title: Virtual mutation and directional evolution of anti-amoxicillin ScFv antibody for immunoassay of penicillins in milk.
abstract: In this study, an anti-amoxicillin single chain variable fragment (ScFv) antibody was evolved by directional mutagenesis of a contact amino acid residue based on the analysis of virtual mutation. Comparison with its parental ScFv, the mutant showed highly improved affinity for 11 penicillins with up to 6-folds increased sensitivity. Then, its recognition mechanisms for the 11 drugs were studied by using molecular docking. Results showed that the mutant-penicillins intermolecular forces increased and the total binding energies decreased dramatically, which were responsible for the improvement of antibody sensitivity. The ScFv mutant was used to develop an indirect competitive enzyme linked immunosorbent assay for determination of the 11 drugs in milk. The limits of detection were in the range of 0.2-3.0 ng/mL, the crossreactivities were in the range of 31%-132%, and the recoveries from standards fortified blank milk were in the range of 65.7%-92.4%. This is the first study reporting the directional evolution of a ScFv antibody based on virtual mutation and the use of ScFv antibody for determination of penicillins in foods of animal origin.
authors: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, Hebei, 071000, China.
authors: Xin
authors: X
authors: He
authors: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, Hebei, 071000, China.
authors: Chang Fei
authors: CF
authors: Duan
authors: College of Animal Science, Henan Institute of Science and Technology, Xinxiang Henan, 453003, China.
authors: Yong Hua
authors: YH
authors: Qi
authors: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, Hebei, 071000, China.
authors: Jun
authors: J
authors: Dong
authors: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, Hebei, 071000, China.
authors: Geng Nan
authors: GN
authors: Wang
authors: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, Hebei, 071000, China.
authors: Guo Xian
authors: GX
authors: Zhao
authors: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, Hebei, 071000, China.
authors: Jian Ping
authors: JP
authors: Wang
authors: College of Veterinary Medicine, Agricultural University of Hebei, Baoding, Hebei, 071000, China. Electronic address: 444937594@qq.com.
authors: Jing
authors: J
authors: Liu
mesh_terms: D019943:Amino Acid Substitution; D000658:Amoxicillin / Q000032:analysis* / Q000737:chemistry; D000818:Animals; D002417:Cattle; D019020:Directed Molecular Evolution / Q000379:methods*; D005504:Food Analysis / Q000379:methods*; D008892:Milk / Q000737:chemistry*; D020125:Mutation, Missense; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics
chemical_list: D057127:Single-Chain Antibodies; D000658:Amoxicillin
keywords: Directional evolution; Milk; Mutant; Penicillins; ScFv; Virtual mutation