Deantonio Cecilia, Cotella Diego, Macor Paolo, Santoro Claudio, Sblattero Daniele
Department of Health Sciences and Interdisciplinary Research Center for Autoimmune Diseases (IRCAD), University of Eastern Piedmont, Novara, Italy.
Methods Mol Biol. 2014;1060:277-95. doi: 10.1007/978-1-62703-586-6_14.
During the last 15 years in vitro technologies opened powerful routes to combine the generation of large libraries together with fast selection procedures to identify lead candidates. One of the commonest methods is based on the use filamentous phages. Antibodies (Abs) can be displayed successfully on the surface of phage by fusing the coding sequence of the antibody variable (V) regions to the phage minor coat protein pIII. By creating large libraries, antibodies with affinities comparable to those obtained using traditional hybridomas technology can be selected by a series of cycles of selection on antigen. As in this system antibody genes are cloned simultaneously with selection they can be easily further engineered for example by increasing their affinity (to levels unobtainable in the immune system), modulating their specificity or their effector function (by recloning into a full-length immunoglobulin scaffold). This chapter describes the basic protocols for antibody library construction, handling, and selection.
在过去15年中，体外技术开辟了强大的途径，可将大型文库的构建与快速筛选程序相结合，以鉴定潜在的候选物。最常见的方法之一是基于丝状噬菌体的使用。通过将抗体可变（V）区的编码序列与噬菌体次要外壳蛋白pIII融合，抗体（Abs）可以成功地展示在噬菌体表面。通过创建大型文库，可以通过一系列对抗原的筛选循环来选择亲和力与使用传统杂交瘤技术获得的抗体相当的抗体。由于在该系统中抗体基因在筛选的同时被克隆，它们可以很容易地进一步改造，例如通过提高其亲和力（达到免疫系统中无法获得的水平）、调节其特异性或效应功能（通过重新克隆到全长免疫球蛋白支架中）。本章描述了抗体文库构建、处理和筛选的基本方案。
