Evaluation of the fluorometric protein phosphatase inhibition assay in the determination of okadaic acid in mussels

The protein phosphatase inhibition assay for okadaic acid, the major DSP toxin, modified to use the fluorescence substrates methylumbelliferyl phosphate (MUP) and fluorescein diphosphate (FDP), was compared to the assay using p-nitrophenylphosphate ( p-NPP) and the bioluminescence assay using luciferin phosphate (L–P). Under the standard assay conditions used okadaic acid inhibited the enzyme activity dose-dependently with IC 50 values of 1.5 nM (MUP) and 1.2 nM (FDP). This compares to IC 50 values of 0.9 and 6 nM using L–P and p-NPP respectively. CDP- star, a chemiluminescence substrate, was not hydrolysed by the enzyme. Decreasing the enzyme concentration lowered the IC 50 for the colorimetric method (IC 50=2 nM [ p-NPP], 0.75 nM enzyme) but no shift was observed with fluorimetry. However at enzyme concentrations ELISA>PP-2A>LC–MS. The fluorimetric assay was both more sensitive and accurate than the colorimetric assay (the latter showed a propensity towards false positives in the region 20 μg/100 g), and the moderate increase in equipment cost appears to be outweighed by the performance of the method.