ABSTRACT
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 signiï¬cantly against pefloxacin as predicted. These may provide useful insights for studying antigen-antibody interaction mechanisms to improve antibody affinity maturation in vitro.
Subject(s)
Anti-Bacterial Agents/analysis , Antibodies, Monoclonal/chemistry , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin Variable Region/chemistry , Pefloxacin/analysis , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/immunology , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/isolation & purification , Antibody Affinity , Antibody Specificity , Binding Sites , Binding, Competitive , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Hybridomas/chemistry , Hybridomas/immunology , Immunization , Immunoconjugates/administration & dosage , Immunoconjugates/chemistry , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/isolation & purification , Limit of Detection , Mice , Mice, Inbred BALB C , Molecular Docking Simulation , Mutation , Pefloxacin/chemistry , Pefloxacin/immunology , Protein Binding , Protein Structure, Secondary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/immunology , Structural Homology, ProteinABSTRACT
Pefloxacin has been increasingly used in veterinary medicine to treat microbial infections. To avoid using a labor-intensive instrumental method to detect the residue of pefloxacin in food, a simple and convenient indirect competitive enzyme-linked immunosorbent assay method has been developed in this study. The antibody generated from immunogen cationized bovine serum albumin-pefloxacin showed high sensitivity toward pefloxacin with an IC50 value of 6.7 ppb in buffer and was suitable for a screening assay to detect the residue of pefloxacin in food products. The antibody has been assessed using rapid enzyme immunoassays to exploit its specificity. The antibody prepared shows cross-reactivity with a few other (fluoro)quinolones including fleroxacin (116%), enrofloxacin (88%), and ofloxacin (10%). The assay measured drug residue in chicken liver spiked with pefloxacin with an interassay coefficient of variation of 13.6% or less and an intra-assay coefficient of variation of 10.9% or less. The average recovery rates at 0.5, 5, 10, 50, and 100 ppb were in the range of 86-106% for interassay and in the range of 87-103% for intra-assay, respectively.
