Isolation and characterization of partially purified leptospiral antigens

Koury, M.C.; Rangel, H.A.

doi:10.1590/S0036-46651991000500009

Abstracts

The methanol extract of Leptospira interrogans serovar canicola was purified by precipitation with acetone or acetone and chloroform. The antigenicity of the antigen was not altered by heating or treatment with pepsin and pronase. However the antigenicity was lost when the antigen was treated with periodic acid. Chemical analysis revealed the presence of 40% carbohydrate (22% methylpentose, 28%; hexoses),4% protein, 20% lipid and 2,7% phosphate. The complement fixation test with sera from patients with leptospirosis agreed with the microscopic agglutination reaction.

Leptospira; Antigens; Purification; Diagnosis

Extrato metílico de Leptospira interrogans sorovar canicola foi purificado por precipitação com acetona ou acetona e clorofórmio. A antigenicidade não foi alterada por aquecimento ou tratamento com pepsina e pronase, entretanto foi perdida quando o antígeno foi tratado com ácido periódico. Análise química revelou a presença de 40% de carboidrato (22% de metilpentose, 28% de hexose), 4% de proteína, 20% de lípide e 2,7% de fosfato. Reação de fixação de complemento realizada com soros de pacientes com leptospirose apresentou concordância com a reação de aglutinação microscópica.

IMMUNOLOGY

Isolation and characterization of partially purified leptospiral antigens

Isolamento e caracterização de antígenos parcialmente purificados de Leptospira

M.C. Koury^I; H.A. Rangel^II

^IDepartment of Microbiology, ICB/UFMg, CP 24S6, CEP 30161 Belo Horizonte, MG, Brazil

^IIDepartment of Microbiology and Immunology, Unicamp, Campinas, Brazil

SUMMARY

The methanol extract of Leptospira interrogans serovar canicola was purified by precipitation with acetone or acetone and chloroform. The antigenicity of the antigen was not altered by heating or treatment with pepsin and pronase. However the antigenicity was lost when the antigen was treated with periodic acid. Chemical analysis revealed the presence of 40% carbohydrate (22% methylpentose, 28%; hexoses),4% protein, 20% lipid and 2,7% phosphate. The complement fixation test with sera from patients with leptospirosis agreed with the microscopic agglutination reaction.

Key words: Leptospira; Antigens; Purification; Diagnosis.

RESUMO

Extrato metílico de Leptospira interrogans sorovar canicola foi purificado por precipitação com acetona ou acetona e clorofórmio. A antigenicidade não foi alterada por aquecimento ou tratamento com pepsina e pronase, entretanto foi perdida quando o antígeno foi tratado com ácido periódico. Análise química revelou a presença de 40% de carboidrato (22% de metilpentose, 28% de hexose), 4% de proteína, 20% de lípide e 2,7% de fosfato. Reação de fixação de complemento realizada com soros de pacientes com leptospirose apresentou concordância com a reação de aglutinação microscópica.

ACKNOWLEDGEMENTS

M.C. Koury wishes to acknowledge financial support from FINEP. The authors would like to thank Dr. Enio Cardillo Vieira (ICB/UFMG) for critical evaluation of the manuscript.

Recebido para publicação em 08/03/1991.

Aceito para publicação em 29/07/1991

This work was supported by FINEP.

