PURIFICATION AND QUANTIFICATION OF FUSOBACTERIUM-NECROPHORUM LEUKOTOXIN BY USING MONOCLONAL-ANTIBODIES

Monoclonal antibodies (Mabs) were produced to the leukotoxin of Fusoba cterium necrophorum. Two mAbs (F7B10 and E12E9) partially neutralized leukotoxin activity, as determined by a tetrazolium (MTT)-dye reductio n assay with bovine polymorphonuclear neutrophils as target cells. Imm unoblot analysis showed that both clones reacted with antigens of 110 and 131 kilodaltons. Epitope analysis showed that the two mAbs recogni zed the same epitope. An affinity column containing immobilized mAb F7 B10 was used to purify leukotoxin from crude toxin. Affinity chromatog raphy of 1 ml of culture supernatant resulted in 0.67 mu g or 1350 uni ts of leukotoxin. Leukotoxin was quantitated by a sandwich enzyme-link ed immunosorbent assay using mAb F7B10 as the capture antibody and as the biotinylated indicator. The minimal detectable level was approxima tely 1 ng, corresponding to 2 leukotoxin units in the sample.