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Articles |
1
HyTest Ltd., Tykistokatu 6A, FIN-20520 Turku, Finland.
2
Departments of Biochemistry and Bioorganic
Chemistry, School of Biology, Moscow State University, 119899 Moscow,
Russia.
3
Institute of Medical Ecology, Simpheropolskiy bull.
8, Moscow 113149, Russia.
4
Department of Biotechnology, University of Turku,
Tykistokatu 6A, FIN-20520 Turku, Finland.
5
Turku University Hospital, Central Laboratory,
Kiinanmyllynkaty 4–8, FIN-20520 Turku, Finland.
a Author for correspondence. Fax +358-2-3338070; e-mail hytest{at}utu.fi
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: indexing terms: AMI diagnosis • immunoassay
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ten years ago cardiac TnI (cTnI) was introduced as a marker of acute myocardial infarction (AMI) (7)(8). cTnI has since proved to be one of the most specific and sensitive markers of AMI (9)(10)(11)(12), perioperative myocardial infarction (12), cardiac contusion (13), and other kinds of myocardial tissue damage. Although cTnI has been used as an AMI marker for a decade and several commercial assays are currently available, many important questions remain open. The crucial question of high relevance for assay development and calibration is: In what form is cTnI released into the bloodstream? For instance, cTnI is known to be highly susceptible to proteolysis. (a) What, then, do differently configured immunoassays measure in serum—the whole molecule or various products of the proteolytic degradation? (b) What is the half-life of cTnI in the bloodstream? (c) cTnI has been shown to be phosphorylated by a cAMP-dependent protein kinase (14) and by Ca2+–phospholipid-dependent protein kinase (protein kinase C) (15)(16) at different sites. The phosphorylation of Ser-23 and Ser-24 changes the conformation of the TnI molecule and affects interaction of TnI with certain monoclonal antibodies (mAbs) (17)(18), but it is unknown in what form—phospo- or dephospho—-cTnI is released in the bloodstream. (d) Human cTnI contains two Cys residues (Cys-80 and Cys-97) (19), and oxidation of SH-groups of TnI affects its interaction with troponin components (20) and may also interfere with its binding to mAbs. (e) Whether cTnI is released into the circulation in a free form (as an isolated protein) or as a complex with other troponin components is also unknown. mAbs are usually generated against purified TnI; therefore we can suppose that not all of these antibodies will interact with cTnI complexed with cTnC or cTnT. This means that the sensitivity and the cutoff values for cTnI will depend on the nature of the mAb used for detection of cTnI. Utilization of cTnI as AMI marker will be successful and reproducible only when cTnI circulating in blood is well determined. In this study we analyzed cTnI in the serum of AMI patients with a large collection of mAbs generated against purified cTnI and found that the predominant part of cTnI circulates not in a free form but in the form of a complex.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mab preparation
Human cTnI, human skeletal troponin I (skTnI), and human cTnC were
purified by a previously described method (21) and
provided by HyTest (Turku, Finland).
BALB/c mice were immunized with purified human cTnI and a standard protocol. Briefly, mice were injected intraperitoneally on day 1 with 0.1 mg of cTnI reconstituted in 0.1 mL of 150 mmol/L NaCl and 10 mmol/L potassium phosphate, pH 7.5 (PBS), and mixed with equal volume of complete Freund's adjuvant. On days 31 and 61, the mice were boosted intraperitoneally with 0.1 mg of cTnI in PBS mixed with incomplete Freund's adjuvant. The final boosts were administered on days 91 and 93 with 0.05 mg of the antigen in PBS delivered both intraperitoneally and intravenously. On day 96 the mice were killed, their spleens removed, and splenocytes isolated for fusion. Splenocytes were fused with a nonsecretor cell line sp2/0 and plated into Dulbecco's modified Eagle's medium containing hypoxanthine, aminopterin, and thymidine with 150 mL/L fetal bovine serum.
Microplate wells exhibiting hybridoma growth were screened for production of anti-TnI antibodies. For this purpose, hybridoma supernatants were incubated for 30 min at 37 °C in micro-ELISA plates coated with cTnI (300 ng/well) and, after washing, incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibodies. After washing, HRP activity was determined by using o-phenylenediamine/hydrogen peroxide as substrate. Positive hybridomas were retested for antibody specificity by using micro-ELISA wells coated with skTnI.
