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

本研究的目的是利用噬菌体展示系统建立制备可用于预防或治疗的人单克隆抗体的常规方法。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。采用RT-PCR方法从2例轮状病毒Wa（HRV Wa）高血清滴度健康人（ON和AO）外周血淋巴细胞中扩增出免疫球蛋白Fd和轻链基因片段，构建了噬菌体抗体库。用兔Ab捕获的HRV对这些文库进行五轮淘选，在洗脱的噬菌体中得到约30倍的富集。通过在夹心ELISA中筛选，然后通过Bst NI指纹图谱，最终从这些文库中分离出8个Fab克隆。所有Fab克隆均以浓度依赖性方式与HRV Wa反应，与一组Ag无交叉反应性。ON文库中的Fab克隆与Wa有交叉反应性，AO文库中的Fab克隆与Wa有特异性。尽管免疫印迹分析显示来自两个单独文库的Fab与相同的Ag（VP 6蛋白）反应，但由于它们不同的菌株特异性和不同的VH基因使用，它们将识别不同的表位。从这些结果来看，噬菌体展示系统将是制备临床上有用的人单克隆抗体的有力工具，并且分离的Fab将用于在临床实验室测试中从生物样品中检测或鉴定轮状病毒。

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.