Enzyme-Linked Immunosorbent Assay for Microcystins in Blue-Green Algal Blooms

Abstract

Abstract A direct competitive enzyme-linked immunosorbent assay (ELISA) for the freshwater blue-green algal toxin mlcrocystln (MCYST) In algae and water was developed. The assay Involves coating antl-MCYST-variant leuclne-arglnine (LR) antibody to the ELISA plate and the use of MCYST-LRperoxidase as the enzyme marker. The linear portion of the standard curve for MCYST in phosphate buffer containing saline (PBS) was 0.5-10.0 ng/mL (25-500 pg/assay). The minimum detection level for MCYST-LR was 0.20 ng/mL (10 pg/assay). Contaminated water could be directly used In the ELISA. The overall analytical recoveries for MCYST-LR added to water at levels of 1-20 ng/mL was 83.4%. For analysis of cellular MCYST, the toxin was first extracted from the algae with 0.1M ammonium bicarbonate, diluted with PBS to less than 0.5 mg dried algae/mL (<5.0 mg wet welght/mL) and directly used in the ELISA. C-18 reverse-phase Sep-Pak cartridges effectively adsorbed MCYST from the toxln-containlng solutions. The toxin could be recovered from the cartridge by elutlng with 60% methanol. Using this approach, an algae extract that was relatively free of MCYST was prepared and was used in a recovery study. The overall analytical recovery of MCYST added to the algae extract In the range of 0.25-20 ppm was 83% with a coefficient of variation of 11.9%. The detection limit for MCYST In dried algae was about 0.25-0.5 pg/g (0.25-0.5 ppm) lyophlllzed algae sample. This method was applied for the analysis of several naturally occurring algal blooms. Limited samples were also analyzed for MYCST by liquid chromatography. ELISA data were in general agreement with those obtainedby liquid chromatography. MCYST concentrations from 0.006 to 2.9 fig/g (6 to 2900 ppb) and from 26 to 5200 /ig/g (26 ppm to 5200 ppm) were found In water and algae (dried weight), respectively