Quantitative cell-adhesion assay for Clostridium difficile cytotoxin

A quantitative assay for Clostridium difficile cytotoxin has been developed, based on the observation that suspended fibroblasts exposed to cytotoxin fail to adhere to plastic. A dye-binding technique was used to quantitate adherent cells, in order to obviate microscopy. Adherent BHK cells were fixed with glutaraldehyde and cell protein was stained with Coomassie blue R-250. Cell-bound dye was eluted and estimated spectrophotometrically. The amount of eluted dye was proportional to the number of adherent cells and cell staining was time dependent. Cytotoxin was purified by gel-permeation and ion-exchange chromatography and migrated as a single band on SDS-PAGE. After exposure of suspended BHK cells to purified cytotoxin, their adhesion to plastic was inhibited in a manner which depended on concentration of cytotoxin and on time and temperature of exposure. This study provides the basis for a C. difficile cytotoxin assay that is quantitative, rapid and reproducible and may have wider applicability in the study of other toxins or agents that inhibit cell adhesion.