋzed" is meant that the particles on or in the support material are not capable of substantial movement to positions elsewhere within the support material. The particles can be selected by one skiUed in the art from any suitable type of particulate material and include those composed of polystyrene, polymethylacrylate, polypropylene, latex, polytetrafluoroethylene, polyacrylonitrile, polycarbonate, or similar materials. The size of the particles is not critical, although it is preferred that the average diameter of the particles be smaUer than the average pore size of the support material being used. Thus, embodiments which utilize various other soϋd phases also are contemplated an are within the scope of this invention. For example, ion capture procedures for immobilizing an immobϋizable reaction complex with a negatively charged polymer, described in co-pending U. S. Patent Application Serial No. 150,278 corresponding to EP Pubϋcation No. 0326100, and U. S. Patent Appϋcation Serial No. 375,029 (E PubUcation No. 0406473), can be employed according to the present invention to effect a fast solution-phase immunochemical reaction. An immobiϋzable immune complex is separated from the rest of the reaction mixture by ionic interactions between the negatively charged polyanion/immune complex and the previously treate positively charged porous matrix and detected by using various signal generating systems previously described, including those described in chemiluminescent signal measurements as described in co-pending U.S. Patent Application Serial No. 921,97 corresponding to EPO Pubϋcation No. 0 273,115. Also, the methods of the present invention can be adapted for use in systems which utiϋze microparticle technology including' automated and semi-automated systems wherein the solid phase comprises a microparticle. Such systems include those described in pending U. S. Patent Application 425,651 and U. S. Patent No. 5,089,424, which correspond to published EPO applications Nos. EP O 425 633 and EP 0 424 634, respectively, and U.S. Patent No. 5,006,309. One aspect of the invention is a confirmatory assay for antibody to T. cruzi. the agent of Chagas' Disease. This assay involves coating a first solid phase with a first T. cruzi antigen, coating a second solid phase with a second T. cruzi antigen, and coating a third soϋd phase with a third T. cruzi antigen. An aϋquot of a test sample, previously screened for T. cruzi antibody and repeat reactive in a screening test is placed in contact with each soϋd phase. Thus, an aϋquot of a test sample is separately placed in contact with each solid phase and reacted separately. The T. cruzi antigen(s) preferred for use in this confirmatory assay are Gp90, Gp 60/50 and LPPG-BSA. The resultant mixture is incubated for a time and under conditions sufficient to form antigen/antibody complexes. Then, an indicator reagent specific for each antibody and attached to a label capable of generating a measurable signal is contacted with the antigen/antibody complexes and incubated for a time and under conditions sufficient to form antigen/antibody/indicator reagent complexes. The signal generated from each soϋd phase is determined. The presence of X. cruzi is determined as a function of the signal generated from the soϋd phase indicator reagent The presence of antibody to at least two of the three T. cruzi antigens indicates the confirmed presence of T. cruzi antibody in the test sample. In a second aspect of the present invention, at least one antigen of the three antigens disclosed above is coated on a soϋd support. A test sample is contacted with the soϋd phase and the resultant mixture is incubated for a time and under conditions sufficient to form antigen antibody complexes. Then, an indicator reagent which specificaUy reacts with the specific binding member(s) now attached to the soϋd support and which is capable of generating a measurable signal, is added to the complexes, and the resultant second mixture is incubated for a time and under conditions sufficient to form antigen/antibody/indicator reagent complexes. The signal generated from the solid phase/indicator reagent is measured; a signal generated which is greater than a pre-determined known negative control cut-off value is considered reactive, and thus, indicative of the presence of antibody to T. cruzi. The eukaryotic organism T. cruzi has greater than 30,000 proteins; there is conservation of epitopes between members of the famUy of Trypanosomidae/Kinetoplastida. D. E. Lanar et al.. Mol. Biochem. Parasitol. 