WO1997005468A2 - A confirmatory serodiagnostic assay for infectious diseases - Google Patents

A confirmatory serodiagnostic assay for infectious diseases Download PDF

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WO1997005468A2
WO1997005468A2 PCT/BR1996/000032 BR9600032W WO9705468A2 WO 1997005468 A2 WO1997005468 A2 WO 1997005468A2 BR 9600032 W BR9600032 W BR 9600032W WO 9705468 A2 WO9705468 A2 WO 9705468A2
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strain
antigen
cruzi
per
solution
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PCT/BR1996/000032
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French (fr)
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WO1997005468A3 (en
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Pereira Rodolfo Mendes
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Pereira Rodolfo Mendes
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention object of this refers to a confirmatory serodiagnostic technique for infectious and/or parasitic disease, including the preparing process of the antigen derived from an infectious and/or parasitic disease-causing microorganism, the process for the preparation of an antigen from T. cruzi. the antigen derived from infectious and/or parisitic disease-causing microorganism, the antigen derived from
  • T. cruzi the composition with an antigen derived from an infectious and/or parasitic disease-causing microorganism
  • the composition with an antigen from T. cruzi the use of the antigen derived from infectious and/or parasitic disease-causing microorganism in serological tests and vaccines, the process for the detection of anti-antigen antibodies from infectious and/or parasitic disease-causing microorganism and a confirmatory serodiagnostic kit for infectious and/or parasitic disease.
  • the enzyme immunoelectrotransfer blot (EITB), or immunoblot, or western blot, has been standardized for application in viral diseases and is used as a confirmatory test for diagnosing infection by HIV (Centers for Disease Control, MM R 38 (1989) 1-7).
  • FIGURE 01 we can observe the serum reactivity of chagasic individuals defined by the frequency of bands showing with different molecular weights produced by the 06 (six) antigens referred to above, fractioned by electrophoresis in SDS-PAGE and obtained by western blot reaction as described hereinafter.
  • FIGURE 1 The preparing process of the antigens eliminates, in some of the antigenic preparations, a large part of the epitopes, this due to all of the 06 antigens having been submitted to the same technique (western blot), under the same conditions, and using the same sera. 2 - All antigens studied except one - antigen (4) - were not able to react with all the antibodies existing in face of all the T. cruzi bands or epitopes.
  • cruzi with formol at 1%, in accordance with the invention, reduces the possibility of crossed reaction of this antigen with sera from individuals with mucocutaneous Leishmaniasis augmenting the specificity of the reaction, which reached 100%, and simultaneously obtaining a sensitivity of 100%.
  • Paul A. Rebers and Richard B. Rimler Carbohydrate Research
  • 1 - Define a process for the production of a 7. cruzi antigen of Y strain or of any other T. cruzi strain, or of other microorganisms, which, due to the characteristics of the process itself, can maintain or preserve the totality of the 7. cruzi epitopes or of other microorganisms which will serve to disclose the totality of the anti-7. cruzi antibodies or any anti-other microorganism antibodies through the western blot technique SDS-PAGE or any other diagnostic technique.
  • 2 Define a confirmatory serodiagnostic technique for the Chagas' disease and for other infectious and/or parasitic diseases, which technique uses the respective and specific antigen obtained through the production process referred to under 1 above.
  • 3 Define the use of the antigen under 1 above as a better immunizing agent, as it is capable of reacting "in vitro" with the entire spectrum of specific antibodies directed against it.
  • the above objectives are designed to eliminate the inconveniences of the technique in the state it is found nowadays.
  • step (b) Take an aliquot of the infectious and/or parasitic disease-causing microorganism cultivated under step (a) above and transfer it to a test tube adding thereto a formol solution of 0.4 to 15% in such a volume as will be sufficient to provide transmitance of 20 to 95% in the Coleman Jr.
  • spectrophotometer in wave length of 660 nm when read in cuvettes of 10/75 or similar, thus obtaining a suspension of homogeneous concentration of microorganisms, and maintaining said suspension from 02 to72 hours under an room temperature and with intermitent stirring, and washing the suspension 02 to 05 times at 500g to 100,000g at 4°C for 05 minutes to 10 hours subsequently in NaCl 0.15 M + phosphate buffer 0.005 to 0.300 M, pH 6.8 to 7.5 (PBS) or other usual buffer as dictated by the technique, thus obtaining a sediment of microorganisms.
  • PBS pH 6.8 to 7.5
  • step (c) Take an aliquot of the microorganisms sediment referred to in step (b) above and prepare a suspension of lxlO 3 to 3xlO n microorganisms per ml; wash said suspension three times consecutively with solution NaCl 0.15 M + TRIS 0.012 to 0.500 M, pH 6.8 to 7.6 (TBS) or other similar buffer, as dictated by the technique, at 500g to 100,000g at 4° C for a time of from 05 minutes to 10 hours, thus obtaining a sediment of microorganisms, and
  • step (b) Take an aliquot of the suspension contained in step (a) above during the exponential phase of growing of the epimastigotes and transfer it to a test tube, adding to it a formol solution at 0.5 to 10% in such a volume as will be sufficient to provide transmittance of 70 to 95% in the Coleman Jr. spectrophotometer or similar, to a wave length of 660 nm when read in 10/75 mm cuvettes, thus obtaining a microorganism suspension of homogeneous concentration and maintaining said suspension for a period of 12 to 24 hours at room temperature and with intermitent stirring, and washing it two times consecutively with NaCl 0.15 M + phosphate buffer 0.005 to 0.300 M. pH 6.8 to 7.5 (PBS) or other usual buffer, as dictated by the technique, at 500g to 3.000g, at 4°C for a period of 06 to 15 minutes, thereby obtaining a sediment of microorganisms.
  • PBS pH 6.8 to 7.5
  • step (c) Take an aliquot of the microorganism sediment in step (b) above and prepare a suspension of 5xl0 7 to lxlO 9 microorganism per ml; wash said suspension three times subsequently with NaCl 0.15 M + TRIS 0.012 to 0.500 M, pH 6.8 to 7.6 (TBS) or with such other usual buffer, as dictated by the technique, of 500g to 3,000g at 4°C for a period of 06 to 15 minutes, thereby obtaining a sediment of microorganisms.
  • TRIS pH 6.8 to 7.6
  • step (d) Resuspend the microorganisms sediment of step (c) above in 0.3 to 1.0 ml of solution TRIS 0.010 to 0.500 M, pH 6.4 to 7.6 containing 0.5 to 6.0% (p/v) SDS or other natural or synthetic anionic, cationic or non-ionic detergent, EDTA solution at 2.0 to 4.0 mM, and bromophenol blue solution at 0.2 to 1.2 ppm.
  • the mixture is to be heated for a period of 1.0 to 10.0 minutes in water bath at 100°C. the T. cruzi antigen or the antigen (4) of EXAMPLE I thereby being obtained and immediately applied to the polyacrylamide gel containing SDS, should this be the case.
  • step (a) cultivation of the protozoa is maintained preferably at a temperature of 28°C for 3 days.
  • formol is preferably added at 1% in such volume as will be sufficient to provide transmittance of 90%, the suspension being maintained at room temperature for 18 hours and being washed two times subsequently with NaCl 0.15 M + phosphate buffer 0.01 M, pH 7.2 (PBS) at 1300g at 4°C for 10 minutes.
  • step (c) a suspension of lxlO 8 microorganism per ml is preferably prepared with washing being carried out in a solution NaCl 0.15 M + TRIS 0.05 M, pH 7.2 at 1300g at 4°C for 10 minutes.
  • step (d) the microorganism sediment is resuspended, preferably in 0.5 ml of solution TRIS 0.100 M. pH 6.8, containing 2.5% (p/v) SDS, EDTA solution at 2.0 mM and bromophenol blue solution at 0.5 ppm.
  • the mixture was heated for 3 minutes in water bath at 100°C and immediately applied to the polyacrylamide gel containing SDS.
  • This invention also refers to the antigen derived from infectious and/or parasitic disease-causing microorganism, said antigen having the following characteristics:
  • the solid support containing antigen can be blocked by a skimmed milk solution.
  • This invention also refers to a 7. cruzi antigen of Y strain or any other 7. cruzi strain, or antigen (4) of EXAMPLE I, with the following characteristics:
  • the solid support containing antigen can be blocked by a skimmed milk solution.
  • composition made of an antigen of infectious and/or parasitic disease-causing microorganism comprising: 0.2 mg/ml to 20.0 mg/ml of a complete solution of proteins of said microorganisms in TRIS 0.005 to 0.300 M, pH6.5 to 7.9 or another usual buffer as a diluting agent, and timerosol 1/1,000 to 1/30,000 and/or another usual conservant used in the technique.
  • the complete solution of proteins of said microorganisms lies within the range of 0.08 mg/ml to 9.00 mg/ml and the composition obtained is used for serodiagnostic tests.
  • Another form prevailing over others is that whereby a complete solution of proteins of said microorganisms within the range of 10.00 mg/ml to 19.00 mg/ml is used employing aluminum hydroxide and or anoiher usual adjuvant as dictated by the technique.
  • the composition thus obtained is used for vaccines.
