Human monoclonal antibodies from a patient with primary biliary cirrhosis that recognize two distinct autoepitopes in the E2 component of the pyruvate dehydrogenase complex
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 heteromyel...
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Veröffentlicht in: | Hepatology (Baltimore, Md.) Md.), 1993-11, Vol.18 (5), p.1069-1077 |
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Zusammenfassung: | 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 × 10−11, 2.3 × 10−10 and 2.6 × 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. (HEPATOLOGY 1993;18:1069‐1077). |
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ISSN: | 0270-9139 1527-3350 |
DOI: | 10.1002/hep.1840180509 |