3:327- 341 (1981) and S. P. Craig et al., Comp. Biochem. Phvsiol. 95B:657-662 (1990). Further diversity of T. cruzi includes zymodemes or strain variabiUty from one geographic location to the next. Xenodiagnosis must be performed with care to rule out chance of cross-reaction with T. rangeli. a parasite which has similar insect vecto but different locations of metacycϋc trypomastigotes. The antigens used for the abov described assay formats are purified to homogeneity from either the amastigote or epimastigote stages of T. cruzi and subsequently attached (coated) onto a soϋd support. The Gp90 and LPPG antigens previously have been characterized and the Gp90 antigen (s) has been shown to be immunogenic. See, for example, M. Schechter et al., Lancet 2:939-941 (1983); L. V. Kirchhoff et al., J. Inf. Pis. 155:561-564 (1987) and J. O. Previato et al.. J. Biol. Chem. 265:2518-2526 (1990) However, the use of the combination of the three preferred T. cruzi antigens has not been described heretofore for use in a confirmatory or screening assay for antibody t T. cruzi. In the confirmatory assay, if the absorbance of a suspected sample is abov the cutoff value in three-of-three or two-of -three determinations using T. cruzi antigens, it is considered as a confirmed positive sample for the disease. However, i the absorbance is not above the cutoff in any or in only one of three antigens utilized, the test sample is subjected to radioimmunoprecipitation assay ("RIPA"). In RIPA, diagnostic bands exhibit characteristic banding patterns at 32, 34 and 90 kO. The tite of a positive test sample is reflected in 19 and 25 kD banding pattern. Thus, the test sample can faU into three categories: confirmed reactive, indeterminate reactive or negative. The present invention wiU now be described by way of examples, which are meant to iUustrate, but not to limit the spirit and scope of the present invention. EXAMPLES Example 1. Culture of Parasites Stock cultures of T. cruzi epimastigotes (avaϋable from the American Type Culture CoUection, Rockvϋle, MD) were maintained in modified NNN medium (disclosed in C. Pan, AΠL H Trop. Med. Hvg. 17:823-832 (1968) with an overlay UM-55 medium (as disclosed in P. M. Rainey et al., Mol. Biochem. Parasitol. 49:111-118 (1991); A. Pan. Exp. Parasitol. 58:72-80 (1984) at 24°C following the procedures described in S. C. Pan. Bull. World Health Organ. 60:101-107 (1982); M. Rainev et al.. Mol. Biochem. Parasitol. 41 : 111 - 118 ( 1991 ) : and A. A. Pan et al. , J. Immunol. 143:1001-1008 (1989). For experiments, epimastigotes were inoculate into 5 ml of UM-55 medium and passaged once. Five miUiliters of this culture in mi log phase of growth was used to inoculate a spinner flask containing 6 liters (1) of UM-55 medium and then was incubated for seven to ten days at 26°C. Example 2. Production of T. cruzi Membranes Epimastigotes or amastigotes of T. cruzi were grown to log phase, harvested and washed three times with phosphate buffered saline (PBS) (pH 7.4) by centrifugation at 6000 x g. The final peUet of cells was resuspended in lysis buffer (consisting of 20 mM Tris-HCl [pH 7.3], 40 mM NaCl, 10 mM EDTA, 2mM phenylmethyl sulfonyl fluoride ([PMSF] and 1 mM iodoacetamide). The organisms (50 ml packed epimastigotes or 20 ml packed amastigotes) were disrupted either by nitrogen cavitation (1500 psi for 15 minutes on ice) or by dounce homogenization. The lysates were subfractioned by differential centrifugation at 48,000 x g for 30 minutes at 4°C, following the method described by A. A. Pan et al., supra. Example 3. Preparation of T. cruzi Antigens for Assay Use Gp 60/50. The Gp 60/50 glycoprotein of T. cruzi was purified from epimastigotes as foUows. Briefly, the membrane-enriched pellet was solubϋized for two