Receptor binding assay for the detection of paralytic shellfish poisoning toxins: comparison to the mouse bioassay and applicability under regulatory use

ABSTRACT

The receptor-binding assay (RBA) method for the detection of paralytic shellfish poisoning (PSP) toxins was evaluated for its overall performance in comparison with the mouse bioassay (MBA). An initial study to evaluate the effects of filtering shellfish extracts prior to running the RBA indicated no significant difference between filtered and unfiltered extracts on the determined saxitoxin (STX) concentrations. Next, we tested the RBA assay on 295 naturally contaminated mussel tissue samples, ranging in concentrations from 320 µg STX equiv. kg⁻¹ to 13,000 µg STX equiv. kg⁻¹ by MBA. An overall trend was observed with the RBA giving higher results (256 µg STX equiv. kg⁻¹ on average) than the MBA; however, at low concentrations (< 500 µg STX equiv. kg⁻¹) the RBA results were marginally lower. A third study was conducted using spiked mussel tissue analysed by three independent laboratories, two of which performed the RBA and one the MBA. This multi-laboratory study again showed the RBA to give higher results than the MBA; however, it also revealed that STX determination was accurate by the RBA, unlike the MBA. To optimise the assay for efficient usage under regulatory practice, three suggestions have been made: the use of an initial screening plate to separate those samples that exceed the alert level; use of rapid PSP test kits in the field and in the laboratory for screening negative samples and for early detection of toxicity; and use of an alternate commercially available porcine membrane in place of the laboratory-prepared rat membrane homogenate. The large number of samples analysed and the diversity of the tests conducted in this study further support the RBA as an affordable rapid method for STX detection that is also free of the routine sacrifice of live animals.

KEYWORDS:

• Paralytic shellfish poisoning
• receptor binding assay
• saxitoxins
• mouse bioassay
• comparability
• porcine

Acknowledgments

The authors would like to thank Roger Ho for helping with the Graphpad Prism data analysis; American Radiolabeled Chemicals Inc. for providing ³H-STX reagents; CDPH Microbial Diseases Laboratory for extracting the shellfish tissue samples; Vanessa Zubkousky-White for conducting confirmatory RBA studies; Christina Morales and Stephanie Abromaitis (supported in part by Grant Number 2B01OT009006 from the Centers for Disease Control and Prevention) for performing confirmatory porcine membrane comparisons. Thank you to our collaborators, Leanne Flewelling from Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute and Darcie Couture from Maine Department of Natural Resources. Special thanks to Sherwood Hall of the US Food and Drug Administration’s Office of Regulatory Science for USFDA STX reference standards and helpful discussions on this study and to Fran Van Dolah and Greg Doucette from the Center for Coastal Environmental Health and Biomolecular Research, NOAA Marine Biotoxins Program for their guidance in the RBA method. From CDPH-DWRL, thanks to William Draper for careful review of the paper and many helpful editorial suggestions, Raimund Roehl and Jian Yao for helpful discussions on the binding curve, and Dadong Xu for developing the spreadsheet for outlier testing; Rita Brenden and Greg Inami from CDPH-MDL for providing the SRT-AOAC data; Peter Miller from UC-Santa Cruz for the Scotia Rapid Test extraction data from the field pilot sites.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the US National Oceanic and Atmospheric Administration [MERHAB Grant NA04NOS4780239];