New ELISA for Detecting Primary Biliary Cirrhosis–Specific Antimitochondrial Antibodies Cornelia Da ¨ hnrich, 1 Albert Pares, 2 Llorenc ¸ Caballeria, 2 Anke Rosemann, 1 Wolfgang Schlumberger, 1 Christian Probst, 3 Maria Mytilinaiou, 4 Dimitrios Bogdanos, 4† Diego Vergani, 4 Winfried Sto ¨ cker, 3 and Lars Komorowski 3*† BACKGROUND: Antimitochondrial antibodies specific for primary biliary cirrhosis (PBC) target the E2 sub- units of 2-oxo acid dehydrogenase complexes, in particular the pyruvate dehydrogenase complex (PDC)-E2. Their antigen-specific detection relies on conventional ELISA using purified PDC. More re- cent assays have employed a hybrid containing the 3 E2-subunits (MIT3). Some PBC sera react with one or the other preparation, suggesting the presence of nonoverlapping epitopes. METHODS: We have developed an ELISA (anti-M2-3E) using a mixture of purified PDC and MIT3 as antigenic targets. We compared this assay to anti-MIT3 alone, conventional anti-PDC, and indirect immunofluores- cence using 173 PBC and 247 disease controls. RESULTS: The anti-M2-3E ELISA showed a 93.6% diag- nostic sensitivity compared with 91.3%, 83.8%, and 87.3% for MIT3, purified PDC, or indirect immuno- fluorescence, respectively, when all specificities are set to 98.8%. By immunoblotting, anti-M2-3E–positive sera unreactive to purified PDC recognized recombi- nant E2-subunits of the other 2 complexes, whereas those with no reactivity to MIT3 immunofixed PDC subunits E1 or E1. CONCLUSIONS: The diagnostic accuracy of the anti-M2- 3E ELISA for detection of antibodies to 2-oxo acid de- hydrogenase complexes exceeds that of conventional ELISA and IFL; its novelty derives from the combina- tion of the MIT3 hybrid and purified PDC. © 2009 American Association for Clinical Chemistry The serological marker of primary biliary cirrhosis (PBC), 5 an immune-mediated chronic inflammatory cholestatic liver disease of unknown etiology, is the presence of high-titer antimitochondrial antibodies (AMAs) directed against members of the 2-oxo acid dehydrogenase complex (OADC), also known as mito- chondrial 2 (M2) antigen (1–3 ). Anti-M2 AMAs rec- ognize mainly the E2 subunits of the pyruvate dehy- drogenase complex (PDC), branched-chain oxo acid dehydrogenase complex (BCOADC), and the oxoglu- tarate dehydrogenase complex (OGDC), and to a lesser extent the E1 and E3 subunits of OADC (1, 4 –7 ). The pathogenic role and the mechanisms responsible for the development of anti-M2 AMA are not clear (8 –12 ); their presence, however, is so closely linked to PBC that the disease is to be questioned in the absence of these antibodies (1, 4, 13 ). AMAs can also predict future de- velopment of PBC for those seropositive cases at early stages with no abnormal cholestatic liver function tests and no symptoms suggestive of cholestatic disease (14, 15 ). Accurate detection of AMAs, therefore, is impor- tant for establishing diagnosis or predicting disease on- set (1, 4 ). Indirect immunofluorescence (IFL) using rodent kidney/stomach/liver tissue sections or HEp-2 cells as substrates is the gold standard for the detection of liver-related autoantibodies (16 ), but is time con- suming, laborious, and observer dependent and cannot be fully automated (5). IFL patterns are also difficult to interpret in the presence of concurrent antibody reac- tivity and are unable to provide information regarding the antigen specificity of the observed AMA reactivity (5, 16 ). To overcome these limitations, ELISAs mainly based on purified PDC from porcine or bovine heart 1 EUROIMMUN AG, Lu ¨ beck, Germany; 2 Liver Unit, Institut de Malalties Digestives CIBEREHD, Hospital Clinic, IDIBAPS, University of Barcelona, Barcelona, Spain; 3 Department of Immunobiochemical Research, Institute for Experimental Immunol- ogy affiliated to EUROIMMUN AG, Lu ¨ beck, Germany; 4 Institute of Liver Studies, King’s College London School of Medicine at King’s College Hospital, London, U.K. † D. Bogdanos and L. Komorowski contributed equally to this work. * Address correspondence to this author at: Department of Immunobiochemical Research, Institute for Experimental Immunology affiliated to EUROIMMUN AG, Seekamp 31, 23560 Lu ¨ beck, Germany. Fax +49 451 5855391; e-mail l.komorowski@euroimmun.de. Received September 26, 2008; accepted January 22, 2009. Previously published online at DOI: 10.1373/clinchem.2008.118299 5 Nonstandard abbreviations: PBC, primary biliary cirrhosis; AMA, antimitochon- drial antibody; OADC, 2-oxo acid dehydrogenase complex; M2, mitochondrial 2; PDC, pyruvate dehydrogenase complex; BCOADC, branched-chain OADC; OGDC, 2-oxoglutarate dehydrogenase complex; IFL, immunofluorescence; MIT3, mitochondrial-3 fusion protein of lipoyl domains of BCOADC, PDC, and OGDC; AIH, autoimmune hepatitis. Clinical Chemistry 55:5 978–985 (2009) Clinical Immunology 978 Downloaded from https://academic.oup.com/clinchem/article/55/5/978/5631765 by guest on 31 January 2024