Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting)

Stolf, Anna Maria Simonsen; Umezawa, Eufrosina Setsu; Zingales, Bianca

doi:10.1590/S0036-46651990000500013

Abstracts

A radioactive Western-blotting technique was developed by which the reactivity of Immunoglobulins (Igs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse T. cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgG specific antibodies. A different pattern of reactivity with acute Chagas' disease patients sera was thus obtained.

Trypanosoma cruzi; Chagas' disease; Surface antigens; Western-blot; Immunoblotting; IgG classes

Classes e subclasses de anticorpos apresentam diferentes funções, influenciando a resposta imune humoral de um hospedeiro, frente a um agente infeccioso. Na maioria dos sistemas, o alvo principal é representado pelos antígenos de membrana do parasita. Entretanto, a identificação de antígenos de superfície de parasitas, reativos para classe (e subclasse) de imunoglobulinas que não se ligam a proteína-A implica em imunoprecipitações sucessivas, que levam a perda de antígenos e/ou reações inespecíficas. Visando esse estudo, foi desenvolvida uma técnica denominada "radio-immunoblotting", através da qual a reatividade de imunoglobulinas de diferentes classes para antígenos de membrana (e/ou internos) foi analisada simultaneamente. O método constitui na marcação prévia da superfície dos parasitas por radioiodação, fracionamento dos polipeptídeos por SDS/PA-GE, transferência das frações para nitrocelulose, reação com soros e conjugados anti-Igs - peroxidase e autoradiografia dos mesmos, a análise é feita comparando-se os antígenos comuns evidenciados na autoradiografia e nas tiras de nitrocelulose coradas com o substrato da peroxidase. Essa técnica foi utilizada para analisar antígenos de superfície de formas tripomastigotas de T. cruzi reativas para IgG, IgM e IgA provenientes de soros de pacientes com doença de Chagas na fase aguda. Obtiveram-se distintos padrões de reatividade para as diferentes classes de anticorpos provenientes de um mesmo soro humano.

BRIEF REPPORT

Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting)

Identificação simultânea de antígenos internos e de superfície de Trypanosoma cruzi reativos para diferentes classes de imunoglobulinas (radio-immunoblotting)

Anna Maria Simonsen Stolf^I; Eufrosina Setsu Umezawa^II; Bianca Zingales^III

^IDepartamento de Parasitologia, Instituto de Ciências Biomédicas. Universidade de São Paulo. São Paulo, SP, Brasil

^IIInstituto de Medicina Tropical de São Paulo da Faculdade de Medicina da Universidade de São Paulo. São Paulo, SP, Brasil

^IIIDepartamento de Bioquímica, Instituto de Química, Universidade de São Paulo. São Paulo, SP, Brasil

^{Address for correspondence} Address for correspondence: Dra. Anna Maria S. Stolf Departamento de Parasitologia. Instituto de Ciências Biomédicas, Universidade de São Paulo Caixa Postal 4365 01051 São Paulo. SP. Brasil

SUMMARY

A radioactive Western-blotting technique was developed by which the reactivity of Immunoglobulins (Igs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse T. cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgG specific antibodies. A different pattern of reactivity with acute Chagas' disease patients sera was thus obtained.

Key words: Trypanosoma cruzi; Chagas' disease; Surface antigens; Western-blot; Immunoblotting; IgG classes

RESUMO

Classes e subclasses de anticorpos apresentam diferentes funções, influenciando a resposta imune humoral de um hospedeiro, frente a um agente infeccioso. Na maioria dos sistemas, o alvo principal é representado pelos antígenos de membrana do parasita. Entretanto, a identificação de antígenos de superfície de parasitas, reativos para classe (e subclasse) de imunoglobulinas que não se ligam a proteína-A implica em imunoprecipitações sucessivas, que levam a perda de antígenos e/ou reações inespecíficas.

