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

A novel conjugation method was developed by linking the hapten, the cyanoba cterial hepatotoxin microcystin-LR, via 2-mercaptoethylamine to keyhole lim pet haemocyanin. Polyclonal antisera were raised against this conjugate and an indirect competitive immunoassay (ELISA) developed which can detect pur ified microcystin-LR and the toxin in extracts of cyanobacteria From fresh- , brackish and marine waters. The cross-reactivity of the microcystin-LR an tibodies was investigated with a range of purified microcystin variants (-L R, -LA, -LY, -LW, -LF, -D-Asp(3)-RR, and -Asp(3)(Z)-Dhb(7)-HtyR) and nodula rin. The antibodies cross-reacted well with all microcystin and nodularin v ariants. The microcystin-LA was the most readily detectable, followed by mi crocystins-LR, -LF, -LW, -D-Asp(3)-RR, -LY, nodularin and microcystin-Asp(3 )(Z)-Dhb(7)-HtyR. Extracts from several genera of cyanobacteria were invest igated by the ELISA and the results compared to high performance liquid chr omatography with diode array detection (HPLC-DAD). By the ELISA procedure, 60% were positive, compared to detection of microcystins in 45% of the samp les by HPLC-DAD. Analysis of microcystin-LR equivalents by ELISA and HPLC-D AD showed good correlation (R = 0.96, p < 1 x 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. (C) 2000 Elsevier Science Ltd. All rights reserved.