Development of a protein phosphatase-based assay for the detection of phosphatase inhibitors in crude whole cell and animal extracts.

Diarrhetic shellfish poisoning (DSP) is a serious and globally widespread phytoplankton-related seafood illness. Although DSP is rarely life-threatening, it causes incapacitating diarrhea and vomiting with no known medical treatments. In addition, phytoplankton producing DSP toxins have been identified in temperate coastal waters worldwide, and their numbers may be increasing as a result of coastal eutrophication. The toxic effects of the major DSP toxins, okadaic acid and dinophysistoxin-1 (35-methylokadaic acid), appear to originate from their inhibitory activity against a family of structurally related serine/threonine protein phosphatases (PSPases). In particular, the inhibition of essential PSPases (e.g. PP1 and PP2A) has catastrophic consequences in most eukaryonic cells. Exploiting the potent inhibitory property of the DSP toxins, we have developed an enzyme-based assay (PP2A assay) capable of detecting both okadaic acid and dinophysistoxin-1 in nanogram amounts. The assay employs purified PP2A, which has an extremely high affinity for both DSP toxins. This provides the PP2A assay with a level of sensitivity comparable to, or surpassing, that of most monoclonal antibody probes. To evaluate the PP2A assay as a means of detecting contaminated shellfish, a series of spike recovery experiments was conducted. The findings from these studies suggest that the PP2A assay has the potential for development into a rapid and relatively simple method for detecting PSPase inhibitors in crude extracts produced from shellfish.