1887

Abstract

Antigens of found in the supernatant of heat-treated cultures were characterized in order to explore whether antigens from this source could be used for the development of a serological test. Culture supernatants and sonicates of 12, 25 and 39 d cultures were analysed by SDS-PAGE. In culture supernatant, major protein bands of 65, 24, and 12 kDa were visible after Coomassie brilliant blue staining. Using murine monoclonal antibodies in Western blots, a pattern of protein bands distinct from that of the corresponding sonicates was found in all the culture supernatants. Gel permeation chromatography, in the presence of SDS, was used to separate the major protein bands in the culture supernatant. In ELISA, sera from 20 of 26 patients with tuberculosis reacted with fractions containing mainly 24 kDa or 12 kDa proteins, whereas none of the control sera reacted. In Western blots, each patient serum had its own characteristic banding pattern with culture supernatant, but all the sera from tuberculosis patients and control subjects reacted with protein bands of 65, 61, 58, 30 and 24 kDa. The 12 kDa protein was recognized only by sera from patients with tuberculosis in both Western blots and ELISA. This suggests that different kinds of epitopes on proteins of are detected by human antibodies in Western blots and ELISA. We assume that epitopes recognized in Western blots by patients with tuberculosis and control subjects are ubiquitous and are also present on normal commensal bacteria. Epitopes recognized by only some patients with tuberculosis in Western blots may be linear and specific. Epitopes recognized by tuberculosis patients but by none of the control subjects in ELISA may be conformation related and specific. The major protein bands found in supernatants of heat-treated cultures, 24 and 12 kDa, possess epitopes that may be specific and are potentially valuable for the development of a serological test.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-136-5-955
1990-05-01
2021-05-13
Loading full text...

Full text loading...

/deliver/fulltext/micro/136/5/mic-136-5-955.html?itemId=/content/journal/micro/10.1099/00221287-136-5-955&mimeType=html&fmt=ahah

