Leishmania braziliensis: isolation of carbohydrate-containing antigen and possibility of its use in the immunodiagnosis of American Cutaneous Leishmaniasis

Silveira, T. G. V.; Kemmelmeier, C

doi:10.1590/S0036-46651995000300011

Abstracts

Leishmania braziliensis is a causative agent of American Cutaneous Leishmaniasis (ACL). The 034-JCG strain, isolated from a patient from the northern region of Paraná State, Brazil, was cultivated in Blood Agar Base medium, lyophilized and submitted to phenol-water extraction. The extract was treated with RNase I. The carbohydrate containing-antigen (Ag-CHO) was immunogenic to rabbits and showed at least a fraction with some negative charge at pH 8.2. This antigen showed cross-reactivity with the phenol-water extract of the growth medium used for the culture of promastigotes and with the surface antigens of promastigotes. Its composition is: 24.3% of total sugars, from which 11.2% of galactose, 7.5% of mannose and 5.6% of ribose. Protein content was 5.4% and phosphate 18.5%. The antigenic activity was maintained after: repeated freezing-thawing; lyophilization; heating at 100ºC for 30 minutes; treatment with RNase, trichloroacetic acid and sodium metaperiodate. The precipitin line obtained is Periodic Acid Schiff positive. The application of the Ag-CHO in counterimmunoelectrophoresis reaction for the immunodiagnosis of ACL showed 60% sensitivity, and no cross-reaction with the five sera of Chagas' disease patients tested. The use of this antigen in a more sensitive technique, with more samples of sera, may improve these results.

Leishmania braziliensis; Carbohydrate-containing antigen; Immunodiagnosis

A Leishmania braziliensis é um dos agentes causadores da Leishmaniose Tegumentar Americana (LTA). A cepa 034-JCG, isolada de paciente da região norte do estado do Paraná, Brasil, foi cultivada em meio Blood Agar Base, liofilizada e submetida a extração com fenol-água e o extrato obtido foi tratado com RNase I. O antígeno contendo carboidrato (Ag-CHO) mostrou-se imunogênico para coelhos e apresentou pelo menos uma fração com poucas cargas negativas em pH 8,2. Este antígeno apresenta reação cruzada com extrato fenólico do meio de cultura usado para o cultivo de promastigotas e com antígenos de superfície de promastigotas. É composto por 24,3% de açúcares totais, dos quais 11,2% de galactose, 7,5% de manose e 5,6% de ribose. As proteínas foram estimadas em 5,4% e fosfato em 18,5%. A atividade antigênica foi mantida após: congelamentos e descongelamentos repetidos; liofilização; aquecimento a 100ºC durante 30 minutos; tratamento com RNase, ácido tricloroacético e periodato de sódio. As linhas obtidas nas reações de precipitação coram-se pelo Ácido Periódico de Schiff. A utilização do Ag-CHO na reação de contraimunoeletroforese para o imunodiagnóstico de LTA mostrou 60% de sensibilidade, e nenhuma reação cruzada com os 5 soros de pacientes com Doença de Chagas testados. O uso deste antígeno com técnicas mais sensíveis, e com um maior número de amostras poderá melhorar estes resultados.

DIAGNOSTIC STUDIES

Leishmania braziliensis: isolation of carbohydrate-containing antigen and possibility of its use in the immunodiagnosis of American Cutaneous Leishmaniasis

Leishmania braziliensis: isolamento de antígeno contendo carboidrato e a possibilidade de sua aplicação no imunodiagnóstico da Leishmaniose Tegumentar Americana

T. G. V. Silveira^I; C. Kemmelmeier^II

^IDepartamento de Análises Clinicas - Universidade Estadual de Maringá. Maringá, Pr., Brasil

^IIDepartamento de Bioquímica - Universidade Estadual de Maringá. Maringá, Pr., Brasil

^{Correspondence to} Correspondence to: T. G. V. Silveira Lab. Imunologia Clínica, Departamento de Análises Clínicas - Universidade Estadual de Maringá Av. Colombo, 5790 87.020-900 Maringá, Pr., Brasil

