Biochemical Factors Influencing Measurement of Cardiac Troponin I ...

Clin Chem Lab Med 1999; 37(11/12):1091–1095 © 1999 by Walter de Gruyter · Berlin · New YorkBiochemical Factors Influencing Measurement of Cardiac Troponin Iin SerumA. Katrukha 1 , A. Bereznikova 1 , V. Filatov 2 andT. Esakova 31HyTest Ltd., Turku, Finland2 Department of Bioorganic Chemistry, Moscow StateUniversity, Moscow, Russia3Moscow Institute of Medical Ecology, Moscow, RussiaTroponin I (cTnI), a sensitive and reliable marker ofdamaged cardiac tissue, is now widely used in clinics.But the existence of different cTnI assays with a widevariety of cut-off values and discrepancies betweenthe results of measurements of one and the same sampleby different assays is puzzling for clinicians. Themost urgent issue at the moment is the developmentof the international standard, which can be used forthe calibration of different assays, thus decreasing betweenassay biases. But another important item,which should be considered by manufacturers, is thestandardisation of the epitopes of the antibodies usedfor the assay development. The importance of suchstandardisation originates from the complicated biochemicalnature of cTnI. Here we briefly try to analysethe main factors that can influence antigen recognitionby different antibodies and formulate principles of antibodyselection for assay development.Key words: Cardiac troponin I; Measurement; Standardisation;Immunodetection; Acute myocardial infarction;Cardiac marker.IntroductionAntibody recognition of a protein can be affected byconformational changes of the antigen, a wide variety ofpost-translational modifications, or epitope blocking bydifferent macromolecules. The complicated biochemistryof cTnI in the living cell and long-term incubation(≥3 hours) in necrotic cardiac tissue after acute myocardialinfarction (AMI) can result in significant changes inp r i m a r y, secondary and ternary structure of the cTnImolecule and we can suppose that protein in the patients’blood is different from newly synthesised or recombinantprotein. To understand what factors can beimportant for accurate cTnI immunodetection, we willtry to analyse some biochemical features of this protein.Complex FormationcTnI is a component of the troponin complex, responsiblefor the regulation of muscle contraction. The troponincomplex consists of three different molecules:troponin C (TnC) with molecular weight 18 kDa and pI4.1, cTnI, with molecular weight 23 kDa and pI 9.9, andtroponin T (cTnT), with molecular weight 34 kDa and pI5.1. All three components interact with each other withdifferent strengths. The strongest interaction wasdemonstrated for cTnI – cTnT complex (K A = 4.4 x 10 –5M –1 ) and especially for cTnI – TnC complex (in the presenceof Ca 2+ ) K A = 1.5 x 10 –8 M –1 ) (1). Complex formationbetween cTnI and TnC and possibly between cTnI andcTnT can make some epitopes unrecognisable bysome anti-cTnI antibodies.Recently it was demonstrated (2, 3) that formation ofthe cTnI-TnC binary complex is actually very importantfor the recognition of the antigen by some monoclonalantibodies (MAbs) and for the immunodetection of thecTnI in AMI blood samples. Bodor et al. (2), studying apanel of cTnI-specific MAbs, noted that some MAbsbetter recognise the complexed than the free form ofthe antigen. Katrukha et al. (3) noticed that amongtested monoclonal antibodies one MAb was unable torecognise the complexed form of the antigen whereasseveral MAbs better recognised the free form of theprotein; others were not affected by complex formationat all. In the same article, the authors demonstratedthat the main part of cTnI in the blood of AMI patients iscomplexed with TnC and only 5–10% of the antigen existsas a free molecule. This finding was confirmed laterby other scientists (4, 5). If the assay utilises antibodiesspecific to the free form of the antigen and not recognisingcTnI in binary complex with troponin C, it willmiss the major part of cTnI in the patient’s blood sample.Wu et al. (4) described the effect of cTnI-cTnT complexformation on several commercially available assays.Some of the tested assays better recognised thefree form of the antigen, others, the complexed; forsome of them, there was no difference. Segura et al.,studying the effect of chelating agents on three commerciallyavailable assays (6), conclude that the Accessassay better recognises the free form of the proteincomparing with the complexed.Two different assays, one of which better recognisesthe free form of the protein and another, the complexed,will give different results with one and thesame patient’s sample, thus confusing clinicians.So it can be concluded that cTnI-TnC complex formationis a very important factor influencing immunochemicaldetection of cTnI. For precise cTnI immunodetectionin patient’s blood, the antibodies used in theassay should be insensitive to cTnI-TnC binary complexformation. To decrease the discrepancy between existingassays, it is a matter of great importance to calibrateassays against the standard representing the naturalform of the antigen. As was demonstrated recently (7),

