From Computationally Guided Hapten Design to Monoclonal Antibody Production: A Novel Strategy for Rapid and Sensitive Detection of Flusilazole Residues

Flusilazole, a widely used triazole fungicide, poses potential risks through its residues in food and the environment. However, rapid surveillance of flusilazole residue remains limited by the lack of reported high-performance antibodies. Herein, we report a high-performance monoclonal antibody (mAb) against flusilazole using a computationally guided hapten design strategy. Among the four candidate haptens evaluated through molecular simulation of three-dimensional conformation, electrostatic potential mapping, and electronic structure descriptors, Hapten 01 exhibited the highest structural and electrostatic similarity to flusilazole. This hapten yielded a high-sensitivity monoclonal antibody (mAb-6H9) with excellent sensitivity (IC 50 value of 1.99 μg/L) and specificity (cross-reactivity rate <0.01%). On this basis, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a colloidal gold test strip (CGTS) were developed for rapid residue analysis, and the detection limits of the two assays were 0.46 and 1.18 μg/L in buffer, respectively. In spiked cucumber, strawberry, and orange samples, satisfactory recoveries (71–108%) and good precision were achieved. This study highlights the effectiveness of computationally guided hapten design and establishes a monoclonal antibody-enabled rapid immunoassay dual-platform format for flusilazole detection.