1. ADLER, B. & FAINE, S. - Species and genus specific antigens in Leptospira revealed by monoclonal antibodies and enzyme-immunoassay. Zbl. Bakt. Hyg. Abt. Orig. A., 255: 317-322, 1983.
2. ANSON, M.L. - The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J. gen. Physiol., 22: 79-91, 1938.
3. AVRAMEAS, S. & TERNYNCK, T. - The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents. Immunochemistry, 6: 53-63, 1969.
4. BALLARD, S.A.; FAINE, S. & ADLER, B. - Purification and characterization of a protein antigen from Leptospira interrogans serovar hardjo, common to a wide range of bacteria. J. gen. Microbiol., 136: 1849-1857, 1990.
5. CHAPMAN, A.J.; ADLER, B. & FAINE, S. - Genus-specific antigens in Leptospira revealed by immunoblotting. Zbl. Bakt. Hyg. A., 264: 279-293, 1987.
6. DISCHE, Z. - Spectrophotometrie method for the determination of free pentose and pentose in nucleotides. J. biol. Chem., 181: 379-392, 1969.
7. DISCHE, Z. & SHETTELS, L.B. - A specific color reaction of methyl pentoses and a spectrophotometric micro-method for their determination, J. biol. Chem., 175: 595-603, 1948.
8. DUBOIS, M.; GILLES, K.A.; HAMILTON, J.K.; REBERS, P.A. & SMITH - Colorimetric methods for determination of sugars and related substances. Anal. Chem., 28: 350-356, 1956.
9. FAIRBANKS, G.; STECK, T.L. & WALLACH, D.F.H. - Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry, 10: 2606-2617, 1971.
10. FISKE, C.H. & SUBARROW, Y. - The colorimetric determination of phosphorus. J. biol. Chem., 66: 375-400, 1925.
11. HARTMAN, L. & LAGO, R.C.A. - Rapid preparation of fatty acid methyl esters from lipids. Lab. Pract., 22: 475-476, 1973.
12. HARTREE, E.F. - Determination of protein: a modification of Lowry method that gives a linear photometric response. Anal. Biochem., 48: 122-127, 1972.
13. KABAT, N. & MAYER, M.M. - Experimental immunochemistry. 2nd. ed. Springfield, Charles C. Thomas, 1961.
14. KASAY, N. & YANAGAWA, R. - Studies on the antigenic determination group of the type specific antigen of Leptospira canicola. Zbl. Bakt. Hyg. I. Abt. Orig. A., 228: 533-541, 1974.
15. KOURY, M.C. & CISALPINO, E.O. - Complement fixation and haemagglutination reaction with methanol extracts of icterohaemorrhagiae, canicola and patoc leptospires serovars. Rev. lat.-amer. Microbiol., 29: 283-286, 1987.
16. LUDERITZ, O.; STAUB, A.M. & WESTPHAL, O. - Immunochemistry of O and R antigens of Salmonella and related Enterobacteriaceae. Bact. Rev., 30: 192-255, 1966.
17. MAYER, M.M.; OSLER, A.G.; BIER, O.G. & HEILDERBERG, M. - Quantitative studies of complement fixation. I. A method. J. Immunol., 59: 195-206, 1948.
18. MOORE, S. & STEIN, W.H. Chromatographic determination of aminoacids by the use of automatic recording equipment. Meth. Enzimol., 6: 819-831, 1963.
19. ONO, E.; TAKASE, H.; NAIKI, M. & YANAGAWA, R. - Purification, characterization, and serological properties of a glycolipid antigen reactive with a serovar. Specific monoclonal antibody against Leptospira pomona serovar canicola. J. gen. Microbiol., 133: 1329-1336, 1987.
20. OUCHTERLONY, O. - Immunodifusion and immunoelectrophoresis. In: WEIR, B.M., ed. HANDBOOK OF EXPERIMENTAL IMMUNOLOGY. Oxford, Blackwell Sci. Publ., 1967. p. 665.
21. RANDALL, R.; WETMORE, P.W. & WARNER Jr., A.R. - Sonic vibrated leptospirae as antigens in the complement fixation test for the diagnosis of leptospirosis. J. Lab. clin. Med., 34: 1411-1415, 1949.
22. SAKAMOTO, N.; ONO, E.; KIDA, H. & YANAGAWA, R. - Characterization of a partially purified leptospiral genus specific protein antigen. Zbl. Bakt. Hyg. A., 259: 507-519, 1985.
23. SHENBERG, E. - Growth of pathogenic leptospira in chemically defined media. J. Bact., 93: 1598-1606, 1967.
24. SHINAGAWA, M. & YANAGAWA, R. - Isolation and characterization of a leptospiral type specific antigen. Infect. Immun., 5: 12-19, 1972.
25. TERPSTRA, W.G.; LIGTHART, G.S. & SCHOONE, G.J. - Serodiagnosis of human leptospirosis by enzyme linked-immunosorbent assay (Elisa). Zbl. Bakt. Hyg. I. Abt. Orig. A., 247: 400-405, 1980.
26. THIERMANN, A.B. & GARRET, L.A. - Enzyme-linked immunosorbent assay for the detection of antibodies to Leptospira interrogans serovar hardjo and pomona in cattle. Amer. J. vet. Res., 44: 884-887, 1983.
27. TREVELYAN, W.W. & HARRISON, S. - Studies on yeast metabolism. I. Fractionation and microdetermination of cell carbohydrates. Biochem. J., 50: 298-303, 1952.
28. TREVELYAN, W.A.; PROCTER, D.P. & HARRISON, J.S. - Detection of sugars on paper chromatograms. Nature, 166: 444-445, 1950.
29. WORLD HEALTH ORGANIZATION (WHO). Current problems in leptospirosis research. Wld. Hlth. Org. techn. Rep. Ser., 380: 5-25, 1967.

Publication Dates

Publication in this collection
06 Sept 2006
Date of issue
Oct 1991

History

Accepted
29 July 1991
Received
08 Mar 1991

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

[1] 1. ADLER, B. & FAINE, S. - Species and genus specific antigens in Leptospira revealed by monoclonal antibodies and enzyme-immunoassay. Zbl. Bakt. Hyg. Abt. Orig. A., 255: 317-322, 1983.

[2] 2. ANSON, M.L. - The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J. gen. Physiol., 22: 79-91, 1938.

[3] 3. AVRAMEAS, S. & TERNYNCK, T. - The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents. Immunochemistry, 6: 53-63, 1969.

[4] 4. BALLARD, S.A.; FAINE, S. & ADLER, B. - Purification and characterization of a protein antigen from Leptospira interrogans serovar hardjo, common to a wide range of bacteria. J. gen. Microbiol., 136: 1849-1857, 1990.

[5] 5. CHAPMAN, A.J.; ADLER, B. & FAINE, S. - Genus-specific antigens in Leptospira revealed by immunoblotting. Zbl. Bakt. Hyg. A., 264: 279-293, 1987.

