1887

Abstract

We examined 10 monoclonal antibodies (mAbs) directed against proteins of 200, 170, 67, 46 and 42 kDa, and one mAb directed against a glycolipid component. The membrane association of the antigens reacting with our mAbs was investigated, in particular by phase-fractionation involving use of the detergent Triton X-114. The 170 kDa protein was shown to be membrane-associated, and surface exposure of this antigen was demonstrated by its disappearance from SDS-PAGE patterns after treatment of intact mycoplasmas with proteolytic enzymes. Cross-reactions with protein antigens of were also shown. A mAb directed against a component of a lipid extract, prepared by the methods used for preparation of the antigen used in the complement fixation (CF) test for serological diagnosis of infection, reacted with one major and a few minor bands in thin-layer chromatography (TLC) of the crude extract. The glycolipid character of this major antigen was demonstrated by treatment of the extract with sodium periodate, and by development of the TLC with orcinol/ferric chloride. These reactive bands were the same as those detected by the use of polyclonal mouse antiserum and a human convalescent serum, a result showing that the CF antigen contains a glycolipid moiety reacting with our mAb. The surface exposure of this antigen was demonstrated by binding of mAbs to intact cells.

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1989-03-01
2024-03-28
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References

  1. Baseman J.B., Dallo S.F., Tully J.G., Rose D.L. 1988; Isolation and characterization of Mycoplasma genitalium strains from the human respiratory tract. Journal of Clinical Microbiology 26:2266–2269
    [Google Scholar]
  2. Boorsma D.M., Streefkerk J.G. 1979; Periodate or glutaraldehyde for preparing peroxidase conjugates. Journal of Immunological Methods 30:245–255
    [Google Scholar]
  3. Bordier C. 1981; Phase separation of integral membrane proteins in Triton X-l14 solution. Journal of Biological Chemistry 256:1604–1607
    [Google Scholar]
  4. Brodeur B.R., Tsang P.S. 1986; High yield monoclonal antibody production in ascites. Journal of Immunological Methods 86:239–241
    [Google Scholar]
  5. Cimolai N., Bryan L.E, Woods D.E. 1987; Immunological cross-reactivity of a Mycoplasma pneumoniae membrane associated protein antigen with Mycoplasma genitalium and Acholeplasma laidlawii . Journal of Clinical Microbiology 25:2136–2139
    [Google Scholar]
  6. Clyde W.A., Hu P.C. 1986; Antigenic determinants of the attachment protein of Mycoplasma pneumoniae shared by other pathogenic Mycoplasma species. Infection and Immunity 51:690–692
    [Google Scholar]
  7. Fazekas De St Groth J., Scheidegger D. 1980; Production of monoclonal antibodies: strategy and tactics. Journal of Immunological Methods 35:1–21
    [Google Scholar]
  8. Hirschberg L., Holme T., Krook A., Vikerfors T. 1988; IgG response to Mycoplasma pneumoniae in patients with community-acquired pneumonia determined by ELISA. Acta pathologica, microbiolo-gica et immunologica scandinavica 96:605–610
    [Google Scholar]
  9. Hu P.C., Collier A.M., Baseman J.B. 1977; Surface parasitism by Mycoplasma pneumoniae of respiratory epithelium. Journal of Experimental Medicine 145:1328–1343
    [Google Scholar]
  10. Hu P.C., Cole R.M., Huang Y.S., Graham J.A., Gardner D.E., Collier A.M., Clyde W.A. 1982; Mycoplasma pneumoniae infection: role of surface protein in the attachment organelle. Science 216:313–315
    [Google Scholar]
  11. Jacin H., Mishkin A.R. 1965; Separation of carbohydrates on borate-impregnated silica gel G plates. Journal of Chromatography 18:170–173
    [Google Scholar]
