High sensitive single chain variable fragment screening from a microcystin-LR immunized mouse phage antibody library and its application in immunoassay

Abstract Microcystin-LR (MC-LR) is one of common high-toxic biotoxins produced by cyanobacteria in waterbody. A high sensitive and convenient detection method is necessary for monitoring for MC-LR. To establish a high sensitive indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on single chain variable fragment (scFv) for detecting MC-LR, 16 positive anti-MC-LR phage scFv particles were screened out from a MC-LR-immunized mouse phage scFv library, which was successfully constructed with the capacity of 8.67 × 10 7 CFU/mL. The most positive anti-MC-LR phage scFv (MscFv7) was successfully expressed in Escherichia coli ( E.coli ) HB2151. The molecular weight (M.W.) of expressed protein was about 30 kDa, and the concentration of purified protein was 512.6 μg/mL analyzed by SDS-PAGE and protein quantitative respectively. The IC-ELISA based on MscFv7-scFv for MC-LR shows a half-maximum inhibition (IC 50 ) of 0.471 μg/L and a limit of detection (LOD) of 0.044 μg/L, which is below the maximum residue limit standard (MRLs) of 1.0 μg/L in drinking water. The MscFv7-scFv has a strong cross-recognition for MC-RR and MC-YR with cross-reactivity (CRs) of 93.1% and 85.9%, respectively, but weak for MC-LW with that of 9.7%, even non-recognition for MC-WR, MC-LF and MC-LY. The recovery rates of IC-ELISA to detect MC-LR spiked in different cleanliness of water samples were 81.2–106.3% with CVs of 2.62–10.22% at intra-assay and inter-assay. The results showed that we obtained a high sensitive anti-MC-LR scFv, and the established IC-ELISA based on MscFv7-scFv should be promising for ultrasensitive monitoring MC-LR, MC-RR and MC-YR in water samples.