Hybridomas selected on the basis of specificity were cloned by two rounds of limiting dilution into aminopterin-free medium. Stable hybridoma clones were cultured as ascites tumors in BALB/c mice. The specificity of mAbs was checked once more with human cTnI- and skTnI-coated micro-ELISA plates and different dilutions of ascitic fluid. mAbs were purified from ascitic fluids by protein A–Sepharose affinity chromatography. Antibody concentrations were determined by the Lowry et al. (22) method with mouse serum IgG (Calbiochem, La Jolla, CA) as a calibrator.
The specificity of mAbs was confirmed by Western blotting. Human cTnI, skTnI, or crude skeletal or cardiac tissue homogenate were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (23) on 7.5–15% gradient polyacrylamide gels. Proteins were blotted to the nitrocellulose (24) and incubated with purified mAbs (3 mg/L) for 1 h at 37 °C. After washing and 1 h of incubation with HRP-labeled goat anti-mouse antibodies at 37 °C, protein bands were visualized by incubation with 4-chloro-1-naphthol/hydrogen peroxide substrate.
labeling antibodies with eu chelate
Stable fluorescent Eu chelate used to label the detection
antibodies was obtained from Wallac (Turku, Finland). We used an Eu
(III) chelate of
2,2',2'',2'''-{{4-[4-(iodoacetamido)phenylethynyl]pyridine-2,6-diyl}bis(methylenenitrolo)}tetrakis(acetic
acid) (25). Labeling of mAbs with the Eu chelate was
performed overnight at 4 °C with 200-fold molar excess of the
chelate in 50 mmol/L sodium carbonate buffer, pH 9.8. Labeled
antibodies were separated from free chelate by gel filtration on NAP-5
and NAP-10 columns (Pharmacia, Uppsala, Sweden). For different mAbs,
3–9 mol of the chelate coupled to each mole of the antibody.
biotinylation of antibodies
Antibody solutions (1–3 g/L) in 0.1 mol/L borate buffer pH 8.8
were mixed with isothiocyanate derivative of biotin dissolved in
dimethyl sulfoxide. About 100 µg of biotin ester were added per
milligram of antibody. The reaction mixture was incubated for 4 h
at room temperature. Twenty microliters of 1 mol/L
NH4Cl were added per 250 µg of ester and incubation
was continued for another 10 min. The mixture was subjected to
exhaustive dialysis against 50 mmol/L Tris-HCl (pH 7.8), 150 mmol/L
NaCl, and 1 g/L sodium azide, and modified antibodies were stored at
3 °C. These antibodies were used as the coating antibodies.
immunoassays
cTnI calibrator preparation.
cTnI is poorly soluble at
neutral pH and physiological ionic strength. For the preparation of the
stock solution of cTnI (0.1 g/L), we used a high concentration of urea
in the diluent buffer (7 mol/L urea, 5 mmol/L EDTA, 10 mmol/L
mercaptoethanol, 20 mmol/L Tris-HCl, pH 7.5). TnI calibrators were
prepared by making several dilutions of the cTnI stock solution in
normal male serum (Scantibodies Lab., Santee, CA). Because we used a
very low concentration of cTnI in our immunological experiments (<100
µg/L), the final concentration of urea in samples was not more than 6
mmol/L and did not affect the assay.
cTnI–cTnC complex formation.
cTnC was purified from
human heart tissue (21). cTnC migrated as a single band on
SDS-PAGE and did not contain proteins detected by anti-cTnI antibodies
in immunoblotting. In vitro formation of cTnI–cTnC complexes was
carried out by adding human cTnC (130 µg/L) to the final dilution of
cTnI (30 µg/L) in normal male serum (already containing 0.6 mmol/L
CaCl2) and incubating the mixture for 30 min on ice with
gentle shaking. Sixfold molar excess of cTnC was added to shift the
equilibrium towards TnI–TnC complex formation.
Assay protocols.
