Production of Antigen-Binding Fragment against O,O-Diethyl Organophosphorus Pesticides and Molecular Dynamics Simulations of Antibody Recognition.

Immunoassay for pesticides is an emerging analytical method since it is rapid, efficient, sensitive, and inexpensive. In this study, a recombinant antigen-binding fragment (Fab) against a broad set of O,O-diethyl organophosphorus pesticides (DOPs) was produced and characterized. The κ chain and Fd fragment were amplified via PCR and inserted into the vector pComb3XSS and the soluble Fab on phagemid pComb3XSS was induced by isopropyl β-d-thiogalactoside in E. coli TOP 10F’. SDS-PAGE, Western blotting, and indirect competitive ELISA results indicated that Fab maintained the good characteristics of the parental mAb. To better understand antibody recognition, the three-dimensional (3D) model of Fab was built via homologous modeling and the interaction between Fab and DOPs was studied via molecular docking and dynamics simulations. The model clearly explained the interaction manner of Fab and DOPs, and showed that the Arg-L96 and Arg-H52 were mainly responsible for antibody binding. This work provided a foundation for further mutagenesis of Fab to improve its characteristics.

Production and characterization of a broad-specificity polyclonal antibody for O,O-diethyl organophosphorus pesticides and a quantitative structure-activity relationship study of antibody recognition.

Polyclonal antibody (PAb) with broad-specificity for O,O-diethyl organophosphorus pesticides (OPs) against a generic hapten, 4-(diethoxyphosphorothioyloxy)benzoic acid, was produced. The obtained PAb showed high sensitivity to seven commonly used O,O-diethyl OPs in a competitive indirect enzyme-linked immunosorbent assay (ciELISA) using a heterologous coating antigen, 4-(3-(diethoxyphosphorothioyloxy)phenylamino)-4-oxobutanoic acid. The 50% inhibition value (IC50) was 348 ng mL(-1) for parathion, 13 ng mL(-1) for coumaphos, 22 ng mL(-1) for quinalphos, 35 ng mL(-1) for triazophos, 751 ng mL(-1) for phorate, 850 ng mL(-1) for dichlofenthion, and 1301 ng mL(-1) for phoxim. The limit of detection (LOD) met the ideal detection criteria of all the seven OP residues. A quantitative structure-activity relationship (QSAR) model was constructed to study the mechanism of antibody recognition using multiple linear regression analysis. The results indicated that the frontier-orbital energies (energy of the highest occupied molecular orbital, E(HOMO), and energy of the lowest unoccupied molecular orbital, E(LUMO)) and hydrophobicity (log of the octanol/water partition coefficient, log P) were mainly responsible for the antibody recognition. The linear equation was log(IC50) = -63.274E(HOMO) + 15.985E(LUMO) + 0.556 log P-25.015, with a determination coefficient (r2) of 0.908.