Development, Screening, and Validation of Camelid-Derived Nanobodies for Neuroscience Research.
Development, Screening, and Validation of Camelid-Derived Nanobodies for Neuroscience Research.
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DOI：
10.1002/cpns.107
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发表时间：
2020-12
影响因子：
--
通讯作者：
Trimmer JS
中科院分区：
文献类型：
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作者：
Gavira-O'Neill CE;Dong JX;Trimmer JS
纳米抗体（nAb）是从仅重链免疫球蛋白获得的重组抗原结合可变结构域片段。在哺乳动物中，这些是骆驼科动物（骆驼、美洲驼、羊驼等）所独有的。纳米抗体在生物医学研究中有很大的用途，因为它们在各种条件下的有效折叠和稳定性，以及它们的小尺寸。后一特征对于用作免疫标记试剂的nAb特别重要，因为与常规抗体相比，这可以改善细胞和组织样品的穿透，以及减少信号和靶标之间的差距距离，从而改善成像分辨率。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，以及使用完善的细菌表达系统（如本文所述的IPTG诱导方法）容易且廉价的生产。该单元将回顾开发，筛选和验证针对神经元靶蛋白的新型nAb的基本工作流程和过程。所述方案利用最常见的nAb开发方法，其中通过噬菌体展示技术筛选来自免疫美洲驼的免疫库。然后可以通过验证测定来获取所选择的nAb以用作免疫标记或用作神经元中的胞内抗体。
Nanobodies (nAbs) are recombinant antigen binding variable domain fragments obtained from the 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, as well as 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 unit 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.