Insights into the Binding Profile of Anti-chlorpyrifos Recombinant Antibodies: From Computational Simulation to Immunoassay Validation

作者：Rubing Zou, Yuanhao Guo, Yuanhao Guo, Yan Wang, Xinying Lu, Zhongjie Ma, Linfei Shou, Yihua Liu, Guonian Zhu, Yirong Guo, Yirong Guo · 发表于：Analytical Chemistry · 年份：2023 · DOI：10.1021/acs.analchem.3c01355 · 被引用次数：19 · 研究领域：Protein purification and stability、Pesticide Residue Analysis and Safety、Monoclonal and Polyclonal Antibodies Research

A novel virtual screening strategy was proposed for the profiling and discovery of active variable regions (VRs) that encode hapten-specific recombinant antibodies (rAbs). Chlorpyrifos, a hazardous organophosphorus pesticide, was selected as the target. First, a VR model-14G4 from anti-chlorpyrifos hybridoma was built via homology modeling. Its binding pattern toward seven organophosphorus analogues was assessed through virtual screening by performing molecular docking. Based on energy scoring, visual examination, and molecular interaction analysis, chlorpyrifos-methyl was also inferred as the high-affinity target for model-14G4 and was then confirmed via a non-competitive surface plasmon resonance (SPR) assay. Subsequently, we attempted to discover hapten-specific VRs by creating a collection of VR models for anonymous testing. Chlorpyrifos and model-14G4 were employed as the known hit and active VRs, respectively. After molecular docking, a novel anti-chlorpyrifos VR (model-1) was identified due to its satisfactory energy scoring and a similar binding pattern to the reference model-14G4. Expressed by HEK293(F) mammalian cells, the newly prepared full-length rAb-model-1 and rAb-14G4 exhibited high sensitivities for detecting chlorpyrifos by the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), with IC 50 of 3.01 ng/mL and 42.82 ng/mL, respectively. They recognized chlorpyrifos-methyl with a cross-reactivity (CR) of 2.5–17.3%. Moreover, the binding properties...