AN IMPROVED METHOD OF TISSUE-CULTURE BIOASSAY FOR TETRODOTOXIN

A tissue culture bioassay, using the mouse neuroblastoma cell line (Ne uro2A), was improved to provide a simple and sensitive bio assay for T TX. The tetrazolium salt, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) was applied to replace the time-consuming a nd incomplete procedure of the original method with automatic measurem ent, using a microplate reader. TTX was used as a standard and an S-sh aped dose-response curve was obtained. In addition to the relative eas e of use and objectivity, sensitivity was found to be several times hi gher 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 w ith a known concentration of TTX and used to determine the effect of t his addition to the sample. Although the highly concentrated extract s howed cytotoxicity, the effect was reduced when the extract was dilute d or purified appropriately. The tetrazolium-based tissue culture bioa ssay described here may be useful for monitoring or screening for sodi um channel blockers (SCB).