Pesquisa de anticorpos IgM anti-Toxoplasma gondii por meio de técnica imunoenzimática reversa

Mineo, José Roberto; Camargo, Mário Endsfeldz; Ferreira, Antonio Walter; Almeida, Gastão

doi:10.1590/S0036-46651986000100002

Resumos

Um teste imunoenzimático reverso foi padronizado utilizando-se como fase sólida, microplacas de polivinil sensibilizadas com anticorpos anti-IgM.7 Estas foram incubadas seqüencialmente com alíquotas de soros de pacientes com suspeita de toxoplasmose aguda, antígeno solúvel de Toxoplasma gondii, conjugado peroxidase F (ab')2 anti-toxoplasma e substrato enzimático. A atividade enzimática foi determinada por leitura espectrofotométrica, considerando-se como títulos dos soros a máxima diluição fornecendo valores de absorbância maiores que os obtidos com a menor diluição do soro padrão não-reativo. Em 69 amostras de soros de pacientes com toxoplasmose aguda, a média geométrica dos títulos no teste ELISA-Reverso IgM foi superior à de todos os outros testes para anticorpos IgM, não se observando resultados negativos falsos devidos a altos títulos de IgG específica. Não foi encontrada, também, reatividade cruzada em nenhuma das 104 amostras de soros de pacientes com outras patologias, inclusive em amostras contendo fator reumatóide IgM.

Toxoplasmose; Diagnóstico; Sorologia; Imunoenzimologia reversa

A Reverse IgM enzyme — linked immunosorbent assay (Reverse-IgM ELISA) was described in which polyvinyl microplates were sensitized with anti-immunoglobulin M (IgM) antibodies and then sequentially allowed to react with patient's serum, Toxoplasma gondii soluble antigen, peroxidase — labeled specific F(ab')2 fragment and substrat. Measurement of activity of the solid phase bound enzyme conjugate was done by spectrofotometric reading of the final developed color. In a group of 69 sera samples from individuals with recently acquired toxoplasmosis, Reverse-IgM ELISA was positive in every case, showing higher geometric mean titer than others conventional serological tests. Furthermore false-negative IgM samples presented positive Reverse-IgM ELISA results even without adsorption with Staphylococcus aureus protein A . In a group of 104 sera samples from individuals with others pathologies, no interference was observed, even in the false-positive IgM samples due to the presence of IgM rheumatoid factor.

ARTIGOS ORIGINAIS

Pesquisa de anticorpos IgM anti-Toxoplasma gondii por meio de técnica imunoenzimática reversa

Reverse IgM enzyme-linked immunosorbent assay (Reverse IgM-ELISA) for toxoplasmosis serodiagnosis

José Roberto Mineo; Mário Endsfeldz Camargo; Antonio Walter Ferreira; Gastão Almeida

Departamento de Ciências Fundamentais para a saúde, Universidade Federal de Uberlândia

^{Presente endereço} Presente endereço: Departamento de Ciências Fundamentais para a saúde Universidade Federal de Uberlândia MG, Brasil, CEP 38400

RESUMO

Um teste imunoenzimático reverso foi padronizado utilizando-se como fase sólida, microplacas de polivinil sensibilizadas com anticorpos anti-IgM.₇ Estas foram incubadas seqüencialmente com alíquotas de soros de pacientes com suspeita de toxoplasmose aguda, antígeno solúvel de Toxoplasma gondii, conjugado peroxidase F (ab')₂ anti-toxoplasma e substrato enzimático. A atividade enzimática foi determinada por leitura espectrofotométrica, considerando-se como títulos dos soros a máxima diluição fornecendo valores de absorbância maiores que os obtidos com a menor diluição do soro padrão não-reativo. Em 69 amostras de soros de pacientes com toxoplasmose aguda, a média geométrica dos títulos no teste ELISA-Reverso IgM foi superior à de todos os outros testes para anticorpos IgM, não se observando resultados negativos falsos devidos a altos títulos de IgG específica. Não foi encontrada, também, reatividade cruzada em nenhuma das 104 amostras de soros de pacientes com outras patologias, inclusive em amostras contendo fator reumatóide IgM.

Unitermos: Toxoplasmose Diagnóstico Sorologia Imunoenzimologia reversa.

