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

摘要

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 x 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 mu g/mL analyzed by SDS-PAGE and protein quantitative respectively. The IC-ELISA based on MscFv7-scFv for MC-LR shows a half-maximum inhibition (IC50) of 0.471 mu g/L and a limit of detection (LOD) of 0.044 mu g/L, which is below the maximum residue limit standard (MRLs) of 1.0 mu g/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.