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Licensed Unlicensed Requires Authentication Published by De Gruyter March 8, 2014

Novel sensitive cardiac troponin I immunoassay free from troponin I-specific autoantibody interference

  • Tanja Savukoski EMAIL logo , Jenna Jacobino , Päivi Laitinen , Bertil Lindahl , Per Venge , Noora Ristiniemi , Saara Wittfooth and Kim Pettersson


Background: Cardiac troponins (cTnI and cTnT) are the recommended biomarkers of myocardial infarction. As cTn-specific autoantibodies (cTnAAb) can interfere with the cTn detection by state-of-the-art cTnI assays, our objective was to develop a sensitive cTnI immunoassay free from this analytical interference.

Methods: The assay used antibody-coated spots containing three capture Mabs/Fabs directed against the N-terminus, midfragment and C-terminus of cTnI and a europium chelate-labeled tracer Mab against the C-terminus. Following a 3-h sample incubation and washing, cTnI was quantified by time-resolved fluorometry.

Results: The limit of detection (LoD) was 2.9 ng/L and the assay was linear up to 50,000 ng/L. The total precision of 10% CV was not reached, but 20% CV was reached at 10 ng/L. Mean cTnI (10–50,000 ng/L) recoveries were 100% and 119% in three cTnAAb-positive and two cTnAAb-negative individuals, respectively, verifying the interference resistance of the antibody design used. On average, Architect hs-cTnI assay gave seven-fold higher cTnI concentrations than the new assay but the correlation between the assays was good (r=0.958). Of apparently healthy individuals (n=159), 18% had measurable cTnI values (>LoD) and 10% were cTnAAb-positive. The proportion of measurable cTnI values, however, was significantly higher in cTnAAb-positive individuals (13/16, median cTnI 8.5 ng/L) than in cTnAAb-negative individuals (15/143, median cTnI <LoD) (p<0.001).

Conclusions: Although the developed sensitive cTnI assay without cTnAAb interference takes too long for diagnostic purposes, it could serve as an important analytical tool for exploring the impact of cTnAAbs for cTn testing and for unraveling the etiology behind cTn-related autoimmune responses.

Corresponding author: Tanja Savukoski, Department of Biotechnology, University of Turku, Tykistökatu 6A 6th floor 20520 Turku, Finland, Phone: +358 2 3338091, Fax: +358 2 3338050, E-mail:


We gratefully acknowledge technical support from Liisa Hattara and Petri Saviranta (VTT Technical Research Centre of Finland) and statistical support from Tuukka Pölönen and Tero Vahlberg (University of Turku).

Conflict of interest statement

Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding played no role in thestudy design; in the collection, analysis, and interpretationof data; in the writing of the report; or in the decision tosubmit the report for publication.

Research funding: T. Savukoski, DIA-NET, the Graduate School of Advanced Diagnostic Technologies and Applications.

Employment or leadership: None declared.

Honorarium: None declared.

Patent: K. Pettersson, coinventor on patent for troponin I determinations (US2008153109/EP1473567).


1. Thygesen K, Alpert JS, Jaffe AS, Simoons ML, Chaitman BR, White HD, et al. Third universal definition of myocardial infarction. Eur Heart J 2012;33:2551–67.10.1093/eurheartj/ehs184Search in Google Scholar PubMed

2. Christ M, Bertsch T, Popp S, Bahrmann P, Heppner HJ, Muller C. High-sensitivity troponin assays in the evaluation of patients with acute chest pain in the emergency department. Clin Chem Lab Med 2011;49:1955–63.10.1515/CCLM.2011.695Search in Google Scholar PubMed

3. Korley FK, Jaffe AS. Preparing the United States for high-sensitivity cardiac troponin assays. J Am Coll Cardiol 2013;61:1753–8.10.1016/j.jacc.2012.09.069Search in Google Scholar PubMed

4. Wu AH, Christenson RH. Analytical and assay issues for use of cardiac troponin testing for risk stratification in primary care. Clin Biochem 2013;46:969–78.10.1016/j.clinbiochem.2013.04.013Search in Google Scholar PubMed

5. Savukoski T, Twarda A, Hellberg S, Ristiniemi N, Wittfooth S, Sinisalo J, et al. Epitope specificity and IgG subclass distribution of autoantibodies to cardiac troponin. Clin Chem 2013;59:512–8.10.1373/clinchem.2012.194860Search in Google Scholar PubMed

6. Panteghini M, Gerhardt W, Apple FS, Dati F, Ravkilde J, Wu AH. Quality specifications for cardiac troponin assays. Clin Chem Lab Med 2001;39:175–9.10.1515/cclm.2001.39.2.175Search in Google Scholar PubMed

7. Eriksson S, Halenius H, Pulkki K, Hellman J, Pettersson K. Negative interference in cardiac troponin I immunoassays by circulating troponin autoantibodies. Clin Chem 2005;51:839–47.10.1373/clinchem.2004.040063Search in Google Scholar PubMed

8. Tang G, Wu Y, Zhao W, Shen Q. Multiple immunoassay systems are negatively interfered by circulating cardiac troponin I autoantibodies. Clin Exp Med 2012;12:47–53.10.1007/s10238-011-0141-xSearch in Google Scholar PubMed

