Graduate Thesis Or Dissertation

 

Monoclonal antibodies against P-1059 strain of Pasteurella Multocida Public Deposited

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  • Monoclonal antibodies were developed against serotype 3, P-1059 strain of Pasteurella multocida. Enzyme-linked immunosorbent assays (ELISA) were used to detect antibodies in supernatants of hybridoma cells to either a surface protective (2.5 S) antigen prepared from a 2.5% saline extract or lipopolysaccharide (LPS) prepared by the phenol-water method. Forty two hybridoma cells were developed by fusing Sp 2/0 myeloma cells with spleen cells obtained from mice immunized three times with inactivated whole cells of P-1059 strain. The supernatant of four hybridomas, designated as 6EE11, D7H10, E11E3, and C11H2 were positive for antibody activity against the 2.5 S antigen, and two of them, E11E3 and C11H2, were positive for the LPS antigen. Hybridomas producing antibodies were cloned by the limiting dilution method, and four clones were obtained. The isotype of monoclonal antibodies were determined by an ELISA, indicating 6EE11 to be a IgG2b and the three others being of the IgG3 subisotype. Antigens 2.5 S or LPS were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane which was subsequently reacted with the monoclonal antibody (Western blot). 6EE11 and D7H10 monoclonal antibodies showed similar patterns in the Western blot, while E11E3 and C11H2 produced an identical pattern, albeit different from those produced by the other two. Monoclonal antibodies 6KE11 and D7H10 reacted with a major band having an apparent molecular weight of 35,500 d and at least one minor band of 78,000 d, whereas E11E3 and C11H2 recognized a band with a molecular weight of 12,500 d in the 2.5 S antigen preparation. After binding of the 2.5 S antigen to the microtiter plate, 10 mM periodic acid treated antigen reveald no remaining reactivity with E11E3 monoclonal antibody, while 6EE11 still maintained reactivity. Monoclonal antibody 6EE11 did not recognize any band in the Western blot after Proteinase K (250 μg/100 ml) treatment of the 2.5 S antigen, whereas antibody activity of E11E3 did not change. Monoclonal antibody 6EE11 did not recognize any band in the Western blot on the LPS antigen, while E11E3 had the ability to recognize a pattern of multiple bands with molecular weights varying from 12,500 to 39,000 d. These antigens were sensitive to periodate oxidation and resistant to proteinase K treatment. All the four monoclonal antibodies failed to agglutinate P-1059 or T-325 strains of P. multocida. Colonies of 15 serotypes of Pasteurella multocida were subjected to the indirect immunofluorescence assay employing the monoclonal antibodies, 6EE11 or E11E3, and fluorescein-labeled goat anti-mouse IgG. Monoclonal antibody, 6EE11, gave positive reactions with serotypes 3, 4, 9, 10, or 11, and, to a lesser extent, with type 12 organisms, but it failed to react with type 1, 5, 6, 7, 8, 13, 14, 15, or 16. E11E3 was positive only with serotype 3 strains or a type 10 strain. Both monoclonal antibodies were negative with the control organisms, P. haemolytica, Escherichia coli, Staphylococcus epidermidis or Salmonella typhimurium.
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