Quantitation and purification of the Pasteurella multocida toxin by using monoclonal antibodies

Pasteurella multocida toxin (PMT), derived from a toxigenic strain of P. multocida originally isolated from a pig with clinical atrophic rhinitis, was used to immunize BALB/c mice. Ninety-two hybridomas secreting monoclonal antibodies (MAbs) against PMT were produced by fusion of spleen cells from these mice with P3-X63-Ag8.653 myeloma cells. The specificity for PMT of the MAbs was ascertained by enzyme-linked immunosorbent assay and immunoblotting analysis. The interrelationship of a panel of 10 MAbs and their respective epitopes was characterized by a competitive enzyme-linked immunosorbent assay based on the biotin-avidin system and by an in vitro neutralization assay based on the cytopathic effect of PMT on embryonic bovine lung cells. In vivo neutralization of the lethal effect of PMT in mice was obtained by passive immunization with an anti-PMT MAb 2 days before challenge with PMT. PMT was quantified by a sandwich enzyme-linked immunosorbent assay with a lower detection limit of approximately 50 pg of PMT. Application of supernatant or bacterial extract from cultivation of toxigenic P. multocida to an affinity column containing immobilized MAb resulted in purification of PMT with a yield of 78 to 93% of the PMT applied.