Neutralizing monoclonal antibodies to Pasteurella haemolytica leukotoxin affinity-purify the toxin from crude culture supernatants

Abstract

The leukotoxin of Pasteurella haemolytica is a major virulence factor of the organism. It is an unstable protein which has proven very difficult to purify using traditional techniques. Hybridomas secreting monoclonal antibodies (mAbs) to P. haemolytica leukotoxin were derived from spleen cells of a mouse immunized with crude culture supernatant. Five hybridomas secreting mAbs specific for the leukotoxin were stabilized. Each of the mAbs reacted with a protein of approximately 100 kDa in toxic culture supernatants, and two of them completely neutralized the toxin in vitro. Affinity chromatography of crude culture supernatant on a column prepared with one of the neutralizing mAbs resulted in the isolation of biologically active toxin.