Steukers Mieke, Schaus Jean-Michel, van Gool Reinoud, Hoyoux Anne, Richalet Pascale, Sexton Daniel J, Nixon Andrew E, Vanhove Marc
Dyax s.a., Building 22, Sart-Tilman, Liège, Belgium.
J Immunol Methods. 2006 Mar 20;310(1-2):126-35. doi: 10.1016/j.jim.2006.01.002. Epub 2006 Jan 31.
Human antibodies able to bind with high affinity and specificity to numerous targets have been successfully identified from Fab phage display libraries. A key step in the library selection screening process is the early characterization of library isolates in order to determine which of these isolates to pursue further. Here we describe a Biacore assay that allows isolated clones expressed as soluble Fab fragments in E. coli to be screened and ranked based on their affinity against the target. The assay takes advantage of our ability to measure Fab concentrations in crude bacterial extracts in Biacore using very high density Protein A chips. The procedure allows up to 100 clones per week to be screened and permits the identification of a small number of high-affinity Fabs from a large batch obtained following library selection or affinity maturation.
已经从Fab噬菌体展示文库中成功鉴定出能够以高亲和力和特异性结合众多靶标的人源抗体。文库筛选过程中的一个关键步骤是对文库分离株进行早期表征，以确定哪些分离株值得进一步研究。在此，我们描述了一种Biacore检测方法，该方法可对在大肠杆菌中表达为可溶性Fab片段的分离克隆进行筛选，并根据它们与靶标的亲和力进行排序。该检测方法利用了我们使用超高密度蛋白A芯片在Biacore中测量粗细菌提取物中Fab浓度的能力。该程序每周最多可筛选100个克隆，并能够从文库筛选或亲和力成熟后获得的大量克隆中鉴定出少数高亲和力的Fab。
