Highly sensitive assay of okadaic acid using protein phosphatase and paranitrophenyl phosphate

Abstract

A colorimetric phosphatase-inhibition bioassay was developed for the quantitative measurement of okadaic acid (OA) the main diarrhetic toxin responsible for diarrhetic shellfish poisoning. The assay used an artificial substrate, paranitrophenylphosphate, and a semi-purified protein phosphatase PP2Ac containing extract prepared from rabbit muscle. Calibration dose-inhibition curves were constructed using standard OA and they permitted easy determination of the enzyme concentration Et in their linear portion. In the range of linearity, the slope increased when Et decreased, thus giving a detecting limit of 0.04 pmol in the reaction mixture (1 ml). The lowest assayable concentration of OA was 4 ng/ml in aqueous solutions and 40 ng/ml (i.e., 100 ng of OA per g of mussel tissue) in crude methanol mussels extracts. The intra and interassay coefficients of variation in the measurement of OA for the toxin spiked aqueous samples averaged, respectively, 7.7% and 3.7%, and interexperiments coefficients of variation for the toxin spiked mussel extracts averaged 4.6%. The presence of OA was ascertained by a method in which one assay was performed at two or three different levels of enzyme concentration. The rapidity, accuracy, reproducibility, specificity, and simplicity of the procedure provides a simple way to assay okadaic acid in buffered or complex solutions.

Development of a monoclonal antibody binding okadaic acid and dinophysistoxins-1, -2 in proportion to their toxicity equivalence factors

“…This method is under increasing criticism not only from an ethical point of view but also because of its limited sensitivity and inability to distinguish between toxins of different groups (Quilliam and Wright, 1995;Hess et al, 2006). A number of alternative methods have been proposed ranging from cytotoxicity assays (Aune et al, 1991;Amzil et al, 1992.;Tubaro et al, 1996a), protein phosphatase assays (Luu et al, 1993;Honkanen et al, 1996;Simon and Vernoux, 1994;Tubaro et al, 1996b;Isobe et al, 1995;Vieytes et al, 1997), liquid chromatography (LC) coupled with fluorescence detection and LC coupled with mass spectrometry, capillary electrophoresis and numerous immunosorbent methods (James et al, 2000). Some of these methods lack the required sensitivity and others require long sample preparation.…”

Functional assays in marine biotoxin detection

“…In a first version, the spectrophotometric PPase assay was based on the use of semi-purified preparations of phosphoprotein phosphatases from rabbit muscle (Simon and Vernoux, 1994), whereas its further development was obtained by the use of purified type 2A phosphoprotein phosphatase from human red blood cells (Tubaro et al, 1996a;Della Loggia et al, 1999). The fluorimetric detection of PPase activity has eventually introduced an improved sensitivity to the assay (Vieytes et al, 1997).…”

“…Thus, another methodological choice regarding the experimental design to develop a functional assay for algal toxins relates to the biological characteristics of the system that will be probed with the sample. For instance, the absolute gain of simplicity of the PPase assay performed by the use of an enzyme under cell-free conditions (Simon and Vernoux, 1994;Tubaro et al, 1996a;Vieytes et al, 1997) has demanded the inclusion of sample pre-treatment by an hydrolytic step (Mountfort et al, 2001), in order to allow the detection of the products derived from DTX3, which are hydrolyzed in living animals yielding toxic analogues.…”

Use of immunoaffinity columns for clean-up of diarrhetic toxins (okadaic acid and dinophysistoxins) extracts from shellfish prior to their analysis by HPLC/fluorimetry

“…They include enzymatic PP inhibition assays based on the OA molecular activity (Simon andVernoux 1994, Tubaro et al 1996) and cytotoxicity assays (Croci et al 1997). But the enzymatic assay did not allow one toxin to be di erentiated from another and some interference might provoke false positive results.…”