Adaptation of a colorimetric microtitration assay for quantifying Pasteurella haemolytica A1 leukotoxin and antileukotoxin

A colorimetric microtitration assay was adapted to quantify the cytotoxicity of Pasteurella haemolytica A1 leukotoxin to bovine neutrophils used as target cells. The viability of leukotoxin-treated target cells was detected by use of a tetrazolium dye that living cells reduced to dark blue formazan. The amount of formazan formed (which was quantified by use of an ELISA plate reader) was directly proportional to the number of viable target cells. This assay system also was used to measure leukotoxin-neutralization antibody titers of bovine serum and lung lavage specimens obtained during vaccination experiments. The major advantages of this assay over other methods such as the 51Cr-release and trypan blue-exclusion assays are precision, rapidity, and low cost; it also does not use radioisotopes.