Visando esse estudo, foi desenvolvida uma técnica denominada "radio-immunoblotting", através da qual a reatividade de imunoglobulinas de diferentes classes para antígenos de membrana (e/ou internos) foi analisada simultaneamente. O método constitui na marcação prévia da superfície dos parasitas por radioiodação, fracionamento dos polipeptídeos por SDS/PA-GE, transferência das frações para nitrocelulose, reação com soros e conjugados anti-Igs peroxidase e autoradiografia dos mesmos, a análise é feita comparando-se os antígenos comuns evidenciados na autoradiografia e nas tiras de nitrocelulose coradas com o substrato da peroxidase.

Essa técnica foi utilizada para analisar antígenos de superfície de formas tripomastigotas de T. cruzi reativas para IgG, IgM e IgA provenientes de soros de pacientes com doença de Chagas na fase aguda. Obtiveram-se distintos padrões de reatividade para as diferentes classes de anticorpos provenientes de um mesmo soro humano.

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ACKNOWLEDGMENTS

This investigation was supported by grants from the Financiadora de Estudos e Projetos (PI-NEP) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) to A.M.S.S. and to B.Z.; the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) to A.M.S.S., B.Z. and E.S.U. and the U.N.D.P./World Bank/World Health Organization Special Programme for Research and Trainning in Tropical Diseases to B.Z.

Recebido para publicação em 19/12/1989.

Abbreviations

M.W. Molecular Weight; Igs Immunoglobulins, SDS Sodium Dodecyl Sulfate; TBS- 50 mM Tris HCl, pH 8,0, 150 mM NaCl; TCT Tissue Culture Trypomastigotes.

1. ALMOND, N.M. & PARKHOUSE, R.M.E. Immunoglobulin class-specific responses to biochemically defined antigens of Trichinella spiralis. Parasite Immunol., 8: 391-406, 1986.
2. ANDERS, R.F.; HOWARD, R.J. & MITCHEL, G.F. Parasite antigens and methods of analysis. In: COHEN, S. & WARREN, K. ed. Immunology of parasitic infections. Oxford, Blackwell Scient. Publ., 1982. p. 28-73.
3. ANDREWS, N.W.; KATZIN, A.M. & COLLI, W. - Mapping of surface glycoproteins of Trypanosoma cruzi by two-dimensional electrophoresis. A correlation with the cell invasion capacity. Europ. J. Biochem., 140: 599-601, 1984.
4. BUTTERWORTH, A.E. & HAGAN, P. Immunity in human schistosomiasis. Parasit. today, 3: 11-16, 1987.
5. CABRERA, Z.; COOPER, M.D. & PARKHOUSE, R.M.E. Differential recognition patterns of human immunoglobulin classes to antigens of Onchocerca gibsoni. Tropenmed. Parasit., 37: 113-116, 1986.
6. GRZYCH, J.M.; CAPRON, M.; DISSONS, C. & CAPRON, A. Blocking activity of rat monoclonal antibodies in experimental schistosomiasis. J. Immunol., 133: 998-1004, 1984.
7. HOFSTETTER, M.; POINDEXTER, R.W.; RUIZ-TIBEN, E. & OTTENSEN, E.A. Modulation of the host response in human schistosomiasis. III. Blocking antibodies specifically inhibit immediate hipersensitivity responses to parasite antigens. Immunology, 46: 777-783, 1982.
8. KHALIFE, J.; CAPRON, M.: CAPRON, A.; GRZYCH, J.; BUTTERWORTH, A.E.; DUNNE, D.W. & OUMA, J.H. Immunity in human schistosomiasis mansoni. J. exp. Med., 164: 1626 1640, 1986.
9. KESSLER, S.W. Use of Protein A bearing Staphylococci for the immunoprecipitation and isolation of anti gens from cells. Meth. Enzymol., 73: 442- 459, 1981.
10. KURTNER, S.; PELLERIN, P.; BRENIERE, S.F.; DESJEUSE, P. & DEDET, J.P. Identification and purification of a 72kDa antigen of Leishmania brasiliensis brasiliensis present on the surface and in the cytoplasm of the promastigotes and its specific recognition by sera from mucocutaneous leishmaniasis patients. Mem. Inst. Oswaldo Cruz, 83(Supl. 1): 431-434, 1988.
11. LAEMMLI, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 237: 680-685, 1970.
12. LANAR, D.E. & MANNING, J.E. - Major surface proteins and antigens on the different in vivo and in vitro forms of Trypanosoma cruzi. Mol. Biochem. Parasit., 11: 119-131, 1984.
13. NOGUEIRA, N.; CHAPLAN, S.; TYDINGS, J.D.; UNKELESS, J. & COHN, Z. Trypanosoma cruzi: surface antigens of blood and culture forms. J. exp. Med., 153: 629-639, 1981.
14. PIRAS, R.; PIRAS, M.M. & HENRIQUEZ, D. Trypanosoma cruzi -fibroblastic cell interactions necessary for cellular invasion. In: Cytopathology of parasitic disease. London, Pitman, 1983. p. 31-51. (Ciba Foundation Symposium 99).
15. PRIMAVERA, K.S.C.; HOSHINO-SHIMIZU, S.; UMEZAWA, E.S.; PERES, B.A.; MANIGOT, D.A. & CAMARGO, M.E. Immunoglobulin A antibodies to Trypanosoma cruzi antigens in digestive forms of Chagas' disease. J. clin. Microbiol., 26: 2101-2104, 1988.
16. SÁ FERREIRA, J.A.; GALVĂO CASTRO, B.; MACEDO, W. & CASTRO, C. Immunoglobulins and other serological parameters in Chagas' disease: evidence for increased IgA levels in the chronic digestive form. Clin. exp. Immunol., 52: 266-270, 1983.
17. SCHECHTER, M. & NOGUEIRA, N. Variations induced by different methodologies in Trypanosoma cruzi surface antigen profiles. Mol. Biochem. Parasit., 29: 37-46, 1988.
18. SHAKIB, F. & STANWORTH, D.R. Human IgG subclasses in health and disease. Ric. clin. lab., 10: 463-580, 1980.
19. TOWBIN, H.; STAEHELIN, T. & GORDON, J. Electrophoresis transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. nat. Acad. Sci. (Wash.), 76: 4350-4354, 1979.
20. ZINGALES, B.; ANDREWS, N.W.; KUWAJIMA, V.Y. & COLLI, W. Cell surface antigens of Trypanosoma cruzi: possible correlation with the interiorization process in mammalian cells. Mol. Biochem. Parasit., 6: 111-124, 1982.

