Leung P S, Krams S, Munoz S, Surh C P, Ansari A, Kenny T, Robbins D L, Fung J, Starzl T E, Maddrey W
University of California, School of Medicine, Davis 95616.
J Autoimmun. 1992 Dec;5(6):703-18. doi: 10.1016/0896-8411(92)90187-u.
Further to define the epitopes of PDC-E2, the major autoantigen in primary biliary cirrhosis (PBC), we have developed and characterized five human monoclonal antibodies. These antibodies were derived by fusing a regional hepatic lymph node from a patient with PBC with the mouse human heterohybrid cell line F3B6. Previous studies of epitope mapping of PDC-E2 have relied on whole sera and have suggested that the immunodominant epitope lies within the inner lipoyl domain of the molecule. However, selective absorption studies using whole sera and a series of overlapping recombinant peptides of PDC-E2 have suggested that the epitope may also include a large conformational component. Moreover, several laboratories have suggested that autoantibodies against the 2-oxo acids dehydrogenase autoantigens are cross-reactive. The five monoclonal antibodies generated included three IgG2a and two IgM antibodies and were studied for antigen specificity using recombinant PDC-E2, recombinant BCKD-E2, histone, dsDNA, IgG (Fc), collagen and a recombinant irrelevant liver specific control, the F alloantigen. The antibodies were also used to probe blots of human, bovine, mouse and rat mitochondria. Finally, fine specificity was studied by selective ELISA and absorption against overlapping expressing fragments of PDC-E2. All five monoclonals, but none of the other mitochondrial autoantigens were specific for PDC-E2. In fact, although affinity purified antibodies to PDC-E2 from patients with PBC cross-reacted with protein X, the human monoclonals did not, suggesting that protein X contains an epitope distinct from that found on PDC-E2. Additionally, all three IgG2 monoclonals recognized distinct epitopes within the inner lipoyl domain of PDC-E2.
为进一步确定原发性胆汁性肝硬化（PBC）中的主要自身抗原——丙酮酸脱氢酶复合物E2（PDC-E2）的表位，我们制备并鉴定了5种人源单克隆抗体。这些抗体是通过将一名PBC患者的局部肝淋巴结与小鼠-人异源杂交细胞系F3B6融合而获得的。先前关于PDC-E2表位定位的研究依赖于全血清，提示免疫显性表位位于该分子的内部硫辛酰结构域内。然而，使用全血清和一系列重叠的PDC-E2重组肽进行的选择性吸收研究表明，该表位可能还包括一个较大的构象成分。此外，几个实验室提示，针对2-氧代酸脱氢酶自身抗原的自身抗体具有交叉反应性。所产生的5种单克隆抗体包括3种IgG2a和2种IgM抗体，使用重组PDC-E2、重组支链α-酮酸脱氢酶复合物E2（BCKD-E2）、组蛋白、双链DNA、IgG（Fc）、胶原蛋白以及一种重组无关肝特异性对照——F同种抗原研究了其抗原特异性。这些抗体还用于探测人、牛、小鼠和大鼠线粒体的印迹。最后，通过选择性ELISA以及针对PDC-E2重叠表达片段的吸收来研究精细特异性。所有5种单克隆抗体均对PDC-E2具有特异性，而其他线粒体自身抗原均无此特异性。事实上，尽管从PBC患者中亲和纯化的抗PDC-E2抗体与蛋白X发生交叉反应，但人源单克隆抗体却不会，这表明蛋白X含有与PDC-E2上发现的表位不同的表位。此外，所有3种IgG2单克隆抗体均识别PDC-E2内部硫辛酰结构域内不同的表位。
