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. 1995 Apr;33(4):952–957. doi: 10.1128/jcm.33.4.952-957.1995

Identification and characterization of outer membrane proteins of Pasteurella multocida serotype D by using monoclonal antibodies.

M V Marandi 1, K R Mittal 1
PMCID: PMC228074  PMID: 7790467

Abstract

Monoclonal antibodies (MAbs) against Pasteurella multocida serotype D were obtained by fusion of spleen cells from BALB/c mice immunized with outer membrane proteins (OMPs) with SP2/0-Ag 14 murine myeloma cells. Desirable MAbs were selected by enzyme-linked immunosorbent assay (ELISA) with OMP as the antigen. MAbs MT1 and MT2 identified two different proteins (H [heavy] and W [weak]), each with a molecular mass of 32 kDa, in Western blots (immunoblots). Treatment of the OMPs with proteolytic enzymes and sodium periodate indicated that the binding sites of MAbs MT1 and MT2 are of protein and glycoprotein natures, respectively. The epitopes reactive with MAbs were surface exposed, as visualized by immunoelectron microscopy. Among field isolates of P. multocida serotype D, two distinct OMP patterns were recognized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and these patterns were designated types I and II. In both the ELISA and the Western blot, MAb MT1 recognized only type I isolates, whereas MAb MT2 recognized both type I and II isolates. Neither MAb MT1 nor MAb MT2 reacted with either reference strains of capsular serotypes A, B, E, and F or field isolates of capsular serotype A of P. multocida. This is the first report of MAbs identifying the serotype D-specific OMP of P. multocida.

