Production and characterisation of monoclonal antibodies to outer-membrane-protein antigens of O1 Free

Abstract

Summary

Monoclonal antibodies (MAbs) were raised against the major, 46–48-Kda outer-membrane proteins of O1. The hybridoma clones were screened by enzyme-linked immunosorbent assay (ELISA) with cell-surface proteins of O1 as the coating antigen. Four hybridomas, which secreted anti- cell-surface-protein antibodies, were subcloned by limiting dilution and obtained as ascites A MAb of the IgG subclass was isolated in good yield from the murine ascites by affinity chromatography with recombinant protein G-Sepharose 4B. It gave positive reactions, as determined by ELISA, against cell-surface proteins prepared from both biotypes (classical and E1 Tor) and both serotypes (Ogawa and Inaba) of O1. The MAb did not have any reactivity towards lipopolysaccharide preparations. Immunoblotting studies were performed on cell-surface proteins separated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE) and also by two-dimensional (2D) electrophoresis with iso-electric focusing in the first dimension followed by SDS-PAGE in the second dimension. When proteins were separated by 1D SDS-PAGE, only one band at 46–48 Kda reacted with the MAb. This protein appeared to consist of two narrowly-spaced and cross-reactive bands when a nitrocellulose blot, obtained by 2D SDS-PAGE, was exposed to the MAb.

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1991-03-01
2024-03-29
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References

  1. Kabir S. Composition and immunochemical properties of outermembrane proteins of Vibrio cholerae. J Bacteriol 1980; 144:382–389
    [Google Scholar]
  2. Kabir S. Antigenic analysis of Vibrio cholerae Ol by crossed-immunoelectrophoresis. Zentralbl Bakteriol Mikrobiol Hyg 1989; A270:361–372
    [Google Scholar]
  3. Kabir S. Composition and immunochemical properties of the cell surface proteins of Vibrio cholerae. J Gen Microbiol 1986; 132:2235–2242
    [Google Scholar]
  4. Richardson K., Parker C. D. Identification and characterization of Vibrio cholerae surface proteins by radioiodination. Infect Immun 1985; 48:87–93
    [Google Scholar]
  5. Kabir S. Immunological responses of rabbits to various somatic and secreted antigens of Vibrio cholerae after intraduodenal inoculation. J Med Microbiol 1987; 24:29–40
    [Google Scholar]
  6. Sciortino C. V., Yang Z., Finkelstein R. A. Monoclonal antibodies to outer membrane antigens of Vibrio cholerae. Infect Immun 1985; 49:122–131
    [Google Scholar]
  7. Kabir S. The serological properties of the cell surface proteins of Vibrio cholerae. J Gen Microbiol 1983; 129:2199–2206
    [Google Scholar]
  8. Schrier M., Kohler G., Hengartner H., Berek C., Trucco M., Fomi L. Hybridoma techniques, 1st edn. Cold Spring Harbor; New York, Cold Spring Harbor Laboratory: 1980
    [Google Scholar]
  9. Oi V. T., Herzenberg L. A. Immunoglobulin-producing hybrid cell lines. In Mishell B. B., Shiigi S. M. (eds) Selected methods in cellular immunology New York: W. H. Freeman and Company; 1980351–372
    [Google Scholar]
  10. Ouchterlony Ö. Antigen-antibody reactions in gels. Acta Pathol MicrobiolScand 1949; 26:507–515
    [Google Scholar]
  11. King J., Laemmli U. K. Polypeptides of the tail fibres of bacteriophage T4. J Mol Biol 1971; 62:465–477
    [Google Scholar]
  12. Kabir S. Charge heterogeneity of cholera toxin and its subunits. FEMS Microbiol Lett 1986; 37:155–162
    [Google Scholar]
  13. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem 1984; 136:175–179
    [Google Scholar]
  14. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265–275
    [Google Scholar]
  15. Dubois M., Gilles K. A., Hamilton J. K., Rebers P. A., Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956; 28:350–356
    [Google Scholar]
  16. Ohlson S., Wieslander J. High-performance liquid affinity chromatographic separation of mouse monoclonal antibodies with protein A silica. J Chromatogr 1987; 397:207–212
    [Google Scholar]
  17. Björck L., Kronvall G. Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J Immunol 1984; 133:969–974
    [Google Scholar]
  18. Bruck C., Portelle D., Glineur C., Bollen A. One-step purification of mouse monoclonal antibodies from ascitic fluid by DEAE Affi-gel blue chromatography. J Immunol Methods 1982; 53:313–319
    [Google Scholar]
  19. Rosenbusch J. P. Characterization of the major envelope protein from Escherichia coli. J Biol Chem 1974; 249:8019–8029
    [Google Scholar]
  20. Nakae T. Outer membrane of Salmonella. Isolation of protein complex that produces transmembrane channels. J Biol Chem 1976; 215:2176–2178
    [Google Scholar]
  21. Reithmeier R. A. F., Bragg P. D. Molecular characterization of a heat-modifiable protein from the outer membrane of Escherichia coli. Arch Biochem Biophys 1977; 178:527–534
    [Google Scholar]
  22. Overbeeke N., Van Scharrenburg G., Lugtenberg B. Antigenic relationships between pore proteins of Escherichia coli K12. Eur J Biochem 1980; 110:247–254
    [Google Scholar]
  23. Hofstra H., Dankert J. Porin from the outer membrane of Escherichia coli: immunological characterization of native and heat-dissociated forms. J Gen Microbiol 1981; 125:285–292
    [Google Scholar]
  24. Nikaido H., Wu H. C. P. Amino acids sequence homology among the major outer membrane proteins of Escherichia coli. Proc Natl Acad Sci USA 1984; 81:1048–1052
    [Google Scholar]
  25. Sciortino C. V., Finkelstein R. A. Vibrio cholerae expresses iron-regulated outer membrane proteins in vivo. Infect Immun 1983; 42:990–996
    [Google Scholar]
  26. Kelley J. T., Parker C. D. Identification and preliminary characterization of Vibrio cholerae outer membrane proteins. J Bacteriol 1981; 145:1018–1024
    [Google Scholar]
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