9. Savukoski T, Engstrom E, Engblom J, Ristiniemi N, Wittfooth S, Lindahl B, et al. Troponin-specific autoantibody interference in different cardiac troponin I assay configurations. Clin Chem 2012;58:1040–8.10.1373/clinchem.2011.179226Search in Google Scholar PubMed

10. Savukoski T, Ilva T, Lund J, Porela P, Ristiniemi N, Wittfooth S, et al. Autoantibody prevalence with an improved immunoassay for detecting cardiac troponin-specific autoantibodies. Clin Chem Lab Med 2014;52:273–9.10.1515/cclm-2013-0310Search in Google Scholar PubMed

11. Ylikotila J, Valimaa L, Vehniainen M, Takalo H, Lovgren T, Pettersson K. A sensitive TSH assay in spot-coated microwells utilizing recombinant antibody fragments. J Immunol Methods 2005;306:104–14.10.1016/j.jim.2005.08.002Search in Google Scholar

12. Ylikotila J, Hellstrom JL, Eriksson S, Vehniainen M, Valimaa L, Takalo H, et al. Utilization of recombinant Fab fragments in a cTnI immunoassay conducted in spot wells. Clin Biochem 2006;39:843–50.10.1016/j.clinbiochem.2006.04.023Search in Google Scholar

13. Valimaa L, Ylikotila J, Kojola H, Soukka T, Takalo H, Pettersson K. Streptavidin-coated spot surfaces for sensitive immunoassays using fluorescence surface readout. Anal Bioanal Chem 2008;391:2135–44.10.1007/s00216-008-2120-ySearch in Google Scholar

14. Vylegzhanina AV, Katrukha IA, Kogan AE, Bereznikova AV. Epitope specificity of anti-cardiac troponin I monoclonal antibody 8I-7. Clin Chem 2013;59:1814–6.10.1373/clinchem.2013.209528Search in Google Scholar

15. von Lode P, Rosenberg J, Pettersson K, Takalo H. A europium chelate for quantitative point-of-care immunoassays using direct surface measurement. Anal Chem 2003;75:3193–201.10.1021/ac0340051Search in Google Scholar

16. Eriksson S, Junikka M, Laitinen P, Majamaa-Voltti K, Alfthan H, Pettersson K. Negative interference in cardiac troponin I immunoassays from a frequently occurring serum and plasma component. Clin Chem 2003;49:1095–104.10.1373/49.7.1095Search in Google Scholar

17. Korpimäki T, Hagren V, Brockmann E, Tuomola M. Generic lanthanide fluoroimmunoassay for the simultaneous screening of 18 sulfonamides using an engineered antibody. Anal Chem 2004;76:3091–8.10.1021/ac049823nSearch in Google Scholar

18. Simoons ML, GUSTO IV-ACS Investigators. Effect of glycoprotein IIb/IIIa receptor blocker abciximab on outcome in patients with acute coronary syndromes without early coronary revascularisation: the GUSTO IV-ACS randomised trial. Lancet 2001;357:1915–24.10.1016/S0140-6736(00)05060-1Search in Google Scholar

19. Koerbin G, Abhayaratna WP, Potter JM, Apple FS, Jaffe AS, Ravalico TH, et al. Effect of population selection on 99th percentile values for a high sensitivity cardiac troponin I and T assays. Clin Biochem 2013;46:1636–43.10.1016/j.clinbiochem.2013.08.004Search in Google Scholar PubMed

20. Apple FS, Ler R, Murakami MM. Determination of 19 cardiac troponin I and T assay 99th percentile values from a common presumably healthy population. Clin Chem 2012;58:1574–81.10.1373/clinchem.2012.192716Search in Google Scholar PubMed

21. Hyytia H, Jarvenpaa ML, Ristiniemi N, Lovgren T, Pettersson K. A comparison of capture antibody fragments in cardiac troponin I immunoassay. Clin Biochem 2013;46:963–8.10.1016/j.clinbiochem.2013.01.012Search in Google Scholar PubMed

22. Jaffe AS, Apple FS, Morrow DA, Lindahl B, Katus HA. Being rational about (im)precision: A statement from the biochemistry subcommittee of the joint European society of cardiology/American college of cardiology foundation/American heart association/World heart federation task force for the definition of myocardial infarction. Clin Chem 2010;56:941–3.10.1373/clinchem.2010.143958Search in Google Scholar PubMed

23. Apple FS, Simpson PA, Murakami MM. Defining the serum 99th percentile in a normal reference population measured by a high-sensitivity cardiac troponin I assay. Clin Biochem 2010;43:1034–6.10.1016/j.clinbiochem.2010.05.014Search in Google Scholar PubMed

24. Thygesen K, Mair J, Katus H, Plebani M, Venge P, Collinson P, et al. Recommendations for the use of cardiac troponin measurement in acute cardiac care. Eur Heart J 2010;31:2197–204.10.1093/eurheartj/ehq251Search in Google Scholar PubMed

