1993年，Hamers等人意外发现了骆驼科物种中自然产生的重链抗体（HCAbs）。自那时以来，基于HCAbs的抗体片段受到了研究人员和生物技术公司的广泛关注。由于其在生物物理化学方面的优势，骆驼科单域抗体（sdAbs，VHHs，纳米抗体）正越来越多地被用作可行的免疫治疗方法。目前，有多个以VHH为基础的治疗药物处于不同阶段的临床试验中，采用了多种格式，例如双价和多价、双特异性和多特异性、CAR-T和抗体药物联合物。第一个获批的VHH药物是一种双价的人源化VHH（caplacizumab），在2018年被EMA批准用于治疗罕见的血栓性疾病，FDA在2019年批准。随后，在2022年批准了一种以抗BCMA VHH为基础的CAR-T细胞产品（ciltacabtagene autoleucel；CARVYKTI™），最近还在日本批准了一种以三价抗肿瘤坏死因子α为基础的VHH药物（ozoralizumab；Nanozora®），用于治疗类风湿性关节炎。在本章中，我们提供了描述分离特异性抗原VHH的最新方法，包括短毛驢免疫、噬菌体展示文库构建、噬菌体筛选和通过ELISA筛选可溶性VHH，通过表面等离子共振测定亲和性，通过流式细胞术进行功能性细胞结合，并通过免疫沉淀进行额外验证的方法。我们提出并讨论了分离和表征特异性抗原VHH的全面、逐步方法。这包括表达、生物素化、纯化和表征已分离的VHH的方法。为了证明整个策略的可行性，我们提供了我们实验室以前分离和表征的VHH的示例。© 2023. 作者，斯普林格自然科学出版集团的一部分，独家许可授权给Springer Science+Business Media, LLC.

Naturally occurring heavy chain antibodies (HCAbs) in Camelidae species were a surprise discovery in 1993 by Hamers et al. Since that time, antibody fragments derived from HCAbs have garnered considerable attention by researchers and biotechnology companies. Due to their biophysico-chemical advantages over conventional antibody fragments, camelid single-domain antibodies (sdAbs, VHHs, nanobodies) are being increasingly utilized as viable immunotherapeutic modalities. Currently there are multiple VHH-based therapeutic agents in different phases of clinical trials in various formats such as bi- and multivalent, bi- and multi-specific, CAR-T, and antibody-drug conjugates. The first approved VHH, a bivalent humanized VHH (caplacizumab), was approved for treating rare blood clotting disorders in 2018 by the EMA and the FDA in 2019. This was followed by the approval of an anti-BCMA VHH-based CAR-T cell product in 2022 (ciltacabtagene autoleucel; CARVYKTI™) and more recently a trivalent antitumor necrosis factor alpha-based VHH drug (ozoralizumab; Nanozora®) in Japan for the treatment of rheumatoid arthritis. In this chapter we provide protocols describing the latest developments in isolating antigen-specific VHHs including llama immunization, construction of phage-displayed libraries, phage panning and screening of the soluble VHHs by ELISA, affinity measurements by surface plasmon resonance, functional cell binding by flow cytometry, and additional validation by immunoprecipitation. We present and discuss comprehensive, step-by-step methods for isolating and characterization of antigen-specific VHHs. This includes protocols for expression, biotinylation, purification, and characterization of the isolated VHHs. To demonstrate the feasibility of the entire strategy, we present examples of VHHs previously isolated and characterized in our laboratory.© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.