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Preparation and Directed Evolution of Anti-Ciprofloxacin ScFv for Immunoassay in Animal-Derived Food

文献类型： 外文期刊

作者： Wang, Fangyu 1 ; Li, Ning 2 ; Zhang, Yunshang 1 ; Sun, Xuefeng 1 ; Hu, Man 1 ; Zhao, Yali 2 ; Fan, Jianming 3 ;

作者机构： 1.Henan Acad Agr Sci, Key Lab Anim Immunol, 116 Huayuan Rd, Zhengzhou 450002, Peoples R China

2.Henan Agr Univ, Coll Food Sci & Technol, Dept Food Nutr & Hlth, 63 Agr Rd, Zhengzhou 450000, Peoples R China

3.Zhengzhou Univ, China Coll Publ Hlth, 100 Kexue Ave, Zhengzhou 450001, Peoples R China

关键词： scFv; ciprofloxacin; recognition mechanism; directional mutagenesis; IC-ELISA

期刊名称：FOODS （ 影响因子：4.35； 五年影响因子：4.957 ）

ISSN：

年卷期： 2021 年 10 卷 8 期

页码：

收录情况： SCI

摘要： An immunized mouse phage display scFv library with a capacity of 3.34 x 10(9) 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.

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