Ito Yuji
Department of Chemistry and Bioscience, Graduate School of Science and Engineering, Kagoshima University.
Yakugaku Zasshi. 2017;137(7):823-830. doi: 10.1248/yakushi.16-00252-2.
As an alternative to hybridoma technology, the antibody phage library system can also be used for antibody selection. This method enables the isolation of antigen-specific binders through an in vitro selection process known as biopanning. While it has several advantages, such as an avoidance of animal immunization, the phage cloning and screening steps of biopanning are time-consuming and problematic. Here, we introduce a novel biopanning method combined with high-throughput sequencing (HTS) using a next-generation sequencer (NGS) to save time and effort in antibody selection, and to increase the diversity of acquired antibody sequences. Biopannings against a target antigen were performed using a human single chain Fv (scFv) antibody phage library. VH genes in pooled phages at each round of biopanning were analyzed by HTS on a NGS. The obtained data were trimmed, merged, and translated into amino acid sequences. The frequencies (%) of the respective VH sequences at each biopanning step were calculated, and the amplification factor (change of frequency through biopanning) was obtained to estimate the potential for antigen binding. A phylogenetic tree was drawn using the top 50 VH sequences with high amplification factors. Representative VH sequences forming the cluster were then picked up and used to reconstruct scFv genes harboring these VHs. Their derived scFv-Fc fusion proteins showed clear antigen binding activity. These results indicate that a combination of biopanning and HTS enables the rapid and comprehensive identification of specific binders from antibody phage libraries.
作为杂交瘤技术的替代方法，抗体噬菌体文库系统也可用于抗体筛选。该方法能够通过一种称为生物淘选的体外筛选过程分离抗原特异性结合物。虽然它有几个优点，如避免动物免疫，但生物淘选的噬菌体克隆和筛选步骤既耗时又有问题。在此，我们引入一种结合高通量测序（HTS）的新型生物淘选方法，使用新一代测序仪（NGS）来节省抗体筛选的时间和精力，并增加获得的抗体序列的多样性。使用人单链Fv（scFv）抗体噬菌体文库对目标抗原进行生物淘选。在每轮生物淘选时，对混合噬菌体中的VH基因通过NGS上的HTS进行分析。对获得的数据进行修剪、合并，并翻译成氨基酸序列。计算每个生物淘选步骤中各个VH序列的频率（%），并获得扩增因子（通过生物淘选的频率变化）以估计抗原结合潜力。使用具有高扩增因子的前50个VH序列绘制系统发育树。然后挑选形成簇的代表性VH序列，用于重建含有这些VH的scFv基因。它们衍生的scFv-Fc融合蛋白显示出明显的抗原结合活性。这些结果表明，生物淘选和HTS的结合能够从抗体噬菌体文库中快速、全面地鉴定特异性结合物。