  • an antigen-based composition i.e., a T. cruzi antigen of Y strain or any other T. cruzi strain, comprising 0.10 mg/ml to 15.00 mg/ml of a complete solution of T. cruzi proteins, as per claim 6, in TRIS 0.005 to 0.300 M, pH 6.5 to 7.9 or other usual buffer as a diluting agent, and timerosol 1 : 1,000 to 1 :30,000 and/or another usual conservant as dictated by the technique.
  • This composition is used for vaccines and for serodiagnostic tests.
  • the composition has 0.12 mg/ml to 7.50 mg/ml of a complete solution of proteins of T. cruzi, Y strain or any other T.
  • the composition has 8.0 mg/ml to 14 mg/ml of a complete solution of proteins of T. cruzi, Y strain or any other 7.
  • This invention also refers to the use of the antigen derived from infecto- contagious and parasitic disease-causing microorganism in serological test, said antigen acting as a reagent for the diagnosis of infectious and/or parasitic diseases through such different serodiagnostic techniques as western blot, ELISA, immunoenzymatic, agglutination, immunnoprecipitation, complement fixation, precipitation, hemagglutination, flocculation and immunofluorescence, and acting also as an immunizing agent against infectious and/or parasitic diseases in vaccines.
  • serodiagnostic techniques as western blot, ELISA, immunoenzymatic, agglutination, immunnoprecipitation, complement fixation, precipitation, hemagglutination, flocculation and immunofluorescence, and acting also as an immunizing agent against infectious and/or parasitic diseases in vaccines.
  • the invention refers also to the use of the antigen derived from T. cruzi, of Y strain or any other T. cruzi strain, causing the Chagas' disease, in serological tests, acting as reagent in the diagnosis of the Chagas' disease through such different techniques as western blot, ELISA, immunoenzymatic, agglutination, immunnoprecipitation, complement fixation, precipitation, hemagglutination, flocculation and immunofluorescence, and acting also as an immunizing reagent for the Chagas' disease in vaccines.
  • Another aspect of this invention is related to the different processes of detection of anti-antigen antibodies of infectious and/or parasitic disease-causing microorganisms, including the Y strain T. cruzi or any other strain T. cruzi in fluids to be tested using such different serological techniques as western blot, ELISA, immunoenzymatic, agglutination, immunnoprecipitation, complement fixation, precipitation, hemagglutination, flocculation and immunofluorescence, and having as one of its reagents the antigen and composition previously mentioned hereinabove.
  • kits for confirmatory serodiagnosis through western blot SDS-PAGE for infectious and/or parasitic diseases with a specific antigen of infectious and/or parasitic disease-causing microorganism containing:
  • the last aspect of the invention is related to a kit similar to the one above, which can be applied for confirmatory diagnosis of Chagas' disease, using the Y strain or other strain T. cruzi antigen absorbed in the nitrocellulose strip in the epimastigote, trypomastigote and amastigote forms.
  • the T. cruzi antigen or antigen (4) was submitted to electrophoresis in polyacrylamide gel in the presence of SDS, as described in Laemmli, U.K., Nature, 227.690-685,1970.
  • the gels were assembled on 14 cm x 16 cm glass plates linked by 1 mm thick spacers.
  • the separation gel was prepared in the concentration of 10% of acrylamide and the mixture, still in liquid form, was immediately applied between two glass plates mounted vertically to a height of 8.5 cm from the lower spacer. Onto this gel a butanol saturated solution was carefully placed and the gel was maintained at room temperature until polymerization occurred, which took place between 30 and 60 minutes.
  • Electrophoresis was run under a constant amperage (25 mA) for approximately 4 hours, that is, until the indicating dye reached the end of the gel. After the run, the plates were carefully separated, the gel removed and the proteins contained in the separation gel fixed and dyed or else transferred to nitrocellulose membranes. Both the fixation and the dyeing were performed during 30 minutes in the solutions: fixing (methanol 50% and acetic acid 7%) and dyeing (amido black 1% in fixing solution). IgG (150 kDa), bovine serum albumin (66 kDa) and ovalbumin (43 kDa) were used as molecular weight markers.
  • the antigenic fractions, separated by western blot SDS-PAGE were transferred by electrophoresis from the gel to nitrocellulose membranes as described in Towbin et alii, Proc. Natl. Acad. Sci. USA, 76: 350-4, 1979 and Bumette, W.N., Anal. Biochem., 112: 195-203, 1981.
  • a transference buffer in the following order: perforated plastic support, foam sheet, 10 (ten) 1 mm filter paper sheets, gel, nitrocellulose membranes, 10 (ten) 1 mm paper filter sheets, foam sheet and perforated plastic support.
  • the assembly was immediately placed in the transference vat already containing the buffer, with the nitrocellulose membrane facing the anode (+). Electrophoresis was run under a constant amperage of 200 mA for 18 hours. The efficiency of the transference was verified through the colouring of part of the membrane in Ponceau-S solution. The antigens present in the remaining part of the membrane are immediately submitted to immunoenzymatic characterization, or they can be stored in vacuum closed plastic bags and preserved at 4°C. The immunoenzymatic analysis ofthe transference is made in accordance with the following steps:
  • Titration of the conjugate is carried out using growing dilutions against the sera used in the standard dilution 1/50. Select the highest dilution of the conjugate which can still reveal all of the bands in a clear cut way with no non-specific antigenic fractions in the presence of normal sera. Said dilution shall be of ⁇ 1:800 for a good conjugate of human anti-IgG peroxidase.
  • the reading criterion for defining the reaction results is: POSITIVE REACTION: - complete profile of 12 bands;
  • the antigen used and the technique employed were the ones mentioned above.
  • the analysis of the data for assessing the diagnosis performance was made by calculating the co-positive and co-negative values, the agreement index, the positive-predictive and negative-predictive values obtained from the double blind study data according to Galeno & Galeno, 1975.
  • the assessment indices of the diagnosis performance ofthe reaction are: 100% co-positivity, 100% co-negativity, agreement index 1, 100% positive predictive value and 100% negative predictive value.
  • the invention in question is intended for the medical area, in particular for the diagnosis, therapy and prophylaxy sectors and for the veterinaty area, particularly the diagnosis, therapy and prophilaxy sectors.
  • the invention in question can also be applicable to the agricultural area.

Abstract

Invention Patent of a confirmatory serodiagnostic assay for infectious diseases. The present invention refers to an antigen to be applied to diagnostic techniques and vaccines. The antigen, derived from Y strain or any other strain T. cruzi of epimastigote, or trypomastigote or amastigote forms, or from any other microorganism, is preserved in formol, fractioned in polyacrylamide gel (SDS-PAGE) and applied to a solid support. The diagnostic technique, derived from the application of same, is presented as confirmatory for the diagnosis of the Chagas' disease and of other human and animal infectious and/or parasitic diseases.

Description

A CONFIRMATORY SERODIAGNOSTIC ASSAY FOR INFECTIOUS DISEASES
Descriptive report on the Invention Patent of "A confirmatory serodiagnostic assay for infectious diseases''.
The invention object of this refers to a confirmatory serodiagnostic technique for infectious and/or parasitic disease, including the preparing process of the antigen derived from an infectious and/or parasitic disease-causing microorganism, the process for the preparation of an antigen from T. cruzi. the antigen derived from infectious and/or parisitic disease-causing microorganism, the antigen derived from
T. cruzi, the composition with an antigen derived from an infectious and/or parasitic disease-causing microorganism, the composition with an antigen from T. cruzi. the use of the antigen derived from infectious and/or parasitic disease-causing microorganism in serological tests and vaccines, the process for the detection of anti-antigen antibodies from infectious and/or parasitic disease-causing microorganism and a confirmatory serodiagnostic kit for infectious and/or parasitic disease.
Various infectious or parasitic diseases are endemic and considered to be a major medical and health problem all over the world, and so is the Chagas' disease in the Americas (Grant, I.H., et al. Ann. Int. Med. I l l: 849-851, 1989; Nickerson.
P.. et al. Ann. Int. Med. I l l: 851-853, 1989; Schmunis, G.A. Transfusion 31: 547- 557, 1991 : Wendel, S.; Gonzaga, A.L. Vox Sang [in press 1992]).
Cronically infected and asymptomatic individuals are potentially capable of transmitting any of these said diseases. Consequently, the urgent need towards developing a technique for a quick, specific and accurate diagnosis for all of them (Kirchhoff, L.V. Ann. Int. Medicine 111: 773-775, 1989; Skolnick, A. JAMA 262: 1433, 1989; Ferreira, A.W., et al. Rev. Inst. Med. Trop. S. Paulo 33: 123-128, 1991: Frasch, A.C.C.; Reyes, M.B. Parasitol Today 6: 137-139, 1990; Pan, A.A., et al. J. Infect. Dis. 165: 585-588, 1992).
Detection of specific antibodies through serological tests is the most commonly used procedure for deteπnining the existence of infection by T. cruzi or for screening Chagas' disease in blood donors, the same applying to other blood transmissible diseases such as hepatitis. AIDS, syphilis and malaria, etc. These tests are in great number, are used successfully, but do also pose a number of problems, the main ones being false positive results, false negative results, doubtful or inconclusive results and such other problems as are related with the stability and reproductibility of said tests. These problems all led to strong debates on the subject (Shulman. I.A. Transfusion 31: 479-480, 1991; Skolnick, A. JAMA 265: 173, 1991).
