Development of a single-chain variable fragment-alkaline phosphatase fusion protein-based direct competitive enzyme-linked immunosorbent assay for furaltadone metabolite in ctenopharyngodon idellus

To determine furaltadone metabolites 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ) residual levels in ctenopharyngodon idellus, direct competitive enzyme-linked immunosorbent assay (dcELISA) was developed based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein. Firstly, VH and VL gene sequences were cloned from hybridoma cell lines secreting monoclonal antibody against AMOZ derivative, and then the productive VH and VL genes were assembled to a scFv gene and engineered to produce scFv-AP fusion protein. The fusion protein was further identified as a bifunctional reagent for immunoassay, then a sensitive one-step dcELISA against AMOZ derivative was developed with a half-maximal inhibitory concentration (IC50) and limit of detection (LOD) of 10.62 and 4.83 ng/mL, respectively. The recovery values of AMOZ from spiked fish samples determined by dcELISA were in 71.31%∼82.88%. The results suggested that the prepared scFv-AP fusion protein in this work can be used for rapid and convenient immunoassays to detect AMOZ residues in fish samples.