An Improved Method of Tissue Culture Bioassay for Tetrodotoxin

Abstract

A tissue culture bioassay, using the mouse neuroblastoma cell line (Neuro2A), was improved to provide a simple and sensitive bioassay for TTX. The tetrazolium salt, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) was applied to replace the time-consuming and incomplete procedure of the original method with automatic measurement, using a microplate reader. TTX was used as a standard and an S-shaped dose-response curve was obtained. In addition to the relative ease of use and objectivity, sensitivity was found to be several times higher than the original (half-maximal inhibition was 12.9 nM TTX). The applicability of this method for detection of TTX produced by bacteria was investigated. Extracts prepared from E. coli cells were amended with a known concentration of TTX and used to determine the effect of this addition to the sample. Although the highly concentrated extract showed cytotoxicity, the effect was reduced when the extract was diluted or purified appropriately. The tetrazolium-based tissue culture bioassay described here may be useful for monitoring or screening for sodium channel blockers (SCB).