Generation of monoclonal antibodies to Pasteurella haemolytica leukotoxin : characterization of a neutralizing monoclonal antibody

Pasteurella haemolytica produces a potent leukotoxin that is believed to play a major role in bovine pneumonic pasteurellosis. Very little is understood regarding the antigenic and functional domains of the leukotoxin molecule. This study was undertaken to further understanding of antigenic domains of P. haemolytica leukotoxin. Two approaches were taken to define antigenic determinants. One approach was to predict potential antigenic regions in the leukotoxin sequence by hydrophilicity and antigenicity algorithms and synthesize peptides for the generation of polyclonal antisera that would recognize native polypeptide. The other approach was to generate monoclonal antibodies (MAbs) to leukotoxin using electrophoretically purified antigen. Three peptides were identified and synthesized but only one peptide successfully generated rabbit antisera reactive with leukotoxin. Unfortunately, the epitope identified did not appear to be a neutralizing epitope and antisera to the epitope appeared to preferentially recognize denatured leukotoxin. However, six MAbs reactive with P. haemolytica leukotoxin were derived from mice immunized with leukotoxin excised from SDS-polyacrylamide gels. Of the six MAbs, only one, Ltx-2, neutralized leukotoxin in a BL-3 cytotoxicity assay. MAb Ltx-2 blocked binding of A1 leukotoxin to BL-3 cells as measured by flow cytometry. The potential epitope for MAb Ltx-2 was mapped with CNBr cleavage fragments, mutant deletion proteins and alkaline phosphatase/leukotoxin fusion proteins and found to be between amino acids 918 and 939. MAb Ltx-2 was further tested for neutralizing activity against leukotoxin produced by P. haemolytica serotypes 1-12. The MAb neutralized leukotoxin from all of the A biotypes 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, TIO). The results indicate that MAb Ltx-2 reacts with a leukotoxin epitope associated with the membrane binding function of leukotoxin and the epitope is either not present or expressed noncritically for function in leukotoxin produced by P. haemolytica serotypes A2, T3, T4 and TIO.