HUMAN MONOCLONAL-ANTIBODIES FROM A PATIENT WITH PRIMARY BILIARY-CIRRHOSIS THAT RECOGNIZE 2 DISTINCT AUTOEPITOPES IN THE E2 COMPONENT OF THEPYRUVATE-DEHYDROGENASE COMPLEX

Peripheral B lymphocytes from a patient with primary biliary cirrhosis were infected with Epstein-Barr virus, and Epstein-Barr virus-transfo rmed B lymphocytes producing large amounts of IgG antibodies to pyruva te dehydrogenase complex were selected, expanded and fused with the hu man-mouse heteromyeloma cell line F3B6. The resulting Epstein-Barr vir us--transformed B-cell hybrids were repeatedly cloned by limiting dilu tion, and three stable hybridoma clones producing human monoclonal ant ibodies to pyruvate dehydrogenase complex were generated. These monocl onal antibodies, designated M18GP8, M37GP11 and M82GP8, specifically b ound to pyruvate dehydrogenase complex, and their dissociation constan t with pyruvate dehydrogenase complex was calculated to be 2.4 x 10(-1 1), 2.3 x 10(-10) and 2.6 x 10(-11) mol/L, respectively. These three m onoclonal antibodies stained the mouse stomach/kidney cryostat section s in a typical immunofluorescence pattern of antimitochondrial antibod y. Furthermore, the enzymatic activity of pyruvate dehydrogenase compl ex was almost completely inhibited by the three monoclonal antibodies. Western blotting analysis revealed that M18GP8 and M82GP8 reacted wit h only pyruvate dehydrogenase complex-E2 in contrast to M37GP11, which reacted with both pyruvate dehydrogenase complex-E2 and protein X. Th e binding of monoclonal antibody M37GP11 to solid-phase pyruvate dehyd rogenase complex was partially inhibited by two different synthetic pe ptides corresponding to both the inner and outer lipoyl-binding domain s of pyruvate dehydrogenase complex-E2. These monoclonal antibodies, w hich are the first human monoclonal antibodies to pyruvate dehydrogena se complex generated from a patient with primary biliary cirrhosis, wi ll be a valuable tool for studying the B-cell autoepitopes in PDC and the mechanism of autoantibody production in primary biliary cirrhosis.