Address for correspondence:

Dra. Anna Maria S. Stolf

Departamento de Parasitologia. Instituto de Ciências Biomédicas, Universidade de São Paulo

Caixa Postal 4365

01051 São Paulo. SP. Brasil

Publication Dates

Publication in this collection
12 Sept 2006
Date of issue
Oct 1990

History

Received
19 Dec 1989

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. ALMOND, N.M. & PARKHOUSE, R.M.E. Immunoglobulin class-specific responses to biochemically defined antigens of Trichinella spiralis. Parasite Immunol., 8: 391-406, 1986.

[2] 2. ANDERS, R.F.; HOWARD, R.J. & MITCHEL, G.F. Parasite antigens and methods of analysis. In: COHEN, S. & WARREN, K. ed. Immunology of parasitic infections. Oxford, Blackwell Scient. Publ., 1982. p. 28-73.

[3] 3. ANDREWS, N.W.; KATZIN, A.M. & COLLI, W. - Mapping of surface glycoproteins of Trypanosoma cruzi by two-dimensional electrophoresis. A correlation with the cell invasion capacity. Europ. J. Biochem., 140: 599-601, 1984.

[4] 4. BUTTERWORTH, A.E. & HAGAN, P. Immunity in human schistosomiasis. Parasit. today, 3: 11-16, 1987.

[5] 5. CABRERA, Z.; COOPER, M.D. & PARKHOUSE, R.M.E. Differential recognition patterns of human immunoglobulin classes to antigens of Onchocerca gibsoni. Tropenmed. Parasit., 37: 113-116, 1986.

