Purification and quantification of Fusobacterium necrophorum leukotoxin by using monoclonal antibodies

Monoclonal antibodies (Mabs) were produced to the leukotoxin of Fusobacterium necrophorum. Two mAbs (F7B10 and E12E9) partially neutralized leukotoxin activity, as determined by a tetrazolium (MTT)-dye reduction assay with bovine polymorphonuclear neutrophils as target cells. Immunoblot analysis showed that both clones reacted with antigens of 110 and 131 kilodaltons. Epitope analysis showed that the two mAbs recognized the same epitope. An affinity column containing immobilized mAb F7B10 was used to purify leukotoxin from crude toxin. Affinity chromatography of 1 ml of culture supernatant resulted in 0.67 microgram or 1350 units of leukotoxin. Leukotoxin was quantitated by a sandwich enzyme-linked immunosorbent assay using mAb F7B10 as the capture antibody and as the biotinylated indicator. The minimal detectable level was approximately 1 ng, corresponding to 2 leukotoxin units in the sample.