Nanobodies (Nbs) have shown great potential in immunodetection of small-molecule contaminants in food and environmental monitoring. However, the limited knowledge of the mechanism of Nbs binding to small molecules has hampered the development of high-affinity Nbs and assay improvement. We previously reported two homologous nanobodies Nb26 and Nb28 specific to aflatoxin B1 (AFB1), with the former exhibiting higher sensitivity in ELISA. Herein, Nb26 was selected as the model antibody to resolve its solution nuclear magnetic resonance (NMR) structure, and investigate its AFB1 recognition mechanism. The results revealed that Nb26 exhibits a typical immunoglobulin fold and its AFB1-binding interface is uniquely located in complementarity-determining region 3 (CDR3) and framework region 2 (FR2). This finding was applied to improve the binding activity of Nb28 against AFB1 by constructing two Nb28-based mutants A50V and S102D, resulting in 2.3- and 3.3-fold sensitivity enhancement over the wild type, respectively. This study develops an NMR-based strategy to analyze the underlying mechanism of Nb against AFB1, and successfully generated two site-modified Nbs with improved detection sensitivity. It is believed that this work could greatly expand the applications of Nbs by providing a way to enhance the binding activity.
Keywords: Aflatoxin B(1); Nanobody; Structure-guided modification.
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