Lectins for the detection of IgM antibodies to T. gondii in the diagnosis of acute toxoplasmosis by immunofluorescence test

Nojimoto, I. T. I.; Hoshino-Shimizu, S.; Nagasse-Sugahara, T. K.; Camargo, M. E.

doi:10.1590/S0036-46651987000600004

Abstracts

Lectins were labeled with fluorescein and tried as conjugates in the immunofluorescence (IP) test for the detection of IgM antibodies to T. gondii, in the diagnosis of acute toxoplasmosis. This approach was an attempt to find alternative reagents for anti-human IgM fluorescent conjugates (AHIgMFC), which contain quite frequently anaibcdies to toxoplasma, as contaminants, due to natural T. gondii infections among animals used for imunization. Lentil (Lens culinaris) lectin fluorescence conjugates (LcFC) provided most satisfactory results. The evaluation of LcFC carried out in a total of 179 sera from patients with acute and chronic toxoplasmosis, with non-related infections or healthy subjects, gave high values of relative efficiency, co-positivity and co-negativity indices, respectively 0.989, 0.969 and 1.000, in reference to the conventional AHIgMFC. Moreover, three batches of LcFC successively prepared gave reproducible test results. The advantage of LcFC as an alternative reagent for the serodiagnosis of acute toxoplasmosis is supported by practical aspects of its preparation.

Anti-IgM detection; Lectins; Acute toxoplasmosis; Lectin conjugate

Lectinas foram marcadas com fluoresceína e testadas na reação de imunofluorescência para a detecção de anticorpos anti-T. gondii, no diagnóstico da toxoplasmose aguda. Neste estudo tentou-se encontrar reagentes alternativos para conjugados fluorescentes anti-IgM humano (CFAIgMH), que com freqüência contém anticorpos antitoxoplasma, como contaminantes, devido às infecções naturais por T. gondii, entre os animais utilizados para imunização. Conjugados fluorescentes de lectina de lentilha (CFL) (Lens culinaris) forneceram resultados satisfatórios. Avaliação do CFL efetuada em total de 179 amostras de soros de pacientes com toxoplasmose aguda e crônica, com infecções não relacionadas e de indivíduos sadios, mostrou valores altos nos índices de eficiência relativa, co-positividade e co-negatividade, respectivamente de 0,989, 0,969 e 1,000, em relação ao CFAIgMH convencional. Além disso, três partidas de CFL preparadas sucessivamente forneceram resultados reprodutíveis. A vantagem de se empregar o LFC como reagente alternativo no diagnóstico sorológico da toxoplasmose aguda consiste nos aspectos práticos da sua preparação.

ORIGINAL ARTICLES

Lectins for the detection of IgM antibodies to T. gondii in the diagnosis of acute toxoplasmosis by immunofluorescence test

Lectinas para a detecção de anticorpos IgM anti-T. gondii no diagnóstico da toxoplasmose aguda pela reação de imunofluorescência

I. T. I. Nojimoto^I; S. Hoshino-Shimizu^II; T. K. Nagasse-Sugahara^I; M. E. Camargo^I

^IInstituto de Medicina Tropical de São Paulo, Brasil

^IIFaculdade de Ciências Farmacêuticas da Universidade de São Paulo, Brasil

^{Address for correspondence} Address for correspondence: Dra. Sumie Hoshiro-Shimizu Faculdade de Ciências Farmacêuticas da USP Av. Prof. Lineu Prestes, 580 - Bloco A Conjunto das Químicas, Cidade Universitária CEP 05508 São Paulo, SP, Brasil

SUMMARY

Lectins were labeled with fluorescein and tried as conjugates in the immunofluorescence (IP) test for the detection of IgM antibodies to T. gondii, in the diagnosis of acute toxoplasmosis. This approach was an attempt to find alternative reagents for anti-human IgM fluorescent conjugates (AHIgMFC), which contain quite frequently anaibcdies to toxoplasma, as contaminants, due to natural T. gondii infections among animals used for imunization. Lentil (Lens culinaris) lectin fluorescence conjugates (LcFC) provided most satisfactory results. The evaluation of LcFC carried out in a total of 179 sera from patients with acute and chronic toxoplasmosis, with non-related infections or healthy subjects, gave high values of relative efficiency, co-positivity and co-negativity indices, respectively 0.989, 0.969 and 1.000, in reference to the conventional AHIgMFC. Moreover, three batches of LcFC successively prepared gave reproducible test results. The advantage of LcFC as an alternative reagent for the serodiagnosis of acute toxoplasmosis is supported by practical aspects of its preparation.