A two-step fluoroimmunoassay was
performed in streptavidin-coated 96-well microtitration plates
(Wallac). First, biotinylated antibodies (coating antibodies, 400 ng
per well) were immobilized to the streptavidin surface by incubating at
room temperature in 0.2 mL of DelfiaTM assay buffer
(Wallac). After 30 min of incubation, the plates were washed twice with
the washing solution containing 9 g/L NaCl, 0.25 g/L Tween 20, 0.5 g/L
sodium azide, and 10 mmol/L Tris-HCl pH 7.4. We then added 50 µL (200
ng/well) of detection antibodies in Delfia assay buffer to the well,
followed by 50 µL of cTnI calibrators or cTnI–cTnC mixture. The
final concentration of cTnI in the cTnI calibrators and in the
cTnI–cTnC mixture was equal. The same protocol was used in the
experiments with serum samples collected from AMI patients. If
necessary (to decrease the strength of cTnI–cTnC interaction) the
incubation buffer contained 5 mmol/L EDTA. The mixture of antigen with
Eu-labeled detection antibodies was incubated for 30 min at room
temperature. After washing by Delfia wash solution (six times), Lanfia
enhancement solution (26) (0.2 mL/well) was added, and
incubation was continued for 3 min at room temperature with gentle
shaking. The fluorescent signal (cps) was measured with the 1234 Delfia
Plate Fluorometer (Wallac).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All antibodies (196 combinations) were tested in sandwich fluoroimmunoassay as coating and detection antibodies to find optimal pairs for TnI fluoroimmunoassay development. Ten two-site combinations of six mAbs, highly sensitive for human cTnI, were chosen for further experiments.
Interaction of TnC with TnI results in significant changes in the
structure of both TnC and TnI (1)(2)(3)(4). Therefore, it was
reasonable to analyze the effect of cTnC on the binding of mAbs to
cTnI. Six-molar excess of cTnC was added to the TnI solution in normal
human serum (already containing 0.6 mmol/L CaCl2) to form
cTnI–cTnC complex (see Materials and Methods). Isolated
cTnI and cTnI–cTnC complex were tested in 10 sandwich immunoassays,
utilizing six mAbs in different combinations. For cTnI–cTnC complex,
the immunofluorescence experiments were performed both in the presence
and absence of 5 mmol/L EDTA, thus modifying (decreasing in the
presence of EDTA) the strength of cTnI–cTnC interaction. The data
presented in Fig. 1
indicate that, as a rule, the addition of cTnC negatively
affected the immunoreactivity of cTnI. In one case (7F4–10B11
antibodies, case 1), complexation with cTnC increased the fluorescent
signal. This can be attributable to cTnC-induced exposure of antigenic
determinants of cTnI interacting with 7F4 or 10B11 antibodies. In two
cases (7F4–2A3, case 3 and 2A3–10B11, case 4), complexation of cTnI
had practically no effect on the interaction of mAbs with cTnI. This
indicates that the accessibility of the epitopes of these antibodies
does not depend on the binding of cTnC to cTnI. In the remaining
combinations (seven of 10 analyzed pairs of mAbs), addition of cTnC
significantly reduced the fluorescent signal, reflecting changes in the
interaction of mAbs with cTnI. However, addition of EDTA to the assay
buffer resulted in partial (cases 2, 6, 8, 9, and 10) or complete
(cases 5 and 7) reversal of the inhibitory effect of cTnC on cTnI–mAb
interaction.
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Assuming that a part of cTnI can be released into the circulation in
the form of a complex with cTnC, we tested how addition of EDTA affects
the cTnI measurements in the serum samples collected from AMI patients.
Three assays (2A3–10B11, 10F4–7F4, and 7F4–414) were chosen for this
purpose. All three systems were insensitive to addition of EDTA when
tested with isolated cTnI (Fig. 2
, the data for 2A3–10B11 not shown). This means that if cTnI is
liberated into the bloodstream in the free form, the signal level for
all three systems will not depend on the presence of EDTA in the assay.
