Development of a peptidomimetic-based competitive immunoassay for endosulfan: Insights into a distinct recognition...

Peptidomimetics have emerged as promising non-toxic alternatives to traditional hapten-based antigens for detecting small-molecule pollutants (SMPs) in food samples. However, the competitive binding mechanism between SMPs and peptidomimetics remains poorly understood, hindering their rational optimization and broader application. Here, we isolated 13 peptidomimetics specific to anti-endosulfan mAb, analyzed and experimentally verified their binding mechanisms through AlphaFold 3 and site-directed mutagenesis. Our analysis revealed that although these peptidomimetics compete with endosulfan for antibody binding, they adopt distinct interaction modes involving both the central aromatic residue and additional peripheral contacts. Based on the most sensitive peptidomimetic, we optimized a P-ELISA for endosulfan detection, achieving an IC₅₀ of 7.10 ng/mL and LOD of 1.55 ng/mL. The method was validated in apple, orange and soil samples, with recoveries of 74.4 %-90.6 % and good correlation with GC-MS (R² = 0.9715). Our study provides a reliable method for endosulfan detection and offers valuable insights for peptidomimetics optimization.