hours at 4°C in Buffer A (consisting of 20 mM Tris-HCl, [pH 7.2]) containing 150 mM NaCl, 2.0 mM iodoacetamide, 1 mM EDTA, 0.5 mM PMSF, 77 μM Aprotonin and two percent (2%) NP-40, and cleared by centrifugation at 48,000 x g for 30 minutes at 4°C. The resultant supernatant fluid was appϋed to a 20 ml Gananthus nivulus (GNA) lectin column (available from E. Y. Laboratories, San Mateo, CA), equiϋbrated in Buffer A which contained 0.5 M NaCl, 0.1% NP-40 and 1 mM EDTA. The column was eluted with 0.3M alpha-methyl pyranoside in the sam buffer. This eluate was applied to a monoclonal anti-Gp 60/50 (A. A. Pan et al., supra) IgG Sepharose column in phosphate-buffered saϋne (PBS, pH 7.2), washed with PBS, and eluted with 50 mM diethylamine as described in M. A. Winkler et al. Proc. Natl. Acad. Sci. 81:30543058 (1984). The antigen was analyzed by ten percent (10%) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (U. K. Laemmli, Nature 227:680-685 [1970]) and subjected to silver- staining (available from Bio-Rad, Richmond, CA), as described in M. A. Winkler et al., supra. This novel process produced a homogeneous glycoprotein (at least 20% carbohydrate). The Gananthus nivulus column isolated the glycoprotein in 1-2% yie from the majority of membrane-extracted proteins. Gp 90. The Gp 90 kD antigen was isolated from axenically grown amastigot membranes using a similar technique as for GP 60/50, with the foϋowing modifications. A lentil lectin Sepharose 4B column (avaϋable from Pharmacia-LKB, Piscataway, NJ) was substituted for the GNA lectin column, and the Buffer A used i the application, washing, and elution of the column contained 50 mM Tris-HCl (pH 7.5),0.5 mM CaC-2, 0.5 mM MnCfc., 0.5 M NaCl and 0.1% NP-40. The eluted material was affinity purified by a monoclonal anti-Gp 90 kD IgG2b (A. A. Pan et al supra) Sepharose 4B column as described above. Protein concentration was measur for the membranes and purified antigens by the Pierce Coomassie Blue G-250 dye binding assay (available from Pierce, Rockford, LL). This antigen also was analyzed by SDS-PAGE, but stained by Coomassie BriUiant Blue R-250. The novel process produced a homogeneous glycoprotein for use in capturing and detecting T. cruzi antibodies. It was determined that about 24 ng gave a high signal in assays. Also, th yield of glycoprotein was at least 300 g per 18 1 of Amastigotes. Example 4. Preparation of Lipophosphonopeptidoglycan (LPPG)Antigen for Assay Use LLPG was isolated from the whole epimastigotes by the procedure of J. O. Previato et al., T. Biol. Chem. 265:2518-2526 (1990). Briefly, epimastigotes were thawed, washed three times in water, and extracted with 45 percent phenol in water. The aqueous phase was lyophiUzed, appϋed to a P-100 gel filtration column in water and the excluded peak lyophiUzed. The powder was extracted with chloroform:methanol:water (10:3:1), dried, then dissolved in water and precipitated with five (5) volumes of methanol at -20°C. LPPG was quantitated by orcinal-surfu acid assay for carbohydrates, following the method of C. A. White et al., "OUgosaccharides" in Carbohydrate analysis. A Practical Approach. M. F. Chaplin e al., eds. Washington DC: IRL Press, pp. 37-54 (1987). This glycolipid coated poo onto the soϋd supports used (polystyrene beads) and therefore, a process was developed for Unking the LPPG antigenic glycolipid from T. cruzi to a protein carrie so that the conjugate could be purified by affinity chromatography on blue dextran a subsequently coated onto soϋd phases for diagnostic assay use. LPPG was linked to bovine serum albumin (BSA) with ethyl diaminopropylcarbodϋmide (ED AC, avaϋab from Sigma Chem. Co., St. Louis, MO), following the methods disclosed in S. Bauminger et al., Methods Enzvmol.70:151-159 (1980) and modified as follows. LPPG was Unked to bovine serum albumin (BSA) with EDAC in a two-step procedure, using a 1:2 mass ratio of LPPG to BSA. In the first step, LPPG was reacted at room temperature with ED AC, and then the activated mixture was reacted overnight with BSA. This material was purified by applying the mixture to a Blue dextran Sepharose (available from Sigma Chem. Co., St. Louis, MO) affinity