Quantitative microtiter cytotoxicity assay for Shigella toxin

Abstract

The cytotoxic activity of Shigella dysenteriae 1 was assayed by exposing HeLa cells in microtiter cultures to dilutions of toxin. Exposure to toxin caused either failure of cells in suspension to attach or detachment of cells from established monolayers. Estimates of toxin potency were made by staining residual cells with crystal violet and visually inspecting the stained plates. Quantitation of the cytotoxic effect was made possible by eluting and spectrophotometrically measuring the stain. The dilution of toxin causing 50% cell detachment, the endpoint chosen for the assay, was estimated from plots of dye absorbance versus toxin dilution. The 50% cell detachment dilution of toxin varied as a function of cell concentration, incubation of toxin with cells in suspension or as established monolayers, and the cell line used for assay. The HeLa cell line was the most sensitive of the cell lines examined. The method was easily utilized to monitor toxin purification and to measure antitoxin neutralization of toxin activity.