He Qiyi, Pan Junkang, Zhang Yijia, Xu Zhihao, Morisseau Christophe, Hammock Bruce D, Li Dongyang
College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, People's Republic of China.
Zhejiang Key Laboratory of Intelligent Sensing and Robotics for Agriculture, Zhejiang University, Hangzhou 310058, People's Republic of China.
Trends Analyt Chem. 2025 Nov;192. doi: 10.1016/j.trac.2025.118404. Epub 2025 Aug 2.
Nanobodies, derived from camelid heavy-chain antibodies, offer exceptional specificity, stability, and solubility, enabling rapid and sensitive immunoassays. Their small size makes them ideal scaffolds for genetic fusion with reporter proteins, functional peptides, and signal amplification modules. In contrast to conventional antibodies, nanobodies can be precisely engineered at the gene level, allowing directional conjugation and efficient incorporation of signal-enhancing elements. This tunability simplifies assay design and reduces processing steps. Additionally, recombinant expression systems enable stable, scalable, and standardized production of nanobody-based immunoreagents, ensuring reproducibility in diagnostics. In this review, we summarize recent advances in the engineering and application of nanobody fusion proteins in immunoassays, with a particular focus on three main strategies: signal reporting, site-oriented strategies, and multivalency. We highlight how these approaches enhance sensitivity, shorten assay time, and enable multiplex detection. Finally, we discuss future perspectives and remaining challenges in the design and application of nanobody-based platforms for diagnostics.
源自骆驼科动物重链抗体的纳米抗体具有卓越的特异性、稳定性和溶解性，可用于快速灵敏的免疫测定。其小尺寸使其成为与报告蛋白、功能肽和信号放大模块进行基因融合的理想支架。与传统抗体不同，纳米抗体可在基因水平上进行精确工程改造，允许定向缀合和高效掺入信号增强元件。这种可调性简化了测定设计并减少了处理步骤。此外，重组表达系统能够稳定、可扩展且标准化地生产基于纳米抗体的免疫试剂，确保诊断的可重复性。在本综述中，我们总结了纳米抗体融合蛋白在免疫测定中的工程和应用方面的最新进展，特别关注三种主要策略：信号报告、位点导向策略和多价性。我们强调这些方法如何提高灵敏度、缩短测定时间并实现多重检测。最后，我们讨论了基于纳米抗体的诊断平台设计和应用的未来前景及尚存的挑战。
