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We have raised a monoclonal antibody (PBC-MoAb) directed against mitochondria which resembles patent anti-mitochondrial autoantibodies (AMA) (M2 type) in several respects. The reaction pattern of PBC-MoAb was characterized by western blot experiments, immunoaffinity purification and enzyme inhibition studies. PBC-MoAb reacts specifically with an epitope on the E2 subunit of pyruvate dehydrogenase (dihydrolipoamide acyltransferase) which is essential for enzymatic activity. This was shown as follows: (1) PBC-MoAb, like PBC-AMA, completely inhibited PDH enzyme activity and reacted weakly with OGDH; (2) PBC-MoAb bound strongly to the E2 subunit in western blots, with weaker binding to a doublet of about 56 kDa; and (3) in immunosorbent experiments, PBC-MoAb absorbed most (greater than 95%) of the AMA reactive material found in solubilized mitochondria. The present data together with earlier findings that the majority of PBC patient autoantibodies bind to epitopes defined by the PBC-MoAb, makes this antibody a valuable tool for characterizing the major PBC-associated epitope on PDH-E2 and localizing this epitope in liver tissue.

Primary biliary cirrhosis (PBC)-specific antigens were purified from beef heart mitochondria by immunoaffinity chromatography. Three major polypeptides (75, 60, and 40 kDa) were detected in the purified antigen fraction both by Coomassie blue staining and by western blot analysis. The 75 kDa antigen was identified as a subunit of Complex I (NADH-ubiquinone reductase) by the following criteria: (1) antibodies against the purified 75 kDa subunit of beef heart Complex I react with the immunoaffinity-purified 75 kDa antigen. (2) the 75 kDa subunit present in isolated Complex I, like that in the immunoaffinity-purified antigen, reacts with PBC sera only after reduction with mercaptoethanol, and (3) the 75 kDa antigen is enriched in isolated Complex I. A relationship between the 75 kDa and the 60 and 40 kDa antigens is suggested, since optimal binding of anti-mitochondrial autoantibodies (AMA) to the latter antigens also requires prior reduction with mercaptoethanol. A fourth major antigen (70 kDa) was also detected by western blot analysis, but only in samples that had not been boiled prior to electrophoresis. This antigen, which is also present in isolated Complex I, resembles the 75, 60, and 40 kDa antigens in its response to mercaptoethanol and its reaction with antibodies against the 75 kDa subunit of Complex I. A scheme is presented which relates all of the PBC antigens to the parent 75 kDa subunit of Complex I, probably as proteolytic products of the latter.

A monoclonal antibody specific for the major primary biliary cirrhosis (PBC)-associated mitochondrial antigen (subunit I of NADH-ubiquinone reductase) was produced and used to study the binding sites recognized by anti-mitochondrial autoantibodies (AMA) in PBC sera. Immunization of mice with purified beef heart mitochondrial inner membranes resulted in one monoclonal antibody which reacted with mitochondrial proteins. This antibody (PBC-MoAb), which was of the IgG2b subclass with kappa light chains, exhibited a pattern of immunofluorescence reactivity with rat kidney, human thyroid, and cultured human epithelial cells (Hep-2) similar to that obtained with sera from PBC patients. Similar binding patterns between PBC-MoAb and AMA were also found in western blot analysis using mitochondria as antigen. Both types of antibodies revealed a major antigen of 75 kDa, a minor antigen of 60 kDa, and a third antigen (70 kDa), which was detected only in samples that had not been boiled prior to electrophoresis. Furthermore, optimal binding of the PBC-MoAb and AMA to the 75 and 70 kDa antigens required reduction of the antigen with mercaptoethanol prior to electrophoresis. Competition ELISA experiments were conducted to compare the epitopes recognized by PBC-MoAb and AMA. Of 28 PBC sera tested, 27 inhibited the binding of PBC-MoAb to mitochondrial inner membranes by almost 100% and one serum inhibited binding by 50%, indicating that most PBC sera contain autoantibodies reactive with the same or a closely related antibody binding site as the PBC-MoAb. PBC-MoAb inhibited AMA binding to the inner membrane by more than 80% in 10 sera, 60-80% in 11 sera, and 40-59% in seven sera, with an average inhibition of 71%. Our observations strongly indicate that anti-mitochondrial autoantibody binding sites are restricted to a highly immunogenic epitope on the major PBC-specific antigen (NADH-ubiquinone reductase subunit I), and that the anti-mitochondrial monoclonal antibody obtained has a specificity identical with the human PBC-specific M2 type anti-mitochondrial autoantibody.

Anti-mitochondrial autoantibodies (AMA) from patients with primary biliary cirrhosis (PBC) were analysed for fine specificity by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Inhibition ELISA showed that complex I (NADH-ubiquinone reductase) from beef heart mitochondria completely inhibited the binding of AMA to mitochondrial inner membranes (SMP), indicating that the major mitochondrial antigens are located in complex I. Immunoblot analysis of beef heart SMP, complex I and the iron sulphur (IP) subfraction of complex I revealed several antigens, one of which (75 kDa) reacted with all PBC sera but not with the additional autoimmune sera tested. Resolution of SMP or complex I by two-dimensional electrophoresis yielded in both preparations a polypeptide of 75 kDa with an isoelectric point of 6.4, which reacted with PBC serum and with rabbit antisera raised against the 75,000 subunit of complex I. In immunoblot experiments, the antigenicity of the 75,000 polypeptide in SMP, complex I, and the IP subfraction is increased by prior reduction of the sample with mercaptoethanol. This suggests a similarity to the PBC-specific 'M-2' antigen, which is also sensitive to sulphur reagents. The data indicate that the 75 kDa polypeptide of complex I is a major mitochondrial antigen binding AMA in PBC sera, and allows us to identify the location and probable function of the PBC antigen.