SUMMARY

Leishmania braziliensis is a causative agent of American Cutaneous Leishmaniasis (ACL). The 034-JCG strain, isolated from a patient from the northern region of Paraná State, Brazil, was cultivated in Blood Agar Base medium, lyophilized and submitted to phenol-water extraction. The extract was treated with RNase I. The carbohydrate containing-antigen (Ag-CHO) was immunogenic to rabbits and showed at least a fraction with some negative charge at pH 8.2. This antigen showed cross-reactivity with the phenol-water extract of the growth medium used for the culture of promastigotes and with the surface antigens of promastigotes. Its composition is: 24.3% of total sugars, from which 11.2% of galactose, 7.5% of mannose and 5.6% of ribose. Protein content was 5.4% and phosphate 18.5%. The antigenic activity was maintained after: repeated freezing-thawing; lyophilization; heating at 100ºC for 30 minutes; treatment with RNase, trichloroacetic acid and sodium metaperiodate. The precipitin line obtained is Periodic Acid Schiff positive. The application of the Ag-CHO in counterimmunoelectrophoresis reaction for the immunodiagnosis of ACL showed 60% sensitivity, and no cross-reaction with the five sera of Chagas' disease patients tested. The use of this antigen in a more sensitive technique, with more samples of sera, may improve these results.

Keywords:Leishmania braziliensis; Carbohydrate-containing antigen; Immunodiagnosis.

RESUMO

A Leishmania braziliensis é um dos agentes causadores da Leishmaniose Tegumentar Americana (LTA). A cepa 034-JCG, isolada de paciente da região norte do estado do Paraná, Brasil, foi cultivada em meio Blood Agar Base, liofilizada e submetida a extração com fenol-água e o extrato obtido foi tratado com RNase I. O antígeno contendo carboidrato (Ag-CHO) mostrou-se imunogênico para coelhos e apresentou pelo menos uma fração com poucas cargas negativas em pH 8,2. Este antígeno apresenta reação cruzada com extrato fenólico do meio de cultura usado para o cultivo de promastigotas e com antígenos de superfície de promastigotas. É composto por 24,3% de açúcares totais, dos quais 11,2% de galactose, 7,5% de manose e 5,6% de ribose. As proteínas foram estimadas em 5,4% e fosfato em 18,5%. A atividade antigênica foi mantida após: congelamentos e descongelamentos repetidos; liofilização; aquecimento a 100ºC durante 30 minutos; tratamento com RNase, ácido tricloroacético e periodato de sódio. As linhas obtidas nas reações de precipitação coram-se pelo Ácido Periódico de Schiff. A utilização do Ag-CHO na reação de contraimunoeletroforese para o imunodiagnóstico de LTA mostrou 60% de sensibilidade, e nenhuma reação cruzada com os 5 soros de pacientes com Doença de Chagas testados. O uso deste antígeno com técnicas mais sensíveis, e com um maior número de amostras poderá melhorar estes resultados.

Full text available only in PDF format.

Texto completo disponível apenas em PDF.

ACKNOWLEDGMENT

The authors wish to thank Dr. Adelar Bracht for the English review of the manuscript.

Recebido para publicação em 07/06/1994.

Aceito para publicação em 02/05/1995.