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Selected References

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  1. BOBBITT J. M. Periodate oxidation of carbohydrates. Adv Carbohydr Chem. 1956;48(11):1–41. doi: 10.1016/s0096-5332(08)60115-0. [DOI] [PubMed] [Google Scholar]
  2. Barenkamp S. J., Munson R. S., Jr, Granoff D. M. Subtyping isolates of Haemophilus influenzae type b by outer-membrane protein profiles. J Infect Dis. 1981 May;143(5):668–676. doi: 10.1093/infdis/143.5.668. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Buchanan T. M., Hildebrandt J. F. Antigen-specific serotyping of Neisseria gonorrhoeae: characterization based upon principal outer membrane protein. Infect Immun. 1981 Jun;32(3):985–994. doi: 10.1128/iai.32.3.985-994.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. CARTER G. R. Studies on Pasteurella multocida. I. A hemagglutination test for the identification of serological types. Am J Vet Res. 1955 Jul;16(60):481–484. [PubMed] [Google Scholar]
  6. Carter G. R., Rundell S. W. Identification of type A strains of P multocida using staphylococcal hyaluronidase. Vet Rec. 1975 Apr 12;96(15):343–343. doi: 10.1136/vr.96.15.343. [DOI] [PubMed] [Google Scholar]
  7. Carter G. R., Subronto P. Identification of type D strains of Pasteurella multocida with acriflavine. Am J Vet Res. 1973 Feb;34(2):293–294. [PubMed] [Google Scholar]
  8. Cloeckaert A., Zygmunt M. S., de Wergifosse P., Dubray G., Limet J. N. Demonstration of peptidoglycan-associated Brucella outer-membrane proteins by use of monoclonal antibodies. J Gen Microbiol. 1992 Jul;138(7):1543–1550. doi: 10.1099/00221287-138-7-1543. [DOI] [PubMed] [Google Scholar]
  9. Confer A. W. Immunogens of Pasteurella. Vet Microbiol. 1993 Nov;37(3-4):353–368. doi: 10.1016/0378-1135(93)90034-5. [DOI] [PubMed] [Google Scholar]
  10. Gill J., Stellwag E., Dworkin M. Monoclonal antibodies against cell-surface antigens of developing cells of Myxococcus xanthus. Ann Inst Pasteur Microbiol. 1985 Jan-Feb;136A(1):11–18. doi: 10.1016/s0769-2609(85)80015-6. [DOI] [PubMed] [Google Scholar]
  11. Hamel J., Brodeur B. R., Belmaaza A., Montplaisir S., Musser J. M., Selander R. K. Identification of Haemophilus influenzae type b by a monoclonal antibody coagglutination assay. J Clin Microbiol. 1987 Dec;25(12):2434–2436. doi: 10.1128/jcm.25.12.2434-2436.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Heddleston K. L., Gallagher J. E., Rebers P. A. Fowl cholera: gel diffusion precipitin test for serotyping Pasteruella multocida from avian species. Avian Dis. 1972 Jul-Sep;16(4):925–936. [PubMed] [Google Scholar]
  13. Ireland L., Adler B., Milner A. R. Proteins and antigens of Pasteurella multocida serotype 1 from fowl cholera. Vet Microbiol. 1991 Apr;27(2):175–185. doi: 10.1016/0378-1135(91)90009-5. [DOI] [PubMed] [Google Scholar]
  14. Johnson R. B., Dawkins H. J., Spencer T. L. Electrophoretic profiles of Pasteurella multocida isolates from animals with hemorrhagic septicemia. Am J Vet Res. 1991 Oct;52(10):1644–1648. [PubMed] [Google Scholar]
  15. Khosraviani M., Nunoya T., Matsumoto M. Monoclonal antibodies against surface antigens of Pasteurella multocida strain P-1059. Avian Dis. 1990 Jan-Mar;34(1):163–173. [PubMed] [Google Scholar]
  16. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Lariviere S., Leblanc L., Mittal K. R., Martineau G. P. Characterization of Pasteurella multocida from nasal cavities of piglets from farms with or without atrophic rhinitis. J Clin Microbiol. 1992 Jun;30(6):1398–1401. doi: 10.1128/jcm.30.6.1398-1401.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Li Z., Jensen N. S., Bélanger M., L'Espérance M. C., Jacques M. Molecular characterization of Serpulina (Treponema) hyodysenteriae isolates representing serotypes 8 and 9. J Clin Microbiol. 1992 Nov;30(11):2941–2947. doi: 10.1128/jcm.30.11.2941-2947.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lu Y. S., Lai W. C., Pakes S. P., Nie L. C. A monoclonal antibody against a Pasteurella multocida outer membrane protein protects rabbits and mice against pasteurellosis. Infect Immun. 1991 Jan;59(1):172–180. doi: 10.1128/iai.59.1.172-180.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lugtenberg B., Van Alphen L. Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteria. Biochim Biophys Acta. 1983 Mar 21;737(1):51–115. doi: 10.1016/0304-4157(83)90014-x. [DOI] [PubMed] [Google Scholar]
  22. Lugtenberg B., van Boxtel R., Evenberg D., de Jong M., Storm P., Frik J. Biochemical and immunological characterization of cell surface proteins of Pasteurella multocida strains causing atrophic rhinitis in swine. Infect Immun. 1986 Apr;52(1):175–182. doi: 10.1128/iai.52.1.175-182.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lugtenberg B., van Boxtel R., de Jong M. Atrophic rhinitis in swine: correlation of Pasteurella multocida pathogenicity with membrane protein and lipopolysaccharide patterns. Infect Immun. 1984 Oct;46(1):48–54. doi: 10.1128/iai.46.1.48-54.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mutharia L. M., Hancock R. E. Characterization of two surface-localized antigenic sites on porin protein F of Pseudomonas aeruginosa. Can J Microbiol. 1985 Apr;31(4):381–386. doi: 10.1139/m85-073. [DOI] [PubMed] [Google Scholar]
  25. Ogden J. R., Leung K. Purification of murine monoclonal antibodies by caprylic acid. J Immunol Methods. 1988 Jul 22;111(2):283–284. doi: 10.1016/0022-1759(88)90139-1. [DOI] [PubMed] [Google Scholar]
  26. Overbeeke N., Lugtenberg B. Major outer membrane proteins of Escherichia coli strains of human origin. J Gen Microbiol. 1980 Dec;121(2):373–380. doi: 10.1099/00221287-121-2-373. [DOI] [PubMed] [Google Scholar]
  27. Rimler R. B., Rhoades K. R. Serogroup F, a new capsule serogroup of Pasteurella multocida. J Clin Microbiol. 1987 Apr;25(4):615–618. doi: 10.1128/jcm.25.4.615-618.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Springer W. R., Barondes S. H. Monoclonal antibodies block cell-cell adhesion in Dictyostelium discoideum. J Biol Chem. 1983 Apr 25;258(8):4698–4701. [PubMed] [Google Scholar]
  29. Thomson M. S., Lauerman L. H., Wilt G. R. Monoclonal antibody in the identification of Haemophilus somnus. J Vet Diagn Invest. 1990 Apr;2(2):116–119. doi: 10.1177/104063879000200206. [DOI] [PubMed] [Google Scholar]
  30. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wijewardana T. G., Wilson C. F., Gilmour N. J., Poxton I. R. Production of mouse monoclonal antibodies to Pasteurella multocida type A and the immunological properties of a protective anti-lipopolysaccharide antibody. J Med Microbiol. 1990 Dec;33(4):217–222. doi: 10.1099/00222615-33-4-217. [DOI] [PubMed] [Google Scholar]
  32. Woodward M. P., Young W. W., Jr, Bloodgood R. A. Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J Immunol Methods. 1985 Apr 8;78(1):143–153. doi: 10.1016/0022-1759(85)90337-0. [DOI] [PubMed] [Google Scholar]

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