Isolating and engineering human antibodies using yeast surface display
Isolating and engineering human antibodies using yeast surface display
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DOI：
10.1038/nprot.2006.94
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Wittrup, K. Dane
中科院分区：
文献类型：
--
作者：
Chao, Ginger;Lau, Wai L.;Wittrup, K. Dane
该协议描述了使用酵母表面展示分离和工程化抗体或蛋白质以提高亲和力和稳定性的过程。首先使用磁激活细胞分选选择从酵母表面展示的现有非免疫人类文库中分离出单链抗体片段 (scFv)，然后使用流式细胞术进行选择。然后对这个富集的群体进行诱变，并进行连续几轮的随机诱变和流式细胞术选择，以通过定向进化获得所需的 scFv 特性。还描述了弱结合 scFv 的标记策略，以及表征和“滴定”酵母上展示的 scFv 克隆的程序。遵循该方案的最终结果是一组 scFv 对感兴趣的抗原具有更高的稳定性和亲和力。
This protocol describes the process of isolating and engineering antibodies or proteins for increased affinity and stability using yeast surface display. Single-chain antibody fragments (scFvs) are first isolated from an existing nonimmune human library displayed on the yeast surface using magnetic-activated cell sorting selection followed by selection using flow cytometry. This enriched population is then mutagenized, and successive rounds of random mutagenesis and flow cytometry selection are done to attain desired scFv properties through directed evolution. Labeling strategies for weakly binding scFvs are also described, as well as procedures for characterizing and 'titrating' scFv clones displayed on yeast. The ultimate result of following this protocol is a panel of scFvs with increased stability and affinity for an antigen of interest.