1. ALBERSHEIM, P.; NEVINS, D.J.; ENGLISH, P.D. & KARR, A. - A method for the analysis of sugars in plant cell wall polysaccharides by gas-liquid chromatography. Carbohydr. Res., 5: 340-345, 1967.
2. AMES, B.N. - Assay of inorganic phosphate, total phosphate and phosphatases. Meth. Enzymol., 8: 115-118, 1966.
3. AYESTA, C.; ARGÜELLO, C. & HERNANDEZ, A.G. - Leishmania braziliensis: cell surface differences in promastigotes of pathogenic and nonpathogenic strains. Exp. Parasit., 59: 185-191, 1985.
4. BEHMER, O.A.; TOLOSA, E.M.C. & FREITAS NETO, A.G. - Manual de técnicas para histología e patologia. Săo Paulo, EDUSP, 1976.
5. BURNS Jr., J.M.; SHREFFLER, W.G.; BENSON, D.R. et al. - Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis. Proc. nat. Acad. Sci. (Wash.), 90: 775-779, 1993.
6. CAMARGO, M.E. - Introduçăo ŕs técnicas de imunofluorescęncia. Săo Paulo, Instituto de Medicina Tropical de Săo Paulo, 1973. p. 60-61.
7. CLINTON, B.A.; PALCZUK, N.C. & STAUBER, L.A. - Leishmania donovani: partial characterization of some flagellate cytoplasmic immunogens. J. Immunol., 108: 1570-1577, 1972.
8. DAWIDOVICZ, K.; HERNANDEZ, A.G. & INFANTE, R.B. - The surface membrane of Leishmania. I. The effects of lectins on different stages of Leishmania braziliensis. J. Parasit., 61: 950-953, 1975.
9. DECKER-JACKSON, J.E. & HONIGBERG, B.M. - Glycoproteins released by Leishmania donovani: immunologic relationship with host and bacterial antigens and preliminary biochemical analysis. J. Protozool., 25: 514-525, 1978.
10. DISCHE, A. - Color reactions of 2-deoxysugars. In: WHISTLER, R.L. & WOLFROM, M.L., ed. - Methods in Carbohydrate Chemistry. New York, Academic Press, 1962a. v. 1, p. 503-507.
11. DISCHE, A. - Color reactions of pentoses. In: WHISTLER, R.L. & WOLFROM, M.L., ed. - Methods in Carbohydrate Chemistry. New York, Academic Press, 1962b. v. 1, p.484-488.
12. DUBOIS, M.; GILLES, K.A.; HAMILTON, J.K.; REBERS, P.A. & SMITH, F. - Colorimetric method for determination of sugars and related substances. Analyt. Chem., 28: 350-356, 1956.
13. DWYER, D.M. - Leishmania donovani: surface membrane carbohydrates of promastigotes. Exp. Parasit., 41: 341-358, 1977.
14. EL-ON, J. & SCHNUR, L.F. - Purification and preliminary characterization of leishmanial excreted factors (EF). J. Protozool., 21: 463-464, 1974.
15. EL-ON, J.; SCHNUR, L.F. & GREENBLATT, C.L. - Leishmania donovani: physicochemical, immunological, and biological characterization of excreted factor from promastigotes. Exp. Parasit., 47: 254-269, 1979.
16. FURTADO, T. - Critérios para o diagnóstico da leishmaniose tegumentar americana. An. bras. Derm., 55: 81-86, 1980.
17. GARDINER, P.R.; JAFFE, C.L. & DWYER, D.M. - Identification of cross-reactive promastigote cell surface antigens of some leishmanial stocks by ¹²⁵I labeling and immunoprecipitation. Infect. Immun., 43: 637-643, 1984.
18. GOTTLIEB, M. - A carbohydrate-containing antigen from Trypanosoma cruzi and its detection in the circulation of infected mice. J. Immunol., 119: 465-470, 1977.
19. GUIMARĂES, M.C.S.; GIOVANNINI, V.L. & CAMARGO, M.E. - Antigenic standardization for mucocutaneous leishmaniasis immunofluorescence test. Rev. Inst. Med. trop. S. Paulo, 16: 145-148, 1974.
20. GUIMARĂES, M.C.S.; CELESTE, B.J. & FRANCO, E.L. - Evaluation of dot enzyme-linked immunosorbent assay for mucocutaneous leishmaniasis and comparison with microplate enzyme immunoassay. J. clin. Microbiol., 24: 364-367, 1986.
21. GUIMARĂES, M.C.S.; CELESTE, B.J.; CORRALES, E.M. & ANTUNES, C.M.F. - Comparison on the performance of Leishmania major-like and Leishmania braziliensis braziliensis as antigen for new world leishmaniasis IgG-immunofluorescence test. Rev. Inst. Med. trop. S. Paulo, 33: 503-508, 1991.
22. JACOBSON, R.L.; SLUTZKY, G.M.; GREENBLATT, C.L. & SCHNUR, L.F. - Surface reaction of Leishmania. I. Lectin-mediated agglutination. Ann. trop. Med. Parasit., 76: 45-52, 1982.
23. KANESHIRO, E.S.; GOTTLIEB, M. & DWYER, D.M. - Cell surface origin of antigens shed by Leishmania donovani during growth in axenic culture. Infect. Immun., 37: 558-567, 1982.
