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。

基本信息

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

项目摘要

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.

该研究项目的目的是建立传统程序，以使人类单克隆抗体适用于使用噬菌体显示系统适用于预防或治疗。我们选择了轮状病毒作为靶标，因为它们被认为是人类和动物中严重病毒性胃炎的最重要原因。使用RT-PCR从两个健康个体（ON和AO）的PBL上放大了具有高血清滴度的PBL（ON和AO），使用RT-PCR将免疫球蛋白FD和轻链基因片段放大，然后构建了组合lgg1，k Fab Phage phage phage Phage展示库。这些图书馆的五轮平移，带有兔子AB捕获的HRV在洗脱噬菌体中大约30倍。最终，通过在三明治ELISA中筛选八个库克隆，然后筛选出BST NI指纹。所有Fab克隆都以关注的方式与HRV WA反应，对AGS小组没有交叉反应。来自ON文库的Fab克隆显示了应变交叉反应性，相反，AO库的晶球克隆对WA是特异性的。尽管免疫印迹分析表明，来自两个单个文库的FAB与相同的AG反应VP6蛋白，但由于其独特的应变特异性和不同的VH基因使用情况，它们会识别不同的表位。从这些结果中，噬菌体显示系统将是使临床上有用的人类单克隆抗体的强大工具，并且分离的Fabs对于在临床实验室测试中从生物标本中检测或鉴定轮状病毒很有用。