Cha S, Leung P S, Gershwin M E, Fletcher M P, Ansari A A, Coppel R L
Division of Rheumatology, Allergy and Clinical Immunology, University of California School of Medicine, Davis 95616.
Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2527-31. doi: 10.1073/pnas.90.6.2527.
mRNA from a regional lymph node of a patient with primary biliary cirrhosis (PBC) was used to construct a combinatorial immunoglobulin library in the lambda phage vector system. Six human monoclonal IgG Fab clones (LC1-LC6) specific for the major autoantigen of PBC--dihydrolipoamide acetyltransferase, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2)--were isolated, appearing at a frequency of 0.01% in the combinatorial immunoglobulin library. These Fab clones recognize human PDC-E2 with high affinity (Ka = 10(-7)-10(-9) M-1). Using both immunoblotting and ELISA, LC1-LC6 showed little cross-reactivity to any of the other autoantigens commonly recognized by PBC sera or to other antigens commonly recognized by PBC sera or to other antigens such as histone, calf thymus DNA, and bovine serum albumin. The Fab monoclonal antibodies show a typical anti-mitochondrial staining pattern in HEp-2 cells but react strongly with the luminal aspect of biliary epithelial cells of patients with PBC. Our results demonstrate that a recombinant combinatorial immunoglobulin library can be used to isolate high-affinity Fabs against a specific autoantigen. Such reagents will facilitate the analysis of immunoglobulin gene structure, idiotype, and antigen-antibody interactions in autoimmune disease.
取自原发性胆汁性肝硬化（PBC）患者区域淋巴结的mRNA，被用于在λ噬菌体载体系统中构建组合免疫球蛋白文库。分离出了6个对PBC主要自身抗原——二氢硫辛酰胺乙酰转移酶（丙酮酸脱氢酶复合体E2亚基，PDC-E2）具有特异性的人单克隆IgG Fab克隆（LC1-LC6），在组合免疫球蛋白文库中的出现频率为0.01%。这些Fab克隆以高亲和力（Ka = 10(-7)-10(-9) M-1）识别人类PDC-E2。通过免疫印迹和ELISA检测，LC1-LC6对PBC血清中常见的任何其他自身抗原，或对组蛋白、小牛胸腺DNA和牛血清白蛋白等其他抗原几乎没有交叉反应。Fab单克隆抗体在HEp-2细胞中呈现典型的抗线粒体染色模式，但与PBC患者胆管上皮细胞的管腔面有强烈反应。我们的结果表明，重组组合免疫球蛋白文库可用于分离针对特定自身抗原的高亲和力Fab。此类试剂将有助于分析自身免疫性疾病中免疫球蛋白基因结构、独特型以及抗原-抗体相互作用。