Key words: Anti-IgM detection; Lectins; Acute toxoplasmosis; Lectin conjugate.

RESUMO

Lectinas foram marcadas com fluoresceína e testadas na reação de imunofluorescência para a detecção de anticorpos anti-T. gondii, no diagnóstico da toxoplasmose aguda. Neste estudo tentou-se encontrar reagentes alternativos para conjugados fluorescentes anti-IgM humano (CFAIgMH), que com freqüência contém anticorpos antitoxoplasma, como contaminantes, devido às infecções naturais por T. gondii, entre os animais utilizados para imunização.

Conjugados fluorescentes de lectina de lentilha (CFL) (Lens culinaris) forneceram resultados satisfatórios.

Avaliação do CFL efetuada em total de 179 amostras de soros de pacientes com toxoplasmose aguda e crônica, com infecções não relacionadas e de indivíduos sadios, mostrou valores altos nos índices de eficiência relativa, co-positividade e co-negatividade, respectivamente de 0,989, 0,969 e 1,000, em relação ao CFAIgMH convencional. Além disso, três partidas de CFL preparadas sucessivamente forneceram resultados reprodutíveis. A vantagem de se empregar o LFC como reagente alternativo no diagnóstico sorológico da toxoplasmose aguda consiste nos aspectos práticos da sua preparação.

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ACKNOWLEDGEMENTS

We are grateful for the support received from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and from Conselho de Desenvolvimento Científico e Tecnológico (CNPq-PIDE V). Also, we thank to Miss Maria Inês Cardillo and to Almir Robson Ferreira for helping us to prepare the manuscript.

Recebido para publicação em 1/6/87.