SUMMARY

A Reverse IgM enzyme linked immunosorbent assay (Reverse-IgM ELISA) was described in which polyvinyl microplates were sensitized with anti-immunoglobulin M (IgM) antibodies and then sequentially allowed to react with patient's serum, Toxoplasma gondii soluble antigen, peroxidase labeled specific F(ab')₂ fragment and substrat. Measurement of activity of the solid phase bound enzyme conjugate was done by spectrofotometric reading of the final developed color. In a group of 69 sera samples from individuals with recently acquired toxoplasmosis, Reverse-IgM ELISA was positive in every case, showing higher geometric mean titer than others conventional serological tests. Furthermore false-negative IgM samples presented positive Reverse-IgM ELISA results even without adsorption with Staphylococcus aureus protein A . In a group of 104 sera samples from individuals with others pathologies, no interference was observed, even in the false-positive IgM samples due to the presence of IgM rheumatoid factor.

Texto completo disponível apenas em PDF.

Full text available only in PDF format.

Recebido para publicação em 28-2-1985

Trabalho realizado no Laboratório de Imunologia do Instituto de Medicina Tropical de São Paulo.

1. AMBROISE-THOMAS, P.; FRANESIO, J.; SIMON, J.; MICOUIN, C. & PIERSON, Y. Les facteurs rheumatoids. Cause de non-especificité de l'immunofluorescence anti-IgM dans la toxoplasmose. Ann. Biol. Clin. 38: 315-319, 1980.
2. ARAÚJO, F. G.; BARNETT, E. V. & GENTRY, L. O. False-positive antitoxoplasma fluorescent-antibody tests in patients with antinuclear antibodies. Appl. Microbiol. 22: 270-275, 1971.
3. AVRAMEAS, S. & TERNYNCK, T. The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents. Immunochemistry 6: 53-66, 1969.
4. CAMARGO, M. E.; FERREIRA, A. W.; MINEO, J. R.; TAKIGUTI, C. K. & NAKAHARA, O. S. Immunoglobulin G and immunoglobulin M enzyme-linked immunosorbent assays and defined toxoplasmosis serological patterns. Infect. & Immun. 21: 55-58, 1978.
5. CAMARGO, M. E. & LESER, P. G. Diagnostic information from serological tests in human toxoplasmosis. II. Evolutive study of antibodies and serological patterns in acquired toxoplasmosis, as detected by hemagglutination, complement fixation, igG and IgM-immunofluorescence tests. Rev. Inst. Med. trop. São Paulo 18: 227-238, 1976.
6. CAMARGO, M. E.; LESER, P. G. & LESER, W. S. P. Diagnostic information from serological tests in human toxoplasmosis. I. A comparative study of hemagglutination, complement fixation, IgG and IgM-lmmunofluorescence tests in 3,752 serum samples. Rev. Inst. Med. trap. São Paulo 18: 215-226, 1976.
7. CAMARGO, M. E.; LESER, P. G. & ROCCA, A. Rheumatoid factors as a cause for false positive IgM anti-toxoplasrna fluorescent tests. A technique for especific results. Rev. Inst. Med. trop. São Paulo 14: 310-313, 1972.
8. CHANTLER, S.; DEVRIES, E.; ALLEN, P. R. & HURN, B. A. L. A rapid immunofluorescent procedure for the detection of specific IgG and IgM antibody in sera using Staphylococcus aureus and latex-IgG as absorbents. J. Immunol. Meth. 13: 367-380, 1976.
9. FRANCO, E. L.; WALLS, K. W. & SULZER, A. J. Reverse enzyme immunoassay for detection of specific antitoxoplasma immunoglobulin M. antibodies. J. Clin. Microbiol. 13: 859-864, 1981.
10. FRANCO, E. L.; WALLS, K. W.; SULZER, A. J. & SOTO, J. C. Diagnosis of acute acquired toxoplasmosis with the enzyme labelled antigen Reversed Immunoassay for immunoglobulin M antibodies. J. Immunoassay 4: 373-393, 1983.
11. MICHELI, A. & ISLDKER, H. Cleavage of Gamma. M-globulin by means of reducing enzyme systems. Immunochemistry 3: 385-392, 1966.
12. MINEO, J. R.; CAMARGO, M. E. & FERREIRA, A. W. Enzyme-linked immunosorbent assay for antibodies to Toxoplasma gondii polissaccharides in human toxoplasmosis. Infect. & Immun. 27: 283-287, 1980.
13. NAOT, Y. & REMINGTON. J. S. An enzyme-linked immunosorbent assay in detection of IgM antibodies to Toxoplasma gondii: use for diagnosis of acute acquired toxoplasmosis. J. Infect. Dis. 142: 757-766, 1980.
14. WILSON, M. B. & NAKANE, P. K. Recent developments in the periodate-method or conjugating horseradish peroxidase (HRPO) to antibodies. In: KNAPP, HOLOBAR, K. & WICK, G. ed. Immunofluorescence and Related Techniques. Amsterdam, NortHolland Biomedical, 1978, pag. 215.

