Malmborg A C, Dueñas M, Ohlin M, Söderlind E, Borrebaeck C A
Department of Immunotechnology, Lund University, Sweden.
J Immunol Methods. 1996 Oct 30;198(1):51-7. doi: 10.1016/0022-1759(96)00159-7.
In this report we show that phage displayed antibodies can be selected based on dissociation rate constants, using a BIAcore biosensor. To demonstrate the principle, two Fab phage stocks displaying antibodies specific for hen egg lysozyme or phenyloxazolone were mixed in a ratio of 1:10 and injected over the biosensor chip containing immobilized lysozyme. Antigen-specific bound phages were eluted and analysed for specificity and phage titer. This procedure enriched for phages carrying specific antibodies. Selection of high affinity binders from phage libraries was then demonstrated with the BIAcore when phages were eluted and collected at different time points. Soluble antibody fragments were subsequently expressed and their kinetic parameters were determined. The time of elution was directly proportional to the affinity, due to decreased dissociation rate constants. This procedure offers a rapid and simple approach for selecting binders from phage libraries differing in antibody dissociation rate constants.
在本报告中，我们展示了使用BIAcore生物传感器，可基于解离速率常数来筛选噬菌体展示抗体。为证明该原理，将展示对鸡蛋清溶菌酶或苯恶唑酮具有特异性抗体的两种Fab噬菌体原液按1:10的比例混合，并注入含有固定化溶菌酶的生物传感器芯片上。洗脱抗原特异性结合的噬菌体，并分析其特异性和噬菌体效价。该程序富集了携带特异性抗体的噬菌体。当在不同时间点洗脱并收集噬菌体时，BIAcore随后证明了从噬菌体文库中筛选高亲和力结合物的过程。随后表达了可溶性抗体片段，并测定了其动力学参数。由于解离速率常数降低，洗脱时间与亲和力直接成正比。该程序为从抗体解离速率常数不同的噬菌体文库中筛选结合物提供了一种快速且简单的方法。