The clinical or laboratorial diagnosis having been made for the Chagas' disease, there is a need for its immediate confirmation. However, the technical resources currently available, such as demonstration of the parasite by direct microscopic examination or xenodiagnosis, are difficult, time-consuming, have reduced sensitivity and are expensive even in individuals confirmedly infected. The altemative resorted to, one which is also expensive and time-consuming, is that whereby two different techniques are parallelly used for diagnosmg the Chagas' disease, in which case, there will still be sera with a positive or doubtful result in one technique or negative results in both techniques - the so-called doubtful or inconclusive sera. Regarding the Chagas' disease, the incidence of said problem can reach up to or go over 1% of the exams made, depending on the laboratory.
Once the techniques to be used have been defined, there remains the problem of which antigen to use. Nowadays, following the traditional techniques of diagnosis, all antigens, the use of which is possible, are resorted to, such as: highly complex mixes of glycoproteins, carbohydrates and other proteic mixes (Hoshino- Shimizu. S., Rev. Inst. Med. Trop. Sao Paulo, 24: 63-8, 1982), molecules extracted from one-piece or fractioned parasites and specific antigens of T. cruzi only (Kirchhoff. L.V., et al. J. Infect. Dis. 155: 561-564, 1987; Scharfstein, J., et al. J. Immunol. 131: 972-976, 1983; Schechter, M., et al. Lancet ii: 939-941, 1983) and specific antigens of T. cruzi obtained through molecular biology technique (Frasch, A.C.C.; Reyes, M.B. Parasitol. Today 6: 137-39, 1990; Lorca, M.; et al. Am. J. Trop. Med. Hyg. 46: 44-49, 1992; Requena, J.M., et al. Mol. Biochem. Parasitol. 51: 271-280, 1992; Vergara, U.. et al. Am. J. Trop. Med. Hyg. 46: 39-43, 1992). Nowithstanding the type of antigen chosen (these antigens have less and less molecular weight, a pattern following a scientific trend, namely, Reductionism), the problems remain the same, namely, (i) false positive results arising out of the crossed reaction with other antigens of microorganisms such as: Leishmania sp., and T. rangeli (Schmunis, G.A. Transfusion 31: 547-557, 1991; Guhl, F., et al. Parasitol. 94: 475-484, 1987); (ii) non-specific reactions and false negative results (Vergara, U., et al. Am. J. Trop. Med. Hyg. 46: 39-43, 1992; Schmunis, G.A. Transfusion 31: 547-557, 1991 ); (iii) stability and reproductibility (Hoshino- Shimizu, S.; Camargo, M.E.; Shimizu, T.; Nagasse-Sugahara, T.K. Rev. Inst. Med. Trop. S. Paulo 24: 63-68, 1982; Luquetti. A.O. Mem. Inst. Oswaldo Cruz 85: 497- 505, 1990), all of which despite the increasing and high sensitivities obtained from these different techniques. e.g., 93% to 99% (Levin, M.J.; Franco da Silveira, J.; Frasch, A.C.C.; Camargo, M.E.; Lafon, S.; Degrave, W.M.; Rangel-Aldao, R. FEMS Microb. Immunol., 89: 11-21, 1991; Moncayo, A.; Luquetti, A.O., Mem. Inst. Oswaldo Cruz 85: 489-495, 1990).
In brief, there is not as yet an agreement as to which would be the relevant or ideal antigen(s) of T. cruzi to be applied in diagnostic techniques and the best diagnostic technique for Chagas' disease. The same lack of agreement is found when searching for an antigen of T. cruzi for the production of vaccines. And the same applies to which would be the best technique for detecting an antigen of parasite in the blood or tissue, or for detecting a parasite in the blood. These techniques are not sufficiently sensitive to offer conclusive diagnoses, although the definitive confirmation for the infection is the demonstration of the parasite proper in the host (Brofen et al., Mem. Inst. Oswaldo Cruz 84(2): 237-240, 1989, and S. Wendel et al. Chagas' Disease (American Trypanosomiasis): Its Impacts on Transfusion and Clinical Medicine, Sao Paulo: ISBT Brazil '92, Sao Paulo, Brazil year 1992, p. 271).
The enzyme immunoelectrotransfer blot (EITB), or immunoblot, or western blot, has been standardized for application in viral diseases and is used as a confirmatory test for diagnosing infection by HIV (Centers for Disease Control, MM R 38 (1989) 1-7).
In the patents European Patent Application 0397 129 A2, WO-A-8 707 647, WO-A-8 504 903, WO-A-8 900 207 and EP-A-0 314 066, a mixture of different recombining antigens of the HIV itself, with different molecular weights and in large concentrations, is used as an antigen in the western blot reaction for diagnosing the infection by HIV. In these tests a positive result does not necessarily mean the disclosure or presence of all antigens or bands used; two (2) or three (3) of the more specific bands of the virus, among the 5 to 8 shown in the nitrocellulose strips or solid supports are enough as antigens to be tested. Even so, depending on the stage of the disease, this technique, presented as a confirmatory test does not confirm the diagnosis in some sera. A good explanation for using this technique shortcut is that the HIV virus is, antigenically speaking, 300 times smaller than the T. cruzi, which facilitates the execution of said shortcut. For parasitic infections, the western blot is used for the diagnosis (not for confirmatory assay) of cysticercosis (Tsang et al., J. Infect. Dis. 159 (1989) 50-59) and other diseases (Partanem et al., FEBS Lett. 158 (1983) 252-256; Burnie and Mathews, J. Immunol. Meth. 100 ( 1987) 41-47; Tsang and Wilkins, Clin. Lab. MED. 11 (1991) 1029-1039; Gross et al., J. Clin. Microbiol. 30 (1992) 1436-1441; Varastequi et al. J. Clin. Microbiol. 30 (1992) 1557-1561).
Analyses of T. cruzi proteins by western blot have been described but have not been applied to the development of a serological diagnostic test for the Chagas' disease (Araύjo, Infect. Immun. 53 (1986) 179-185 and Chiller et al., Am. J. Trop. Med. Hyg. 43 (1990) 650-656). M.G.M. Teixeira et al.. Trop. Med. Parasitol. 45 (1994) 308-312, developed an EITB test for the diagnosis of the Chagas' disease, which showed sensitivity of 99.3% and specificity of 100%, using a lysate of T. cruzi, Y strain, maintained in a mixture of protease inhibitors and obtained by freezing and thawing the T. cruzi, followed by sonication and centrifuging. In this author's work, the extract used by him does not succeed in having all 16 identified and specific T. cruzi bands reacting with 100% of the sera used, and in order to obtain a positive reactivity pattern he specifies a group of 7 bands with a reactivity of 75%. and considers that a serum is positive when it reacts with at least three (3) bands out of a group of seven (7) bands. With regard to the stability and reproductibility of the western blot technique,
Stolf, A.M.S. and Araύjo, F., in an as yet unpublished work, succeeded in maintaining the reproductibility of the technique for 8 years with T. cruzi antigens fractioned by the western blot technique, SDS-PAGE and fixed in nitrocellulose membranes. To this day all of the 7. cruzi antigens used in different diagnosis techniques have used antigenic preparations which are just one more extraction from or a change to or a purification of a raw antigen from the Chagas' disease-causing parasite, that is, 7. cruzi, in its different forms. That is to say, to this date only the different types or compositions of antigens have been dealt with or deserved any concern.
The author of the present invention - Rodolfo Pereira Mendes - has been studying the serum reactivity from individuals with the T. cruzi parasite using different types of antigen, including those used under the traditional techniques of diagnosis for the Chagas' disease. Data fro this experience have shown that:
1 - All individuals bearing Chagas' disease are likely to produce antibodies against all epitopes or antigenic determinants existing in the T. cruzi, all of which can be revealed by the western blot technique regardless of the stage ofthe disease, age and sex. among other factors, ofthe individual infected. 2 - The antigen likely to reveal all of these antibodies should preferably derive from a one-piece T. cruzi parasite, Y strain, in the epimastigote form, derivation from other strains and other forms of T. cruzi also being possible. 3 - The above antigen will only reveal 100% of the anti-r. cruzi antibodies depending on the process of preparation thereof. 4 - The revelation ofthe overall spectrum ofthe epitopes associated with the use of all of the specific antibodies for T. cruzi produced by chagasic individuals increases the specificity and sensitivity of a reaction and reduces the problems with inconclusive sera, thus rendering such reaction into a confirmatory reaction.
The above observations have been possible only thanks to the study of different preparations ofT. cruzi antigens, namely (see Figure 01):
1 - antigen for the complement-fixation reaction (FC)-(antigen (1)),
2 - antigen for the immunoenzymatic reaction (ELISA)-(antigen (2)), 3 - antigen for the hemagglutination reaction (HA)-(antigen (3)),
4 - antigen for the immunofluorescence reaction (IFI)-(antigen (4)),
5 - antigen for the intradermal reaction (ID)-(antigen (5)), and
6 - raw antigen for control of Y strain epimastigotes without any treatment or preservative (RAW)-(antigen (6)). In FIGURE 01 we can observe the serum reactivity of chagasic individuals defined by the frequency of bands showing with different molecular weights produced by the 06 (six) antigens referred to above, fractioned by electrophoresis in SDS-PAGE and obtained by western blot reaction as described hereinafter. We can thus conclude, observing FIGURE 1, that: 1 - The preparing process of the antigens eliminates, in some of the antigenic preparations, a large part of the epitopes, this due to all of the 06 antigens having been submitted to the same technique (western blot), under the same conditions, and using the same sera. 2 - All antigens studied except one - antigen (4) - were not able to react with all the antibodies existing in face of all the T. cruzi bands or epitopes.
