title: Development of a biotinylated broad-specificity single-chain variable fragment antibody and a sensitive immunoassay for detection of organophosphorus pesticides.
abstract: Organophosphorus pesticides (OPs) are the most widely used pesticides in agriculture, and OP residues have been broadly reported in food and environmental samples. The aim of this study is to develop a recombinant antibody-based broad-specificity immunoassay for OPs. A phage display library was prepared from a mouse pre-immunized with a generic immunogen of OPs, and a single-chain variable fragment (scFv) antibody was selected. The selected scFv antibody was fused with biotin acceptor domain (BAD) and overexpressed as an inclusion body in Escherichia coli BL21 (DE3). Then, the protein was refolded by stepwise urea gradient dialysis and biotinylated in vitro by E. coli biotin ligase (BirA). Subsequently, the scFv-BAD protein was purified from the biotinylated system with high yield (66.7 mg L(-1)) and confirmed by SDS-PAGE and Western blot. Based on the biotinylated scFv-BAD, a sensitive and broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for detection of OPs was developed. The cross-reactivity (CR) studies demonstrated that the ciELISA described here exhibited the broadest detection spectrum for OPs up to now, and 30 OPs could be determined with 50 % inhibition value (IC50) values ranging from 19.4 to 515.2 ng mL(-1). Moreover, the developed ciELISA was used for the recovery study of the spiked samples and showed satisfactory recoveries. Graphical Abstract Schematic diagram of the development of biotinylated broad-specificity single-chain variable fragment antibody-based immunoassay for organophosphorus pesticides.
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: College of Life Science, Taishan Medical University, Taian, 271016, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Tian
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: Jiye
authors.initials: J
authors.lastname: Liu
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. zhyouyang@sdau.edu.cn.
authors.forename: Zhengyou
authors.initials: Z
authors.lastname: Yang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D019921:Biotinylation; D061505:Cell Surface Display Techniques; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005504:Food Analysis / Q000379:methods*; D008807:Mice, Inbred BALB C; D009943:Organophosphorus Compounds / Q000032:analysis*; D010575:Pesticides / Q000032:analysis*; D011994:Recombinant Proteins / Q000737:chemistry; D057127:Single-Chain Antibodies / Q000737:chemistry*; D014675:Vegetables / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D009943:Organophosphorus Compounds; D010575:Pesticides; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies
keywords: Biotinylation; Organophosphorus pesticides; Phage display; Refolding; ScFv