A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies
A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies
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DOI：
10.1074/jbc.274.26.18218
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发表时间：
1999-06-25
影响因子：
4.8
通讯作者：
Hoogenboom, HR
中科院分区：
文献类型：
--
作者：
de Haard, HJ;van Neer, N;Hoogenboom, HR
我们报道了首个大容量非免疫Fab格式噬菌体抗体文库的设计、构建和使用，该文库能够快速分离和分析抗原特异性人抗体片段的亲和力。通过高效的两步克隆法将单独克隆的重链和轻链可变区文库进行组合，从而能够克隆出总共3.7×10¹⁰个独立的Fab克隆。通过对6种测试抗原平均成功筛选出14种不同的Fab，确定了该文库的性能。这些抗原包括破伤风类毒素、半抗原苯噁唑酮、与乳腺癌相关的MUC1抗原以及三种高度相关的糖蛋白激素：人绒毛膜促性腺激素、人黄体生成素和人卵泡刺激素。在后一类中，鉴定出了一组激素特异性或交叉反应性抗体。该文库的设计允许用多克隆噬菌体制剂监测筛选过程，并利用BIAcore上未纯化的Fab片段对抗体解离速率进行大规模筛选。回收了解离速率在10⁻²到10⁻⁴ s⁻¹数量级且亲和力高达2.7 nM的抗体。这些噬菌体抗体的动力学与二次免疫反应相关抗体处于同一数量级。这个新的噬菌体抗体文库将成为针对许多不同靶点的抗体的重要来源，并在功能基因组学领域的靶点发现和验证中发挥至关重要的作用。
We report the design, construction, and use of the first very large non-immunized phage antibody library in Fab format, which allows the rapid isolation and affinity analysis of antigen-specific human antibody fragments. Individually cloned heavy and light chain variable region libraries were combined in an efficient two-step cloning procedure, permitting the cloning of a total of 3.7 x 10(10) independent Fab clones. The performance of the library was determined by the successful selection of on average 14 different Fabs against 6 antigens tested. These include tetanus toroid, the hapten phenyl-oxazolone, the breast cancer-associated MUC1 antigen, and three highly related glycoprotein hormones: human chorionic gonadotropin, human luteinizing hormone, and human follicle-stimulating hormone. In the latter category, a panel of either homone-specific or crossreactive antibodies were identified. The design of the library permits the monitoring of selections with polyclonal phage preparations and to carry out large scale screening of antibody off-rates with unpurified Fab fragments on BIAcore. Antibodies with off-rates in the order of 10(-2) to 10(-4) s(-1) and affinities up to 2.7 nM were recovered. The kinetics of these phage antibodies are of the same order of magnitude as antibodies associated with a secondary immune response. This new phage antibody library is set to become a valuable source of antibodies to many different targets, and to play a vital role in target discovery and validation in the area of functional genomics.