Ciprofloxacin (CIP) is a synthetic third-generation fluoroquinolone (FQ) antibiotic that has been developed and is widely used to treat bacterial infections in humans and animals. This antibiotic exerts effects by inhibiting DNA gyrase or topoisomerase II in susceptible bacteria and exhibits high activity against a broad spectrum of Gram-negative and Gram-positive bacteria. However, the unreasonable and extensive use of antibiotics has resulted in the potential for residual antibiotics in food of animal origin, which can damage multiple systems in the body and cause bacterial resistance. Therefore, the European Union, the Joint FAO/WHO Expert Committee on Food Additives (JECFA, Rome, Italy) and China established maximum residue limits of CIP in animal-derived food to prevent the accumulation of antimicrobial residues, e.g., 100 µg/kg in milk and meat.
An immunized mouse phage display scFv library with a capacity of 3.34 × 109 CFU/mL was constructed and used for screening of recombinant anti-ciprofloxacin single-chain antibody for the detection of ciprofloxacin (CIP) in animal-derived food. After four rounds of bio-panning, 25 positives were isolated and identified successfully. The highest positive scFv-22 was expressed in E. coli BL21. Then, its recognition mechanisms were studied using the molecular docking method. The result showed the amino acid residue Val160 was the key residue for the binding of scFv to CIP. Based on the results of virtual mutation, the scFv antibody was evolved by directional mutagenesis of contact amino acid residue Val160 to Ser. After the expression and purification, an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on the parental and mutant scFv was established for CIP, respectively. The IC50 value of the assay established with the ScFv mutant was 1.58 ng/mL, while the parental scFv was 26.23 ng/mL; this result showed highly increased affinity, with up to 16.6-fold improved sensitivity. The mean recovery for CIP ranged from 73.80% to 123.35%, with 10.46% relative standard deviation between the intra-assay and the inter-assay. The RSD values ranged between 1.49% and 9.81%. The results indicate that we obtained a highly sensitive anti-CIP scFv by the phage library construction and directional evolution, and the scFv-based IC-ELISA is suitable for the detection of CIP residue in animal-derived edible tissues.
2. Phage Display Technology
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