colum (10 x 1 cm), washing in PBS and eluting the LPPG-BSA conjugate with 0.5 M potassium thiocyanate. Titration of antigen coating (100 ng to 20 μg per bead) showed that 30 ng per 1/4" bead was preferred. The resultant eluate can be diluted and directly coated onto beads. Example 5. Three Bead Confirmatory Enzyme Immunoassay Bead Coating Procedure Each of the above antigens were separately coated onto polystyrene beads (0.625 cm in size), as follows: Polystyrene beads (2000 beads, 0.635 cm, available from Abbott Laboratories, Abbott Park, IL) were washed with isopropanol-water (71:400) for 20 minutes at 22°C. The fluid was removed by aspiration. 400 ml of PBS containing antigen (either 60 μg of LPPG-BSA prepared as in Example 4 or 60ug of Gp60/50 prepared as in Example 3, or 48 μg of Gp90 prepared as in Examp 3) was added to the beads. The beads and antigens were contacted with agitation for hours at 40°C. Following agitation, the fluid was removed, and 400 ml of a blockin solution (3% bovine serum albumin in PBS) was added. The resultant mixture was agitated for 1 hour at 40°C. Then the fluid was removed, and 400 ml of an over-coat solution (5% sucrose and 0.5% gelatin in water) was added, the resultant mixture wa incubated with agitation for 20 minutes at 22°C. The fluid was drained, and the bead were dried at 22°C with nitrogen gas. The coated beads were stored dessicated at 4° Immunoassay Enzyme Immunoassay was performed as follows. 5 μl of a serum sample wa diluted with 200 μl of specimen dϋuent in three separate reaction weUs of a tray, and then incubated with each of three antigen coated beads (prepared as described hereinabove) for one (1) hour at 40°C. The beads then were washed with distiUed water (Abbott Quikwash®, available from Abbott Laboratories, Abbott Park, IL), an incubated with conjugate (goat anti-human IgG [heavy and Ught chain] conjugated to horseradish peroxidase [available from Kierkegaard & Perry, Gaithersburg, MD]) fo thirty (30) minutes at 40°C. The beads then were washed, transferred to tubes and incubated with 300 μl of OPD tablet (Abbott Laboratories, Abbott Park, IL) dissolve in OPD diluent (comprising 50 mM citrate-phosphate containing 0.02% H2O2) for thirty (30) minutes at room temperature. The reaction was stopped by adding 1 ml o 1 N H2SO4, and the absorbance was analyzed using an Abbott Quantum® spectrophotometer at 492 nm. In all experiments, a negative control (recalcified human plasma) and a positive control (inactivated human plasma, positive for antibody to T. cruzi) were included and assayed in triplicate. The cutoff value was determined from inspection of the sample absorbances in a negative population (SE Wisconsin; N=289). The cutoff value (S/N) was 3.3 for LPPG; 3.5 for Gp60/50; and 3.5 for Gp 90. The presence or absence of antibody to T. cruzi in an unknown sample was determined by relating the S/N to the cutoff value. Example 6. Radioimmunoprecipitation (RTPA) The epimastigote membrane enriched fraction obtained from Gp 60/50 purification was solubilized in 10 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM iodoacetamide, and 2.0% NP-40. The resultant mixture was equiϋbrated for one hour at 4°C, and the particulate material was separated from the soluble membrane protein by centrifugation at 20,000 x g for thirty (30) minutes at 4°C. The resultant material was radiolabeled with Na 125l by chloramine-T method as described by W. M. Hunter et al., Nature 194:495-496 (1962). The labeled antigen was pre-absorbed with normal human serum-coated protein A Sepharose C1-4B for one (1) hour at 4°C, and then it was centrifuged at 1000 x g for ten (10) minutes. The unbound material (at 10^ cpm in a total volume of 20 to 25 μl) was incubated with 10 μl of the test serum overnight on ice. A 50 μl aUquot of 50% protein-A Sepharose CL-4B (Sigma Chemical Co., St. Louis, MO) suspension in PBS was added to the mixture and vortexed for 30 minutes at 4°C. The sample then was washed three (3) times in PBS containing 1% NP-40 and once in PBS containing 1% NP-40 and 0.05% sodium dodecyl sulfate (SDS). The sample was boϋed for 5 minutes in SDS- loading buffer (2.3% SDS, 10% glycerol, 62.5 mM Tris-HCl, pH 6.8), centrifuged (12,500 x g for 5 minutes) then the supernatant fluid was removed for analysis. The SDS-polyacrylamide-gel-electrophoresis was carried out according to the procedure of Laemmϋ (Nature 227:680-685 [1970]) in a 12.5% polyacrylamide gel. Autoradiography was conducted for 7-10 days at -70°C with X-ray film (Kodak XAR 5) and Lightnight Plus intensifying screen (E. I. DuPont de Nemours & Co., Wilmington, DE). Example 7. Comparative Testing Samples Xenodiagnosis-positive samples were obtained from Institute Fatala Chaben, Buenos Aires, Argentina. Malaria sera were obtained from Africa (the Gambia) and India (Madras, India). African leishmaniasis sera were obtained from Sudan; schistosomiasis sera were obtained from Brazil. Negative samples, obtained from a "low risk" area of the United States (Milwaukee, WI), systemic lupus erythematosus (SLE) (Milwaukee, WI) and syphilis sera (Detroit MI) also were assayed with the confirmatory assay of the invention. Dilution Panel of Positive Serum Several sera positive for antibody to T. cruzi. as determined by the confirmatory assay of the invention set forth in Example 5, were dϋuted appropriately to produce a dϋution panel for determination of sensitivity. The positive control (human plasma heat inactivated at 56°C) was dϋuted into normal human plasma to giv an absorbance at 492 nm of 0.500 to 1.999, and was further dϋuted to 1:22 and 1:100. Several other positive samples were similarly dϋuted to 1:4 (high positive); 1: (medium positive); 1:9 (borderline positive); 1:13.5 (low positive); and 1:22 (non- reactive positive). Positive and negative controls also were tested (Abbott Laboratories, Abbott Park, IL). All sera were analyzed by the confirmatory assay of the invention and RIPA as described above. Evaluation A study was performed to evaluate the confirmatory assay of the invention an RIPA. Samples previously tested with a screening assay (Chagas Antibody EIA for Chagas' Disease, Abbott Laboratories, Abbott Park, IL) were used in the evaluation. These samples were obtained from eight field sites selected from the southwestern United States; the samples were unlinked except for classification at each specific location into Hispanic or non-Hispanic surname groups. Specimens were identified only by number for testing, the assays were performed, and the assay results were decoded at a later date. Total number of samples screened was 13,109. These samples were obtained from the foUowing sites (Hispanic/non-Hispanic): Albuquerque, NM (224/224); Houston, TX (986/1326); McAUen, TX (664/259); Sa Antonio, TX (2396/1599); Los Angeles, CA (1050/1050); Sacramento, CA (1899/600); and San Diego (416/416). Samples were S/N greater than 3.0 (N=112) using the screening assay described above and were evaluated by the confirmatory assay of the invention by foUowing the procedure detailed in Example 5 and RIPA according to the procedure of Example 6. Results 1. Accuracy. The abϋity of the confirmatory assay of the invention to detect antibody to T. cruzi was estabUshed by comparison to consensus positive samples (samples in which there were positive results by both hemagglutination and indirect immunofluorescent antibody assays for Chagas' Disease). The assay of the invention was utilized on these 82 consensus positive samples. Fifty-six (56) samples demonstrated an absorbance value above the cutoff in three of three beads (sensitivity of 68.3% [24/82]). Twenty-four (24) samples were above the cutoff in only two of three antigens (29.3%). Two samples were positive for only one of three antigens; these had to be confirmed by RLPA. As seen in Table 1, the overall performance of the three-bead confirmatory assay was 97.56% (80/82) on consensus positive samples. TABLE 1 Sample Sensitivit Type (%)* ♦ Sensitivity (%) = (No. Chagas-EIA Positive/No. Xenodiagnosis Positive) x 100; ** Consensus Positive are reactive by Hemagglutination and Immunofluorescence assays Two hundred eighty-nine negative samples from a "low risk" region (southeast Wisconsin) also were assayed by the confirmatory EIA. As seen in FIGURE 1A, FIGURE IB and