亲和层析法用于噬菌体抗体库的筛选

本研究报道一种基于固定化金属亲和层析(IMAC)的噬菌体抗体库液相筛选方法。将纯化的带有His标签的抗原与噬菌体抗体库混合,噬菌体抗体与抗原充分结合后再加入亲和介质,使噬菌体抗体抗原复合物通过His标签与介质结合,然后通过充分洗涤去除非特异性噬菌体抗体,最后将特异性噬菌体抗体洗脱下来,感染TG1,进行下一轮筛选。整个筛选过程中抗原与抗体的结合在液相中完成,不仅消除了固相介质对抗原表位的影响,也更有利于噬菌体抗体与抗原的充分作用。将此方法应用于HEV NE2蛋白特异性人源噬菌体抗体的筛选,抗原竞争ELISA,阳性血清阻断,可溶性单链抗体表达检测及测序结果表明,最终获得2个特异性人源抗体。

Affinity Chromatography Applied to Screening Phage Antibody Library

In this study,a new method based on immobilized metal affinity chromatography(IMAC) for screening phage antibody library against antigen in solution was reported.The purified antigen with His-tag was mixed with phage antibody library.After well combining,affinity resin was added to the mixture.Then the non-specific phage antibodies were removed by washing and the eluted specific bound phage antibodies were used to infect TG1 and then stepped into the next round.The binding process of antigen and antibody completed in the solution,which doesn't have the shortage of conformational changes encountered in the immobilized screening.We applied the method to screen human phage antibodies specific to HEV NE2.The results of antigen competitive ELISA,sera-blocking ELISA,scFv expression and sequence analysis indicated that two specific human phage antibodies were obtained.