Highly sensitive assay for Clostridium perfringens enterotoxin that uses inhibition of plating efficiency of Vero cells grown in culture

Abstract

A highly sensitive and reproducible biological assay for Clostridium perfringens enterotoxin is described that uses Vero (African green monkey kidney) cells grown in tissue cultures. Very small doses of the enterotoxin inhibited the plating efficiency of the cells. This inhibition of plating efficiency could be used to detect as little as 0.1 ng (1 ng/ml) of enterotoxin, and a linear dose-response curve was obtained with 0.5 to 5 ng (5 to 50 ng/ml). A nonlinear, but reproducible, curve was obtained with a dose range from 0.1 to 100 ng (1 to 1,000 ng/ml). A new unit of biological activity, called the plating efficiency unit, was defined as that amount of enterotoxin that caused a 25% inhibition of the plating of 200 cells inoculated into 100 microliters of medium in a microwell culture system. One milligram of highly purified enterotoxin contained about 400,000 plating efficiency units. Additional studies demonstrated that the biological and serological activities of the enterotoxin molecule were not equally labile.