24. LOWRY, O.H.; ROSEBROUGH, N.J.; FARR, A.L. & RANDALL, R.J. - Protein measurement with the folin phenol reagent. J. biol. Chem., 193: 265-275, 1951.
25. McCONVILLE, J.L.; HOMANS, S.W.; THOMAS-OATES, J.E.; DELL, A. & BACIC, A. - Structures of the glycoinositolphospholipids from Leishmania major: a family of novel galactofuranose-containing glycolipids. J. biol. Chem., 265: 7385-7394, 1990.
26. PALATNIK, C.B.; PREVIATO, J.O.; GORIN, P.A.J. & PREVIATO, L.M. - Partial chemical characterization of the carbohydrate moieties in Leishmania adleri glycoconjugates. Molec. Biochem. Parasit., 14: 41-54, 1985.
27. PAPPAS, M.G.; McGREEVY, P.B.; HAJKOWSKI, R. et al. - Evaluation of promastigote and amastigote antigens in the indirect fluorescent antibody test for American cutaneous leishmaniasis. Amer. J. trop. Med. Hyg., 32: 1260-1267, 1983.
28. ROFFI, J.; DEDET, J.P.; DESJEUX, P. & GARRÉ, M.T. - Detection of circulating antibodies in cutaneous leishmaniasis by enzyme-linked immunosorbent assay (ELISA). Amer. J. trop. Med. Hyg., 29: 183-189, 1980.
29. ROSEN, G.; LONDNER, M.V.; GREENBLATT, C.L.; MORSY, T.A. & EL-ON, J. - Leishmania major: solid phase radioimmunoassay for antibody detection in human cutaneous leishmaniasis. Exp. Parasit., 62: 79-84, 1986.
30. ROSEN, G.; LONDNER, M.V.; SEVLEVER, D. & GREENBLATT, C.L. - Leishmania major: glycolipid antigens recognized by immune human sera. Molec. Biochem. Parasit., 27: 93-100, 1988.
31. SANTOS, J.I.; MORGADO, M.G. & GALVĂO-CASTRO, B. - Human visceral leishmaniasis: analysis of the specificity of humoral immune response to polypeptides of Leishmania donovani chagasi. Amer. J. trop. Med. Hyg., 37: 263-270, 1987.
32. SAWARDEKER, J.S.; SLONEKER, J.H. & JEANES, A. - Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography. Analyt. Chem., 37: 1602-1604, 1965.
33. SCHNUR, L.F.; ZUCKERMAN, A. & GREENBLATT, C.L. - Leishmanial serotypes as distinguished by the gel diffusion of factors excreted "in vitro" e "in vivo". Israel J. med. Sci, 8: 932-942, 1972.
34. SEMPREVIVO, L.H. - Exometabolites of Leishmania donovani promastigotes. I. Isolation and initial characterization. Proc. Soc. exp. Biol. Med., 159: 105-110, 1978.
35. SHAW, J.J. & LAINSON, R. - A simply prepared amastigote leishmanial antigen for use in the indirect fluorescent antibody test for Leishmaniasis. J. Parasit., 63: 384-385, 1977.
36. SILVEIRA, T.G.V.; ARRAES, S.M.A.A.; PEREIRA, D.S. et al. - Avaliaçăo da reaçăo de imunofluorescęncia indireta para leishmaniose tegumentar americana em pacientes da regiăo noroeste do estado do Paraná-Brasil. Rev. Unimar, 12: 177-188, 1990.
37. WALTON, B.C.; BROOKS, W.H. & ARJONA, I. - Serodiagnosis of American Leishmaniasis by indirect fluorescent antibody test. Amer. J. trop. Med. Hyg., 21: 296-299, 1972.
38. WALTON, B.C.; SHAW, J.J. & LAINSON, R. - Observations on the "in vitro" cultivation of Leishmania hraziliensis. J. Parasit., 63: 1118-1119, 1977.
39. WARR, G.W. - Purification of antibodies. In: MARCHALONIS, J.J. & WARR, G.W, ed. Antibody as a toll: the applications of immunochemistry. New York, The Pitman Press, 1982. p. 62.
40. WESTPHAL, O. & JANN, K. - Bacterial lipopolysaccharides: extraction with phenol-water and further applications of the procedure. In: WHISTLER, R.L. & WOLFROM, M.L., ed. Methods in carbohydrate chemistry. New York, Academic Press, 1965. v. 5, p. 83-91.
41. WILSON, M.E. & PEARSON, R.D. - Stage-specific variations in lectin binding to Leishmania donovani. Infect. Immun., 46: 128-134, 1984.
42. YAMAMOTO, Y.I. - Leishmaniose cutâneo-mucosa: reaçőes de hemaglutinaçăo passiva e de contraimunoeletroforese com antígeno proteico de Leishmania braziliensis Vianna, 1911. Săo Paulo, 1980. (Dissertaçăo de Mestrado - Faculdade de Cięncias Farmacęuticas da Universidade de Săo Paulo).
43. ZEHAVI, U.; ABRAHAMS, J.C.; GRANOTH, R. et al. - Leishmanial excreted factors (EFs): purification by affinity chromatography. Z. Parasitenk., 69: 695-701, 1983.

