A FLUORESCENT MICROPLATE ASSAY FOR DIARRHEIC SHELLFISH TOXINS

A fluorescent enzyme inhibition assay for okadaic acid using 4-methylu mbelliferyl phosphate and fluorescein diphosphate as substrates for th e enzyme phosphatase 2A was developed. In the inhibition assay, perfor med in a microtiter plate, the PP2A was inhibited by adding okadaic ac id and the resulting fluorescence enhancement derived from enzymatic h ydrolysis of the substrate was quantified in a fluorescence plate read er. The measurable range of okadaic acid was 3.2 to 3200 pg/ml with an IC50 = 0.1 nM. The detection limit of okadaic acid was 2.56 pg/well i n buffer solutions and 12.8 ng/g hepatopancreas in shellfish extracts. The coefficient of variation (CV, n = 22) for each point ranged from 18.80 to 37.90% (mean 28.35%). The proposed method is very convenient, rapid, and sensitive by using the enzyme inhibition assay system and fluorescent reaction as a detection system. This work demonstrates tha t the fluorescent assay can be used to quantify the amount of okadaic acid in shellfish samples and also is valid for very dilute samples, s uch as phytoplankton samples. (C) 1997 Academic Press.