Enhancing the detection sensitivity of nanobody against aflatoxin B1 through structure-guided modification
Enhancing the detection sensitivity of nanobody against aflatoxin B1 through structure-guided modification
复制标题
通过结构引导修饰提高纳米抗体对黄曲霉毒素B(1)的检测灵敏度。
DOI：
10.1016/j.ijbiomac.2021.11.182
复制
发表时间：
2021-12-05
影响因子：
8.2
通讯作者：
Liu, Maili
中科院分区：
文献类型：
--
作者：
He, Ting;Nie, Yao;Liu, Maili
纳米小体在食品和环境监测中对小分子污染物的免疫检测显示出巨大的潜力。然而，对Nbs与小分子结合机制的有限了解阻碍了高亲和力Nbs的发展和检测的改进。我们之前报道了两个同源纳米体Nb26和Nb28对黄曲霉毒素B-1 （AFB(1)）的特异性，前者在ELISA中表现出更高的敏感性。本文选择Nb26作为模型抗体，解析其溶液核磁共振（NMR）结构，研究其AFB(1)识别机制。结果表明，Nb26具有典型的免疫球蛋白褶皱，其AFB(1)结合界面独特地位于互补决定区3 （CDR3）和框架区2 （FR2）。利用这一发现，通过构建两个基于Nb28的突变体A(50)V和(SD)- d -102来提高Nb28对AFB(1)的结合活性，其敏感性分别比野生型提高2.3倍和3.3倍。本研究开发了一种基于核磁共振的策略来分析Nb对抗AFB的潜在机制(1)，并成功生成了两个具有更高检测灵敏度的位点修饰的Nbs。相信本研究可以提供一种增强Nbs结合活性的方法，从而极大地扩展Nbs的应用。
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 B-1 (AFB(1)), 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 AFB(1) recognition mechanism. The results revealed that Nb26 exhibits a typical immunoglobulin fold and its AFB(1)-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 AFB(1) by constructing two Nb28-based mutants A(50)V and (SD)-D-102, 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 AFB(1), 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.