Correspondence to:

T. G. V. Silveira

Lab. Imunologia Clínica, Departamento de Análises Clínicas - Universidade Estadual de Maringá

Av. Colombo, 5790

87.020-900 Maringá, Pr., Brasil

Publication Dates

Publication in this collection
21 Sept 2006
Date of issue
June 1995

History

Accepted
02 May 1995
Received
07 June 1994

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

[1] 1. ALBERSHEIM, P.; NEVINS, D.J.; ENGLISH, P.D. & KARR, A. - A method for the analysis of sugars in plant cell wall polysaccharides by gas-liquid chromatography. Carbohydr. Res., 5: 340-345, 1967.

[2] 2. AMES, B.N. - Assay of inorganic phosphate, total phosphate and phosphatases. Meth. Enzymol., 8: 115-118, 1966.

[3] 3. AYESTA, C.; ARGÜELLO, C. & HERNANDEZ, A.G. - Leishmania braziliensis: cell surface differences in promastigotes of pathogenic and nonpathogenic strains. Exp. Parasit., 59: 185-191, 1985.

[4] 4. BEHMER, O.A.; TOLOSA, E.M.C. & FREITAS NETO, A.G. - Manual de técnicas para histología e patologia. Săo Paulo, EDUSP, 1976.

[5] 5. BURNS Jr., J.M.; SHREFFLER, W.G.; BENSON, D.R. et al. - Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis. Proc. nat. Acad. Sci. (Wash.), 90: 775-779, 1993.

[6] 6. CAMARGO, M.E. - Introduçăo ŕs técnicas de imunofluorescęncia. Săo Paulo, Instituto de Medicina Tropical de Săo Paulo, 1973. p. 60-61.

[7] 7. CLINTON, B.A.; PALCZUK, N.C. & STAUBER, L.A. - Leishmania donovani: partial characterization of some flagellate cytoplasmic immunogens. J. Immunol., 108: 1570-1577, 1972.

[8] 8. DAWIDOVICZ, K.; HERNANDEZ, A.G. & INFANTE, R.B. - The surface membrane of Leishmania. I. The effects of lectins on different stages of Leishmania braziliensis. J. Parasit., 61: 950-953, 1975.

[9] 9. DECKER-JACKSON, J.E. & HONIGBERG, B.M. - Glycoproteins released by Leishmania donovani: immunologic relationship with host and bacterial antigens and preliminary biochemical analysis. J. Protozool., 25: 514-525, 1978.

[10] 10. DISCHE, A. - Color reactions of 2-deoxysugars. In: WHISTLER, R.L. & WOLFROM, M.L., ed. - Methods in Carbohydrate Chemistry. New York, Academic Press, 1962a. v. 1, p. 503-507.

[11] 11. DISCHE, A. - Color reactions of pentoses. In: WHISTLER, R.L. & WOLFROM, M.L., ed. - Methods in Carbohydrate Chemistry. New York, Academic Press, 1962b. v. 1, p.484-488.