1092 Katrukha et al.: Measurement of cTnI in serumthe best results can be achieved with ternary cTnI-cTnT-TnC complex purified from human cardiac tissue.In cardiac muscle, cTnI may form a complex withcTnT but with significantly lower affinity comparedwith the cTnI-TnC binary complex. For the detection ofthe cTnI-cTnT binary complex in patient samples, itwas suggested to use a ”mixed“ sandwich immunoassay(5). Capture MAb in such an assay is specific tocTnI; detection, to cTnT. Measurements of the binarycomplex in AMI sera demonstrated (8) that only 50% ofpatients sera contain detectable amounts of such complexand that the concentration of it in these serumsamples does not exceed 5% of the total amount of thecTnI. Comparable results were obtained by a gel filtrationmethod (4). There is no available informationabout anti-cTnI MAbs that are affected by cTnI – cTnTcomplex formation. So we can conclude that the cTnIcTnTcomplex in some cases can be detected in patient’ssamples, but obviously is not as important forcTnI immunodetection as the binary complex with TnC.Stable and Unstable Parts of the MoleculeAnother very important factor for cTnI measurementsis the stability of the cTnI molecule. cTnI is known to bea very unstable protein sensitive to proteolytic degradation.During infarction, cells are suffering from severeischemia and if circulation is not restored, diewithin 30–60 minutes. In necrotic tissue, internal aswell as external cell membranes, are destroyed and differentproteases are released from the lysosomes to intracellularmatrix. It means that 30–60 minutes after onsetof the symptoms and up to its release into the bloodstream, cTnI is incubated in a protease cocktail. It is obviousthat in such conditions cTnI should be (at leastpartially) cleaved by proteases.In in vitro experiments with necrotic human cardiactissue (9), cTnI rapidly undergoes proteolytic degradation.Only 1–3% of the protein remained intact in the tissuesamples after 20 hours of experimental necrosis.Different parts of the cTnI molecule revealed differentstability. N- and C- terminal parts of the molecule wererapidly cleaved by proteases. The central part of cTnIlocated between amino acid residues 28 and 110 hadsignificantly better stability, possibly because of theprotection by troponin C. Extracts of human cardiac tissuethat were incubated for 20 hours at 37°C weretested by several sandwich immunoassays utilisingMAbs with different epitope specificity. Only 2–5% ofinitial immunological activity was observed in suchsamples if the epitopes of MAbs were located at the N-and C-terminal parts of the molecule, whereas morethan 40% of immunological activity was detected in thecase of the central location of the epitopes.Morjana (10) reported isolation of two main cTnIpeptides with molecular weights 18 and 14 kDa frompatient’s blood. The 18 kDa peptide is considered to bea product of cTnI’s proteolytic degradation from the C-terminal region whereas its subsequent degradationfrom the N-terminal region results in the appearance ofthe 14 kDa fragment. So results presented by Morjanaconfirm the assumption about better stability of thecentral part of the molecule.The epitope location can be also very important forbetter sensitivity of the assays and for the standardisationof cTnI assays (9). Serial blood samples from AMIpatients were tested by two different assays, one withMAbs recognising the stable part of the molecule andanother with MAbs specific to the N- and C- terminalparts of the molecule. Within 24 hours after onset of thechest pain, the ratio of concentrations measured by thesecond and first assay was close to one, but 2–3 dayslater it was close to 3 or even more. Four to five daysafter infarction, in some cases there was still no detectablecTnI in the second assay, but significantamounts of the antigen were measured by the assaywith MAbs recognising the stable part of the molecule.We have got the same results with two commerciallyavailable assays, one of which is specific to the stablepart of the molecule, the other to the unstable part (ourunpublished data).So we can conclude that for the better sensitivity andreproducibility of measurements of one and the samesample by different assays, antibodies used in assaysshould recognise the stable central part of the cTnImolecule.Phosphorylation by Protein Kinase AThe cTnI molecule contains two serines in the 22 and23 positions. Both amino acid residues can be phosphorylatedin vivo by protein kinase A (11), so fourforms of protein, one dephospho-, two monophospho-,and one biphospho- can coexist in the cell and theoreticallyall these forms can be released into the patient’sblood after infarction. Phosphorylation changes thestructure and conformation of the cTnI molecule and asa result can affect the interaction of some antibodieswith the antigen (12).We studied the effect of phosphorylation on the interactionof the antigen with anti-cTnI MAbs fromHyTest (Turku, Finland) by Western blotting and insandwich immunoassay. Some MAbs from the anticTnIpanel (22B11, 16F1, 10G4, 17H6) recognise part ofthe molecule, containing sites of phosphorylation – Ser22 and Ser 23. In Figure 1 results of Western blottingFig. 1 Monoclonal antibodies 22B11(A), 10B11(B) and 8E10(C), recognising dephospho- and bisphospho- forms of humancardiac TnI in Western blotting.