In practice it was correct only for the system 2A3–10B11 (Fig. 3
). This system is insensitive to the presence of cTnC in the
incubation mixture (see Fig. 1
, case 4), and in the assay with serum
samples from AMI patients gave identical signals independent of the
presence of EDTA in the incubation mixture. In the model experiments
(Fig. 1
) we have shown that two other systems (7F4–414 and 10F4–7F4)
are sensitive to addition of cTnC (Fig. 1
, cases 2 and 7). Moreover,
EDTA partially (system 7F4–414) or completely (system 10F4–7F4)
reversed the inhibitory action of cTnC on the signal level. Therefore
it is reasonable to use these systems to investigate the effect of EDTA
on the signal level while serum samples from AMI patients are tested.
As shown in Fig. 3
, addition of EDTA significantly increased the signal
level for both assays. Addition of EDTA diminishes the strength of
interaction of troponin components and by this means increases the
level of the fluorescent signal. The data presented correlate with our
suggestion that at least part of cTnI is released to the bloodstream in
the form of a complex with cTnC.
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From the data presented in Fig. 1
we may conclude that with different
pairs of mAbs we can determine both the concentration of free cTnI as
well as the total concentration of cTnI in the sample. Indeed, the
pairs of mAbs that equally react with isolated cTnI and cTnI in the
form of cTnI–TnC complex (7F4–2A3, case 3; 2A3–10B11, case 4) can be
used for determination of the total cTnI concentration in the serum.
The total concentration of cTnI can also be determined by using the
pairs of mAbs that equally recognize isolated cTnI and cTnI complexed
with cTnC in the presence of EDTA (10F4–8E10, case 5 and 10F4–7F4,
case 7). For example, by using biotinylated 10F4 and Eu-labeled 7F4 and
measuring in the presence of excess EDTA, we observed equal signals
both for isolated TnI and its complex with cTnC (Fig. 1
).
For the determination of free TnI concentration, the pairs of mAbs showing virtually complete suppression of the cTnI signal upon complexation with cTnC can be used. This requirement is satisfied by three pairs of mAbs (7F4–414, case 2; 8E10–414, case 8; and 10B11–414, case 10). But assay with 7F4–414 mAbs had the highest signal among the two combinations and >97% reduction of the signal after cTnI complexation with cTnC.
Thus, by using two assays it is possible to measure both concentration
of free TnI and concentration of total TnI. In our experiments we chose
7F4–414 assay for measurement of free TnI, and 10F4–7F4 assay in the
presence of EDTA for measurement of total TnI. This assay had better
sensitivity, kinetics, and range of linearity than all others (7F4–2A3
and 2A3–10B11). The calibration curves of two chosen assays in the
presence and absence of EDTA are shown on Fig. 2
. As exemplified in
Fig. 2
, EDTA does not affect the signal level of both assays.
To investigate the concentrations of free and complexed TnI during AMI,
we assayed serum samples from 30 AMI patients by two assays described
above. The data of Fig. 4
, showing profiles of free and total cTnI in four representative
cases of AMI, indicate that the peak of the total TnI concentration was
observed 15–25 h after onset of chest pain, and the concentration of
total TnI remained increased for at least 80–100 h. The kinetics of
TnI release is similar to that described in the literature
(27). The peak value of total TnI varied from 6 to 50
µg/L and is probably dependent on the size of infarction zone and the
rate of reperfusion. In all cases the concentration of free TnI was
much less than that of the total TnI. The peak of free TnI was less
pronounced and, in many cases, the concentration of free TnI was only
slightly changed during the whole time of observation. At the peak
concentration, cTnI was 5–12 times higher than the corresponding
concentration of free cTnI. The ratio
[cTnI]total/[cTnI]free was changed during
the time of observation. As a rule, at the beginning of observation
this ratio was low; then it increased to its maximal value and at the
end of observation returned to its initial value. At the present stage
of investigation we cannot give a detailed explanation for the complex
kinetics of cTnI liberation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have found that cTnC does significantly modify the interaction of
cTnI with mAbs. In seven of 10 analyzed pairs of antibodies,
complexation with cTnC significantly diminished the fluorescent signal
(Fig. 1
). For certain pairs of antibodies, the effect of cTnC was so
strong that the fluorescent signal was decreased by 97% (7F4–414,
Fig. 1
case 2). Addition of EDTA removes Ca2+ and thus
modifies the strength of the cTnI–cTnC interaction
(1)(2)(4). In the absence of
Ca2+ the apparent dissociation constant of the cTnI–cTnC
complex is increased >1000-fold
(1)(2)(4). Indeed, addition of
EDTA to the cTnI–cTnC complex improves the binding of certain mAb
combinations to cTnI (Fig. 1
, cases 5, 7, and 9). Nevertheless, even in
the presence of 5 mmol/L EDTA, certain pairs of mAbs interact less
effectively with cTnI inside the cTnI–cTnC complex than with isolated
cTnI (Fig. 1
, cases 2, 8, and 10). This shows that only certain pairs
of antibodies could be used for immunodetection of cTnI in the
circulation if it is partly or predominantly released in the form of a
complete troponin complex (TnI–TnT–TnC) or in the form of a binary
cTnI–TnC complex. Until now, the form in which cTnI is released from
damaged cardiac myocytes has not been known. We have tried to answer
this question by using two highly sensitive immunofluorometric assays.
