Department of Physiology and Membrane Biology, University of California Davis School of Medicine, Davis, California.
Current address: Research Center for Innovative Anti-Cancer Drugs, Research Institute of Tsinghua University in Shenzhen, Shenzhen, China.
Curr Protoc Neurosci. 2020 Dec;94(1):e107. doi: 10.1002/cpns.107.
Nanobodies (nAbs) are recombinant antigen-binding variable domain fragments obtained from heavy-chain-only immunoglobulins. Among mammals, these are unique to camelids (camels, llamas, alpacas, etc.). Nanobodies are of great use in biomedical research due to their efficient folding and stability under a variety of conditions, as well as their small size. The latter characteristic is particularly important for nAbs used as immunolabeling reagents, since this can improve penetration of cell and tissue samples compared to conventional antibodies, and also reduce the gap distance between signal and target, thereby improving imaging resolution. In addition, their recombinant nature allows for unambiguous definition and permanent archiving in the form of DNA sequence, enhanced distribution in the form of sequences or plasmids, and easy and inexpensive production using well-established bacterial expression systems, such as the IPTG induction method described here. This article will review the basic workflow and process for developing, screening, and validating novel nAbs against neuronal target proteins. The protocols described make use of the most common nAb development method, wherein an immune repertoire from an immunized llama is screened via phage display technology. Selected nAbs can then be taken through validation assays for use as immunolabels or as intrabodies in neurons. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Total RNA isolation from camelid leukocytes Basic Protocol 2: First-strand cDNA synthesis; V H and V repertoire PCR Basic Protocol 3: Preparation of the phage display library Basic Protocol 4: Panning of the phage display library Basic Protocol 5: Small-scale nAb expression Basic Protocol 6: Sequence analysis of selected nAb clones Basic Protocol 7: Nanobody validation as immunolabels Basic Protocol 8: Generation of nAb-pEGFP mammalian expression constructs Basic Protocol 9: Nanobody validation as intrabodies Support Protocol 1: ELISA for llama serum testing, phage titer, and screening of selected clones Support Protocol 2: Amplification of helper phage stock Support Protocol 3: nAb expression in amber suppressor E. coli bacterial strains.
纳米抗体（nAbs）是从重链仅有免疫球蛋白中获得的重组抗原结合可变结构域片段。在哺乳动物中，这些抗体仅存在于骆驼科动物（骆驼、羊驼、骆马等）中。由于纳米抗体在各种条件下具有高效折叠和稳定性，以及体积小的特点，因此在生物医学研究中具有很大的用途。后一特点对于用作免疫标记试剂的 nAbs 尤为重要，因为与传统抗体相比，这可以提高细胞和组织样本的穿透性，并且还可以减少信号与靶标之间的间隙距离，从而提高成像分辨率。此外，它们的重组性质允许以 DNA 序列的形式明确定义和永久存档，以序列或质粒的形式增强分布，并使用成熟的细菌表达系统（如这里描述的 IPTG 诱导方法）轻松且廉价地生产。本文将综述开发、筛选和验证针对神经元靶蛋白的新型 nAbs 的基本工作流程和过程。所描述的方案利用了最常见的 nAb 开发方法，即通过噬菌体展示技术筛选免疫骆驼的免疫受体库。然后可以对选定的 nAbs 进行验证实验，以用作免疫标记物或神经元内体。© 2020 威立出版社。基本方案 1：从骆驼白细胞中分离总 RNA 基本方案 2：第一链 cDNA 合成；V H 和 V 库 PCR 基本方案 3：噬菌体展示文库的制备 基本方案 4：噬菌体展示文库的淘选 基本方案 5：小量 nAb 表达 基本方案 6：选定 nAb 克隆的序列分析 基本方案 7：nAb 作为免疫标记物的验证 基本方案 8：生成 nAb-pEGFP 哺乳动物表达构建体 基本方案 9：nAb 作为内体的验证 支持方案 1：用于检测骆驼血清、噬菌体效价和筛选选定克隆的 ELISA 支持方案 2：辅助噬菌体库存的扩增 支持方案 3：在琥珀色抑制大肠杆菌菌株中表达 nAb