The patents GB 2 000 968 A and FR 78 20 799 of Behringwerke, which use aldehydes and their derivatives to render inactive the trypanossomes, together with the works and patents GB 38.695 of The Wellcome Foundation Limited, US
4,795,709, and European Patent Office 0 040 572 Bl, which use different chemical substances to better fix their antigens in the preparation of extracts for vaccines, do reinforce the findings of Rodolfo Pereira Mendes when he recommends the need for a larger number of epitopes to render the antigens more immunizing, that is, he reinforces the need for a larger number of epitopes for revealing a larger number of specific antibodies, that is, the obtention of a higher sensitivity antigen. The preparing process of the T. cruzi, with formol at 1%, in accordance with the invention, reduces the possibility of crossed reaction of this antigen with sera from individuals with mucocutaneous Leishmaniasis augmenting the specificity of the reaction, which reached 100%, and simultaneously obtaining a sensitivity of 100%. However, Paul A. Rebers and Richard B. Rimler, Carbohydrate Research,
133 (1984) 83-94, upon studying the effect of the formol on the antigenic capacity of the Pasteurella multocida lipopolysacharide (LPS) observed a reduction of the antigenic capacity with the use of formol, the same occurring with H. Ohnuma (Clin. Exp. Immunol. (1986) 67, 709-715), who studied the hepatitis B virus antigens.
The advantages ofthe invention are:
1 - The obtention of an antigen which has preserved all or most of the original epitopes giving rise to it.
2 - The use or disclosure of all specific antibodies against all epitopes preserved under item (1) above, through the diagnostic techniques whereby the antigen has been or will be applied.
3 - An antigen which, due to the production process, shows a mmimum or a quantity as little as possible of epitopes producing false-positive reactions. Example: the average of bands shown per sera of individuals bearing mucocutaneous Leishmaniasis in a western blot reaction SDS-PAGE with the antigens (1), (2), (3), (4) and (5) is, respectively: 0.9 ± 1.1; 1.4 ± 1.74; 0.7 ± 1.05; 0.5 ± 0.84, and 0.0 ± 0.00 bands per antigen.
4 - A multiplying factor characterized by the fact of using an antigen with the overall spectrum of epitopes of a microorganism associated with the fact that this antigen reveals the overall spectrum of the antibodies existing in the sera of individuals infected. 5 - The fact that this invention was tested in a double blind study when then maximum values were obtained, that is, 100% co-positivity, 100% co- negativity, agreement index 1, positive predictive value 100%. negative predictive value 100%. These technical effects reached were unexpected at the time this invention was being carried out and were confirmed later.
The novelty in regard to this invention is that, after two exhaustive searches, nothing equal or similar to it has as yet been possible to find. The technical effects attained were:
1 - A new methodology for producing an antigen.
2 - A different approach to the "Reductionist Philosophy" with regard to the production, use and application of antigens. 3 - The possibility of using the entire spectrum of specific antibodies of an individual against the antigen proposed. 4 - Obtention of an antigen with a higher antigenic and/or immunizing power, since this same antigen is capable of reacting with the entire spectrum of antibodies specific for it. The objectives of this invention are:
1 - Define a process for the production of a 7. cruzi antigen of Y strain or of any other T. cruzi strain, or of other microorganisms, which, due to the characteristics of the process itself, can maintain or preserve the totality of the 7. cruzi epitopes or of other microorganisms which will serve to disclose the totality of the anti-7. cruzi antibodies or any anti-other microorganism antibodies through the western blot technique SDS-PAGE or any other diagnostic technique.
2 - Define a confirmatory serodiagnostic technique for the Chagas' disease and for other infectious and/or parasitic diseases, which technique uses the respective and specific antigen obtained through the production process referred to under 1 above. 3 - Define the use of the antigen under 1 above as a better immunizing agent, as it is capable of reacting "in vitro" with the entire spectrum of specific antibodies directed against it.
The above objetives are designed to eliminate the inconveniences of the technique in the state it is found nowadays.
The understanding of this invention is enhanced in the following 12 procedural items (processes) described hereinbelow: 1 - Preparation process of an antigen derived from an infectious and/or parasitic disease-causing microorganism.
(a) Cultivate a specific form of infectious and/or parasitic disease-causing microorganism in a medium varying in accordance with the infecto- contagious and parasitic disease-causing microorganism to be cultivated and in accordance with the usual specific technique to be employed.
(b) Take an aliquot of the infectious and/or parasitic disease-causing microorganism cultivated under step (a) above and transfer it to a test tube adding thereto a formol solution of 0.4 to 15% in such a volume as will be sufficient to provide transmitance of 20 to 95% in the Coleman Jr. spectrophotometer or similar, in wave length of 660 nm when read in cuvettes of 10/75 or similar, thus obtaining a suspension of homogeneous concentration of microorganisms, and maintaining said suspension from 02 to72 hours under an room temperature and with intermitent stirring, and washing the suspension 02 to 05 times at 500g to 100,000g at 4°C for 05 minutes to 10 hours subsequently in NaCl 0.15 M + phosphate buffer 0.005 to 0.300 M, pH 6.8 to 7.5 (PBS) or other usual buffer as dictated by the technique, thus obtaining a sediment of microorganisms.
(c) Take an aliquot of the microorganisms sediment referred to in step (b) above and prepare a suspension of lxlO3 to 3xlOn microorganisms per ml; wash said suspension three times consecutively with solution NaCl 0.15 M + TRIS 0.012 to 0.500 M, pH 6.8 to 7.6 (TBS) or other similar buffer, as dictated by the technique, at 500g to 100,000g at 4° C for a time of from 05 minutes to 10 hours, thus obtaining a sediment of microorganisms, and
(d) Resuspend the microorganisms sediment referred to under step (c) above in
0.2 to 5.0 ml of solution TRIS 0.010 to 0.500 M, pH 6.4 to 7.6 or other usual buffer, as dictated by the technique, containing 1.0 to 10.0% (p/v) of SDS or ether natural or synthetic anionic, cationic or non-ionic detergent, EDTA solution of 1.0 to 5.0 mM, and bromophenol blue solution of 0.1 to 1.5 ppm. The mixture is to be heated for a time of from 1.0 to 10.0 minutes in water bath at 100°C, the desired antigen thus being obtained and immediately applied to a polyacrylamide gel containing SDS, should this be the case.
- Preparation process of a 7. cruzi antigen of Y strain or any other T. cruzi strain, or antigen (4) of EXAMPLE I.
(a) Cultivate epimastigote forms of T. cruzi Y strain in LIT (Liver Infusion and Tryptose) medium contained in 50 to 200 ml of the culture medium adequate for the cultivation and growing ofthe protozoa, maintaining it in a incubator at a temperature of 22° to 35°C, with continuous stirring, for a period of 2 to 7 days, thus obtaining a suspension of epimastigotes in its exponencial phase.
(b) Take an aliquot of the suspension contained in step (a) above during the exponential phase of growing of the epimastigotes and transfer it to a test tube, adding to it a formol solution at 0.5 to 10% in such a volume as will be sufficient to provide transmittance of 70 to 95% in the Coleman Jr. spectrophotometer or similar, to a wave length of 660 nm when read in 10/75 mm cuvettes, thus obtaining a microorganism suspension of homogeneous concentration and maintaining said suspension for a period of 12 to 24 hours at room temperature and with intermitent stirring, and washing it two times consecutively with NaCl 0.15 M + phosphate buffer 0.005 to 0.300 M. pH 6.8 to 7.5 (PBS) or other usual buffer, as dictated by the technique, at 500g to 3.000g, at 4°C for a period of 06 to 15 minutes, thereby obtaining a sediment of microorganisms.
(c) Take an aliquot of the microorganism sediment in step (b) above and prepare a suspension of 5xl07 to lxlO9 microorganism per ml; wash said suspension three times subsequently with NaCl 0.15 M + TRIS 0.012 to 0.500 M, pH 6.8 to 7.6 (TBS) or with such other usual buffer, as dictated by the technique, of 500g to 3,000g at 4°C for a period of 06 to 15 minutes, thereby obtaining a sediment of microorganisms.
(d) Resuspend the microorganisms sediment of step (c) above in 0.3 to 1.0 ml of solution TRIS 0.010 to 0.500 M, pH 6.4 to 7.6 containing 0.5 to 6.0% (p/v) SDS or other natural or synthetic anionic, cationic or non-ionic detergent, EDTA solution at 2.0 to 4.0 mM, and bromophenol blue solution at 0.2 to 1.2 ppm. The mixture is to be heated for a period of 1.0 to 10.0 minutes in water bath at 100°C. the T. cruzi antigen or the antigen (4) of EXAMPLE I thereby being obtained and immediately applied to the polyacrylamide gel containing SDS, should this be the case.
