Enzyme Immunoassay for Direct Determination of Microcystins in Environmental Water

Abstract

An enzyme-linked immunosorbent assay (ELISA) was developed for direct quantitation of microcystins (MCs), a group of freshwater cyanobacterial toxins. An anti-MC monoclonal antibody exhibiting broad cross-reactivity to major MC derivatives was used. The detection limit and linear range of the ELISA standard curve with microcystin-(leucine-arginine) (MCLR), a variant of MCs, were 20 and 20-500 pg/mL, respectively. For analysis of MC released from cyanobacterial cells, water sample filtered through a glass fiber filter was applied directly to ELISA. For analysis of total MC (released MC plus intracellular MC), intracellular toxin was extracted by freeze-thawing twice before filtration. Mean recovery of MCLR added to tap water and toxin-free environmental water was 101%, with a coefficient of variation (CV) of 7.3% at toxin levels of 20-500 pg/mL. Mean recovery of MCLR added to toxin-free cyanobacterial extracts was 93%, with a CV of 12.5% at toxin levels of 50-500 pg/mL. At 20 pg/mL, an increasing matrix effect on assay variance was observed; therefore, both released MC and total MC were measured in the range 50-500 pg/ mL. Comparative studies with a liquid chromatographic (LC) method showed that the ELISA gives a reliable correlation with LC for analysis of MC in water extracts of natural blooms and cultured cyanobacterial cells (r = 0.98). The ELISA was applied to water samples collected from lakes and ponds in Japan. In 4 of 13 and 12 of 17 samples, 81-800 pg released MC/mL and 64-94 000 pg total MC/mL were detected, respectively. By LC separation followed by the ELISA analysis, the presence of MCLR, microcystin-arginine-arginine, and microcystin-tyrosine-arginine were confirmed in 4 ELISA-positive samples selected randomly. The newly developed ELISA is a reliable and powerful method for mass monitoring of MC levels in environmental water.