Colorimetric immune-protein phosphatase inhibition assay for specific detection of microcystins and nodularins of cyanobacteria

A novel immunoassay was developed for specific detection of cyanobacterial cyclic peptide hepatotoxins which inhibit protein phosphatases. Immunoassay methods currently used for microcystin and nodularin detection and analysi s do not provide information on the toxicity of microcystin and/or nodulari n variants. Furthermore, protein phosphatase inhibition-based assays for th ese toxins are not specific and respond to other environmental protein phos phatase inhibitors, such as okadaic acid, calyculin A, and tautomycin. We a ddressed the problem of specificity in the analysis of protein phosphatase inhibitors by combining immunoassay-based detection of the toxins with a co lorimetric protein phosphatase inhibition system in a single assay, designa ted the colorimetric immune-protein phosphatase inhibition assay (CIPPIA). Polyclonal antibodies against microcystin-LR were used in conjunction with protein phosphatase inhibition, which enabled seven purified microcystin va riants (microcystin-LR, -D-Asp(3)-RR, -LA, -LF, -LY, -LW, and -YR) and nodu larin to be distinguished from okadaic acid, calyculin A, and tautomycin. A range of microcystin- and nodularin-containing laboratory strains and envi ronmental samples of cyanobacteria were assayed by CIPPIA, and the results showed good correlation (R-2 = 0.91, P < 0.00001) With the results of high- performance liquid chromatography with diode array detection for toxin anal ysis. The CIPPIA procedure combines ease of use and detection of low concen trations with toxicity assessment and specificity for analysis of microcyst ins and nodularins.