Dziegiel M, Nielsen L K, Andersen P S, Blancher A, Dickmeiss E, Engberg J
Department of Clinical Immunology, University Hospital, Copenhagen, Denmark.
J Immunol Methods. 1995 May 11;182(1):7-19. doi: 10.1016/0022-1759(95)00013-z.
A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively.
已开发出一种新型噬菌体展示系统，用于人免疫球蛋白基因Fab片段的PCR扩增和克隆。利用该系统，我们从一株针对红细胞抗原恒河猴D的小鼠-人杂交瘤细胞系中克隆出一种抗体。完整红细胞用于吸附Fab噬菌体。从克隆材料产生的可溶性Fab片段在凝集试验中表现出与亲本抗体相同的性能。凝胶过滤证实Fab片段由κ-Fd异二聚体组成。成功使用完整细胞筛选特异性Fab噬菌体表明，有可能绕过对目标抗原的纯化。与已发表的种系序列比较表明，免疫球蛋白编码区分别与VH 1.9III和VκHumκv325种系基因具有最高同源性。
