Feldhaus Michael, Siegel Robert
Battelle/Pacific Northwest National Laboratory, Richland, WA, USA.
Methods Mol Biol. 2004;263:311-32. doi: 10.1385/1-59259-773-4:311.
A method to screen and isolate antigen specific clones from a library of single-chain antibodies expressed on the surface of yeast cells is presented. Two rounds of magnetic bead enrichment before flow cytometric sorting enables one to screen libraries of far greater diversity than can be screened by just flow cytometry. The strength of flow cytometric sorting is the ability to follow the selection in real time and to isolate easily the highest affinity antigen-specific clones. A major strength of yeast display as a discovery platform is the ability to characterize the binding properties, the affinity of a clone without the need for subcloning, expression, and purification of the scFv. The methodology for directed evolution of single-chain antibodies to increase the affinity of a clone is also described.
本文介绍了一种从酵母细胞表面表达的单链抗体文库中筛选和分离抗原特异性克隆的方法。在进行流式细胞术分选之前，先进行两轮磁珠富集，这使得人们能够筛选比仅通过流式细胞术所能筛选的多样性大得多的文库。流式细胞术分选的优势在于能够实时跟踪筛选过程，并轻松分离出亲和力最高的抗原特异性克隆。酵母展示作为一种发现平台的一个主要优势是能够在无需对单链抗体进行亚克隆、表达和纯化的情况下，对克隆的结合特性、亲和力进行表征。本文还描述了用于单链抗体定向进化以提高克隆亲和力的方法。
