Production and characterization of a single-chain Fab fragment for the detection of O,O-diethyl organophosphorus pesticides

Abstract

A broad-specificity, single-chain antigen-binding fragment (scFab) for the detection of O,O-diethyl organophosphorus pesticides (DPPs) was produced and characterized. A recombinant plasmid encoding an Fab with broad specificity towards a specific class of DPPs was used as a template for amplification of the antibody heavy (Fd) and light (κ) chain genes, which were then cloned in two different orientations with the (Gly₄Ser)₃ linker. SDS-PAGE and western blotting were used to identify expression of the scFabs. Indirect competitive ELISA (icELISA) was used to test the immunology activity of two different κ/Fd orientations. The optimal scFab orientation was determined by characterizing the expression, specificity, and stability in comparison with their homologous scFv and Fab. The 50% inhibition of binding (IC₅₀) values of the κ-linker-Fd scFab for coumaphos and parathion determined by icELISA were ∼1.5 ng mL⁻¹ and 3.1 ng mL⁻¹, respectively, surpassing that of the reverse scFab orientation, Fd-linker-κ (3.7 μg mL⁻¹ and 8.5 μg mL⁻¹, respectively). The IC₅₀ of the κ-linker-Fd scFab was also similar to that of Fab and was relatively low in comparison with that of scFv. The concentration of scFab in the expression extract against the antigen was consistent with that of scFv, while Fab displayed a lower concentration in comparison with both scFv and scFab. After incubation for 9 days, scFab and Fab still exhibited high antigen-binding activity while scFv showed almost no remaining affinity. Analysis of DPP-spiked vegetable samples demonstrated that scFab-based detection using icELISA and gas chromatography by the QuEChERS approach yielded good sensitivity and reproducibility for monitoring food safety.