FIGURE IC, the two hundred eighty-four samples were below the cutoffs in at least two of the three kinds of antigen-coated beads. Five samples demonstrated an absorbance value (S N) above the cutoff in one of three beads. However, since confirmation is determined by reaction above the cutoff values in at least two of three beads, the overall performance of the confirmatory assay of the invention was 100% (289/289). Confirmatory EIA of Xenodiagnosis- Positive Samples A sample positive by xenodiagnosis represented a sample in which there was a greater degree of certainty as to an individual having Chagas' Disease and thus, antibody to T. cruzi. Table 1 summarizes the data on the confirmatory assay of the invention on these samples. As the data from the Table 1 demonstrate, all samples showed an absorbance above the cutoff value with three-of-three or two-of-three beads. No samples had to be retested by RIPA. The overaϋ agreement on the confirmatory assay was 100% (28/28) on xenodiagnosis-positive samples. Evaluation of the Three-Bead Confirmatory Assay with a Dilution Panel The assay of the invention was tested with four consensus positive samples dϋuted two- and three-fold from 1:2 to 1:96. The results of these tests are shown in FIGURE 2A, FIGURE 2B, FIGURE 2C and FIGURE 2D. As can be seen in these figures, aU of the sera tested were positive (above the cutoff value in at least two of three antigen beads) by the confirmatory assay of the invention through a dilution of 1:16. The samples were considered consensus positive when tested by the Chagas US Screen EIA ( Abbott Laboratories, Abbott Park, IL), hemagglutination and indirec immunofluorescent antibody. All samples were dϋuted two- or three-fold to a dϋution of 1:96. Cross-Reactivity Specimens from patients with other parasitic diseases were tested with the confirmatory assay of the invention. As the data in Table 1 demonstrate, absorbances were below the cutoff value, which indicated no cross-reactivity in the assay. Evaluation of Three-Bead Confirmatory Assay with Samples from the U.S. Prevalence Study In an in-house evaluation, 13,109 blood samples donated in southwestern areas of the U.S. were screened with Abbott Chagas Antibody EIA (Abbott Laboratories, Abbott Park, IL). All samples with signal-to-negative (S/N) greater than 3.0 were assayed by the confirmatory assay of the invention as detaϋed in Example 5. Thirty-four (34) samples in the screening assay were "RR" (repeat reactive). As seen in Table 2, nine (9) samples were positive in at least two-of-three beads, two samples were positive in one-of-three beads, and two samples were negative in all three beads. Four samples (two negative in all three beads and two positives in only one bead) were indeterminate and required additional analysis by RJPA. TABLE 2 Confirmatory Assay of the Invention Tested with Positive Sera Positive Sample Type Xenodiagnosis Consensus* In-House Study * Samples in which Hemagglutination and Immunofluorescence Assays were positive in both tests + Samples to be confirmed by RJPA RIPA Figure 3 represents the results of a RIPA of ^--^l-labeled solubiϋzed membrane extract of epimastigotes of T. cruzi with the dilution panel of positive sera for antibody to T. cruzi. Nine (9) major proteins were revealed under reducing conditions; these bands had molecular weights (Mr) of 19, 25, 28, 32, 34„ 44, 58, 69 and 90 kD (kϋodaltons). There appeared to be two (2) bands of Mr 32 and 34 kD which were strongly precipitated, while the 90 kD band was of medium intensity; these three bands appeared to be the most diagnostic proteins. The 19 and 25 kD bands appeared to be dependent on the titer of serum, being present in the positive control 1 :22, 1:100,1:4 (high positive); and the screening test's positive control (previously described above). The 19 and 25 kD bands were absent in the 1:6 (medium positive); 1:9 (borderline positive); 1:13.5 0ow positive); 1:22 (non-reactive positive); and the negative control for the Chagas' Disease screening assay previously described herein. The bands which immunoprecipitated at 28, 44, 58 and 69 kD appeared in various negative samples tested and