Matsui M, Nakamura M, Ishibashi H, Koike K, Kudo J, Niho Y
First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Hepatology. 1993 Nov;18(5):1069-77.
Peripheral B lymphocytes from a patient with primary biliary cirrhosis were infected with Epstein-Barr virus, and Epstein-Barr virus-transformed B lymphocytes producing large amounts of IgG antibodies to pyruvate dehydrogenase complex were selected, expanded and fused with the human-mouse heteromyeloma cell line F3B6. The resulting Epstein-Barr virus-transformed B-cell hybrids were repeatedly cloned by limiting dilution, and three stable hybridoma clones producing human monoclonal antibodies to pyruvate dehydrogenase complex were generated. These monoclonal antibodies, designated M18GP8, M37GP11 and M82GP8, specifically bound to pyruvate dehydrogenase complex, and their dissociation constant with pyruvate dehydrogenase complex was calculated to be 2.4 x 10(-11), 2.3 x 10(-10) and 2.6 x 10(-11) mol/L, respectively. These three monoclonal antibodies stained the mouse stomach/kidney cryostat sections in a typical immunofluorescence pattern of antimitochondrial antibody. Furthermore, the enzymatic activity of pyruvate dehydrogenase complex was almost completely inhibited by the three monoclonal antibodies. Western blotting analysis revealed that M18GP8 and M82GP8 reacted with only pyruvate dehydrogenase complex-E2 in contrast to M37GP11, which reacted with both pyruvate dehydrogenase complex-E2 and protein X. The binding of monoclonal antibody M37GP11 to solid-phase pyruvate dehydrogenase complex was partially inhibited by two different synthetic peptides corresponding to both the inner and outer lipoyl-binding domains of pyruvate dehydrogenase complex-E2. These monoclonal antibodies, which are the first human monoclonal antibodies to pyruvate dehydrogenase complex generated from a patient with primary biliary cirrhosis, will be a valuable tool for studying the B-cell autoepitopes in PDC and the mechanism of autoantibody production in primary biliary cirrhosis.
从一名原发性胆汁性肝硬化患者的外周血B淋巴细胞中分离出细胞，用爱泼斯坦-巴尔病毒（EB病毒）感染，然后筛选出能产生大量针对丙酮酸脱氢酶复合体的IgG抗体的EB病毒转化B淋巴细胞，进行扩增，并与人类-小鼠异源骨髓瘤细胞系F3B6融合。对所得的EB病毒转化B细胞杂交瘤通过有限稀释法反复克隆，获得了三个稳定的产生针对丙酮酸脱氢酶复合体的人单克隆抗体的杂交瘤克隆。这些单克隆抗体命名为M18GP8、M37GP11和M82GP8，它们能特异性结合丙酮酸脱氢酶复合体，计算得出它们与丙酮酸脱氢酶复合体的解离常数分别为2.4×10⁻¹¹、2.3×10⁻¹⁰和2.6×10⁻¹¹mol/L。这三种单克隆抗体以典型的抗线粒体抗体免疫荧光模式对小鼠胃/肾冰冻切片进行染色。此外，这三种单克隆抗体几乎完全抑制了丙酮酸脱氢酶复合体的酶活性。蛋白质印迹分析显示，与M37GP11不同，M18GP8和M82GP8仅与丙酮酸脱氢酶复合体-E2发生反应，而M37GP11与丙酮酸脱氢酶复合体-E2和蛋白X均发生反应。丙酮酸脱氢酶复合体-E2的内部和外部硫辛酰结合域对应的两种不同合成肽部分抑制了单克隆抗体M37GP11与固相丙酮酸脱氢酶复合体的结合。这些单克隆抗体是首例从原发性胆汁性肝硬化患者产生的针对丙酮酸脱氢酶复合体的人单克隆抗体，将成为研究丙酮酸脱氢酶复合体中B细胞自身表位以及原发性胆汁性肝硬化自身抗体产生机制的有价值工具。