1. AGRAWAL, B. B. L. & GOLDSTEIN, I. J. - Protein carbohydrate interaction. VI - Isolation of concanavalin-A by specific absorption on cross-linked dextran gels. Biochim. biophys. Acta. (Amst.), 147: 262-271, 1967.
2. ARDOIN, P.; COUZINNAU, P. & BAUFINE-DUCROCQ, H. - Sur l'utilization du sarcome TG-180 pour l'obtention d'une suspension riche en Toxoplasma gondii extracellulaires. C.R. Soc. Biol. (Paris), 161: 117-119, 1967.
3. CAMARGO, M. E.; LESER, P. G. & ROCCA, A. - Rheumatoid factors as a cause for false positive IgM anti-toxoplasma fluorescent tests. A technique for specific results. Rev. Inst. Med. trop. S. Paulo, 14: 310-313, 1972.
4. CAMARGO, M. E. & LESER, P. G. - Diagnostic information from serological tests in human toxoplasmosis. II - Evolutive study of antibodies and serological pattern in acquired toxoplasmosis, as detected by hemagglutination, complement fixation, IgG and IgM immunofluorescence tests. Rev. Inst. Med. trop. S. Paulo, 18: 227-238, 1976.
5. CAMARGO, M. E.; LESER, P. G. & LESER, W. S. P. - Definição de perfis sorológicos na toxoplasmose. Importância diagnostica e epidemiológica. Rev. bras. Pat. clin., 13: 113-127, 1977.
6. CAMARGO, M. B.; LESER, P. G. & ROCCA. A. - Detection of IgM anti-toxoplasma antibodies in acute acquired and congenital toxoplasmosis after protein A treatment of serum. Rev. Inst. Med. trop. S. Paulo. 25: 201-206, 1983.
7. CHATELAIN, C; LEMOINE, A.; DUGOUYON, J. M.; BLANC, M. & ROUGÉ, P. - Interaction of polyclonal and monoclonal human immunoglobulins G with various lectins (Concanavalin A, lentil and pea lectins). In BOG-HANSEN, T. C, ed. Lectins, biology, biochemistry, clinical biochemistry. New York, Walter de Gruyter, 1982. v. 2, p. 303-402.
8. CLARK, H. F. & SHEPARD, C. C. - A dialysis technique for preparing fluorescent antibody. Virology, 20: 642-644, 1S63.
9. DEUTSCH, H. & MORTON, J. I. - Dissociation of human serum macroglobulins. Science, 125: 600-601, 1957.
10. GALEN, R. S. & GAMBINO, S. R. - Beyond normality. The predictive value and efficiency medical diagnosis. New York, John Wiley, 1975.
11. HEIMBERGER, N.; HEIDE, H.; HAUPT, H. & SCHULTZE, H. E. - Baustein analysen von human serum proteinen. Clin. Chim. Acta, 10: 293-307, 1964.
12. HICKMAN, S.; KORNFELD, R.; OSTERLAND, C. K. & KORNFELD, S. - The structure of the glyco-peptides of a human δ M - immunoglobulin. J. biol. Chem., 247: 2156-2163, 1972.
13. HOSHINO-SHIMIZU, S.; MINEO, J. R. & CAMARGO, M. E. - Lectin used in the purification process of Toxoplasma gondii tachyzoites. J. Parasit., 64: 989-991, 1980.
14. HOWARD, I. K. & SAGE, H. J. - Isolation and characterization of a phyto-hemagglutinin from the lectin. Biochemistry, 8: 2436-2441, 1969.
15. HOWARD, I. K.; SAGE, H. J.; STEIN, M. D.; YOUNG, N. M.; LEON, M. A. & DYCKES, D. F. - Studies on a phytohemagglutinin from the lentil. II - Multiple forms of Lens Culinaris hemagglutinin. J. biol. Chem., 246: 1590-1595, 1971.
16. HUET, M. - Faactors affecting the molecular structure and agglutinating ability of concanavalin A and other lectins. Europ. J. Biochem., 59: 627-632, 1975.
17. LOWRY, O. H.; ROSEBROUGH, N. J.; FARR, A. L. & RANDALL, R. J. R. - Protein measurement with the folin phenol reagent. J. biol. Chem., 193: 265-271, 1951.
18. LUTZ, W. - Statistical methods as applied to immunological data. In: WEIR, D. M., ed. Handbook of experimental immunology. Oxford, Blackwell Scientific Publications, 1973. p. 1163-1202.
19. PEREIRA, M. E. A. - Lectins. In: ATASSI, M. Z. A.; VAN OSS, C. J. & ABSOLON, D. R., ed. Molecular immunology. New York, Marcel Dekker, 1984. p. 319-335.
20. SPENGLER, G. A. & WEBER, R. M. - Interactions of PHA with human normal serum proteins. In: BOG-HANSEN, T. C, ed., Lectins, biology, biochemistry, clinical biochemistry. New York, Walter de Gruyter, 1981. p. 231-240.
21. WEINSTEIN, Y.; GIVOL, D. & STRAUSBAUCH, P. H. - The fraction of immunoglobulins with insolubilized concanavalin A. J. Immunol., 109: 1402-1404, 1972.
22. WHITE, C. - Statistical methods in serums surveys. in: PAUL, J. R. & WHITE, C. - Serological epidemiology. New York, Academic Press, 1973. p. 19-32.
23. YOUNG, N. M.; LEON, M. A. & TAKAHASHI, T. - Studies on a phytohemagglutmin from the lentil. J. biol. Chem., 246: 1596-1601, 1971.

Address for correspondence:

Dra. Sumie Hoshiro-Shimizu

Faculdade de Ciências Farmacêuticas da USP

Av. Prof. Lineu Prestes, 580 - Bloco A

Conjunto das Químicas, Cidade Universitária

CEP 05508 São Paulo, SP, Brasil

Publication Dates

Publication in this collection
17 Feb 2011
Date of issue
Dec 1987

History

Received
01 June 1987

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

[1] 1. AGRAWAL, B. B. L. & GOLDSTEIN, I. J. - Protein carbohydrate interaction. VI - Isolation of concanavalin-A by specific absorption on cross-linked dextran gels. Biochim. biophys. Acta. (Amst.), 147: 262-271, 1967.

[2] 2. ARDOIN, P.; COUZINNAU, P. & BAUFINE-DUCROCQ, H. - Sur l'utilization du sarcome TG-180 pour l'obtention d'une suspension riche en Toxoplasma gondii extracellulaires. C.R. Soc. Biol. (Paris), 161: 117-119, 1967.

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

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

[5] 5. CAMARGO, M. E.; LESER, P. G. & LESER, W. S. P. - Definição de perfis sorológicos na toxoplasmose. Importância diagnostica e epidemiológica. Rev. bras. Pat. clin., 13: 113-127, 1977.

[6] 6. CAMARGO, M. B.; LESER, P. G. & ROCCA. A. - Detection of IgM anti-toxoplasma antibodies in acute acquired and congenital toxoplasmosis after protein A treatment of serum. Rev. Inst. Med. trop. S. Paulo. 25: 201-206, 1983.