were non-specific. As demonstrated, the 32, 34 and 90 kD bands appeared in all of the dϋutions tested. Comparison of RJPA to Xenodiagnosis-Positive Samples AU 28 xenodiagnosis-positive samples were analyzed by RJPA. All samples showed the characteristic diagnostic bands at 32, 34 and 90 kD. Several of these sera also demonstrated high titer bands at 19 and 25 kD. The overaU agreement on the RIPA was 100% (28/28) on xenodiagnosis-positive samples. RIPA of Samples from U.S. Prevalence Study To demonstrate the utility of RIPA, 122 samples from the U.S. prevalence study described above with signal/negative (S/N) greater than 3.0 were assayed as follows. Samples with S/N from 3.0 to 5.0 (N=100) were negative in RIPA. As Table 3 below demonstrates, of the 22 samples having S/N greater than 5.0,13 samples were confirmed positive. Four of the five indeterminate samples from the confirmatory assay confirmed positive, and one sample remained indeterminate. Seven (7) samples demonstrated bands at 19 and 25 kD, which indicated high titer sera. TABLE 3 Com rison of the 3-Bead As a V rs RJPA As shown by the data presented above, both the confirmatory assay of the present invention and RTPA had a cUnical sensitivity of 100% when tested against xenodiagnosis-positive sera. When tested with consensus positive sera, the confirmatory assay of the invention had a sensitivity of 97.56% (80/82). The remaining 2.4% (2/82) showed reactivity with one of the three beads. When assayed against negative samples from a "low risk" region of southeast Wisconsin, the confirmatory assay of the invention had a specificity of >99.99%. The proposed scheme for confirmation is shown in FIGURE 3. Thus, it can be seen that test samples reactive with two-of-three or three-of-three T. cruzi antigens is considered a confirmed reactive seropositive sample. If the absorbance of a sample is only above the cutoff value in one of the three antigens, or the sample is not above a cutoff with aU three antigens, the sample is considered as negative, or it is subjected to RJPA. In RIP A, test samples are confirmed reactive when three-of-three or two-of-three diagnostic bands precipitate. Samples immunoprecipitating only one band are considered indeterminant, and samples which do not immunoprecipitate any bands are considered negative. The confirmatory assay of the invention provides a means of reducing the number of samples which need to be assayed by RJPA. This is beneficia because RJPA is considered a very time-consuming and labor-intensive procedure when compared to the assay of the present invention: the confirmatory assay of the invention requires approximately two hours to perform, whϋe a RJPA may take upwards of ten days for a result. RIPA previously has been used to confirm the presence of T. cruzi antibody, wherein a protein extract of surface radiolabeled parasites was used when testing pedigreed reactive samples. Protein bands at 72 and 90 kD in RIPA appeared to be sensitive and specific. These antigens appeared to be highly conserved across a wide geographical selection of strains of the parasite. However, RJPA has practical limitations in that radioisotopes with short half-life are used, the technique is time-consuming, and the technique is not readϋy adaptable to large scale screening. R C. K. Wong et al., Trans. R. Soc. Trop. Med. Hyg. 80:275 281(1986). It is contemplated that the assay of the invention can be optimized even furthe by varying assay conditions and or incubation times, using various combinations of antigen or antibody capture or probe reagents, and other methods, reagents and conditions known to those skiϋed in the art. The variance of the antibody capture reagent may then require the use of a different antigen capture reagent AU these variations are contemplated to be within the scope of this invention. Also, whϋe som of the assays described in the examples used an automated system, it is weϋ within th scope of the present invention that manual methods or other automated analyzers can be used or adapted to the assay of the present invention. Therefore, the present invention is meant to be limited only by the appended claims.

