Characterization of a neutralizing monoclonal antibody to Pasteurella haemolytica leukotoxin

Abstract

Six hybridoma clones producing monoclonal antibodies (MAbs) reactive with Pasteurella haemolytica A1 leukotoxin were derived from mice immunized with leukotoxin excised from sodium dodecyl sulfate-polyacrylamide gels. Of the six MAbs, only one, Ltx-2, neutralized leukotoxin in a BL-3 cell cytotoxicity assay. MAb Ltx-2 blocked association of A1 leukotoxin to BL-3 cells, as measured by flow cytometric analysis. The epitope recognized by Ltx-2 was localized to the carboxyl half of the native protein, between residues 450 and 939, by Western immunoblot analysis of CNBr fragments. Further analysis with leukotoxin deletion proteins indicated either that the Ltx-2-reactive epitope was localized in the carboxyl portion of the leukotoxin between amino acids 768 and 939 or that this region influences MAb recognition of the epitope. MAb Ltx-2 was tested for neutralizing activity against leukotoxin produced by P. haemolytica serotypes 1 through 12. The MAb neutralized leukotoxin produced by all of the A biotype isolates (serotypes 1, 5, 6, 7, 8, 9, and 12), with the exception of serotype A2, but did not neutralize any T biotype leukotoxin tested (T3, T4, or T10). The results indicate that MAb Ltx-2 neutralizes leukotoxin by interfering with target cell association and that the MAb-specific epitope is either not present or not critical for function in the leukotoxin produced by P. haemolytica serotypes A2, T3, T4, and T10.