Construction and directional evolution of anti-enrofloxacin ScFv antibody for the immunoassay of fluoroquinolones

Abstract A recombinant anti-enrofloxacin single-chain antibody (scFv) was produced for the detection of enrofloxacin. An immunized mouse phage display scFv library with a capacity of 2.35×109 CFU/mL was constructed and used for anti-enrofloxacin scFv screening. After four rounds of bio-panning, 10 positives were isolated and identified successfully. The highest positive scFv was expressed in E. coli BL21. Then, its recognition mechanisms were studied using the molecular docking method. The result showed the amino acid residues Leu121 were the key residues for the binding of ScFv to ENR. Based on the results of virtual mutation, the ScFv antibody was evolved by directional mutagenesis of contact amino acid residue Leu121 to Asn. After the expression and purification, an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on the parental and mutant ScFv were established for enrofloxacin respectively. The IC50 value of the assay established with the ScFv mutant was 1.63 ng/mL, while the parental ScFv was 21.08 ng/mL, this result showed highly increased affinity with up to 12.9-folds improved sensitivity. The mean recovery for ENR ranged from 71.80% to 117.35% with 10.46% relative standard deviation between the intra-assay and the inter-assay. The results indicate that we have obtained a highly sensitive anti-ENR scFv by the phage library construction and directional evolution, and the scFv-based IC-ELISA is suitable for the detection of ENR residue in animal derived edible tissues and milk.