Characterization of Antimitochondrial Antibodies in Healthy Adults ALBERTO MATTALIA, 1 S TEFANO QUARANTA, 2 PATRICK S.C. LEUNG, 1 MARZIA B AUDUCCI, 2 J UDY V AN DE WATER , 1 PIER LUIGI CALVO, 3 FRANCA DANIELLE, 3 MARIO RIZZETTO, 2 AFTAB ANSARI, 4 ROSS L. COPPEL, 5 FLORIANO ROSINA, 2 AND M. ERIC GERSHWIN 1 The detection of antimitochondrial antibodies (AMAs) is an important criterion for the diagnosis of primary biliary cirrhosis (PBC). During the last decade, the mitochondrial autoantigens have been cloned, sequenced, and identified as members of the 2-oxo-acid dehydrogenase pathway, including the E2 subunits of pyruvate dehydrogenase (PDC- E2), branched-chain 2-oxo-acid dehydrogenase (BCOADC- E2), and 2-oxo-glutarate dehydrogenase (OGDC-E2). We have developed a rapid and sensitive diagnostic test for use in PBC based on a triple hybrid recombinant molecule (r-MIT3) that contains the autoepitopes of PDC-E2, BCOADC-E2, and OGDC-E2. To help understand the frequency and antigen specificity of AMAs in an asymptomatic population and to identify patients with early disease, we investigated the prevalence of AMA, by enzyme-linked immunosorbent assay (ELISA), in a cohort of 1,530 people from northern Italy. Positive sera were further analyzed for immunoglobu- lin (Ig) isotypes, subclasses, and epitopes of AMA by a combination of ELISA and immunoblotting. In this cohort of 1,530 people, 9 (0.5%) reacted to r-MIT3 by ELISA. Of the 9 reactive sera, 2 recognized PDC-E2, 2 of 9 recognized BCOADC-E2, 1 of 9 recognized OGDC-E2, 2 of 9 recog- nized both PDC-E2 and BCOADC-E2, and 1 of 9 recognized PDC-E2 and OGDC-E2. AMA reactivity was primarily IgM and IgA. Epitope mapping revealed an AMA pattern of reactivity to PDC-E2 that differed from that found in patients with histologically proven PBC in most of the sera. However, 1 sera of a 72-year-old female with a normal alkaline phosphatase had an AMA profile identical to typical PBC. After a variable follow-up period (8-14 months), sera from 8 of 9 of these people were re-obtained for AMA and relative epitope mapping. Interestingly, the reactivity had a wider AMA pattern than before. (HEPATOLOGY 1998;27: 656-661.) Primary biliary cirrhosis (PBC) is a cholestatic disease characterized by immunological destruction of small- or medium-sized bile ducts, which leads to liver impairment. 1 At least one third of patients with PBC have no specific symptoms; rather, they present with unexplained liver func- tion abnormalities. The presence of antimitochondrial anti- bodies (AMA) may be the first expression of disease and may be found in the absence of any clinical symptoms or abnormal liver enzymes, including alkaline phosphatase and - glutamyl transpeptidase. PBC affects members of all races and accounts for 0.6% to 2.0% of death from cirrhosis world- wide. 1 Estimates of its prevalence range from 19 to 151 cases per million, corresponding to an estimated incidence of from 3.9 to 15 cases per million per year. 2 Although genetic factors play a part in the development of the disorder, PBC is not inherited in any simple recessive or dominant pattern, although its prevalence in families with one affected member is estimated to be 1,000 times higher than in the general population. 3 Since their discovery, the detection of AMA has become a major criterion in the clinical diagnosis of PBC. Although indirect immunofluorescence microscopy is widely used in clinical laboratories for detection, the staining patterns are often nonspecific. During the last decade, the major autoanti- gens recognized in PBC have been identified as members of the 2-oxo acid dehydrogenase complex (2-OADC) family, including the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the E2 subunit of the branched-chain 2-oxo acid dehydrogenase complex (BCOADC-E2), and the E2 subunit of the 2-oxo glutarate dehydrogenase complex (OGDC-E2). 4 The recent cloning, expression, and identifica- tion of commonly recognized B-cell epitopes have greatly facilitated the specific and sensitive detection of AMA. 5 However, what is still to be determined is how this increased sensitivity affects the specificity of the test as a predictor for PBC and the significance of the finding of AMA in an otherwise asymptomatic patient. In this study, we took advantage of the recent cloning of a triple-expression hybrid clone (pML-MIT3) in our laboratory, which includes the immunodominant epitopes or the lipoyl domains of PDC-E2, BCOADC-E2, and OGDC-E2. 6 We determined the reactivity of 1,530 sera collected from volun- teers without a known history of liver disease against r-MIT3. Sera samples showing positive reactivity to r-MIT3 were Abbreviations: PBC, primary biliary cirrhosis; AMA, antimitochondrial antibodies; PDC-E2, pyruvate dehydrogenase dihydrolipoamide acetyltransferase; BCOADC-E2, branched-chain alpha-keto acid dehydrogenase dihydrolipoamide acetyltransferase; OGDC-E2, 2-oxoglutarate dehydrogenase dihydrolipoamide acetyltransferase; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; Ig, immunoglobu- lin; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. From the 1 Division of Rheumatology/Allergy and Clinical Immunology, University of California Davis, One Shields Avenue, TB 192, Davis, CA; 2 Department of Gastroenter- ology, Molinette Hospital, Torino, Italy; 3 Blood Bank, Molinette Hospital, Torino, Italy; 4 Department of Pathology, Emory University, Atlanta, GA; and 5 Department of Microbiology, Monash University, Clayton, Victoria, Australia. Received September 5, 1997; accepted November 5, 1997. Supported by NIH grant DK39588. Address reprint requests to: M. Eric Gershwin, M.D., Division of Rheumatology, Allergy and Clinical Immunology, One Shields Avenue, TB 192, University of California Davis, Davis, CA 95616. Fax: (530) 752-4669. Copyright  1998 by the American Association for the Study of Liver Diseases. 0270-9139/98/2703-0003$3.00/0 656