title: Production and characterization of a biotinylated single-chain variable fragment antibody for detection of parathion-methyl.
abstract: In this article, we reported the development of a biotinylated single-chain variable fragment (scFv) antibody based indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for parathion-methyl (PM) detection. Firstly, a phage display library was generated using a pre-immunized BALB/C mouse against a specific hapten of PM. After four rounds of panning, the scFv gene fragments were transferred into a secreted expression vector. Then, the scFv antibodies were secreted expressed and screened by IC-ELISA against PM. The selected scFv antibody was fused with a biotin acceptor domain (BAD) and inserted into pET-28a(+) vector for high-level expression in Escherichia coli BL2 (DE3). After optimizing expression conditions, the scFv-BAD antibody was expressed as a soluble protein and biotinylated in vitro by the E. coli biotin ligase (BirA). Subsequently, the biotinylated scFv-BAD antibody was purified with a high yield of 59.2 ± 3.7 mg/L of culture, and was characterized by SDS-PAGE and western blotting. Finally, based on the biotinylated scFv-BAD, a sensitive IC-ELISA for detection of PM was developed, and the 50% inhibition value (IC50) of PM was determined as 14.5 ng/mL, with a limit of detection (LOD, IC10) of 0.9 ng/mL. Cross-reactivity (CR) studies revealed that the scFv antibody showed desirable specificity for PM.
authors.affiliation: Department of Microbiology, College of Life Science, Key Laboratory for Agriculture Microbiology, Shandong Agricultural University, Taian 271018, China.
authors.forename: Huimin
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Microbiology, College of Life Science, Key Laboratory for Agriculture Microbiology, Shandong Agricultural University, Taian 271018, China.
authors.forename: Fengchun
authors.initials: F
authors.lastname: Zhao
authors.affiliation: Department of Microbiology, College of Life Science, Key Laboratory for Agriculture Microbiology, Shandong Agricultural University, Taian 271018, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Han
authors.affiliation: Department of Microbiology, College of Life Science, Key Laboratory for Agriculture Microbiology, Shandong Agricultural University, Taian 271018, China. Electronic address: zhyouyang@sdau.edu.cn.
authors.forename: Zhengyou
authors.initials: Z
authors.lastname: Yang
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D019921:Biotinylation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008743:Methyl Parathion / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000737:chemistry*
chemical_list: D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D008743:Methyl Parathion
keywords: Biotinylation; IC-ELISA; Parathion-methyl; Phage display; scFv antibody