Preparation and Clinical application of Human Monoclonal Antibodies using Phage Display System.

噬菌体展示系统人单克隆抗体的制备及临床应用。

• 批准号: 08672601
• 负责人: ITOH Kunihiko
• 金额: $ 1.41万
• 依托单位: Akita University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08672601/
• 关键词: Phage display system; Human antibodies; Combinatorial libraries; Rotaviruses; ファージディスプレイシステム

The goal of this research project is the establishing of the conventional procedure for making human monoclonal antibodies applicable for the prophylaxis or therapy using phage display system. We have selected rotaviruses as a target since they are recognized as the most important cause of severe viral gastroenteritis in humans and animals. The immunoglobulin Fd and light chain gene segments were amplified from PBL of two healthy individuals (ON and AO) with high serum titer to human rotavirus Wa (HRV Wa) using RT-PCR,then the combinatorial lgG1, k Fab phage display library were constructed. Five rounds of panning of these libraries with rabbit Ab-captured HRV Wayielded an approximately 30-fold enrichment in eluted phage. Eight Fab clones were finally isolated from these libraries by screening in the sandwich ELISA followed by the Bst NI finger printing. All Fab clones reacted with HRV Wa in a concerntration dependent manner with no cross-reactivity to a panel of Ags. The Fab clones from ON library showed the strain cross-reactivity, on the contrary, those from AO library were specific for Wa. Although the immunoblotting analysis revealed that Fabs from two individual libraries react with the identical Ag, the VP6 protein, they would recognize the different epitopes because of their distinct strain specificity and the different VH gene usage. From these results, the phage display system would be powerful tool for making clinically useful human monoclonal antibodies, and the isolated Fabs would be useful for detection or identification of rotaviruses from biological specimen in clinical laboratory testing.

本研究的目的是利用噬菌体展示系统建立一种常规的制备人源单抗用于预防或治疗的方法。我们选择轮状病毒作为目标，因为它们被认为是人类和动物严重病毒性胃肠炎的最重要原因。以抗人轮状病毒Wa(HRV Wa)高滴度的健康人外周血为材料，用RT-PCR方法扩增出免疫球蛋白Fd和轻链基因片段，构建了lgG1、k Fab噬菌体展示文库。用兔抗体捕获的HRV对这些文库进行了五轮淘洗，在洗脱的噬菌体中获得了大约30倍的浓缩。通过夹心ELISA法和BST NI指纹图谱筛选，最终从这些文库中分离到8个Fab克隆。所有Fab克隆均与HRV Wa呈浓度依赖性反应，与一组AGS无交叉反应。来自On文库的Fab克隆显示了菌株的交叉反应，而来自Ao文库的Fab克隆则是Wa特异的。虽然免疫印迹分析表明，来自两个单独文库的Fabs与相同的抗原VP6蛋白发生反应，但由于它们不同的菌株特异性和不同的VH基因使用，它们可以识别不同的表位。从这些结果来看，噬菌体展示系统将是制备临床有用的人单抗的有力工具，所分离的抗体将用于临床实验室检测或鉴定生物标本中的轮状病毒。