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, V-H and V-L gene sequences were cloned from hybridoma cell lines secreting monoclonal antibody against AMOZ derivative, and then the productive V-H and V-L 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%similar to 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.