In process 2, above described, step (a), cultivation of the protozoa is maintained preferably at a temperature of 28°C for 3 days. In step (b) formol is preferably added at 1% in such volume as will be sufficient to provide transmittance of 90%, the suspension being maintained at room temperature for 18 hours and being washed two times subsequently with NaCl 0.15 M + phosphate buffer 0.01 M, pH 7.2 (PBS) at 1300g at 4°C for 10 minutes.
In step (c) a suspension of lxlO8 microorganism per ml is preferably prepared with washing being carried out in a solution NaCl 0.15 M + TRIS 0.05 M, pH 7.2 at 1300g at 4°C for 10 minutes.
In step (d) the microorganism sediment is resuspended, preferably in 0.5 ml of solution TRIS 0.100 M. pH 6.8, containing 2.5% (p/v) SDS, EDTA solution at 2.0 mM and bromophenol blue solution at 0.5 ppm. The mixture was heated for 3 minutes in water bath at 100°C and immediately applied to the polyacrylamide gel containing SDS.
- This invention also refers to the antigen derived from infectious and/or parasitic disease-causing microorganism, said antigen having the following characteristics:
(a) total concentration of proteins of 100 μg/ml to 200 mg/ml of the microorganism;
(b) protein bands fractioned by western blot SDS-PAGE with molecular weights varying from 300 to 15 kDa.
(c) a positive reaction to the protein tests: Lowry, extinction at 280 nm, biuret and nihydrin;
(d) the protein content is resistant to the digestion by usual proteases;
(e) it can be transferred to a solid support such as a nitrocellulose membrane or another similar and usual support;
(f) the solid support containing antigen can be blocked by a skimmed milk solution.
- This invention also refers to a 7. cruzi antigen of Y strain or any other 7. cruzi strain, or antigen (4) of EXAMPLE I, with the following characteristics:
(a) Total concentration of proteins, from 100 μg/ml to 200 mg/ml of epimastigote, trypomastigote or amastigote forms of T. cruzi. (b) A positive reaction to the protein tests: Lowry. extinction at 280 mm. biuret and nihydrin.
(c) A reaction in the western blot SDS-PAGE with the same 12 bands revealed by sera of chagasic individuals, with molecular weights of 140, 100, 85, 78, 59, 57, 46, 35, 27, 23, 20, 18 kDa. with bands of 78 and 27 kDa being the broadest and more strongly dyed or reactive when using epimastigote forms of 7. cruzi.
(d) A reaction lacking bands in 55% of the sera of non-chagasic individuals, average of 0.63 bands per serum of non-chagasic individuals: and average of 0.5 bands per serum of individuals with monocutaneous leishmaniasis, bands 78 and/or 35 kDa, when using epimastigote forms of 7. cruzi.
(e) A reaction with the following indices: 100% co-positivity, 100% co- negativity, agreement index of 1, positive predictive value of 100% and negative predictive value of 100%, when using epimastigote forms of 7. cruzi, Y strain.
(f) It is not susceptible to digestion by usual proteases.
(g) It can be transferred to a solid support such as a nitrocellulose membrane or other usual solid support.
(h) The solid support containing antigen can be blocked by a skimmed milk solution.
(i) A reaction producing from 01 to 05 bands per serum out ofthe 08 bands of molecular weight of 18, 20, 23, 27, 35, 46, 57. 78 kDa when the antigen reacts with samples or fluids of non-chagasic organisms, when using epimastigote forms of 7. cruzi. - One other aspect of the invention is that it refers to a composition made of an antigen of infectious and/or parasitic disease-causing microorganism comprising: 0.2 mg/ml to 20.0 mg/ml of a complete solution of proteins of said microorganisms in TRIS 0.005 to 0.300 M, pH6.5 to 7.9 or another usual buffer as a diluting agent, and timerosol 1/1,000 to 1/30,000 and/or another usual conservant used in the technique.
Preferably, the complete solution of proteins of said microorganisms lies within the range of 0.08 mg/ml to 9.00 mg/ml and the composition obtained is used for serodiagnostic tests. Another form prevailing over others is that whereby a complete solution of proteins of said microorganisms within the range of 10.00 mg/ml to 19.00 mg/ml is used employing aluminum hydroxide and or anoiher usual adjuvant as dictated by the technique. The composition thus obtained is used for vaccines.
- It also refers to an antigen-based composition, i.e., a T. cruzi antigen of Y strain or any other T. cruzi strain, comprising 0.10 mg/ml to 15.00 mg/ml of a complete solution of T. cruzi proteins, as per claim 6, in TRIS 0.005 to 0.300 M, pH 6.5 to 7.9 or other usual buffer as a diluting agent, and timerosol 1 : 1,000 to 1 :30,000 and/or another usual conservant as dictated by the technique. This composition is used for vaccines and for serodiagnostic tests. Preferably, the composition has 0.12 mg/ml to 7.50 mg/ml of a complete solution of proteins of T. cruzi, Y strain or any other T. cruzi strain, and is used for diagnostic tests such as western blot, ELISA, immunoenzymatic, agglutination, immunoprecipitation. complement fixation, precipitation, hemagglutination, flocculation and immunofluorescence. Particularly, the composition has 8.0 mg/ml to 14 mg/ml of a complete solution of proteins of T. cruzi, Y strain or any other 7. cruzi strain plus aluminum hydroxide and/or another usual adjuvant as dictated by the technique, and is used for vaccines. Punctually, the composition contains 0.15 mg/ml of a complete solution of proteins of 7. cruzi. Y strain or any other T. cruzi strain in TRIS 0.125 M, pH 7.2 and timerosol 1/1.000, and is used for western blot serodiagnostic test.
- This invention also refers to the use of the antigen derived from infecto- contagious and parasitic disease-causing microorganism in serological test, said antigen acting as a reagent for the diagnosis of infectious and/or parasitic diseases through such different serodiagnostic techniques as western blot, ELISA, immunoenzymatic, agglutination, immunnoprecipitation, complement fixation, precipitation, hemagglutination, flocculation and immunofluorescence, and acting also as an immunizing agent against infectious and/or parasitic diseases in vaccines.
- Particularly, the invention refers also to the use of the antigen derived from T. cruzi, of Y strain or any other T. cruzi strain, causing the Chagas' disease, in serological tests, acting as reagent in the diagnosis of the Chagas' disease through such different techniques as western blot, ELISA, immunoenzymatic, agglutination, immunnoprecipitation, complement fixation, precipitation, hemagglutination, flocculation and immunofluorescence, and acting also as an immunizing reagent for the Chagas' disease in vaccines.
- Another aspect of this invention is related to the different processes of detection of anti-antigen antibodies of infectious and/or parasitic disease-causing microorganisms, including the Y strain T. cruzi or any other strain T. cruzi in fluids to be tested using such different serological techniques as western blot, ELISA, immunoenzymatic, agglutination, immunnoprecipitation, complement fixation, precipitation, hemagglutination, flocculation and immunofluorescence, and having as one of its reagents the antigen and composition previously mentioned hereinabove.
-In particular, when the western blot technique of diagnosis is used, the following procedural steps are carried out: (a) put the fluid containig antibodies in contact with a solid support containing the specific antigen of each microorganism, as characterized in claims 05 and 06. said fluid having previously been blocked with a skimmed milk solution of 1.0 to 10.0 %;
(b) wash the solid support absorbed by the antibodies in a skimmed milk solution of 1.0 to 10.0%,
(c) incubate the solid support with an anti-IgG and/or IgM and or IgA and/or IgE conjugate labeled with an enzyme and or radioactive and/or fluorescent substance, diluted in skimmed milk solution of 1.0 to 10.0 %;
(d) wash the solid support;
(e) develop the reaction with a chromogen substract when using an enzimatic conjugate or use a specific development technique when using a different conjugate;
(f) stop the reaction obtained under (e) above with distilled water; and
(g) proceed to the reading ofthe results obtained.
-Another aspect ofthe invention is related to a kit for confirmatory serodiagnosis through western blot SDS-PAGE for infectious and/or parasitic diseases with a specific antigen of infectious and/or parasitic disease-causing microorganism containing:
(a) 10 to 20 nitrocellulose strips with said antigen absorbed in the strips, all of which tightly vacuum packed in plastic bags;
(b) a glass bottle of adequate volume, containing 50 to 200 mg/ml of nonfat dried milk meant for the washing solution and for the diluting solution of the fluid to be tested; (c) cuvettes for performing the reaction;
(d) a glass bottle containing the anti-IgG and/or IgM and/or IgA and/or IgE enzymatic or radiolabeled or fluorescent conjugate meant for the development ofthe reaction;
(e) a bottle of adequate volume containing 4-chloro-l-naphtol at 0.05% p/v or another usual substract the as dictated by technique to match the conjugate to be used, and methanol at 15% v/v in distilled water;
(f) a glass bottle of adequate volume containing H202 at 10% meant for the preparation ofthe chromogen substract;
(g) standard positive and negative sera, for control purposes.
12 -The last aspect of the invention is related to a kit similar to the one above, which can be applied for confirmatory diagnosis of Chagas' disease, using the Y strain or other strain T. cruzi antigen absorbed in the nitrocellulose strip in the epimastigote, trypomastigote and amastigote forms.
E X A M P L E I Application of the Y strain T. cruzi antigen, epimastigote form, or antigen (4) of FIGURE I, to the western blot technique.
The descriptive example below is intended to better define the techniques employed without, however, restricting the scope ofthe invention.