  12. Jacobs E., Bennewitz A., Bredt W. 1986; Reaction pattern of human anti-Mycoplasma pneumoniae antibodies in enzyme-linked immunosorbent assays and immunoblotting. Journal of Clinical Microbiology 23:517–522
    [Google Scholar]
  13. Kenny G.E., Grayston T.J. 1965; Eaton pleuropneumonia-like organism (Mycoplasma pneumoniae) complement fixing antigen: extraction with organic solvents. Journal of Immunology 95:19–25
    [Google Scholar]
  14. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227:680–685
    [Google Scholar]
  15. Leith D.K., Trevino L.B., Tully J.G., Senterfit L.B., Baseman J.B. 1983; Host discrimination of Mycoplasma pneumoniae proteinaceous immunogens. Journal of Experimental Medicine 157:502–514
    [Google Scholar]
  16. Lind K., Lindhardt B.ø., Schütten H.J., Blom J., Christiansen C. 1984; Serological cross-reactions between Mycoplasma genitalium and Mycoplasma pneumoniae . Journal of Clinical Microbiology 20:1036–1043
    [Google Scholar]
  17. Madsen R.D., Saeed F.A., Fendly B.M., Coates S.R. 1986; Species-specific monoclonal antibody to a 43000-molecular weight membrane protein of Mycoplasma pneumoniae . Journal of Clinical Microbiology 24:680–683
    [Google Scholar]
  18. Mattsby-Baltzer I., Alving C.R. 1984; Lipid A fractions analyzed by a technique involving thin-layer chromatography and enzyme-linked immunosorbent assay. European Journal of Biochemistry 138:333–337
    [Google Scholar]
  19. Morrison-Plummer J., Jones D.H., Baseman J.B. 1983; An ELISA to detect antibodies specific for lipid determinants of Mycoplasma pneumoniae . Journal of Immunological Methods 64:165–178
    [Google Scholar]
  20. Morrison-Plummer J., Leith D.K., Baseman J.B. 1986; Biological effects of anti-lipid and antiprotein monoclonal antibodies to Mycoplasma pneumoniae . Infection and Immunity 53:398–403
    [Google Scholar]
  21. Morrison-Plummer J., Lazell A., Baseman J.B. 1987; Shared epitopes between Mycoplasma pneumoniae major adhesin protein PI and a 140-kilodalton protein of Mycoplasma genitalium . Infection and Immunity 55:49–56
    [Google Scholar]
  22. Plackett P., Marmion B.P., Shaw E.J., Lemcke R.M. 1969; Immunochemical analysis of Mycoplasma pneumoniae. 3. Separation and chemical identification of serologically active lipids. Australian Journal of Experimental Biology and Medical Science 41:171–195
    [Google Scholar]
  23. Ritchie R.F., Smith R. 1976; Immunofixation. I. General principles and application to agarose gel electrophoresis. Clinical Chemistry 22:497–499
    [Google Scholar]
  24. Riethman H.C., Boyer M.J., Wise K.S. 1987; Triton X-114 phase fractionation of an integral membrane surface protein mediating monoclonal antibody killing of Mycoplasma hyorhinis . Infection and Immunity 55:1094–1100
    [Google Scholar]
  25. Svennerholm L. 1956; The quantitative estimation of cerebrosides in nervous tissue. Journal of Neurochemistry 1:42–53
    [Google Scholar]
  26. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76:4350–4354
    [Google Scholar]
  27. Wessel D., Flügge U.I. 1984; A method for quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Analytical Biochemistry 138:141–143
    [Google Scholar]
  28. Woodward M.P., Young W.W., Bloodgood R.A. 1985; Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. Journal of Immunological Methods 78:143–153
    [Google Scholar]
  29. Vu A.C., Foy H.M., Cartwright F.D., Kenny G.E. 1987; The principal protein antigens of isolates of Mycoplasma pneumoniae as measured by levels of immunoglobulin G in human serum are stable in strains collected over a 10-year period. Infection and Immunity 55:1830–1836
    [Google Scholar]
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