Efficient Discovery of Immunoglobulin Genes for Detecting Organophosphorus Pesticides Using a Reverse Virtual Screening Pipeline

Hapten-specific recombinant antibodies (rAbs) are increasingly crucial elements in bioanalytical chemistry for tracing small molecules, but identifying anti-hapten immunoglobulin genes remains challenging. Here, we established a reverse virtual screening pipeline for the efficient discovery of variable regio (VR) sequences encoding hapten-specific rAbs. Parathion, a representative organophosphorus pesticide, was selected as the model compound. First, a set of homology models for the antibody fragment variable (Fv) was constructed by pairing diverse VR sequences obtained from an anti-parathion hybridoma cell line. Based on energy scoring and molecular interaction analysis, Model 6 was predicted to be the anti-parathion receptor without relying on any reference model, and it was top-ranked in high-throughput immune repertoire sequencing. Using the HEK293(F) mammalian cell expression system, corresponding rAb-Model 6 was gained and showed high affinity for parathion in surface plasmon resonance analysis, along with broad specific binding to parathion-methyl and fenitrothion (equilibrium dissociation constant_: 3.84 × 10^-9-1.07 × 10^-8 M). Moreover, characterized by indirect competitive ELISAs, rAb-Model 6 exhibited high and close sensitivities (IC₅₀: 4.94-9.70 ng/mL) for the three EU-banned organophosphorus pesticides. These results confirmed the efficiency and effectiveness of this <i>in silico</i> approach for identifying anti-parathion VR sequences. Finally, the recognition mechanism of rAb-Model 6 was elucidated through molecular dynamics simulations. In summary, this study provides an efficient and cost-effective strategy for discovering anti-parathion immunoglobulin genes. It also offers a promising element for the simultaneous monitoring of parathion, parathion-methyl, and fenitrothion.