the 135 control sera from patients with primary scleros-The detection of antimitochondrial autoantibodies ing cholangitis (PSC), chronic autoimmune hepatitis (AMAs) is critical in the diagnosis of primary biliary cir-(CAH), systemic lupus erythematosus (SLE), or healthy rhosis (PBC). However, conventional laboratory assays volunteers showed significant reactivity against pMLto detect AMA are dependent on the time-consuming MIT3 recombinant fusion protein in the ELISA assay. method of immunofluorescence microscopy, a method
Our results indicate that an ELISA using recombinant, often plagued by problems of nonspecificity. AMAs react cloned autoantigen of pML-MIT3 is a powerful and very against mitochondrial autoantigens including the E2 specific method for the detection of AMA. (HEPATOLOGY components of the pyruvate dehydrogenase complex 1996;24:97-103.) (PDC-E2), the branched-chain 2-oxo-acid dehydrogenase complex (BCOADC-E2), and the 2-oxo glutarate dehydrogenase complex (OGDC-E2). Interestingly, the immunodominant epitopes of PDC-E2, BCOADC-E2, and OGDC-Primary biliary cirrhosis (PBC) is an autoimmune liver E2 are all conformational lipoate binding sites, but disease that results in destruction of intrahepatic bile ducts antibodies against them do not cross-react. Although with progressive inflammatory scarring. 1 A characteristic se-80% to 90% of sera from patients with PBC react to PDCrological feature observed in sera from patients with PBC is E2, approximately 10% patients with PBC react only to the presence of high titers of autoantibodies directed against BCOADC-E2 and/or OGDC-E2. We have taken advantage mitochondrial antigens (AMAs). [5] The major autoantigens of our epitope-mapping studies of the E2 components of recognized by sera from PBC patients have been identified as PDC, BCOADC, and OGDC, and constructed a ''designer'' members of the 2-oxo-acid dehydrogenase complex (2-OADC) hybrid clone, designated as pML-MIT3, that coexpresses family, including the E2 subunit of the pyruvate dehydrogethe immunodominant epitopes within the three distinct nase complex (PDC-E2), the E2 subunit of the branchedlipoyl domains. We examined a total of 321 sera, includchain 2-oxo-acid dehydrogenase complex (BCOADC-E2), the ing 186 sera from patients with PBC, to test the immuno-E2 subunit of the 2-oxo glutarate dehydrogenase complex reactivity of pMIT3. Of 186 sera from patients with PBC, (OGDC-E2), and the E1a subunits of PDC and protein X. [6][8][9][10][11] 152 sera (81.7%) reacted with recombinant fusion protein Among these enzyme components, the E2 component of PDC, of PDC-E2, whereas 171 sera (91.9%) showed positive reor dihydrolipoamide dehydrogenase, is the major autoantigen activities when probed by immunoblotting against the of PBC, because the serum samples of the majority of patients recombinant fusion protein expressed from the pML-(80% to 90%) contain PDC-E2 specific AMA. In addition to MIT3 clone. Of 34 PBC sera that did not react with re-PDC-E2, approximately 60% of patients with PBC are also combinant PDC-E2, 18 sera contained BCOADC-E2-spereactive with BCOADC-E2. 13, Interestingly, 4% to 13% of cific AMA and 1 serum possessed only OGDC-E2-specific sera from patients with PBC recognize only the BCOADC-AMA. We also developed an enzyme-linked immunosor-E2 but not the PDC-E2. [16] The E2 component of OGDC-E2, bent assay (ELISA), using affinity-purified recombinant dihydrolipoamide succinyltransferase, is recognized in 30% fusion protein of pML-MIT3 clone as the antigen source, to 80% of sera from patients with PBC. 13,17, to quantify specific AMAs in patients with PBC. None of
The immunodominant epitopes of PDC-E2 and BCOADC-E2 have been previously mapped to their lipoic acid-binding domains. 20 Although PDC-E2 contains two lipoic acid-Abbreviations: PBC, primary biliary cirrhosis; AMA, antimitochondrial autoantibody; binding domains, the reactivity of AMA is about 100 times 2-OADC, 2-oxo-acid dehydrogenase complex; PDC-E2, pyruvate dehydrogenase complex E2 stronger to the inner lipoyl domain. 10 Recently, by using an component; BCOADC-E2, branched-chain 2-oxo-acid dehydrogenase complex E2 compoexpressing complementary DNA (cDNA) clone of rat OGDCnent; OGDC-E2, 2-oxo glutarate dehydrogenase complex E2 component; cDNA, complemen-E2, we have shown that the immunodominant epitope of tary DNA; ELISA, enzyme-linked immunosorbent assay; PSC, primary sclerosing cholangitis; CAH, chronic autoimmune hepatitis; SLE, systemic lupus erythematosus; IPTG,