Evaluation of 11-[3H]-tetrodotoxin use in a heterologous receptor binding assay for PSP toxins.
This report describes the preparative scale production of 11-[3H]-tetrodotoxin (TTX) and its evaluation as a substitute for [3H]-saxitoxin (STX) as the radioligand in a receptor binding assay for paralytic shellfish poisoning (PSP) toxins. Restrictions on the world-wide distribution of [3H]-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-[3H]-TTX was found to exhibit site-specific binding characteristics in the receptor assay (dissociation constant(K(d))=4.77+/-1.54nM; maximum binding(B(max))=1. 62+/-0.24pmol/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-[3H]-TTX could be used interchangeably with [3H]-STX in the receptor assay for determination of PSP toxicity in shellfish and algal extracts without compromising assay performance. We conclude that the 11-[3H]-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 [3H]-STX as the radioligand.
Doucette
G J
GJ
Marine Biotoxins Program, NOAA/National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC 29412, USA. greg.doucette@noaa.gov
Powell
C L
CL
Do
E U
EU
Byon
C Y
CY
Cleves
F
F
McClain
S G
SG
D005069:Evaluation Studies as Topic; D008387:Marine Toxins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D011869:Radioligand Assay; D012758:Shellfish / Q000032:analysis*; D013779:Tetrodotoxin / Q000378:metabolism*; D014316:Tritium
D008387:Marine Toxins; D008565:Membrane Proteins; D014316:Tritium; D013779:Tetrodotoxin