title: Development and preliminary validation of a microtiter plate-based receptor binding assay for paralytic shellfish poisoning toxins.
abstract: More than 20 countries have either established or proposed regulatory limits for one or more of the paralytic shellfish poisoning (PSP) toxins as they occur in seafood products. PSP toxin levels are generally estimated using the standard AOAC mouse bioassay, yet because of various limitations of this method [e.g. high variability (+/-20%), low sensitivity, limited sample throughput and use of live animals], there remains a need for alternative testing protocols. A sensitive and selective, high capacity assay was developed for the PSP toxins which exploits the highly specific interaction of these toxins with their biological receptor (i.e. voltage-dependent sodium channel) and is thus based on functional activity. This receptor binding assay provides a radioactive endpoint, and is performed in a microtiter filter plate format with results determined by standard liquid scintillation counting within 24 hr. The Ki for the assay is 3.66 +/- 0.86 nM saxitoxin, with a limit of detection of c. 5 ng saxitoxin/ml in a sample extract. Good quantitative agreement of the assay with both mouse bioassay and high-performance liquid chromatographic analysis of crude extracts of contaminated shellfish, as well as PSP toxin-producing algae, was observed. Our findings indicate that the receptor binding assay has a strong predictive value for toxicity determined by mouse bioassay, and that this approach warrants consideration as a rapid, reliable and cost-effective alternative to live animal testing for detection and estimation of PSP-related toxicity in seafood and toxic algae.
authors.affiliation: Marine Biotoxins Program, Charleston Laboratory, National Marine Fisheries Service, SC 29412-9110, USA.
authors.forename: G J
authors.initials: GJ
authors.lastname: Doucette
authors.forename: M M
authors.initials: MM
authors.lastname: Logan
authors.forename: J S
authors.initials: JS
authors.lastname: Ramsdell
authors.forename: F M
authors.initials: FM
authors.lastname: Van Dolah
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid; D056890:Eukaryota / Q000737:chemistry; D008297:Male; D051379:Mice; D008974:Mollusca / Q000737:chemistry; D011869:Radioligand Assay / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012530:Saxitoxin / Q000032:analysis* / Q000378:metabolism; D012758:Shellfish; D015222:Sodium Channels / Q000378:metabolism; D013574:Synaptosomes / Q000737:chemistry / Q000378:metabolism
chemical_list: D015222:Sodium Channels; D012530:Saxitoxin