The T. cruzi antigen or antigen (4) was submitted to electrophoresis in polyacrylamide gel in the presence of SDS, as described in Laemmli, U.K., Nature, 227.690-685,1970. The gels were assembled on 14 cm x 16 cm glass plates linked by 1 mm thick spacers. The separation gel was prepared in the concentration of 10% of acrylamide and the mixture, still in liquid form, was immediately applied between two glass plates mounted vertically to a height of 8.5 cm from the lower spacer. Onto this gel a butanol saturated solution was carefully placed and the gel was maintained at room temperature until polymerization occurred, which took place between 30 and 60 minutes. Following polymerization, the butanol was discarded and the gel surface was washed with distilled H20 and onto this surface a mixture of stacking gel with 4% acrylamide was placed. In the stacking gel mixture, still in liquid form, a teflon comb was inserted and this served as a mold for the application of the antigen. Following polymerization of this second gel, the comb and lower spacer were withdrawn, and the plates were fastened to the electrophoresis vat. The vat was completed with the buffer and the antigen was then applied to the gel.
Electrophoresis was run under a constant amperage (25 mA) for approximately 4 hours, that is, until the indicating dye reached the end of the gel. After the run, the plates were carefully separated, the gel removed and the proteins contained in the separation gel fixed and dyed or else transferred to nitrocellulose membranes. Both the fixation and the dyeing were performed during 30 minutes in the solutions: fixing (methanol 50% and acetic acid 7%) and dyeing (amido black 1% in fixing solution). IgG (150 kDa), bovine serum albumin (66 kDa) and ovalbumin (43 kDa) were used as molecular weight markers.
The transference of proteins to the nitrocellulose membranes is carried out as follows:
The antigenic fractions, separated by western blot SDS-PAGE were transferred by electrophoresis from the gel to nitrocellulose membranes as described in Towbin et alii, Proc. Natl. Acad. Sci. USA, 76: 350-4, 1979 and Bumette, W.N., Anal. Biochem., 112: 195-203, 1981. In brief, the following is assembled to a reservoir with a transference buffer, in the following order: perforated plastic support, foam sheet, 10 (ten) 1 mm filter paper sheets, gel, nitrocellulose membranes, 10 (ten) 1 mm paper filter sheets, foam sheet and perforated plastic support. The assembly was immediately placed in the transference vat already containing the buffer, with the nitrocellulose membrane facing the anode (+). Electrophoresis was run under a constant amperage of 200 mA for 18 hours. The efficiency of the transference was verified through the colouring of part of the membrane in Ponceau-S solution. The antigens present in the remaining part of the membrane are immediately submitted to immunoenzymatic characterization, or they can be stored in vacuum closed plastic bags and preserved at 4°C. The immunoenzymatic analysis ofthe transference is made in accordance with the following steps:
A) Titration ofthe sera to be used as source of antibodies:
Test the growing sera dilutions against lOμg to 30μg of antigens fractioned by western blot SDS-PAGE and transferred to nitrocellulose membranes. Under these conditions, the optimum dilution of the serum, capable of revealing all of the antigenic components without causing a significant non-specific reaction, shall be
1:50.
B) Titration ofthe immunoenzymatic conjugates:
Titration of the conjugate is carried out using growing dilutions against the sera used in the standard dilution 1/50. Select the highest dilution of the conjugate which can still reveal all of the bands in a clear cut way with no non-specific antigenic fractions in the presence of normal sera. Said dilution shall be of ± 1:800 for a good conjugate of human anti-IgG peroxidase.
C) Test development: Following the transference, the nitrocellulose membranes are incubated with a good skimmed dried milk at 1.0 to 10.0% in solution TRIS 0.005 to 0.300 M, pH 6.5 to 7.9 (blocking solution) for 1.0 to 4.0 hours. This step is followed by incubation for 10 to 24 hours with the sera to be tested diluted in blocking solution. Next, the strips are washed 3 times with solution TRIS 0.005 to 0.300 M, pH 6.5 to 7.9, with changes at every 15 minutes and incubated for 1.0 to 5.0 hours with anti-human IgG goat IgG labeled peroxidase (immunoenzymatic conjugate) also diluted in blocking solution. The excessive conjugate is removed with a new cycle of washing and the antigens are then revealed with H20 0.03% (v/v), 4-chloro-l naphtol 0.05% (v/v) and methanol 15 % (v/v) in distilled water (chromogenous substract), the reaction then being interrupted by washing with distilled water. In Figure 2. we can see a band schematic of T. cruzi antigen. Y strain, epimastigote form, or antigen (4) of Figure 1, with bands fractioned and revealed by the western blot SDS-PAGE (10%) technique, as described above, with 20 sera of chagasic individuals, and by positive and negative control sera. IgG (150 kDa), bovine serum albumin (66 kDa) and ovalbumin (43 kDa) were used as molecular weight markers.
In Figure 3, we can see a band schematic of T. cruzi antigen, Y strain, epimastigote form, or antigen (4) of Figure 1, with bands fractioned and developed by the western blot SDS-PAGE (10%) technique, as described above, with 20 sera of non-chagasic individuals, and by positive and negative control sera. IgG (150 kDa), bovine serum albumin (66 kDa) and ovalbumin (43 kDa) were used as molecular weight markers.
The reading criterion for defining the reaction results is: POSITIVE REACTION: - complete profile of 12 bands;
- bands easily identifiable with the unaided eye or by densitometer;
- defmed and reactive bands, with molecular weights 140, 100, 85, 78, 59, 57, 46, 35, 27, 23. 20 and 18 kDa;
- the 78 kDa and/or 27 kDa bands are the broadest and the most dyed or reactive. NEGATIVE REACTION:
- absence of bands (in 55% ofthe sera tested, from non-chagasic individuals); or
- variable profile from 01 to 05 bands not well defined or less reactive, with molecular weights 78, 57, 46, 35, 27. 23, 20 and 18 kDa;
- average of 0.63 bands per serum of non-chagasic individuals. CROSSED REACTION:
Crossed reactivity with sera from mucocutaneous leishmaniasis bearers produces 78 kDa and/or 38 kDa bands, all of which not well-defined though, and with an average of 0.5 band per serum studied. With the process and technique above described, higher efficiency and effectiveness can be obtained in the diagnosis of Chagas' disease and of other diseases caused by other types of microorganisms. Assessment of he technique above described The diagnosis performance of this technique was assessed by Dr. Rodolfo
Pereira Mendes through a double blind test carried out at the W.H.O. Immunology Laboratory located at Instituto Butantan, Sao Paulo, SP, using a battery of sera as a source of primary standard sera. This sera battery originated from the W.H.O. Reference Laboratory for Chagas' disease of Universidade Federal de Goias, Goiania, Go.
The antigen used and the technique employed were the ones mentioned above.
Inteφretation ofthe technique was as above mentioned.
The analysis of the data for assessing the diagnosis performance was made by calculating the co-positive and co-negative values, the agreement index, the positive-predictive and negative-predictive values obtained from the double blind study data according to Galeno & Galeno, 1975. The assessment indices of the diagnosis performance ofthe reaction are: 100% co-positivity, 100% co-negativity, agreement index 1, 100% positive predictive value and 100% negative predictive value.
The reproductivity of the different batches of antigen was verified in the same manner as described above, against standard positive and negative sera, said antigens having provided five times consecutively the same number of antigenic bands. The purpose ofthe example above described is to better illustrate the present invention without however limiting its scope.
As to industrial use, the invention in question is intended for the medical area, in particular for the diagnosis, therapy and prophylaxy sectors and for the veterinaty area, particularly the diagnosis, therapy and prophilaxy sectors. The invention in question can also be applicable to the agricultural area.

