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

Xiuyuan Zhang; Kuo He; Donghao Zhang; Zhihong Huang

doi:10.1080/09168451.2018.1562876

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

Biosci Biotechnol Biochem. 2019 Apr;83(4):633-640. doi: 10.1080/09168451.2018.1562876. Epub 2019 Jan 8.

Authors

Xiuyuan Zhang^{1

2}, Kuo He¹, Donghao Zhang¹, Zhihong Huang¹

Affiliations

¹ a Food Safety Centre of Hebei North University , Hebei North University , Zhangjiakou , China.
² b College of Food Engineering , Henan University of Animal Husbandry & Economy , Zhenzhou , China.

PMID: 30618329
DOI: 10.1080/09168451.2018.1562876

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 signiﬁcantly against pefloxacin as predicted. These may provide useful insights for studying antigen-antibody interaction mechanisms to improve antibody affinity maturation in vitro.

Keywords: ELISA; Pefloxacin; molecular recognition; monoclonal antibody.

MeSH terms

Animals
Anti-Bacterial Agents / analysis*
Anti-Bacterial Agents / chemistry
Anti-Bacterial Agents / immunology
Antibodies, Monoclonal / biosynthesis
Antibodies, Monoclonal / chemistry*
Antibodies, Monoclonal / isolation & purification
Antibody Affinity
Antibody Specificity
Binding Sites
Binding, Competitive
Cloning, Molecular
Enzyme-Linked Immunosorbent Assay / methods*
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 / chemistry*
Immunoglobulin Variable Region / isolation & purification
Limit of Detection
Mice
Mice, Inbred BALB C
Molecular Docking Simulation
Mutation
Pefloxacin / analysis*
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, Protein

Substances

Anti-Bacterial Agents
Antibodies, Monoclonal
Immunoconjugates
Immunoglobulin Variable Region
Recombinant Proteins
Serum Albumin, Bovine
Pefloxacin