Production of novel polyclonal antibodies against the cyanobacterial toxin microcystin-LR and their application for the detection and quantification of microcystins and nodularin

A novel conjugation method was developed by linking the hapten, the cyanobacterial hepatotoxin microcystin-LR, via 2-mercaptoethylamine to keyhole limpet haemocyanin. Polyclonal antisera were raised against this conjugate and an indirect competitive immunoassay (ELISA) developed which can detect purified microcystin-LR and the toxin in extracts of cyanobacteria from fresh-, brackish and marine waters. The cross-reactivity of the microcystin-LR antibodies was investigated with a range of purified microcystin variants (-LR, -LA, -LY, -LW, -LF, -D-Asp3-RR, and -Asp3(Z)-Dhb7-HtyR) and nodularin. The antibodies cross-reacted well with all microcystin and nodularin variants. The microcystin-LA was the most readily detectable, followed by microcystins-LR, -LF, -LW, -D-Asp3-RR, -LY, nodularin and microcystin-Asp3(Z)-Dhb7-HtyR. Extracts from several genera of cyanobacteria were investigated by the ELISA and the results compared to high performance liquid chromatography with diode array detection (HPLC–DAD). By the ELISA procedure, 60% were positive, compared to detection of microcystins in 45% of the samples by HPLC–DAD. Analysis of microcystin-LR equivalents by ELISA and HPLC–DAD showed good correlation (R=0.96, p<1×10−10). The application of the anti-microcystin-LR antibodies for the detection and quantification of microcystins and nodularin in environmental water samples is discussed.