Claims

22
C L A I M S - Preparation process of an antigen derived from infecto-contagious and/or parasitic disease-causing microorganism, said process characterized by and comprising the following steps: (a) Cultivate a specific form of infectious and or parasitic disease-causing microorganism in a medium varying in accordance with the infecto- contagious and or parasitic disease-causing microorganism to be cultivated and in accordance with the usual specific technique to be employed; (b) Take an aliquot of the infectious and/or parasitic disease-causing microorganism cultivated under step (a) above and transfer it to a test tube adding thereto a formol solution of 0.4 to 15% in such a volume as will be sufficient to provide transmittance of 20 to 95% in the Coleman Jr. spectrophotometer or similar, in wave length of 660 nm when read in cuvettes of 10/75 or similar, thus obtaining a suspension of homogeneous concentration of microorganisms, and maintaining said suspension from 02 to 72 hours under an room temperature and with intermitent stirring, and washing the suspension 02 to 05 times at 500g to lOO.OOOg at 4°C for 05 minutes to 10 hours subsequently in NaCl 0.15 M + phosphate buffer 0.005 to 0.300 M, pH 6.8 to 7.5 (PBS) or such other usual buffer as dictated by the technique, thus obtaining a sediment of microorganisms;
(c) Take an aliquot of the microorganisms sediment referred to in step (b) above and prepare a suspension of lxlO3 to 3xl0π microorganisms per ml: wash said suspension three times consecutively with solution NaCl
0.15 M + hydroxymethil aminomethane (TRIS) 0.012 to 0.500 M, pH 6.8 to 7.6 (TBS) or other similar buffer, as dictated by the technique, at 500g to 100,000g, at 4° C, for a time of 05 minutes to 10 hours, thus obtaining a sediment of microorganisms; (d) Resuspend the microorganisms sediment referred to under step (c) above in 0.2 to 5.0 ml of solution TRIS 0.010 to 0.500 M, pH 6.4 to 7.6 or other usual buffer, as dictated by the technique, containing 1.0 to 10.0% (p/v) of SDS or other natural or synthetic anionic, cationic or non-ionic, detergent EDTA solution of 1.0 to 5.0 mM, and bromophenol blue solution of 0.1 to 1.5 ppm, and;
(e) Heat mixture in step (d) at 100°C for 1 to 10 minutes, obtaining the antigen desired. - Process under claim 01 above characterized by the fact that the microorganism referred to in step (a) above is the Y strain or any other strain
7. cruzi in its amastigote and/or trypomastigote forms. - Preparation process of an antigen of Y strain or any other strain T. cruzi, as per claim 01, characterized by and comprising the following steps:
(a) Cultivate epimastigote forms of Y strain or any other strain 7. cruzi in LIT (Liver Infusion and Tryptose) medium or such other medium as dictated by the technique, contained in 50 to 200 ml of the culture medium adequate for the cultivation and growing of the protozoa, maintaining it in an incubator at a temperature of 22° to 35°C. with continuous stirring, for a period of 2 to 17 days, thus obtaining a suspension of epimastigotes in its exponencial phase;
(b) Take an aliquot of the suspension contained in step (a) above during the exponential phase of growing of the epimastigotes and transfer it to a test tube, adding to it a formol solution at 0.5 to 10% in such a volume as will be sufficient to provide transmittance of 70 to 95% in the Coleman Jr. spectrophotometer or similar, to a wave length of 660 nm when read in 10/75 mm cuvettes or similar, thus obtaining a microorganism suspension of homogeneous concentration and maintaining said suspension for a period of 12 to 48 hours at room temperature and with intermitent stirring, and washing it two times consecutively with NaCl 0.15 M + phosphate buffer 0.005 to 0.300 M, pH 6.8 to 7.5 or such other usual buffer as dictated by the technique, at 500g to 3.000g, at 4°C. for a period of 06 to 15 minutes. thereby obtaining a sediment of microorganisms; (c) Take an aliquot of the microorganism sediment in step (b) above and prepare a suspension of 5x107 to lxlO9 microorganism per ml; wash said suspension three times subsequently with NaCl 0.15 M + TRIS 0.012 to 0.500 M, pH 6.8 to 7.6 or with such other buffer as dictated by the technique of 500g to 3,000g, at 4°C, for a period of 06 to 15 minutes, thereby obtaining a sediment of microorganisms; (d) Resuspend the microorganisms sediment of step (c) above in 0.3 to 1.0 ml of solution TRIS 0.010 to 0.500 M, pH 6.4 to 7.6 containing 1.5 to 6.0% (p/v) SDS or other natural or synthetic anionic, cationic or non¬ ionic. detergent EDTA solution at 2.0 to 4.0 mM, and bromophenol blue solution at 0.2 to 1.2 ppm, and; (e) Heat the mixture in step (d) at 100°C for 1.0 to 10.0 minutes, thereby obtaining the T. cruzi (Y strain or any other strain) antigen desired. - Preparation process of a T. cruzi ( Y strain or any other strain) antigen, as per claim 03, said process characterized and comprising the following steps:
(a) Cultivate epimastigote forms of Y strain or any other strain T. cruzi in LIT (Liver Infusion and Tryptose) medium contained in 100 ml of the culture medium or such other medium as dictated by the technique, contained in 100 ml of the culture medium adequate for the cultivation and growing of the protozoa, maintaining it in an incubator at a temperature of 28°C. with continuous stirring, for a period of 3 days, thus obtaining a suspension of epimastigotes in its exponencial phase;
(b) Take an aliquot of the suspension contained in step (a) above during the exponential phase of growing of the epimastigotes and transfer it to a test tube, adding to it a formol solution at 1% in such a volume as will be sufficient to provide transmittance of 90% in the Coleman Jr. spectrophotometer or similar, to a wave length of 660 nm when read in 10/75 mm cuvettes or similar, thus obtaining a microorganism suspension of homogeneous concentration and maintaining said suspension for a period of 18 hours at room temperature and with intermitent stirring, and washing it two times consecutively with NaCl 0.15 M + phosphate buffer 0.01 M, pH 7.2 at l,300g, at 4°C for 10 minutes, thereby obtaining a sediment of microorganisms;
(c) Take an aliquot of the microorganism sediment in step (b) above and prepare a suspension of lxlO8 microorganisms per ml; wash said suspension three times subsequently with solution NaCl 0.15 M + TRIS 0.05 M, pH 7.2 at 1300g, at 4°C for 10 minutes. thereby obtaining a sediment of microorganism;
(d) Resuspend the microorganism sediment of step (c) in 0.5 ml solution TRIS 0.100 M, pH 6.8 containing 2.5% (p. v.) SDS, EDTA solution at 2.0 mM and bromophenol blue solution at 0.5 ppm, thereby obtaining the 7. cruzi (Y strain or any other strain) antigen;
(e) Heat the mixture in step (d) above at 100°C for 3.0 minutes, thereby obtaining the 7. cruzi ( Y strain or any other strain) antigen desired. - Antigen derived from infectious and/or parasitic disease-causing microorganism thus characterized by having the following characteristics: (a) Total concentration of proteins of 200 μg/ml of the microorganism;
(b) Protein bands fractioned by western blot SDS-PAGE with molecular weights varying from 300 to 15 kDa;
(c) A positive reaction to the protein tests: Lowry, extinction at 280 nm, biuret and nihydrin; (d) The protein content is resistant to the digestion by usual proteases;
(e) It can be transfeπed to a solid support such as a nitrocellulose membrane or another similar and usual support;
(f) The solid support containing antigen can be blocked by a skimmed milk solution of 1.0 to 10.0%. Antigen derived from the Chagas' disease-causing T. cruzi (Y strain or any other strain), characterized by having the following characteristics: (a) Total concentration of T. cruzi (Y strain or any other strain) proteins, of 200 μg/ml of epimastigote, trypomastigote or amastigote forms; (b) A positive reaction to the protein tests: Lowry, extinction at 280 nm, biuret and nihydrin.
(c) A reaction in the western blot SDS-PAGE reaction always with the same 12 bands revealed by sera of chagasic individuals, with molecular weights of 140, 100, 85, 78, 59, 57, 46, 35, 27, 23, 20, 18 kDa, with bands of 78 and 27 kDa being the broadest and more strongly dyed or reactive when using the epimastigote form of Y strain 7. cruzi;
(d) A reaction lacking bands in 55% of the sera of non-chagasic individuals, average of 0.63 bands per serum of non-chagasic individuals and 0.5 bands per serum of individuals with monocutaneous leishmaniasis, bands 78 and/or 35 kDa, when using the epimastigote form of Y strain 7. cruzi;
(e) A reaction with the following indices: 100% co-positivity, 100% co- negativity, agreement index of 1, positive predictive value of 100% and negative predictive value of 100%, when using the epimastigote form of Y strain T. cruzi;
(f) It is not susceptible to digestion by usual proteases;
(g) It can be transfeπed to a solid support such as a nitrocellulose membrane or other usual solid support;
(h) The solid support containing antigen can be blocked by a skimmed milk solution of 1.0 to 10.0%;
(i) A reaction producing from 01 to 05 bands out of the 08 bands of molecular weight of 18, 20, 23, 27, 35, 46, 57, 78 kDa when the antigen reacts with samples or fluids of non-chagasic organisms and originates from Y strain T. cruzi epimastigote forms. 7 - Composition consisting of antigen of infectious and/or parasitic disease- causing microorganism, as per claim 05. characterized by and containing 0.2 mg/ml to 20.0 mg/ml of a total solution of proteins from said microorganism in TRIS 0.005 to 0.300 M, pH 6.5 to 7.9 or other usual buffer, as a diluting agent, and timerosol 1/1,000 to 1/30.000 and/or another usual preserving agent, aluminum hydroxide and/or usual adjuvant, as dictated by the technique. - Composition as per claim 07, characterized by and containing 0.08 mg/ml to
9.00 mg/ml of a total solution of proteins from said microorganisms in TRIS 0.005 to 0.300 M, pH 6.5 to 7.9 and/or another usual buffer, as a diluting agent, and timerosol 1/1,000 to 1/30.000 and/or another usual preserving agent as dictated by the technique. - Composition as per claim 07, characterized by and containing 10.0 mg/ml to
19.0 mg/ml of a total solution of proteins from said microorganism in TRIS 0.005 to 0.300 M, pH 6.5 to 7.9 or other usual buffer, as a diluting agent, and timerosol 1/1,000 to 1/30,000 and/or another usual preserving agent, aluminum hydroxide and/or other usual adjuvant, as dictated by the technique.
10 - Composition as per claim 08, characterized by the fact of its being used for serodiagnostic tests. 11 - Composition as per claim 09, characterized by the fact of its being used for vaccines.
12 - Composition made of T. cruzi (Y strain or any other strain) antigen, characterized by and containing 0.10 mg/ml to 15.0 mg/ml of total solution of T. cruzi proteins, as per claim 06, in TRIS 0.005 to 0.300 M, pH 6.5 to 7.9 and/or another usual buffer, as a diluting agent, and timerosol 1:1,000 to
1:30,000 and/or another usual preserving agent, aluminum hydroxide and/or another usual adjuvant, as dictated by the technique.