Presente endereço:

Departamento de Ciências Fundamentais para a saúde

Universidade Federal de Uberlândia

MG, Brasil, CEP 38400

Datas de Publicação

Publicação nesta coleção
16 Out 2012
Data do Fascículo
Fev 1986

Histórico

Recebido
28 Fev 1985

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

[1] 1. AMBROISE-THOMAS, P.; FRANESIO, J.; SIMON, J.; MICOUIN, C. & PIERSON, Y. Les facteurs rheumatoids. Cause de non-especificité de l'immunofluorescence anti-IgM dans la toxoplasmose. Ann. Biol. Clin. 38: 315-319, 1980.

[2] 2. ARAÚJO, F. G.; BARNETT, E. V. & GENTRY, L. O. False-positive antitoxoplasma fluorescent-antibody tests in patients with antinuclear antibodies. Appl. Microbiol. 22: 270-275, 1971.

[3] 3. AVRAMEAS, S. & TERNYNCK, T. The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents. Immunochemistry 6: 53-66, 1969.

[4] 4. CAMARGO, M. E.; FERREIRA, A. W.; MINEO, J. R.; TAKIGUTI, C. K. & NAKAHARA, O. S. Immunoglobulin G and immunoglobulin M enzyme-linked immunosorbent assays and defined toxoplasmosis serological patterns. Infect. & Immun. 21: 55-58, 1978.

[5] 5. CAMARGO, M. E. & LESER, P. G. Diagnostic information from serological tests in human toxoplasmosis. II. Evolutive study of antibodies and serological patterns in acquired toxoplasmosis, as detected by hemagglutination, complement fixation, igG and IgM-immunofluorescence tests. Rev. Inst. Med. trop. São Paulo 18: 227-238, 1976.

[6] 6. CAMARGO, M. E.; LESER, P. G. & LESER, W. S. P. Diagnostic information from serological tests in human toxoplasmosis. I. A comparative study of hemagglutination, complement fixation, IgG and IgM-lmmunofluorescence tests in 3,752 serum samples. Rev. Inst. Med. trap. São Paulo 18: 215-226, 1976.

[7] 7. CAMARGO, M. E.; LESER, P. G. & ROCCA, A. Rheumatoid factors as a cause for false positive IgM anti-toxoplasrna fluorescent tests. A technique for especific results. Rev. Inst. Med. trop. São Paulo 14: 310-313, 1972.

[8] 8. CHANTLER, S.; DEVRIES, E.; ALLEN, P. R. & HURN, B. A. L. A rapid immunofluorescent procedure for the detection of specific IgG and IgM antibody in sera using Staphylococcus aureus and latex-IgG as absorbents. J. Immunol. Meth. 13: 367-380, 1976.

[9] 9. FRANCO, E. L.; WALLS, K. W. & SULZER, A. J. Reverse enzyme immunoassay for detection of specific antitoxoplasma immunoglobulin M. antibodies. J. Clin. Microbiol. 13: 859-864, 1981.

[10] 10. FRANCO, E. L.; WALLS, K. W.; SULZER, A. J. & SOTO, J. C. Diagnosis of acute acquired toxoplasmosis with the enzyme labelled antigen Reversed Immunoassay for immunoglobulin M antibodies. J. Immunoassay 4: 373-393, 1983.

[11] 11. MICHELI, A. & ISLDKER, H. Cleavage of Gamma. M-globulin by means of reducing enzyme systems. Immunochemistry 3: 385-392, 1966.

[12] 12. MINEO, J. R.; CAMARGO, M. E. & FERREIRA, A. W. Enzyme-linked immunosorbent assay for antibodies to Toxoplasma gondii polissaccharides in human toxoplasmosis. Infect. & Immun. 27: 283-287, 1980.

[13] 13. NAOT, Y. & REMINGTON. J. S. An enzyme-linked immunosorbent assay in detection of IgM antibodies to Toxoplasma gondii: use for diagnosis of acute acquired toxoplasmosis. J. Infect. Dis. 142: 757-766, 1980.

[14] 14. WILSON, M. B. & NAKANE, P. K. Recent developments in the periodate-method or conjugating horseradish peroxidase (HRPO) to antibodies. In: KNAPP, HOLOBAR, K. & WICK, G. ed. Immunofluorescence and Related Techniques. Amsterdam, NortHolland Biomedical, 1978, pag. 215.