Production and partial characterization of monoclonal antibodies to Pasteurella haemolytica A1 capsular polysaccharide and lipopolysaccharide

Hybridoma-derived monoclonal antibodies (MAB) against the cell surface antigens of Pasteurella haemolytica serotype 1 were obtained by the fusion of murine myeloma cells (P3 X 63 - Ag 8.653) with splenocytes of BALB/c mice immunized with crude logarithmic growth-phase culture supernatant. Initial screening was performed, using an ELISA, with the same bacterial growth culture supernatant as coating antigens. Further selection was done, using a panel of purified antigens--either capsular polysaccharide or lipopolysaccharide--as the coating antigen in an ELISA, and then performing a leukotoxin-neutralization assay. Two MAB, designated IIB-6 and H-2, reacted specifically with the capsular polysaccharide and the other 3, designated IVG-3, IH-3, and IIC-2, reacted with the lipopolysaccharide. One MAB, designated IH-6, did not react with leukotoxin, capsular polysaccharide, or lipopolysaccharide. The MAB to the capsular polysaccharide (IIB-6 and H-2) were characterized further; both antibodies belonged to the IgM class and were agglutinating. In addition, they promoted neutrophil-mediated opsonophagocytosis and complement-mediated immune bacteriolysis of P haemolytica serotype 1. Results from 3 studies indicated that the MAB IIB-6 and H-2 were specific only to the capsular polysaccharide of serotype 1 of P haemolytica. The MAB to the lipopolysaccharide (IVG-3, IH-3, and IIC-2) were of the IgG1, IgG3, and IgM classes, respectively and were not characterized further. The availability of a MAB identifying a serotype-specific, surface-exposed determinant on the capsule of P haemolytica serotype 1 should facilitate and expand studies concerning the role of the capsular material and lipopolysaccharide in the pathogenicity of P haemolytica infection in cattle.