The data presented in Fig. 4
indicate that in all AMI patients under
investigation, the largest part of cTnI is liberated in the form of a
complex (probably with cTnC) and only a small part of TnI circulates in
the bloodstream in a free form. The ratio of total to free TnI in the
serum varies in the course of observation and is different in serum
samples from different patients. In general, this means that special
precautions should be taken in developing sensitive and reproducible
methods for the determination of TnI in the bloodstream. First, pairs
of antibodies that are used for TnI determination should equally react
with free cTnI and cTnI forming complexes with other troponin
components. In other words, antibodies should recognize epitopes that
are not perturbed or sterically shielded by other troponin components.
Second, for some pairs of antibodies for which the interaction with
cTnI is affected by cTnC in the presence of Ca2+, it
is advisable to add EDTA to the sample or the incubation mixture.
Addition of EDTA decreases the strength of interaction among troponin
components (1)(2) and therefore increases
probability of interaction of certain mAbs with TnI. Obviously, special
control experiments should be performed to exclude probable effects of
EDTA on the detection system.
The cutoff concentration for cTnI in published cTnI immunoassays varies from 0.2 µg/L (12) up to 3.1 µg/L (9). This difference can at least partly be explained by different specificities of mAbs for free and complexed cTnI in the above-mentioned assays. In this investigation we analyzed only the effect of cTnC on the interaction of mAbs with cTnI. Evaluation of an effective and reliable method for cTnI determination will require further investigations directed to analyses of the effect of phosphorylation, partial proteolytic degradation, and oxidation of SH groups of TnI on its interaction with mAbs.
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Acknowledgments |
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Footnotes |
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References |
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 B. J. Freda, W. H. W. Tang, F. Van Lente, W. F. Peacock, and G. S. Francis Cardiac troponins in renal insufficiency: Review and clinical implications J. Am. Coll. Cardiol., December 18, 2002; 40(12): 2065 - 2071. [Abstract] [Full Text] [PDF]
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M. Panteghini Performance of Today's Cardiac Troponin Assays and Tomorrow's Clin. Chem., June 1, 2002; 48(6): 809 - 810. [Full Text] [PDF]
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D. Uettwiller-Geiger, A. H.B. Wu, F. S. Apple, A. W. Jevans, P. Venge, M. D. Olson, C. Darte, D. L. Woodrum, S. Roberts, and S. Chan Multicenter Evaluation of an Automated Assay for Troponin I Clin. Chem., June 1, 2002; 48(6): 869 - 876. [Abstract] [Full Text] [PDF]
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D. A. Colantonio, W. Pickett, R. J. Brison, C. E. Collier, and J. E. Van Eyk Detection of Cardiac Troponin I Early after Onset of Chest Pain in Six Patients Clin. Chem., April 1, 2002; 48(4): 668 - 671. [Full Text] [PDF]
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 T. M. Welsh, G. D. Kukes, and L. M. Sandweiss Differences of Creatine Kinase MB and Cardiac Troponin I Concentrations in Normal and Diseased Human Myocardium Ann. Clin. Lab. Sci., January 1, 2002; 32(1): 44 - 49. [Abstract] [Full Text] [PDF]
