Production and Directional Evolution of Antisarafloxacin ScFv Antibody for Immunoassay of Fluoroquinolones in Milk

A recombinant antisarafloxacin ScFv antibody was produced by direct transformation of its gene into Rosetta-gami(DE3) for expression, and then its recognition mechanisms for 12 fluoroquinolones were studied using the molecular docking method. On the basis of the results of virtual mutation, the ScFv antibody was evolved by directional mutagenesis of contact amino acid residue Tyr99 to His. The ScFv mutant showed highly increased affinity for the 12 drugs with up to sevenfold improved sensitivity. Finally, the mutant was used to develop an indirect competitive enzyme linked immunosorbent assay for determination of the 12 drugs in milk. The limits of detection were in the range of 0.3–8.0 ng/mL; the ties were in the range of 5–106%, and the recoveries from the standard fortified blank milk were in the range of 62.0–89.3%. This is the first study reporting the evolution of an ScFv antibody using a directional mutagenesis strategy based on virtual mutation.