Evaluation of 11-[3H]-tetrodotoxin use in a heterologous receptor binding assay for PSP toxins

This report describes the preparative scale production of 11-[H-3]-tetrodotoxin (TTX) and its evaluation as a substitute for [H-3]-saxitoxin (STX) as the radioligand in a receptor binding assay for paralytic shellfish poisoning (PSP) toxins. Restrictions on the world-wide distribution of [H-3]-STX imposed by the international Chemical Weapons Convention served as the primary impetus for this study. We have incorporated on a preparative scale, a nonexchangeable tritium label into the TTX molecule at a specific activity of 12.90 Ci/mmol and recovered material of high radiochemical purity (98%). The resulting 11-[H-3]TTX was found to exhibit site-specific binding characteristics in the receptor assay (dissociation constant (K-d) = 4.77 +/- 1.54 nM; maximum binding (B-max) = 1.62 +/- 0.24 pmol/mg of synaptosomal protein). The inhibition constant (K-i) for the assay was 1.46 +/- 0.28 nM STX equiv. (n = 6), with an estimated detection limit of ca. 2-4 ng STX equiv./ml in a sample extract. Moreover, quantitative comparisons indicated that 11-[H-3]-TTX could be used interchangeably with [H-3]-STX in the receptor assay for determination of PSP toxicity in shellfish and algal extracts without compromising assay performance. We conclude that the 11-[H-3]-TTX produced and evaluated herein exhibits physical, chemical and biological characteristics suitable not only for use in the PSP receptor binding assay, but likely for other applications employing [H-3]-STX as the radioligand. Published by Elsevier Science Ltd.