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 R. C. Payne, B. I. Bluestein, D. L. Morris, R. Labugger, L. Organ, C. Collier, J. E. Van Eyk, and D. Atar Extensive Troponin I and T Modification Detected in Serum From Patients With Acute Myocardial Infarction Response Circulation, July 31, 2001; 104 (5): e26 - e27. [Full Text] [PDF]
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R. H. Christenson, S. H. Duh, F. S. Apple, G. S. Bodor, D. M. Bunk, J. Dalluge, M. Panteghini, J. D. Potter, M. J. Welch, A. H.B. Wu, et al. Standardization of Cardiac Troponin I Assays: Round Robin of Ten Candidate Reference Materials Clin. Chem., March 1, 2001; 47(3): 431 - 437. [Abstract] [Full Text] [PDF]
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A. Lavoinne, B. Cauliez, H. Eltchaninoff, R. Koning, and A. Cribier Analytical and Clinical Performance of the Immulite Cardiac Troponin I Assay Clin. Chem., December 1, 2000; 46(12): 1989 - 1990. [Full Text] [PDF]
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D. A. Morrow, E. M. Antman, M. Tanasijevic, N. Rifai, J. A. de Lemos, C. H. McCabe, C. P. Cannon, and E. Braunwald Cardiac troponin I for stratification of early outcomes and the efficacy of enoxaparin in unstable angina: a TIMI-11B substudy J. Am. Coll. Cardiol., November 15, 2000; 36(6): 1812 - 1817. [Abstract] [Full Text] [PDF]
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T. Meyer, L. Binder, N. Hruska, H. Luthe, and A. B. Buchwald Cardiac troponin I elevation in acute pulmonary embolism is associated with right ventricular dysfunction J. Am. Coll. Cardiol., November 1, 2000; 36(5): 1632 - 1636. [Abstract] [Full Text] [PDF]
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A. S. Jaffe, J. Ravkilde, R. Roberts, U. Naslund, F. S. Apple, M. Galvani, and H. Katus It's Time for a Change to a Troponin Standard Circulation, September 12, 2000; 102(11): 1216 - 1220. [Full Text] [PDF]
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W. Gerhardt, G. Nordin, A.-K. Herbert, B. Linaker Burzell, A. Isaksson, E. Gustavsson, S. Haglund, M. Muller-Bardorff, and H. A. Katus Troponin T and I Assays Show Decreased Concentrations in Heparin Plasma Compared with Serum: Lower Recoveries in Early than in Late Phases of Myocardial Injury Clin. Chem., June 1, 2000; 46(6): 817 - 821. [Abstract] [Full Text] [PDF]
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B. E. Sobel and M. M. LeWinter Ingenuous interpretation of elevated blood levels of macromolecular markers of myocardial injury: a recipe for confusion J. Am. Coll. Cardiol., April 1, 2000; 35(5): 1355 - 1358. [Abstract] [Full Text] [PDF]
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D. A. Morrow, N. Rifai, M. J. Tanasijevic, D. R. Wybenga, J. A. de Lemos, and E. M. Antman Clinical Efficacy of Three Assays for Cardiac Troponin I for Risk Stratification in Acute Coronary Syndromes: A Thrombolysis In Myocardial Infarction (TIMI) 11B Substudy Clin. Chem., April 1, 2000; 46(4): 453 - 460. [Abstract] [Full Text] [PDF]
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 B. Jurlander, P. Clemmensen, G. S. Wagner, and P. Grande Very early diagnosis and risk stratification of patients admitted with suspected acute myocardial infarction by the combined evaluation of a single serum value of cardiac troponin-T, myoglobin, and creatine kinase MBmass Eur. Heart J., March 1, 2000; 21(5): 382 - 389. [Abstract] [PDF]
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P. Datta, K. Foster, and A. Dasgupta Comparison of Immunoreactivity of Five Human Cardiac Troponin I Assays toward Free and Complexed Forms of the Antigen: Implications for Assay Discordance Clin. Chem., December 1, 1999; 45(12): 2266 - 2269. [Full Text] [PDF]
