Nanobodies (Nbs), the single-domain antigen-binding fragments, have emerged as promising biorecognition elements for immunoassays due to their small size, high stability, strong affinity, and ease of engineering. This review comprehensively summarizes recent advances in Nb-based immunoassay technologies, highlighting their advantages in immunoassay such as phage-displayed Nbs, Nb-reporter fusions, toxin-free substitutes using anti-idiotypic Nbs, reusable immunoaffinity ligands, bispecific Nbs for multi-target detection, and multivalent Nbs to enhance binding avidity. The review further discusses their applications in food safety, clinical diagnostics, and environmental monitoring, highlighting their impact across these fields. Key challenges such as the limited number of available Nbs, low expression levels, and commercialization bottlenecks are discussed, along with emerging solutions like synthetic libraries and computer-aided design. This review aims to provide insights into the development trends and application potential of Nb-based immunoassays, promoting their future advancement in analytical and diagnostic.
Keywords: Biosensors; Clinical biomarkers; Food safety; Immunoassays; Nanobody.
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