The Effects of Sample Matrices on Immunoassays to Detect Microcystin-LR in Water

Immunoassays are widely used biochemical techniques to detect microcystins in environmental samples. The use of immunoassays for the detection of microcystins is vulnerable to matrix components and other interferents. This study is an evaluation of the effects of interfering substances commonly found in drinking and ambient water samples using commercially-available immunoassay kits for microcystin toxins. The microplate and strip test immunoassay formats were tested in the study. For the microplate ELISA, the following were found to inhibit microcystin-LR (MC-LR) detection: 250 μg/mL Ca or Mg, 0.01% ascorbic acid, 0.1% EDTA chelating agent, 0.05 M glycine-HCl, pH 3. The following exhibited no effect: sodium chloride (NaCl, 1% to 4%) and sodium thiosulfate (0.001% and 0.01%), 0.01 to 0.1 M phosphate buffers (PB), pH 7 and 0.067 M PB at pH 5, 6, 7 and 8. Overall, up to 50 μg/mL of standard and reference natural organic matter (NOM) from various sources did not interfere in the assay system (without MC-LR) but diminished the detection of MC-LR at varying degrees. This is the first study evaluating standard and reference humic and fulvic acids from various sources in immunoassays for microcystins. The strip test also showed variable effects on MC-LR detection in the presence of NOM. This assay format was also sensitive to varying pHs and ionic strengths. MC-LR binding was inhibited at low pH (0.05 M glycine-HCl, pH 3), whereas, 0.067 M PB with pH 6, 7 and 8 can yield false positive results. Lower ionic strength of 0.01 M PB, pH 7 showed no interference in MC-LR binding whereas higher ionic strengths can interfere with MC-LR detection. NaCl at 3% and 4% can interfere with the analysis giving false positive results. Mg at 50 and 250 μg/mL showed no effect on the analysis while the same concentration of Ca can yield false positive results. The performance in marine, brackish and hard waters should be tested given the potential sensitivity to salinity. Results of this study may assist in the further refinement of existing assays and the development of practical antibody-based methods to clean-up samples and detect cyanotoxins in water.