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Identification and characterization of outer membrane proteins of Pasteurella multocida serotype D by using
1Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada.
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.
Insights
Monoclonal antibodies (MAbs) were developed to identify specific outer membrane proteins (OMPs) of Pasteurella multocida serotype D. These MAbs offer a novel diagnostic tool for P. multocida serotype D detection.
Area of Science:
- Veterinary Microbiology
- Immunology
- Bacteriology
Background:
- Pasteurella multocida causes significant disease in animals.
- Accurate identification of P. multocida serotypes is crucial for effective disease control.
- Outer membrane proteins (OMPs) are key targets for serological identification.
Purpose of the Study:
- To develop and characterize monoclonal antibodies (MAbs) against Pasteurella multocida serotype D.
- To identify specific OMPs recognized by these MAbs.
- To evaluate the potential of these MAbs for serotype D identification.
Main Methods:
- Hybridoma technology for MAb production.
- Enzyme-linked immunosorbent assay (ELISA) and Western blotting for MAb screening and characterization.
- Immunoelectron microscopy for epitope localization.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for OMP analysis.
Main Results:
- Two MAbs, MT1 and MT2, were generated, recognizing distinct 32 kDa proteins (H and W).
- MAbs MT1 and MT2 target protein and glycoprotein epitopes, respectively, which are surface-exposed.
- MAb MT1 identified type I P. multocida serotype D isolates, while MAb MT2 recognized both type I and II isolates.
- Neither MAb cross-reacted with other P. multocida capsular serotypes (A, B, E, F).
Conclusions:
- This study reports the first MAbs capable of identifying serotype D-specific OMPs of P. multocida.
- These MAbs represent a valuable tool for the specific diagnosis and differentiation of P. multocida serotype D.
- The findings contribute to improved understanding and control of P. multocida infections.