Biochemical Factors Influencing Measurement of Cardiac Troponin I ...

1094 Katrukha et al.: Measurement of cTnI in serumThe clinical importance of the oxidation – reductionof the cysteines and existence of cTnI-specific autoantibodiesshould be clarified. But if both forms – oxidisedand reduced – are found in patient’s samples and existenceof auto-antibodies will be confirmed, these twofactors also should be considered by assay manufacturers.ReferencesFig. 5 Effect of heparin on cTnI recognition by different sandwichimmunoassays. Black columns: samples without heparin,white columns: heparin 50 U/ml, grey columns: heparin200 U/ml.AutoantibodiesSeveral years ago, Bohner (14) reported a 69-year-oldCABG patient with diffuse three-vessel disease whowas measured as by Dade’s cTnI assay but positivewith troponin T and CKMB assays. The authors clearlydemonstrated that cTnI in the samples was not detectedbecause of the presence of auto anti-TnI antibodies.Unfortunately, the authors did not check theepitope specificity of the autoantibodies, so we do notknow what sites are important for the generation ofsuch antibodies.This case is the only one described in the literature ofthe presence of the autoantibodies in patients’ samples,so we can conclude that appearance of autoantibodiesto cTnI is a very rare phenomenon and possiblyhas no practical significance.Oxidation-ReductioncTnI has two cysteine residues, Cys 79 and Cys 96 (15)which can be oxidised or reduced (16) and this oxidationand reduction change the structure and the conformationof the antigen. Wu et al. (4) demonstratedthe effect of oxidation – reduction on the recognition ofthe antigen by different commercially available assays.Some assays better recognised the oxidised form ofthe antigen, but for the majority of assays there was nodifference what form of the protein was tested. Unfortunately,we do not know in what form oxidised or reducedcTnI is present in the blood stream of the patients,it is difficult to estimate the importance oftroponin oxidation-reduction for immunodetection.But in any case, preferably MAbs used in assays shouldrecognise sites different from the sites that can bechanged by oxidation-reduction.To summarise: monoclonal or polyclonal antibodiesused for the development of reliable cTnI assayspreferably should have epitopes that are not affectedby: cTnI – TnC complex formation, phosphorylation,molecules with low pI. Epitopes should be located inthe stable part of the cTnI molecule.1. Reiffert S, Jaquet K, Heilmeyer L, Herberg F. Stepwisesubunit interaction changes by mono- and bisphosphorylationof cardiac troponin I. Biochemistry 1998; 37;13516–5.2. Bodor G, Porter S, Landt Y, Ladenson J. Development ofmonoclonal antibodies for an assay of cardiac troponin -Iand preliminary results in suspected cases of myocardialinfarction. Clin Chem 1992; 38:2203–14.3. Katrukha A, Bereznikova A, Esakova T, Pettersson K, LovgrenT, Severina M, et al. Troponin I is released in bloodstreamof patients with acute myocardial infarction not infree form but as complex. ClinChem 1997; 43:1379–85.4. Wu A, Feng Y-J, Moore R, Apple F, McPherson P, BuechlerK, et al. Characterization of cardiac troponin subunit releaseinto serum after acute myocardial infarction andcomparison of assays for troponin T and I. Clin Chem 1998;44:1198–208.5. Giuliani I, Bertinchant J-P, Granier C, Laprade M, ChocronS, Toubin G, et al. 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Degradation of human cardiac troponin I aftermyocardial infarction. Biotechnol Appl Biochem 1998; 28(part 2):105–1111. Solaro RJ. Protein phosphorylation and the cardiac myofilaments.In: Solaro RJ, ed. Protein phosphorylation in theheart muscle. Boca Raton: CRC Press Inc, 1986:129–56.12. Hillawi E, Chilton D, Trayer I, Cummins P. Phosphorylationspecificantibodies for human cardiac troponin-I. Eur JBiochem 1998; 256: 535–40.13. Filatov V, Katrukha A, Bereznikova AV, Esakova T, BularginaT, Severina M, et al. Epitope mapping of anti-TnImonoclonal antibodies. Biochem Mol Biol Intern 1998;45:1179–87.14. Bohner J, Pape K-W, Hannes W, Stegmann T. False-negativeimmunoassay results for cardiac troponin I probablydue to circulating troponin I autoantibodies. Clin Chem1996; 42:2046.15. Vallins W, Brand N, Dabhade N, Butler-Browne G, Yacoub