Katrukha et al.: Measurement of cTnI in serum 1093with dephosphorylated and phosphorylated forms ofthe protein are presented. cTnI bands were visualisedby three monoclonal antibodies: 22B11 ( 20 RRSSN 24 ),10B11 ( 16 APIRR 20 ) and 8E10 ( 87 LGFAE 91 ) (13). PhosphorylatedcTnI is not recognised by 22B11, whereas thereis no difference in recognition of the two forms in thecase of 10B11 and 8E10 MAbs. Using these three MAbs,we developed two sandwich immunoassays with captureMAb 8E10 and MAbs 22B11 and 10B11 as detection.Both forms of the antigen – phosphorylated anddephosphorylated – were tested by such assays. As followsfrom Figure 2, phosphorylation had no effect onthe signal level in the case of the 8E10–10B11 pair, and,on the contrary, the phosphorylated form was not detectedby the assay utilising 22B11 antibodies. So weconcluded that the first assay can be used for the detectionof both – phosphorylated and dephosphorylated– forms of the antigen and the second assay onlyfor the detection of dephosphorylated cTnI.Fig. 3 Two sandwich immunoassays recognising native(black columns) and dephosphorylated (white columns) cTnIfrom human cardiac tissue.Fig. 2 Two sandwich immunoassays recognising dephospho-(black columns) and bisphospho- (white columns) formsof cTnI.Fig. 4 cTnI measurements by two experimental assays –10B11–8E10 (––) and 22B11–8E10 (––) in serum samplesfrom representative AMI patients.By both assays, we checked in what form cTnI existsin human cardiac tissue. Antigen was purified from thetissue obtained from several different donors 5–8 hpostmortem. Two forms, native and dephosphorylatedby phosphatase from E.coli, were tested. There was nodifference in recognising both forms by the assay utilising10B11 MAbs (Figure 3), but in the assay sensitiveto phosphorylation, the signal level increased up to300%. We concluded that even in several hours postmortemtissue, cTnI is still phosphorylated. So it isquite possible that the phosphorylated form of the proteincan be found in the patients’ blood. To verify thisassumption, both assays were calibrated against thedephosphorylated form of the antigen and cTnI concentrationswere measured in patients’ samples. In alltested samples, the concentration of cTnI determinedby the assay sensitive to phosphorylation was half asmuch compared with the results we have got with theassay that does not discriminate phosphorylated anddephosphorylated forms (Figure 4). Thus our resultssuggest that a significant part of the protein in the patients’blood is phosphorylated.Unfortunately, because of the indirect method weused in our study described above, results should beconsidered only as preliminary. To be more precise,MAbs recognising only the phosphorylated form of theantigen should be used instead of 22B11 antibodies.Effect of HeparinThe cTnI molecule has a very high positive charge (pI9.87) and because of it forms complexes with negativelycharged molecules. Such complexes can interferewith the antibody-antigen interaction. Heparin,widely used in clinical practice, is a mixture of moleculeswith negative charge. Heparin forms complexeswith cTnI thus changing antigen – antibody interaction.We studied the effect of heparin at the concentration of50 and 200 units per ml on the recognition of the antigenby several sandwich immunoassays. As followsfrom Figure 5, in some cases heparin had no effect onthe antigen-antibody interaction, but in two immunoassaysthe signal level in the presence of heparinwas significantly reduced.

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. Determination of cardiac troponin Iforms in the blood of patients with acute myocardial infarctionand patients receiving crystalloid or cold bloodcardioplegia. Clin Chem 1999; 45: 213–22.6. Segura R, Varela E, Marti R, Vidal E, Gonzalez C, Figueras J,et al. Comparison of three assays for cardiac troponin Imeasurement: effect of chelating agents. Clin Chem LabMed 1999; 37 (Special Suppl):457.7. Katrukha A, Bereznikova A, Pettersson K. New approach tostandardisation of human cardiac troponin I (cTnI). ScandJ Clin Lab Invest 1999; 59 (Suppl 230):124–7.8. Katrukha A, Bereznikova A, Filatov V, Pettersson K,Esakova T, Kolosova O, et al. Binary troponin I-troponin Tcomplex in serum of patients with myocardial infarction.Clin Chem Lab Med 1999; 37 (Special Suppl):449.9. Katrukha A, Bereznikova A, Filatov V, Esakova T, KolosovaO, Pettersson K, et al. Degradation of cardiac troponin I:implication for reliable immunodetection. Clin Chem 1998;44:2433–40.10. Morjana N. 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

Katrukha et al.: Measurement of cTnI in serum 1095M, Barton P. Molecular cloning of human cardiac troponinI using polymerase chain reaction. FEBS Lett 1990;270:57–61.16. Ingraham R, Hodges R. Effects of Ca 2+ and subunit interactionon surface accessibility of cysteine residues of cardiactroponin. Biochemistry 1988; 27: 5891–8.Received 15 July 1999; accepted November 1999Corresponding author: Dr. A. Katrukha, Hytest Ltd.,Tykistökatu 2B, Euro-City, 20520 Turku, FinlandTel.: +358-2-512-0900, Fax: Tel.: +358-2-512-0909Email: hytest@hytestfinland.com