Computer-Assisted Hapten Design for High-Performance Recombinant Antibody against Napropamide: Molecular Recognition...

Given the widespread use and potential health risks of Napropamide (Nap), establishing a rapid and sensitive detection method for its residues is imperative. Herein, we designed novel haptens H1 and H2 via a computer-assisted strategy. H1, with a long-chain carboxyl extension, generated a high-sensitivity recombinant antibody (rAb) 2A4 (IC₅₀ = 1.64 ng/mL), outperforming hapten H2-derived rAb 3D7 (IC₅₀ = 5.08 ng/mL). Molecular docking (MD) showed that the enhanced hydrogen bond donor capacity of hapten H1 formed a hydrophilic binding pocket, with key interactions (a strong hydrogen bond between SER A-103 and TYR B-96 and π-π stacking with TRP B-50) ensuring the high sensitivity of rAb 2A4, validating rational hapten design. To validate the reliability of MD predictions, alanine scanning mutagenesis was further performed in this study, with the experimental results further confirming the critical roles of the key amino acid residues in antibody recognition. Based on rAb 2A4, a dual T-line colloidal gold test strip was developed with a calculated limit of detection of 18.72-36.54 μg/kg for cabbage, watermelon, soybean, and peanut samples. The quantitative results were consistent with Liquid Chromatography-Tandem Mass methods, demonstrating high sensitivity and user-friendliness, suitable for high-throughput monitoring. Therefore, this study provides a practical tool for rapidly detecting Nap in food.