Molecular Evolution of Antiparathion Nanobody with Enhanced Sensitivity and Specificity Based on Structural Analysis

作者：Jiadong Li, Xing Shen, Zhenlin Xu, Yifan Liang, Yu‐Dong Shen, Jin‐Yi Yang, Hong Wang · 发表于：Journal of Agricultural and Food Chemistry · 年份：2023 · DOI：10.1021/acs.jafc.3c05176 · 被引用次数：18 · 研究领域：Monoclonal and Polyclonal Antibodies Research、Computational Drug Discovery Methods、Bacteriophages and microbial interactions

Nanobody (Nb) has gained significant attention in immunoassays owing to its numerous advantages, particularly its ease of molecular evolution. However, the limited understanding of how high sensitivity and specificity attained for antihapten Nbs hamper the development of high-performance Nbs. Herein, the antiparathion Nb (Nb9) we prepared previously was chosen as the model, and an approach based on X-ray crystallography, molecular docking, and rational site-directed saturation mutation for constructing a rapid and effective platform for nanobody evolution was described. Based on the structural analysis, two mutants, namely Nb-D5 (IC 50 = 2.4 ± 0.2 ng/mL) and Nb-D12 (IC 50 = 2.7 ± 0.1 ng/mL), were selected out from a six-sites directed saturation mutation library, 3.5-fold and 3.1-fold sensitivity enhancement over Nb9 to parathion, respectively. Besides, Nb-D12 exhibited improved sensitivity for quinalphos, triazophos, and coumaphos (5.4–35.4 ng/mL), indicating its broader detection potential. Overall, our study advances an effective strategy for the future rational evolution of Nbs with desirable performance.