Epitope Specificity and IgG Subclass Distribution of Autoantibodies to Cardiac Troponin Tanja Savukoski, 1* Aleksandra Twarda, 1 Sanna Hellberg, 1 Noora Ristiniemi, 1 Saara Wittfooth, 1 Juha Sinisalo, 2 and Kim Pettersson 1 BACKGROUND: Autoantibodies to cardiac troponins (cTnAAbs) can interfere with the measurement of cardiac troponin I (cTnI) by immunoassays for the di- agnosis of myocardial infarction. Therefore, we deter- mined the cTnI binding sites and IgG subclasses of cir- culating cTnAAbs. METHODS: We studied epitope specificity with sandwich-type immunoassays by measuring the re- covery of troponin complex added to 10 cTnAAb- negative and 10 cTnAAb-positive sera from healthy volunteers. To study the IgG subclasses, we analyzed admission and 3-month follow-up sera from chest pain patients with a reference assay measuring total IgG (14 cTnAAb negative and 14 cTnAAb positive at 3 months) and with 4 subclass-specific assays measuring exclu- sively IgG subclasses 1– 4. RESULTS: Mean recoveries of troponin complex in cTnAAb-positive samples for single cTnI epitopes ranged from 37% to 211%, being lowest for the cTnI midfragment (aa 30 –110). However, the lowest sample-specific recoveries, 4%–92%, showed that none of the studied epitopes completely escaped the cTnAAb-related interference. Eight chest pain pa- tients of the cTnAAb-positive group became positive between sampling points, and according to all 5 cTnAAb assays, specific signals were generally higher at follow-up. IgG4, with the highest prevalence, was detected in 68% of samples in the cTnAAb-positive group. CONCLUSIONS: IgG subclass studies confirm that cTnAAb formation may be triggered/boosted in acute cardiac events. This new information about the epitope speci- ficity of cTnAAbs should be used to reevaluate existing recommendations regarding use of midfragment epitopes in cTnI assays. To circumvent the negative interference of the highly heterogeneous cTnAAbs, use of 3 or more unconventionally selected epitopes should be considered. © 2012 American Association for Clinical Chemistry Ternary troponin complex, consisting of subunits I, T, and C, is part of the myofibril contractile appara- tus in striated muscle cells. Because 2 of these intra- cellular proteins, troponin I and T, exist as different isoforms in skeletal and cardiac muscle, the mea- surement of cardiac-specific isoforms (cardiac tro- ponin) in blood serves as the most sensitive means to prove a recent or persistent myocardial injury. This has made cardiac troponins the recommended bio- markers for the diagnosis of acute coronary syn- drome (ACS) 3 (1). Cardiac troponin–specific autoantibodies (cTnAAbs), however, can interfere with the detection of these clin- ically important biomarkers by cardiac troponin I (cTnI) assays designed according to the IFCC- recommended midfragment approach used in clinical practice (2–4). cTnAAbs have been found in a high proportion (5%–20%) of individuals with or without cardiac diseases (5–10 ), and cardiac troponin–specific autoimmune response leading to autoantibody forma- tion can be triggered by any release of cardiac proteins following myocardial injury, for example, after inflam- mation, ischemia, or cardiotoxic treatments. Never- theless, the mechanisms for their appearance and maintenance are poorly understood. Besides being able to interfere with cardiac tro- ponin detection by immunoassays, cTnAAbs may have clinical significance. Recent studies using a mouse model have associated circulating cTnI-specific 1 Department of Biotechnology, University of Turku, Turku, Finland; 2 Juha Sini- salo, Division of Cardiology, Helsinki University Central Hospital, Helsinki, Finland. * Address correspondence to this author at: Department of Biotechnology, Uni- versity of Turku, Tykistökatu 6A 6th floor, 20520 Turku, Finland. Fax 358-2- 333-8050; e-mail tanja.savukoski@utu.fi. The results of epitope specificity studies have been presented as a poster at the 10th International Congress on Coronary Artery Disease in Venice, 2011, and the results of IgG subclass distribution studies at the 21st International Congress of Clinical Chemistry and Laboratory Medicine/19th IFCC-EFCC European Congress of Clinical Chemistry and Laboratory Medicine in Berlin, 2011. Received August 16, 2012; accepted November 27, 2012. Previously published online at DOI: 10.1373/clinchem.2012.194860 3 Nonstandard abbreviations: ACS, acute coronary syndrome; cTnAAb, cardiac troponin–specific autoantibody; cTnI, cardiac troponin I; MAb, monoclonal antibody; SA, streptavidin; ITC, cardiac troponin complex; TSA, Tris-buffered saline with azide; ILII, insulation layer II. Clinical Chemistry 59:3 512–518 (2013) Proteomics and Protein Markers 512 Downloaded from https://academic.oup.com/clinchem/article/59/3/512/5621979 by guest on 25 October 2022