Claims: WHAT IS CLAIMED IS: 1. A confirmatory assay for the presence of T. cruzi antibody in a test sample, comprising: a. determining the presence of a first £ cruzi antibody in a test sample, comprising: i. contacting a first aϋquot of a test sample with a first T. cruzi antigen attached to a soϋd support to form a mixture and incubating same to form first antigen/antibody complexes; ϋ. contacting said first antigen/antibody complexes with an indicator reagent for a time and under conditions sufficient to form first antigen/antibody /indicator reagent complexes; ϋi. detecting the presence of the first T. cruzi antigen by measuring th signal generated; b. determining the presence of a second £. cruzi antibody in a test sample, comprising: i. contacting a second aliquot of a test sample with a second H cruzi antigen attached to a soϋd support to form a mixture and incubating same to form second antigen/antibody complexes; ϋ. contacting said second antigen/antibody complexes with an indicat reagent for a time and under conditions sufficient to form second antigen/antibody /indicator reagent complexes; ϋi. detecting the presence of the second T. cruzi antigen by measuring the signal generated; c. determining the presence of a third T. cruzi antibody in a test sample, comprising: i. contacting a third aϋquot of a test sample with a third T. cruzi antigen attached to a soϋd support to form a mixture and incubating same to form third antigen/antibody complexes; ϋ. contacting said third antigen/antibody complexes with an indicator reagent for a time and under conditions sufficient to form thϋrd antigen/antibody /indicator reagent complexes; ϋi. detecting the presence of the third J_. cruzi antibody by measuring the sign generated; wherein the presence of at least two antibodies confirms the presence of T. crjjzi antibody in the test sample. 2. The assay of claim 1 wherein the first T. cruzi antigen is selected from the group consisting of Gp90, Gp 60/50 and LPPG, wherein the antigen used as the first antigen is not used for the second antigen in step (b) or the third antigen in step (c). 3. The assay of claim 2 wherein the second T. cruzi antigen is selected from the group consisting of Gp90, Gp 60/50 and LPPG, wherein the antigen used as the second antigen is not used for the first antigen in step (a) or the third antigen in step (c). 4. The assay of claim 3 wherein the third T. cruzi antigen is selected from the group consisting of Gp90, Gp 60/50 and LPPG, wherein the antigen used as the third antigen is not used for the first antigen in step (a) or the second antigen in step (b). 5. The assay of claim 1 wherein said indicator reagent of step (a), step (b) and step (c) comprises a label which selected from the group consisting of a chromogen, a catalyst, a luminescent compound, a chemiluminescent compound, a radioactive element and a direct visual label. 6. The assay of claim 1 wherein the test sample is pre-tested and determined to be reactive prior to performing step (a), (b) or (c). 7. The assay of claim 1 wherein steps (a), (b) and (c) are performed simultaneously. 8. A process for purifying the Gp60/50 antigen of _ cruzi. comprising: a. isolating the membrane of the epimastigote stage of T. cruzi by dounce homogenization; b. extrating the Gp60/50 antigen; c applying the resultant extract of step (b) to a Galanthus nivaϋs lectin affinity column and eluting with a carbohydrate; d. purifying the eluate with an affinity column comprising a monoclonal antibody specific for Gp60/50. 9. The process of claim 8 wherein extraction of step (b) is performed wit a non-ionic detergent. 10. The Gp60/50 antigen according to the process of claim 8. 11. A process for linking an antigenic glycolipid of T. cruzi to a protein carrier, comprising: a. obtaining the glycoϋpid Upophosphonopeptidoglycan (LPPG) from the epimastogote stage of T. cruzi: b. Unking LPPG of step (a) with a protein using ethyldimethylamino-propyl- carbodϋmide (ED AC) by: i. contacting LPPG with ED AC to form a resultant mixture and incubating said mixture for a time and under conditions sufficient to activate the mixture; ii. incubating the activated mixture with the protein for a time and und conditions sufficient to Unk the LPPG with the protein; c. purifying the mixture; d. eluting the LPPG from the mixture. 12. The process of claim 11 wherein the protein is bovine serum albumin (BSA). 13. The process of claim 11 wherein purification of step (c) is performed by passing the mixture over blue dextran Sepharose. 14. The LPPG according to the process of claim 11. 15. A diagnostic reagent for use in an assay for antibody to T. cruzi. wherein said reagent is an antigen selected from the group consisting of Gp90, Gp60/50 and LPPG. 16. A diagnostic kit useful for detection of antibody to H cruzi. comprising: container containing at least one T, cruzi antigen selected from the group consisting o Gp90, Gp60/50 and LPPG. 17. The diagnostic kit of claim 16 wherein said antigen is attached to a soϋd phase. 18. The diagnostic kit of claim 17 wherein said LPPG antigen is Unked to a protein.

Inventors: PAN ALFRED A, WINKLER MARTIN A, PAN, ALFRED, A., WINKLER, MARTIN, A.
Applicants: ABBOTT LAB, ABBOTT LABORATORIES
Application year: 1993
Main classification: G01N33
IPC codes: C07K1, C07K14, C07K16, C07K19, G01N33