Mitigating the Affinity/Specificity Trade-Off: Production of Monoclonal Antibodies with High Affinity and Fine Specificity to Sulfonamides by Ligand- and Receptor-Based Rational Hapten Design

Yingjie Zhang; Chenglong Li; Xiya Zhang; Changfei Duan; Qing Shen; Weilin Wu; Xuezhi Yu; Kai Wen; Jianzhong Shen; Zhanhui Wang

doi:10.1016/j.eng.2026.02.036

Engineering ›› :202602036 DOI: 10.1016/j.eng.2026.02.036

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Mitigating the Affinity/Specificity Trade-Off: Production of Monoclonal Antibodies with High Affinity and Fine Specificity to Sulfonamides by Ligand- and Receptor-Based Rational Hapten Design

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Abstract

The trade-off between affinity and specificity in molecular recognition elements (MREs), including antibodies, protein receptors, and aptamers, represents a well-documented challenge. Achieving MREs that combine high affinity with fine specificity is particularly difficult when targeting structurally similar analytes. To address this limitation, we propose a novel ligand- and receptor-based rational hapten design (LRRHD) strategy to regulate the generation of monoclonal antibodies (mAbs) exhibiting both high affinity and fine specificity, using sulfonamides (SAs) as model target analytes. This strategy enabled the identification of several novel haptens containing rigid spacer arms and led to the generation of mAb 10E6, which unexpectedly exhibited half-maximal inhibitory concentration values ranging from 0.23 to 20 μg·L⁻¹ across 29 tested SAs. The molecular recognition mechanisms underlying 10E6 binding were elucidated through crystal structure determination and molecular dynamics (MD) simulations. The results revealed that the extended complementarity determining region 3 of the heavy chain (CDRH3) of 10E6 forms a broader and more flexible ligand-binding pocket, allowing accommodation of diverse SAs with high affinity. In addition, mAb 10E6 was applied in an immunoassay, demonstrating limits of detection ranging from 0.29 to 4.7 μg·kg⁻¹ in skimmed milk and chicken samples. This study presents an effective rational hapten design strategy to mitigate the affinity/specificity trade-off and provides new insights into antibody discovery and vaccine development targeting small molecules.

Keywords

Molecular recognition elements / Antibody discovery / High affinity / Fine specificity / Rational hapten design / Molecular recognition mechanism / Crystal structure

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Yingjie Zhang, Chenglong Li, Xiya Zhang, Changfei Duan, Qing Shen, Weilin Wu, Xuezhi Yu, Kai Wen, Jianzhong Shen, Zhanhui Wang. Mitigating the Affinity/Specificity Trade-Off: Production of Monoclonal Antibodies with High Affinity and Fine Specificity to Sulfonamides by Ligand- and Receptor-Based Rational Hapten Design. Engineering 202602036 DOI:10.1016/j.eng.2026.02.036

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