Nanobodies: A Promising Toolkit for Diagnostic Applications

ABSTRACT Nanobodies are single‐domain antigen‐binding fragments derived from camelid heavy‐chain‐only antibodies (HCAbs). They combine a small size (~12–15 kDa) with high solubility, favorable stability, and antigen‐binding capability. These properties can facilitate recognition of less accessible epitopes, improve tissue penetration in selected settings, and support assay performance under conditions that may challenge conventional antibodies. In recent years, nanobodies have emerged as versatile molecular recognition tools for diagnostic applications and been integrated into diverse detection platforms—including enzyme‐linked immunosorbent assay (ELISA), lateral flow immunoassays (LFIAs), biosensors, and in vivo imaging techniques such as positron emission tomography (PET), single‐photon emission computed tomography (SPECT), and nanobody–quantum dot conjugates. Advances such as multivalent designs, site‐specific conjugation, and optimized expression systems have further enhanced the performance of nanobodies in clinical diagnostics. Here, we summarize recent progress in nanobody‐based diagnostic technologies, outlining the key structural and biochemical features that contribute to their efficacy, reviewing ongoing clinical trials, and highlighting successful technological developments. Additionally, we discuss emerging strategies aimed at creating scalable, sensitive, and application‐ready nanobody‐enabled diagnostic platforms.