[12] 12. DUBOIS, M.; GILLES, K.A.; HAMILTON, J.K.; REBERS, P.A. & SMITH, F. - Colorimetric method for determination of sugars and related substances. Analyt. Chem., 28: 350-356, 1956.

[13] 13. DWYER, D.M. - Leishmania donovani: surface membrane carbohydrates of promastigotes. Exp. Parasit., 41: 341-358, 1977.

[14] 14. EL-ON, J. & SCHNUR, L.F. - Purification and preliminary characterization of leishmanial excreted factors (EF). J. Protozool., 21: 463-464, 1974.

[15] 15. EL-ON, J.; SCHNUR, L.F. & GREENBLATT, C.L. - Leishmania donovani: physicochemical, immunological, and biological characterization of excreted factor from promastigotes. Exp. Parasit., 47: 254-269, 1979.

[16] 16. FURTADO, T. - Critérios para o diagnóstico da leishmaniose tegumentar americana. An. bras. Derm., 55: 81-86, 1980.

[17] 17. GARDINER, P.R.; JAFFE, C.L. & DWYER, D.M. - Identification of cross-reactive promastigote cell surface antigens of some leishmanial stocks by ¹²⁵I labeling and immunoprecipitation. Infect. Immun., 43: 637-643, 1984.

[18] 18. GOTTLIEB, M. - A carbohydrate-containing antigen from Trypanosoma cruzi and its detection in the circulation of infected mice. J. Immunol., 119: 465-470, 1977.

[19] 19. GUIMARĂES, M.C.S.; GIOVANNINI, V.L. & CAMARGO, M.E. - Antigenic standardization for mucocutaneous leishmaniasis immunofluorescence test. Rev. Inst. Med. trop. S. Paulo, 16: 145-148, 1974.

[20] 20. GUIMARĂES, M.C.S.; CELESTE, B.J. & FRANCO, E.L. - Evaluation of dot enzyme-linked immunosorbent assay for mucocutaneous leishmaniasis and comparison with microplate enzyme immunoassay. J. clin. Microbiol., 24: 364-367, 1986.

[21] 21. GUIMARĂES, M.C.S.; CELESTE, B.J.; CORRALES, E.M. & ANTUNES, C.M.F. - Comparison on the performance of Leishmania major-like and Leishmania braziliensis braziliensis as antigen for new world leishmaniasis IgG-immunofluorescence test. Rev. Inst. Med. trop. S. Paulo, 33: 503-508, 1991.

[22] 22. JACOBSON, R.L.; SLUTZKY, G.M.; GREENBLATT, C.L. & SCHNUR, L.F. - Surface reaction of Leishmania. I. Lectin-mediated agglutination. Ann. trop. Med. Parasit., 76: 45-52, 1982.

[23] 23. KANESHIRO, E.S.; GOTTLIEB, M. & DWYER, D.M. - Cell surface origin of antigens shed by Leishmania donovani during growth in axenic culture. Infect. Immun., 37: 558-567, 1982.

[24] 24. LOWRY, O.H.; ROSEBROUGH, N.J.; FARR, A.L. & RANDALL, R.J. - Protein measurement with the folin phenol reagent. J. biol. Chem., 193: 265-275, 1951.

[25] 25. McCONVILLE, J.L.; HOMANS, S.W.; THOMAS-OATES, J.E.; DELL, A. & BACIC, A. - Structures of the glycoinositolphospholipids from Leishmania major: a family of novel galactofuranose-containing glycolipids. J. biol. Chem., 265: 7385-7394, 1990.

[26] 26. PALATNIK, C.B.; PREVIATO, J.O.; GORIN, P.A.J. & PREVIATO, L.M. - Partial chemical characterization of the carbohydrate moieties in Leishmania adleri glycoconjugates. Molec. Biochem. Parasit., 14: 41-54, 1985.

[27] 27. PAPPAS, M.G.; McGREEVY, P.B.; HAJKOWSKI, R. et al. - Evaluation of promastigote and amastigote antigens in the indirect fluorescent antibody test for American cutaneous leishmaniasis. Amer. J. trop. Med. Hyg., 32: 1260-1267, 1983.

