Biochemical Factors Influencing Measurement of Cardiac Troponin I ...

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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.
Page 1: Clin Chem Lab Med 1999; 37(11/12):1
Page 5: Katrukha et al.: Measurement of cTn

Biochemical Factors Influencing Measurement of Cardiac Troponin I ...

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