References

  1. Aboi-Zeid C., Smith I., Grange J.M., Steele J., Rook G.A.W. 1986; Subdivision of daughter strains of Bacille Calmette-Guerin (BCG) according to secreted protein patterns. Journal of General Microbiology 132:3047–3053
    [Google Scholar]
  2. Aboi-Zeid C., Smith I., Grange J.M., Ratliff T.L., Steele J., Rook G.A.W. 1988a; The secreted antigens of Mycobacterium tuberculosis and their relationship to those recognized by the available antibodies. Journal of General Microbiology 134:531–538
    [Google Scholar]
  3. Abou-Zeid C., Ratliff T.L., Wiker H.G., Harboe M., Bennedsen J., Rook G.A.W. 1988b; Characterization of fibronectin-binding antigens released by Mycobacterium tuberculosis and Mycobacterium bovis BCG. Infection and Immunity 56:3046–3051
    [Google Scholar]
  4. Andersen A.B., Hansen E.B. 1989; Structure and mapping of antigenic domains of protein antigen b, a 38,000-molecular-weight protein of Mycobacterium tuberculosis. Infection and Immunity 57:2481–2488
    [Google Scholar]
  5. Ashbridge K.R., Booth R.J., Watson J.D., Lathigra R.B. 1989; Nucleotide sequence of the 19 kDa antigen gene from Mycobacterium tuberculosis. Nucleic Acids Research 17:1249
    [Google Scholar]
  6. Baird P.N., Hall L.M.C., Coates A.R.M. 1989; Cloning and sequence analysis of the 10 kDa antigen gene of Mycobacterium tuberculosis. Journal of General Microbiology 135:931–939
    [Google Scholar]
  7. Borremans M., De Wit L., Volkaert G., Ooms J., De Bruyn J., Huygen K., Van Vooren J., Stelandre M., Verhofstadt R., Content J. 1989; Cloning, sequence determination, and expression of a 32-kilodalton-protein gene of Mycobacterium tuberculosis. Infection and Immunity 57:3123–3130
    [Google Scholar]
  8. Closs O., Harboe M., Axelsen N.H., Bunch-Christensen K., Magnusson M. 1980; The antigens of Mycobacterium bovis,strain BCG, studied by crossed electrophoresis: a reference system. Scandinavian Journal of Immunology 12:249
    [Google Scholar]
  9. Collins F.M. 1984; Protection against mycobacterial disease by means of live vaccines tested in experimental animals. In The Mycobacteria: a Source Book pp 787–839 Kubica G.P., Wayne L.G. Edited by New York: Marcel Dekker;
    [Google Scholar]
  10. Collins F.M., Lamb J.R., Young D.B. 1988; Biological activity of protein antigens isolated from Mycobacterium tuberculosis culture filtrate. Infection and Immunity 56:1260–1266
    [Google Scholar]
  11. Daniel T.M., Anderson P.A. 1978; The isolation by immunosorbent affinity chromography and physiochemical characterization of Mycobacterium tuberculosis antigen 5. American Review of Respiratory Diseases 117:533–539
    [Google Scholar]
  12. Daniel T.M., Debanne S.M. 1987; The serodiagnosis of tuberculosis and other mycobacterial diseases by enzyme-linked immunosorbent assay. American Review of Respiratory Diseases 135:1137–1155
    [Google Scholar]
  13. De Bruyn J., Bosmans R., Nyabenda J., Van Vooren J.P. 1989; Effect of zinc deficiency on the appearance of two immunodominant protein antigens (32 kDa and 65 kDa) in the culture filtrates of mycobacteria. Journal of General Microbiology 135:79–84
    [Google Scholar]
  14. Engers H.D., Houba V., Bennedsen J., Buchanan T.M., Chaparas S.D., Kadival G., Closs O., David J.R., Vanembden J.D.A., Godal T., Mustafa S.A., Ivanyi J., Young D.B., Kaufmann S.H.E., Khomenko A.G., Kolk A.H.J., Kubin M., Louis J.A., Minden P., Shinnick T.M., Trnka L., Young R.A. 1986; Results of a World Health Organization-Sponsored Workshop to characterize antigens recognized by mycobacterium-specific monoclonal antibodies. Infection and Immunity 51:718–720
    [Google Scholar]
  15. Harboe M. 1981; Antigens of PPD, old tuberculin, and autoclaved Mycobacterium bovis BCG studied by crossed immunoelectrophoresis. American Review of Respiratory Diseases 124:80–87
    [Google Scholar]
  16. Harboe M., Nagai S., Patarroyo M.E., Torres M., Ramirez C., Cruz N. 1986; Properties of proteins MPB64, MPB70, and MPB80 of Mycobacterium bovis BCG. Infection and Immunity 52:293–302
    [Google Scholar]
  17. Ivanyi J., Bothamley G.H., Jackett P.S. 1988; Immunodiagnostic assays for tuberculosis and leprosy. British Medical Bulletin 44:635–649
    [Google Scholar]
  18. Jackett P.S., Bothamley G.H., Batra H.V., Mistry A., Young D.B., Ivanyi J. 1988; Specificity of antibodies to immunodominant mycobacterial antigens in pulmonary tuberculosis. Journal of Clinical Microbiology 26:2313–2318
    [Google Scholar]
  19. Kolk A.H.J., Minh L.H., Klatser P.R., Eggelte T.A., Kuijper S., Dejonge S., Van Leeuwen J. 1984; Production and characterization of monoclonal antibodies to Mycobacterium tuberculosis, M. bovis(BCG) and M. leprae. Clinical and Experimental Immunology 58:511–521
    [Google Scholar]
  20. Kolk A.H.J., Van Leeuwen J., Gilis H., Aglibut S., Sondij S., Kuijper S. 1986; Use of monoclonal antibodies in the identification of mycobacterial antigens. Health Cooperation Papers 7:101–106
    [Google Scholar]
  21. Kolk A.H.J., Evers R., Groothuis D.G., Gilis H., Kuijper S. 1989; Production and characterization of monoclonal antibodies against specific serotypes of Mycobacterium aviumand the Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceumcomplex. Infection and Immunity 57:2514–2521
    [Google Scholar]
  22. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 227680–685
    [Google Scholar]
  23. Minden P., Kelleher P.J., Freed J.H., Nielsen L.D., Brennan P.J., Mcpheron L., Mcclatchy J.K. 1984; Immunological evaluation of a component isolated from Mycobacterium bovisBCG with a monoclonal antibody to M. bovisBCG. Infection and Immunity 46:519–525
    [Google Scholar]
  24. Smith P.K., Krohn R.I., Hermanson G.T., Mallia A.K., Gartner F.H., Provenzano M.D., Fujimoto E.K., Goeke N.M., Olson B.J., Klenk D.C. 1985; Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150:76–85
    [Google Scholar]
  25. Thole J.E.R., Keulen W.J., De Bruyn J., Kolk A.H.J., Groothuis D.G., Berwald L.G., Tiesjema R.H., Van Embden J.D.A. 1987; Characterization, sequence determination, and immunogenicity of a 64-kilodalton protein of Mycobacterium bovisBCG expressed in Escherichia coliK-12. Infection and Immunity 55:1466–1475
    [Google Scholar]
  26. Van Schooten W.C.A., Ottenhof T.H.M., Klatser P.R., Thole J.E.R., De Vries R.R.P., Kolk A.H.J. 1988; T cell epitopes on the 36K and 65K Mycobacterium lepraeantigens defined by human T cell clones. European Journal of Immunology 18:849–854
    [Google Scholar]
  27. Verstijnen C.P.H.J., Schoningh R., Kuijper S., Bruins J., Vanketel R.J., Groothuis D.G., Kolk A.H.J. 1989; Rapid identification of cultured Mycobacterium tuberculosiswith a panel of monoclonal antibodies in western blot and immunofluorescence. Research in Microbiology 140:653–666
    [Google Scholar]
  28. Wiker H.R., Harboe M., Lea T.E. 1986; Purification and characterization of two protein antigens from the heterogeneous BCG85 complex in Mycobacterium bovisBCG. International Archives of Allergy and Applied Immunology 81:298–306
    [Google Scholar]
  29. Wiker H.R., Harboe M., Bennedsen J., Closs O. 1988; The antigens of Mycobacterium tuberculosis,H37Rv, studied by crossed immunoelectrophoresis. Comparison with a reference system for Mycobacterium bovis, BCG. Scandinavian Journal of Immunology 27:223–229
    [Google Scholar]
  30. Yamaguchi R., Matsuo K., Yamazaki A., Nagai S., Terasaka K., Yamada T. 1988; Immunogenic protein MPB57 from Mycobacterium bovis BCG: molecular cloning, nucleotide sequence and expression. FEBS Letters 240:115–117
    [Google Scholar]
  31. Yamaguchi R., Matsuo K., Yamazaki A., Abe Y., Nagai S., Terasaka K., Yamada T. 1989; Cloning and characterization of the gene for immunogenic protein MPB64 of Mycobacterium bovis. Infection and Immunity 57:283–288
    [Google Scholar]
  32. Young D.B., Lathigra R., Hendrix R., Sweetser D., Young R.A. 1988; Stress proteins are immuno targets in leprosy and tuberculosis. Proceedings of the National Academy of Sciences of the United States of America 85:4267–4270
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-136-5-955
Loading
/content/journal/micro/10.1099/00221287-136-5-955
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error