Production and characterization of a monoclonal antibody for Pefloxacin and mechanism study of antibody recognition

In this report, an artificial antigen (PFLX-BSA: Pefloxacin connected bovine serum albumin) was successfully prepared. The monoclonal antibody against pefloxacin was produced and characterized using a direct competitive ELISA. The linear range of detection was 0.115-6.564 µg/L. The limit of detection defined as IC15 was 0.170 ± 0.05 µg/L and the IC50 was 0.902 ± 0.03 µg/L. The antibody variable region genes were amplified, assembled, and sequenced. A three-dimensional structural model of the variable region was constructed to study the mechanism of antibody recognition using molecular docking analysis. Three predicted essential amino acids, Thr53, Arg97 of heavy chain and Thr52 of light chain, were mutated to verify the theoretical model. Three mutants lost binding activity significantly against pefloxacin as predicted. These may provide useful insights for studying antigen-antibody interaction mechanisms to improve antibody affinity maturation in vitro.