Groves Maria, Lane Steven, Douthwaite Julie, Lowne David, Rees D Gareth, Edwards Bryan, Jackson Ronald H
Cambridge Antibody Technology, Milstein Building, Granta Park, Cambridge CB1 6GH, UK.
J Immunol Methods. 2006 Jun 30;313(1-2):129-39. doi: 10.1016/j.jim.2006.04.002. Epub 2006 May 11.
A comparison has been performed, using phage display or ribosome display, of stringent selections on antibody populations derived from three rounds of phage display selection. Stringent selections were performed by reducing concentrations of the antigen, bovine insulin, down to 1 nM. Higher affinity antibodies were isolated using ribosome display in a process that introduces random mutations across the clone population. Whereas the highest affinity antibody produced by phage display, D3, has a K(d) of 5.8 nM as a scFv fragment, ribosome display generated higher affinity variants of this antibody with K(d) values of 189 pM and 152 pM, without or with the use of error prone mutagenesis, respectively. The affinities were further increased for each antibody on conversion of the scFv fragments to whole IgG format, to a K(d) of less than 21 pM for the highest affinity variant of D3. Mutation of VH D101 of antibody D3 to glycine or valine, removing the salt bridge between K94 and D101 at the base of VHCDR3, was responsible for the enhanced affinity observed. In addition to the variants of D3, other unrelated antibodies of comparable or higher affinity for insulin, were isolated by ribosome display, but not phage display, indicating that ribosome display can enrich for different populations of antibodies. Affinity maturation of phage antibody populations using ribosome display is a valuable method of rapidly generating diverse, high affinity antibodies to antigen and should be readily applicable to the isolation of antibodies for the detection and assay of biomarkers.
利用噬菌体展示或核糖体展示，对三轮噬菌体展示筛选所得抗体群体进行了严格筛选的比较。严格筛选是通过将抗原牛胰岛素的浓度降至1 nM来进行的。在一个对整个克隆群体引入随机突变的过程中，利用核糖体展示分离出了亲和力更高的抗体。噬菌体展示产生的亲和力最高的抗体D3，其单链抗体片段（scFv）的解离常数（K(d)）为5.8 nM，而核糖体展示分别在不使用或使用易错诱变的情况下，产生了该抗体的更高亲和力变体，其K(d)值分别为189 pM和152 pM。将scFv片段转化为完整IgG形式后，每种抗体的亲和力进一步提高，D3的最高亲和力变体的K(d)小于21 pM。抗体D3的重链可变区（VH）D101突变为甘氨酸或缬氨酸，消除了VH互补决定区3（VHCDR3）底部K94和D101之间的盐桥，这是观察到亲和力增强的原因所在。除了D3的变体之外, 通过核糖体展示还分离出了其他对胰岛素具有相当或更高亲和力的不相关抗体，但噬菌体展示未分离出这些抗体，这表明核糖体展示可以富集不同群体的抗体。利用核糖体展示对噬菌体抗体群体进行亲和力成熟是一种快速产生针对抗原的多种高亲和力抗体的有价值方法, 并且应该很容易应用于生物标志物检测和分析抗体的分离。