[28] 28. ROFFI, J.; DEDET, J.P.; DESJEUX, P. & GARRÉ, M.T. - Detection of circulating antibodies in cutaneous leishmaniasis by enzyme-linked immunosorbent assay (ELISA). Amer. J. trop. Med. Hyg., 29: 183-189, 1980.

[29] 29. ROSEN, G.; LONDNER, M.V.; GREENBLATT, C.L.; MORSY, T.A. & EL-ON, J. - Leishmania major: solid phase radioimmunoassay for antibody detection in human cutaneous leishmaniasis. Exp. Parasit., 62: 79-84, 1986.

[30] 30. ROSEN, G.; LONDNER, M.V.; SEVLEVER, D. & GREENBLATT, C.L. - Leishmania major: glycolipid antigens recognized by immune human sera. Molec. Biochem. Parasit., 27: 93-100, 1988.

[31] 31. SANTOS, J.I.; MORGADO, M.G. & GALVĂO-CASTRO, B. - Human visceral leishmaniasis: analysis of the specificity of humoral immune response to polypeptides of Leishmania donovani chagasi. Amer. J. trop. Med. Hyg., 37: 263-270, 1987.

[32] 32. SAWARDEKER, J.S.; SLONEKER, J.H. & JEANES, A. - Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography. Analyt. Chem., 37: 1602-1604, 1965.

[33] 33. SCHNUR, L.F.; ZUCKERMAN, A. & GREENBLATT, C.L. - Leishmanial serotypes as distinguished by the gel diffusion of factors excreted "in vitro" e "in vivo". Israel J. med. Sci, 8: 932-942, 1972.

[34] 34. SEMPREVIVO, L.H. - Exometabolites of Leishmania donovani promastigotes. I. Isolation and initial characterization. Proc. Soc. exp. Biol. Med., 159: 105-110, 1978.

[35] 35. SHAW, J.J. & LAINSON, R. - A simply prepared amastigote leishmanial antigen for use in the indirect fluorescent antibody test for Leishmaniasis. J. Parasit., 63: 384-385, 1977.

[36] 36. SILVEIRA, T.G.V.; ARRAES, S.M.A.A.; PEREIRA, D.S. et al. - Avaliaçăo da reaçăo de imunofluorescęncia indireta para leishmaniose tegumentar americana em pacientes da regiăo noroeste do estado do Paraná-Brasil. Rev. Unimar, 12: 177-188, 1990.

[37] 37. WALTON, B.C.; BROOKS, W.H. & ARJONA, I. - Serodiagnosis of American Leishmaniasis by indirect fluorescent antibody test. Amer. J. trop. Med. Hyg., 21: 296-299, 1972.

[38] 38. WALTON, B.C.; SHAW, J.J. & LAINSON, R. - Observations on the "in vitro" cultivation of Leishmania hraziliensis. J. Parasit., 63: 1118-1119, 1977.

[39] 39. WARR, G.W. - Purification of antibodies. In: MARCHALONIS, J.J. & WARR, G.W, ed. Antibody as a toll: the applications of immunochemistry. New York, The Pitman Press, 1982. p. 62.

[40] 40. WESTPHAL, O. & JANN, K. - Bacterial lipopolysaccharides: extraction with phenol-water and further applications of the procedure. In: WHISTLER, R.L. & WOLFROM, M.L., ed. Methods in carbohydrate chemistry. New York, Academic Press, 1965. v. 5, p. 83-91.

[41] 41. WILSON, M.E. & PEARSON, R.D. - Stage-specific variations in lectin binding to Leishmania donovani. Infect. Immun., 46: 128-134, 1984.

[42] 42. YAMAMOTO, Y.I. - Leishmaniose cutâneo-mucosa: reaçőes de hemaglutinaçăo passiva e de contraimunoeletroforese com antígeno proteico de Leishmania braziliensis Vianna, 1911. Săo Paulo, 1980. (Dissertaçăo de Mestrado - Faculdade de Cięncias Farmacęuticas da Universidade de Săo Paulo).

[43] 43. ZEHAVI, U.; ABRAHAMS, J.C.; GRANOTH, R. et al. - Leishmanial excreted factors (EFs): purification by affinity chromatography. Z. Parasitenk., 69: 695-701, 1983.

Brasil