Development of a two-step immunochromatographic assay for microcystin-LR based on fluorescent microspheres

Abstract

Microcystins (MCs) are a class of toxins that are mainly produced by cyanobacteria. Among them, microcystin-leucine arginine (MC-LR) is one of the most toxic and harmful of the freshwater toxins, and it has caused many accidents and threats to human health. In the present study, we report a neoteric lateral flow fluorescent microsphere immunochromatographic assay (FM-ICA) combined with UV light detection to rapidly and quantitatively detect MC-LR in freshwater food, based on direct competitive immunoreactions on chromatographic strips. In the method, MC-LR artificial antigen, MC-LR-bovine serum albumin (MC-LR-BSA), and goat anti-rabbit immunoglobulin G (IgG) were labelled with europium (Eu) nanospheres that functioned as a luminescence tracer. Goat anti-mouse IgG and rabbit IgG were spread on a nitrocellulose (NC) membrane for the test (T) line and control (C) line, respectively. The optimal parameters were 1 mg/mL for the goat anti-mouse IgG and rabbit IgG, 20 μg of MC-LR-BSA conjugated with Eu nanospheres, 1:100 for the dilution of Eu-MC-LR-BSA, and a 1:30000 dilution of anti-MC-LR monoclonal antibody (mAb) with an assay time of 15 min. The working range of the MC-LR standard curve was 0.1–5.0 μg/L with a limit of detection (LOD) of 0.0542 μg/L and a 50% inhibitory concentration (IC50) of 0.5613 μg/L. The average recoveries of the FM-ICA for detecting MC-LR in real samples ranged from 90.1 to 109.3%, with coefficients of variation under 15.8%. The FM-ICA test strips for quantitative determination of MC-LR provide sensitive, simple, and rapid performance, and can be used to monitor freshwater samples.