Production of mouse monoclonal antibodies to Pasteurella multocida type A and the immunological properties of a protective anti-lipopolysaccharide antibody

Abstract

Eight monoclonal antibodies (MAbs) were produced from mice immunised with whole cells of heat-killed Pasteurella multocida type A which had been cultured under iron-restricted conditions. The MAbs were selected by an enzyme-linked immunosorbent assay (ELISA) in which the antigen consisted of whole bacteria of the immunising strain. Their reactivity was investigated further by immunoblotting, indirect haemagglutination, a complement-mediated bactericidal assay and passive protection of mice. One of the eight MAbs was shown by immunoblotting to react with lipopolysaccharide (LPS), was bactericidal, and completely protected mice against homologous challenge with 10 LD50 of live bacteria. This MAb was selected for further study. Its reaction with LPS of 17 type-A strains and of single strains of types B, D and E was investigated by immunoblotting. Strains that reacted with the anti-LPS MAb in immunoblots were susceptible to its bactericidal activity and gave high ELISA absorbances. Those that did not react were not susceptible to its bactericidal activity and gave low ELISA readings. The relation between bactericidal activity and ELISA absorbance was highly significant (p less than 0.001). Five of the strongly reacting heterologous strains and one non-reacting strain were selected as challenge organisms in a passive protection experiment: only the mice receiving the reacting strains were protected.