13 - Composition as per claim 12, characterized by and containing 0.12 mg/ml to
7.50 mg/ml of a total solution of T. cruzi (Y strain or any other strain) proteins in TRIS 0.005 to 0.300 M. pH 6.5 to 7.9 or other usual buffer, as a diluting agent, and timerosol 1 : 1,000 to 1 :30,000 and/or another usual preserving agent, as dictated by the technique.
14 - Composition as per claim 12, characterized by and containing 8.0 mg/ml to
14.0 mg/ml of a total solution of T. cruzi (Y strain or any other strain) proteins from said microorganism in TRIS 0.005 to 0.300 M, pH 6.5 to 7.9 or other usual buffer, as a diluting agent, and timerosol 1/1,000 to 1/30,000 and/or another usual preserving agent, aluminum hydroxide and/or other usual adjuvant, as dictated by the technique.
15 - Composition as per claim 12, characterized by and containing 0.15 mg/ml of a total solution of T. cruzi (Y strain or any other strain) proteins in TRIS 0.125
M, pH 7.2 and timerosol 1: 1,000 and/or another usual preserving agent as dictated by the technique.
16 - Composition as per claim 12, characterized by the fact of its being used for vaccines and serodiagnostic tests. 17 - Composition as per claim 13, characterized by its being used for serodiagnostic tests. 18 - Composition as per claim 14, characterized by its being used for vaccines. 19 - Composition as per claim 15. characterized by its being used for serodiagnostic tests. 20 - Composition as per claim 15, characterized by its being used for the following serodiagnostic tests: western blot, ELISA, immunoenzymatic, agglutination, immunoprecipitation, complement fixation, precipitation, hemagglutination, flocculation and immunofluorescence.
21 - Composition as per claim 15, characterized by its being used for western blot test.
22 - Use of the antigen derived from infectious and/or parasitic disease-causing microorganism, as per claim 05. in serological tests, characterized by its applicability as an immunizing reagent for the diagnosis of infectious and/or parasitic disease. 23 - Use ofthe antigen as per claim 08, characterized by its being employed in the following tests: western blot, ELISA, immunoenzymatic, agglutination, immunoprecipitation, complement fixation, precipitation, hemagglutination. flocculation and immunofluorescence. 24 - Use of the antigen as per claim 05, in vaccines, characterized by its applicability as an immunizing agent for infecto-contagious and/or parasitic diseases.
25 - Use ofthe antigen derived from the Chagas' disease causing T. cruzi (Y strain or any other strain), as per claim 06, in serological tests, characterized by its applicability as a reagent in the diagnosis of Chagas' disease.
26 - Use of the antigen derived from T. cruzi (Y strain or any other strain), as per claim 13, characterized by its being employed in the following tests: western blot, ELISA, immunoenzymatic, agglutination, immunoprecipitation, complement fixation, precipitation, hemagglutination, flocculation and immunofluorescence.
27 - Use of the antigen, as per claim 06, in vaccines, characterized by its applicability as an immunizing agent for Chagas' disease. 28 - Process for detecting anti-antigen antibodies of infectious and or parasitic disease-causing microorganisms, characterized in claim 05, in fluids to be tested through such different techniques as western blot, ELISA, immunoenzymatic, agglutination, immunoprecipitation, complement fixation, precipitation, hemagglutination, flocculation and immunofluorescence, characterized by the application of said antigen to the specific methodology of each one of said techniques. 29 - Process for detecting anti-antigen antibodies of infectious and/or parasitic disease-causing microorganisms in fluids to be tested using the western blot technique comprising the following steps:
(a) put the fluid containig antibodies in contact with a solid support containing the specific antigen of each microorganism, as characterized in claims 05 and 06. said solid support + antigens having previously been blocked with a skimmed milk solution of 5.0 to 10%; (b) wash the solid support absorbed by the antibodies in a skimmed milk solution of 1.0 to 5.0%; (c) incubate the solid support with anti-IgG and or IgM and/ou IgA and/or
IgE conjugate labeled with an enzyme and/or radioactive and/or fluorescent substance, diluted in skimmed milk solution;
(d) wash the solid support;
(e) develop the reaction with a chromogen substract when using an enzymatic conjugate or use a specific development technique when using a different conjugate;
(f) stop the reaction obtained under (e) above with distilled water; and
(g) proceed to the reading ofthe results obtained. - Process as per claim 29, characterized by the fact that the antibodies to be detected are anti-antigen of Y strain or any other strain T. cruzi, and the antigen used is the Y strain or any other strain T. cruzi, characterized in claim 06. - Kit for confirmatory serodiagnosis by western blot SDS-PAGE for infectious and/or parasitic disease with the antigen of claim 05 characterized by and containing:
(a) 10 to 20 nitrocellulose strips with said antigen absorbed in the strips, all of which tightly vacuum packed in plastic bags;
(b) a glass bottle of adequate volume, containing 50 to 200 mg/ml of nonfat dried milk meant for the washing solution and for the diluting solution of the fluid to be tested;
(c) cuvettes for performing the reaction;
(d) a glass bottle containing the anti-IgG and/or IgM and/or IgA and or IgE enzymatic or radiolabeled or fluorescent conjugate meant for the development of the reaction; (e) a bottle of adequate volume containing 4-chloro-l-naphtol at 0.05% p/v or another usual substract as dictated by the technique to match the conjugate to be used, and methanol at 15% v/v in distilled water;
(f) a glass bottle of adequate volume containing H 02 at 10% meant for the preparation ofthe chromogen substract;
(g) standard positive and negative sera, for control purposes. - Kit as per claim 31, characterized by the fact that the antigen absorbed in the nitrocellulose strips is the Y strain or any other strain T. cruzi in the epimastigote or trypomastigote or amastigote forms.
PCT/BR1996/000032 1995-07-25 1996-07-25 A confirmatory serodiagnostic assay for infectious diseases WO1997005468A2 (en)

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BR9503451A BR9503451A (en) 1995-07-26 1995-07-26 Process of preparation of an antigen derived from a microorganism that causes infectious and / or parasitic disease Process of preparation of an antigen from T cruzi antigen derived from a microorganism that causes infectious disease and / or parasitic antigen derived from T cruzi that causes Chagas disease composition the base of an antigen derived from microorganism that causes infectious and / or parasitic disease composition the base of antigen from T cruzi use of the antigen derived from microorganism that causes infectious and / or parasitic disease in serological tests and vaccines use of antigen derived from T cruzi cause of chagas disease in serological tests and vaccines process for detecting antibodies against microorganism antigen causing infectious and / or parasitic disease and confirmatory serodiagnosis kit for infectious and / or parasitic disease

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WO1999005528A1 (en) * 1997-07-28 1999-02-04 Immunetics Assay and device for identifying trypanosoma cruzi infection
US6682900B1 (en) * 1996-08-02 2004-01-27 Fundação Hemocentro de Ribeirão Preto Serological diagnosis of Chagas' disease
US7749717B2 (en) 2006-10-19 2010-07-06 Abbott Laboratories Methods for the detection and diagnosis of Trypanosoma cruzi infection

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EP0138101A1 (en) * 1983-09-30 1985-04-24 Rockefeller University Genetically engineered organisms expressing surface proteins of T. Cruzi
WO1992009895A1 (en) * 1990-11-28 1992-06-11 Fundação Oswaldo Cruz (Fiocruz) - Superintendência De Planejamento Composition and its preparation process using antigen conjugated to enzymatic activity for immunological diagnosis and chagas' disease immunological diagnosis kits, for individual and epidemiological application, based on that composition
WO1994001776A1 (en) * 1992-07-10 1994-01-20 Abbott Laboratories Assay for chagas' disease and reagents for its use
WO1994026899A1 (en) * 1993-05-13 1994-11-24 Institut Français De Recherche Scientifique Pour Le Developpement En Cooperation (Orstom) Method for the culture in vitro of differents stages of tissue parasites
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US3911097A (en) * 1973-08-07 1975-10-07 Research Corp Antigenic preparation suitable for diagnosis of chronic chagas disease
EP0138101A1 (en) * 1983-09-30 1985-04-24 Rockefeller University Genetically engineered organisms expressing surface proteins of T. Cruzi
WO1992009895A1 (en) * 1990-11-28 1992-06-11 Fundação Oswaldo Cruz (Fiocruz) - Superintendência De Planejamento Composition and its preparation process using antigen conjugated to enzymatic activity for immunological diagnosis and chagas' disease immunological diagnosis kits, for individual and epidemiological application, based on that composition
WO1994001776A1 (en) * 1992-07-10 1994-01-20 Abbott Laboratories Assay for chagas' disease and reagents for its use
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US6682900B1 (en) * 1996-08-02 2004-01-27 Fundação Hemocentro de Ribeirão Preto Serological diagnosis of Chagas' disease
WO1999005528A1 (en) * 1997-07-28 1999-02-04 Immunetics Assay and device for identifying trypanosoma cruzi infection
US7749717B2 (en) 2006-10-19 2010-07-06 Abbott Laboratories Methods for the detection and diagnosis of Trypanosoma cruzi infection

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