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.

Construction of an Immunized Rabbit Phage Display Library for Selecting High Activity against Bacillus thuringiensis Cry1F Toxin Single-Chain Antibodies

“…After switching back to the original culture medium, the concentration of RF4-scFv proteins expressed in E. coli HB2151 was 2.93 mg/L, which was better than the 0.8 mg/L reported by Wan et al but could not compare with the 5.0 mg/L reported by Lobova et al Therefore, it is necessary to further optimize the conditions, such as the IPTG concentration, culture temperature, and time, etc. − We established the IC-ELISA for detecting Cry1F toxin based on purified RF4-scFv (Figure B). According to the calculation formula of the standard curve [ y = 10.347 ln­( x ) + 24.98, R 2 = 0.9702], the concentration of RF4-scFv binding to Cry1F toxin causing 50% inhibition (IC 50 ) was 11.56 ng/mL, the working calibration range (IC 20 to IC 80 ) was between 0.92 and 107.36 ng/mL, and the detection limit (IC 10 ) was 0.18 ng/mL.…”

Single‐chain fragment variable produced by phage display technology: Construction, selection, mutation, expression, and recent applications in food safety

“…This was followed by the addition of dithiothreitol for elution, which successfully screened for high‐affinity antibodies (Kobayashi et al., 2005). The scFv obtained by applying biotin–streptavidin screening technology possesses a wide range of specificity for organophosphorus pesticides, and 30 organophosphorus pesticides could be sensitively detected with limits ranging from 4.1 ng/ml to 90.7 ng/ml (Zhao et al., 2016). Chin et al.…”

“…coli (Cai et al., 2014). Another means of increasing the production of scFv is by expressing scFv in the form of inclusion bodies (although this is not recommended); however, to ensure the affinity of scFv, scFv is fused to the biotin receptor domain (BAD) and a more sensitive biotin–streptavidin system is adopted to meet detection needs (Zhao et al., 2016).…”

Glycerol and Antimicrobial Peptide-Modified Natural Latex for Bacteriostasis of Skin Wounds

“…Hydrogen bonds can be formed between the −OH of the plasticizer molecules and the −COOH and −OH of the film material, thereby weakening the force between the molecular chains of the film material and improving the mobility of their molecular chains, but higher concentrations of plasticizers can affect the cross-linking among the molecules of the film material, leading to a decrease in its film-forming properties. 32 Effects of GI and AMP Additions on the Tensile Properties of NRL-GI Films. As a plasticizer, GI can affect the stability of the internal structure of polymer materials, resulting in changes in tensile strength.…”