25. Eriksson S, Ilva T, Becker C, Lund J, Porela P, Pulkki K, et al. Comparison of cardiac troponin I immunoassays variably affected by circulating autoantibodies. Clin Chem 2005;51:848–55.10.1373/clinchem.2004.040089Search in Google Scholar PubMed

26. Hedberg P, Valkama J, Suvanto E, Pikkujamsa S, Ylitalo K, Alasaarela E, et al. Evaluation of Innotrac Aio! second-generation cardiac troponin I assay: The main characteristics for routine clinical use. J Autom Methods Manag Chem 2006;2006:1–7.10.1155/JAMMC/2006/39325Search in Google Scholar PubMed PubMed Central

27. Pagani F, Stefini F, Chapelle JP, Lefevre G, Graine H, Luthe H, et al. Multicenter evaluation of analytical performance of the Liaison troponin I assay. Clin Biochem 2004;37:750–7.10.1016/j.clinbiochem.2004.04.003Search in Google Scholar PubMed

28. Apple FS, Collinson PO, IFCC Task force on clinical applications of cardiac biomarkers. Analytical characteristics of high-sensitivity cardiac troponin assays. Clin Chem 2012;58:54–61.10.1373/clinchem.2011.165795Search in Google Scholar PubMed

29. Sandoval Y, Apple FS. The global need to define normality: the 99th percentile value of cardiac troponin. Clin Chem 2013 Oct 10. [Epub ahead of print].10.1373/clinchem.2013.211706Search in Google Scholar PubMed

30. Okazaki T, Tanaka Y, Nishio R, Mitsuiye T, Mizoguchi A, Wang J, et al. Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice. Nat Med 2003;9:1477–83.10.1038/nm955Search in Google Scholar PubMed

31. Goser S, Andrassy M, Buss SJ, Leuschner F, Volz CH, Ottl R, et al. Cardiac troponin I but not cardiac troponin T induces severe autoimmune inflammation in the myocardium. Circulation 2006;114:1693–702.10.1161/CIRCULATIONAHA.106.635664Search in Google Scholar PubMed

32. Volz HC, Buss SJ, Li J, Goser S, Andrassy M, Ottl R, et al. Autoimmunity against cardiac troponin I in ischaemia reperfusion injury. Eur J Heart Fail 2011;13:1052–9.10.1093/eurjhf/hfr098Search in Google Scholar PubMed

33. Leuschner F, Li J, Goser S, Reinhardt L, Ottl R, Bride P, et al. Absence of auto-antibodies against cardiac troponin I predicts improvement of left ventricular function after acute myocardial infarction. Eur Heart J 2008;29:1949–55.10.1093/eurheartj/ehn268Search in Google Scholar PubMed

34. Shmilovich H, Danon A, Binah O, Roth A, Chen G, Wexler D, et al. Autoantibodies to cardiac troponin I in patients with idiopathic dilated and ischemic cardiomyopathy. Int J Cardiol 2007;117:198–203.10.1016/j.ijcard.2006.04.077Search in Google Scholar PubMed

35. Miettinen KH, Eriksson S, Magga J, Tuomainen P, Kuusisto J, Vanninen EJ, et al. Clinical significance of troponin I efflux and troponin autoantibodies in patients with dilated cardiomyopathy. J Card Fail 2008;14:481–8.10.1016/j.cardfail.2008.02.009Search in Google Scholar PubMed

36. Doesch AO, Mueller S, Nelles M, Konstandin M, Celik S, Frankenstein L, et al. Impact of troponin I-autoantibodies in chronic dilated and ischemic cardiomyopathy. Basic Res Cardiol 2011;106:25–35.10.1007/s00395-010-0126-zSearch in Google Scholar PubMed

37. Dungen HD, Platzeck M, Vollert J, Searle J, Muller C, Reiche J, et al. Autoantibodies against cardiac troponin I in patients with congestive heart failure. Eur J Heart Fail 2010;12:668–75.10.1093/eurjhf/hfq088Search in Google Scholar PubMed

38. Autoantibodies to cardiac troponin in acute coronary syndromes. Clin Chim Acta 2010;411:1793–8.10.1016/j.cca.2010.08.005Search in Google Scholar PubMed

39. Pettersson K, Eriksson S, Wittfooth S, Engstrom E, Nieminen M, Sinisalo J. Autoantibodies to cardiac troponin associate with higher initial concentrations and longer release of troponin I in acute coronary syndrome patients. Clin Chem 2009;55:938–45.10.1373/clinchem.2008.115469Search in Google Scholar PubMed

40. Eggers KM, Lagerqvist B, Venge P, Wallentin L, Lindahl B. Persistent cardiac troponin I elevation in stabilized patients after an episode of acute coronary syndrome predicts long-term mortality. Circulation 2007;116:1907–14.10.1161/CIRCULATIONAHA.107.708529Search in Google Scholar PubMed

Article note: Part of the results has been presented as a poster at the 20th IFCC-EFLM European Congress of Clinical Chemistry and Laboratory Medicine in Milan, 2013.

Received: 2013-12-4
Accepted: 2014-2-17
Published Online: 2014-3-8
Published in Print: 2014-7-1

©2014 by Walter de Gruyter Berlin/Boston

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