Zhou Yu, Marks James D
University of California, San Francisco, CA, USA.
Methods Mol Biol. 2009;525:145-60, xv. doi: 10.1007/978-1-59745-554-1_7.
Phage antibody technology is a powerful approach for generating human antibodies to virtually any target antigen. For many therapeutic applications, it is useful to generate antibodies that bind to cell-surface receptors in a manner where binding results in internalization of the antibody. This allows use of the antibody to deliver toxic payloads intracellularly to achieve a therapeutic effect. Here we describe how phage antibody libraries can be directly selected on tumor cell lines to generate antibodies binding cell-surface receptors and which are rapidly internalized upon binding. Protocols are provided showing how to (1) directly select internalizing antibodies from phage antibody libraries; (2) screen phage antibodies in a high-throughput flow cytometry assay for binding to the tumor cell line used for selection; (3) identify the antigen bound by the phage antibody using immunoprecipitation and mass spectrometry; and (4) verify and quantitate such that phage antibodies are internalized.
噬菌体抗体技术是一种强大的方法，可用于生成针对几乎任何目标抗原的人源抗体。对于许多治疗应用而言，生成以能导致抗体内化的方式结合细胞表面受体的抗体是很有用的。这使得能够利用抗体将有毒载荷递送至细胞内以实现治疗效果。在此，我们描述了如何在肿瘤细胞系上直接筛选噬菌体抗体文库，以生成结合细胞表面受体且在结合后能迅速内化的抗体。文中提供了相关方案，展示了如何：(1) 直接从噬菌体抗体文库中筛选内化抗体；(2) 在高通量流式细胞术检测中筛选噬菌体抗体与用于筛选的肿瘤细胞系的结合情况；(3) 使用免疫沉淀和质谱鉴定噬菌体抗体所结合的抗原；以及 (4) 验证并定量噬菌体抗体的内化情况。
