Development of a biotinylated broad-specificity single-chain variable fragment antibody and a sensitive immunoassay for detection of organophosphorus pesticides
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- 作者：Fengchun Zhao ; Yuan Tian ; Huimin Wang ; Jiye Liu…
- 关键词：Phage display ; ScFv ; Refolding ; Biotinylation ; Organophosphorus pesticides
- 刊名：Analytical and Bioanalytical Chemistry
- 出版年：2016
- 出版时间：September 2016
- 年：2016
- 卷：408
- 期：23
- 页码：6423-6430
- 全文大小：1,375 KB
- 刊物类别：Chemistry and Materials Science
- 刊物主题：Chemistry
Analytical Chemistry
Food Science
Inorganic Chemistry
Physical Chemistry
Monitoring, Environmental Analysis and Environmental Ecotoxicology - 出版者：Springer Berlin / Heidelberg
- ISSN：1618-2650
- 卷排序：408
文摘
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.