[6] 6. DISCHE, Z. - Spectrophotometrie method for the determination of free pentose and pentose in nucleotides. J. biol. Chem., 181: 379-392, 1969.

[7] 7. DISCHE, Z. & SHETTELS, L.B. - A specific color reaction of methyl pentoses and a spectrophotometric micro-method for their determination, J. biol. Chem., 175: 595-603, 1948.

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

[9] 9. FAIRBANKS, G.; STECK, T.L. & WALLACH, D.F.H. - Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry, 10: 2606-2617, 1971.

[10] 10. FISKE, C.H. & SUBARROW, Y. - The colorimetric determination of phosphorus. J. biol. Chem., 66: 375-400, 1925.

[11] 11. HARTMAN, L. & LAGO, R.C.A. - Rapid preparation of fatty acid methyl esters from lipids. Lab. Pract., 22: 475-476, 1973.

[12] 12. HARTREE, E.F. - Determination of protein: a modification of Lowry method that gives a linear photometric response. Anal. Biochem., 48: 122-127, 1972.

[13] 13. KABAT, N. & MAYER, M.M. - Experimental immunochemistry. 2nd. ed. Springfield, Charles C. Thomas, 1961.

[14] 14. KASAY, N. & YANAGAWA, R. - Studies on the antigenic determination group of the type specific antigen of Leptospira canicola. Zbl. Bakt. Hyg. I. Abt. Orig. A., 228: 533-541, 1974.

[15] 15. KOURY, M.C. & CISALPINO, E.O. - Complement fixation and haemagglutination reaction with methanol extracts of icterohaemorrhagiae, canicola and patoc leptospires serovars. Rev. lat.-amer. Microbiol., 29: 283-286, 1987.

[16] 16. LUDERITZ, O.; STAUB, A.M. & WESTPHAL, O. - Immunochemistry of O and R antigens of Salmonella and related Enterobacteriaceae. Bact. Rev., 30: 192-255, 1966.

[17] 17. MAYER, M.M.; OSLER, A.G.; BIER, O.G. & HEILDERBERG, M. - Quantitative studies of complement fixation. I. A method. J. Immunol., 59: 195-206, 1948.

[18] 18. MOORE, S. & STEIN, W.H. Chromatographic determination of aminoacids by the use of automatic recording equipment. Meth. Enzimol., 6: 819-831, 1963.

[19] 19. ONO, E.; TAKASE, H.; NAIKI, M. & YANAGAWA, R. - Purification, characterization, and serological properties of a glycolipid antigen reactive with a serovar. Specific monoclonal antibody against Leptospira pomona serovar canicola. J. gen. Microbiol., 133: 1329-1336, 1987.

[20] 20. OUCHTERLONY, O. - Immunodifusion and immunoelectrophoresis. In: WEIR, B.M., ed. HANDBOOK OF EXPERIMENTAL IMMUNOLOGY. Oxford, Blackwell Sci. Publ., 1967. p. 665.

[21] 21. RANDALL, R.; WETMORE, P.W. & WARNER Jr., A.R. - Sonic vibrated leptospirae as antigens in the complement fixation test for the diagnosis of leptospirosis. J. Lab. clin. Med., 34: 1411-1415, 1949.

[22] 22. SAKAMOTO, N.; ONO, E.; KIDA, H. & YANAGAWA, R. - Characterization of a partially purified leptospiral genus specific protein antigen. Zbl. Bakt. Hyg. A., 259: 507-519, 1985.

[23] 23. SHENBERG, E. - Growth of pathogenic leptospira in chemically defined media. J. Bact., 93: 1598-1606, 1967.

[24] 24. SHINAGAWA, M. & YANAGAWA, R. - Isolation and characterization of a leptospiral type specific antigen. Infect. Immun., 5: 12-19, 1972.

[25] 25. TERPSTRA, W.G.; LIGTHART, G.S. & SCHOONE, G.J. - Serodiagnosis of human leptospirosis by enzyme linked-immunosorbent assay (Elisa). Zbl. Bakt. Hyg. I. Abt. Orig. A., 247: 400-405, 1980.

[26] 26. THIERMANN, A.B. & GARRET, L.A. - Enzyme-linked immunosorbent assay for the detection of antibodies to Leptospira interrogans serovar hardjo and pomona in cattle. Amer. J. vet. Res., 44: 884-887, 1983.

[27] 27. TREVELYAN, W.W. & HARRISON, S. - Studies on yeast metabolism. I. Fractionation and microdetermination of cell carbohydrates. Biochem. J., 50: 298-303, 1952.

[28] 28. TREVELYAN, W.A.; PROCTER, D.P. & HARRISON, J.S. - Detection of sugars on paper chromatograms. Nature, 166: 444-445, 1950.

[29] 29. WORLD HEALTH ORGANIZATION (WHO). Current problems in leptospirosis research. Wld. Hlth. Org. techn. Rep. Ser., 380: 5-25, 1967.

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Isolation and characterization of partially purified leptospiral antigens

Isolamento e caracterização de antígenos parcialmente purificados de Leptospira

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