[6] 6. GRZYCH, J.M.; CAPRON, M.; DISSONS, C. & CAPRON, A. Blocking activity of rat monoclonal antibodies in experimental schistosomiasis. J. Immunol., 133: 998-1004, 1984.

[7] 7. HOFSTETTER, M.; POINDEXTER, R.W.; RUIZ-TIBEN, E. & OTTENSEN, E.A. Modulation of the host response in human schistosomiasis. III. Blocking antibodies specifically inhibit immediate hipersensitivity responses to parasite antigens. Immunology, 46: 777-783, 1982.

[8] 8. KHALIFE, J.; CAPRON, M.: CAPRON, A.; GRZYCH, J.; BUTTERWORTH, A.E.; DUNNE, D.W. & OUMA, J.H. Immunity in human schistosomiasis mansoni. J. exp. Med., 164: 1626 1640, 1986.

[9] 9. KESSLER, S.W. Use of Protein A bearing Staphylococci for the immunoprecipitation and isolation of anti gens from cells. Meth. Enzymol., 73: 442- 459, 1981.

[10] 10. KURTNER, S.; PELLERIN, P.; BRENIERE, S.F.; DESJEUSE, P. & DEDET, J.P. Identification and purification of a 72kDa antigen of Leishmania brasiliensis brasiliensis present on the surface and in the cytoplasm of the promastigotes and its specific recognition by sera from mucocutaneous leishmaniasis patients. Mem. Inst. Oswaldo Cruz, 83(Supl. 1): 431-434, 1988.

[11] 11. LAEMMLI, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 237: 680-685, 1970.

[12] 12. LANAR, D.E. & MANNING, J.E. - Major surface proteins and antigens on the different in vivo and in vitro forms of Trypanosoma cruzi. Mol. Biochem. Parasit., 11: 119-131, 1984.

[13] 13. NOGUEIRA, N.; CHAPLAN, S.; TYDINGS, J.D.; UNKELESS, J. & COHN, Z. Trypanosoma cruzi: surface antigens of blood and culture forms. J. exp. Med., 153: 629-639, 1981.

[14] 14. PIRAS, R.; PIRAS, M.M. & HENRIQUEZ, D. Trypanosoma cruzi -fibroblastic cell interactions necessary for cellular invasion. In: Cytopathology of parasitic disease. London, Pitman, 1983. p. 31-51. (Ciba Foundation Symposium 99).

[15] 15. PRIMAVERA, K.S.C.; HOSHINO-SHIMIZU, S.; UMEZAWA, E.S.; PERES, B.A.; MANIGOT, D.A. & CAMARGO, M.E. Immunoglobulin A antibodies to Trypanosoma cruzi antigens in digestive forms of Chagas' disease. J. clin. Microbiol., 26: 2101-2104, 1988.

[16] 16. SÁ FERREIRA, J.A.; GALVĂO CASTRO, B.; MACEDO, W. & CASTRO, C. Immunoglobulins and other serological parameters in Chagas' disease: evidence for increased IgA levels in the chronic digestive form. Clin. exp. Immunol., 52: 266-270, 1983.

[17] 17. SCHECHTER, M. & NOGUEIRA, N. Variations induced by different methodologies in Trypanosoma cruzi surface antigen profiles. Mol. Biochem. Parasit., 29: 37-46, 1988.

[18] 18. SHAKIB, F. & STANWORTH, D.R. Human IgG subclasses in health and disease. Ric. clin. lab., 10: 463-580, 1980.

[19] 19. TOWBIN, H.; STAEHELIN, T. & GORDON, J. Electrophoresis transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. nat. Acad. Sci. (Wash.), 76: 4350-4354, 1979.

[20] 20. ZINGALES, B.; ANDREWS, N.W.; KUWAJIMA, V.Y. & COLLI, W. Cell surface antigens of Trypanosoma cruzi: possible correlation with the interiorization process in mammalian cells. Mol. Biochem. Parasit., 6: 111-124, 1982.

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