[7] 7. CHATELAIN, C; LEMOINE, A.; DUGOUYON, J. M.; BLANC, M. & ROUGÉ, P. - Interaction of polyclonal and monoclonal human immunoglobulins G with various lectins (Concanavalin A, lentil and pea lectins). In BOG-HANSEN, T. C, ed. Lectins, biology, biochemistry, clinical biochemistry. New York, Walter de Gruyter, 1982. v. 2, p. 303-402.

[8] 8. CLARK, H. F. & SHEPARD, C. C. - A dialysis technique for preparing fluorescent antibody. Virology, 20: 642-644, 1S63.

[9] 9. DEUTSCH, H. & MORTON, J. I. - Dissociation of human serum macroglobulins. Science, 125: 600-601, 1957.

[10] 10. GALEN, R. S. & GAMBINO, S. R. - Beyond normality. The predictive value and efficiency medical diagnosis. New York, John Wiley, 1975.

[11] 11. HEIMBERGER, N.; HEIDE, H.; HAUPT, H. & SCHULTZE, H. E. - Baustein analysen von human serum proteinen. Clin. Chim. Acta, 10: 293-307, 1964.

[12] 12. HICKMAN, S.; KORNFELD, R.; OSTERLAND, C. K. & KORNFELD, S. - The structure of the glyco-peptides of a human δ M - immunoglobulin. J. biol. Chem., 247: 2156-2163, 1972.

[13] 13. HOSHINO-SHIMIZU, S.; MINEO, J. R. & CAMARGO, M. E. - Lectin used in the purification process of Toxoplasma gondii tachyzoites. J. Parasit., 64: 989-991, 1980.

[14] 14. HOWARD, I. K. & SAGE, H. J. - Isolation and characterization of a phyto-hemagglutinin from the lectin. Biochemistry, 8: 2436-2441, 1969.

[15] 15. HOWARD, I. K.; SAGE, H. J.; STEIN, M. D.; YOUNG, N. M.; LEON, M. A. & DYCKES, D. F. - Studies on a phytohemagglutinin from the lentil. II - Multiple forms of Lens Culinaris hemagglutinin. J. biol. Chem., 246: 1590-1595, 1971.

[16] 16. HUET, M. - Faactors affecting the molecular structure and agglutinating ability of concanavalin A and other lectins. Europ. J. Biochem., 59: 627-632, 1975.

[17] 17. LOWRY, O. H.; ROSEBROUGH, N. J.; FARR, A. L. & RANDALL, R. J. R. - Protein measurement with the folin phenol reagent. J. biol. Chem., 193: 265-271, 1951.

[18] 18. LUTZ, W. - Statistical methods as applied to immunological data. In: WEIR, D. M., ed. Handbook of experimental immunology. Oxford, Blackwell Scientific Publications, 1973. p. 1163-1202.

[19] 19. PEREIRA, M. E. A. - Lectins. In: ATASSI, M. Z. A.; VAN OSS, C. J. & ABSOLON, D. R., ed. Molecular immunology. New York, Marcel Dekker, 1984. p. 319-335.

[20] 20. SPENGLER, G. A. & WEBER, R. M. - Interactions of PHA with human normal serum proteins. In: BOG-HANSEN, T. C, ed., Lectins, biology, biochemistry, clinical biochemistry. New York, Walter de Gruyter, 1981. p. 231-240.

[21] 21. WEINSTEIN, Y.; GIVOL, D. & STRAUSBAUCH, P. H. - The fraction of immunoglobulins with insolubilized concanavalin A. J. Immunol., 109: 1402-1404, 1972.

[22] 22. WHITE, C. - Statistical methods in serums surveys. in: PAUL, J. R. & WHITE, C. - Serological epidemiology. New York, Academic Press, 1973. p. 19-32.

[23] 23. YOUNG, N. M.; LEON, M. A. & TAKAHASHI, T. - Studies on a phytohemagglutmin from the lentil. J. biol. Chem., 246: 1596-1601, 1971.

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Brasil

Lectins for the detection of IgM antibodies to T. gondii in the diagnosis of acute toxoplasmosis by immunofluorescence test

Lectinas para a detecção de anticorpos IgM anti-T. gondii no diagnóstico da toxoplasmose aguda pela reação de imunofluorescência

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History