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 M. Hendrikx, H. Jiang, H. Gutermann, J. Toelsie, D. Renard, A. Briers, J. L. Pauwels, and U. Mees RELEASE OF CARDIAC TROPONIN I IN ANTEGRADE CRYSTALLOID VERSUS COLD BLOOD CARDIOPLEGIA J. Thorac. Cardiovasc. Surg., September 1, 1999; 118(3): 452 - 459. [Abstract] [Full Text] [PDF]
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Q. Shi, M. Ling, X. Zhang, M. Zhang, L. Kadijevic, S. Liu, and J. P. Laurino Degradation of Cardiac Troponin I in Serum Complicates Comparisons of Cardiac Troponin I Assays Clin. Chem., July 1, 1999; 45(7): 1018 - 1025. [Abstract] [Full Text] [PDF]
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D. J. Newman, Y. Olabiran, W. D. Bedzyk, S. Chance, E. G. Gorman, and C. P. Price Impact of Antibody Specificity and Calibration Material on the Measure of Agreement between Methods for Cardiac Troponin I Clin. Chem., June 1, 1999; 45(6): 822 - 828. [Abstract] [Full Text] [PDF]
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I. Giuliani, J.-P. Bertinchant, C. Granier, M. Laprade, S. Chocron, G. Toubin, J.-P. Etievent, C. Larue, and S. Trinquier Determination of Cardiac Troponin I Forms in the Blood of Patients with Acute Myocardial Infarction and Patients Receiving Crystalloid or Cold Blood Cardioplegia Clin. Chem., February 1, 1999; 45(2): 213 - 222. [Abstract] [Full Text] [PDF]
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F. S. Apple Clinical and Analytical Standardization Issues Confronting Cardiac Troponin I Clin. Chem., January 1, 1999; 45(1): 18 - 20. [Full Text] [PDF]
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A. G. Katrukha, A. V. Bereznikova, V. L. Filatov, T. V. Esakova, O. V. Kolosova, K. Pettersson, T. Lovgren, T. V. Bulargina, I. R. Trifonov, N. A. Gratsiansky, et al. Degradation of cardiac troponin I: implication for reliable immunodetection Clin. Chem., December 1, 1998; 44(12): 2433 - 2440. [Abstract] [Full Text] [PDF]
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C. Heeschen, B. U. Goldmann, Robert. H. Moeller, and C. W. Hamm Analytical performance and clinical application of a new rapid bedside assay for the detection of serum cardiac troponin I Clin. Chem., September 1, 1998; 44(9): 1925 - 1930. [Abstract] [Full Text] [PDF]
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R. H. Christenson and H. M. E. Azzazy Biochemical markers of the acute coronary syndromes Clin. Chem., August 1, 1998; 44(8): 1855 - 1864. [Abstract] [Full Text] [PDF]
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O. Hetland and K. Dickstein Cardiac troponins I and T in patients with suspected acute coronary syndrome: a comparative study in a routine setting Clin. Chem., July 1, 1998; 44(7): 1430 - 1436. [Abstract] [Full Text] [PDF]
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A. H. B. Wu, Y.-J. Feng, R. Moore, F. S. Apple, P. H. McPherson, K. F. Buechler, G. Bodor, f. t. A. A. for, and C. C. S. o. c. Standardization Characterization of cardiac troponin subunit release into serum after acute myocardial infarction and comparison of assays for troponin T and I Clin. Chem., June 1, 1998; 44(6): 1198 - 1208. [Abstract] [Full Text] [PDF]
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G. Ferrieres, C. Calzolari, J.-C. Mani, D. Laune, S. Trinquier, M. Laprade, C. Larue, B. Pau, and C. Granier Human cardiac troponin I: precise identification of antigenic epitopes and prediction of secondary structure Clin. Chem., March 1, 1998; 44(3): 487 - 493. [Abstract] [Full Text] [PDF]
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R. H. Christenson, S.-H. Duh, L. K. Newby, E. M. Ohman, R. M. Califf, C. B. Granger, S. Peck, K. S. Pieper, P. W. Armstrong, H. A. Katus, et al. Cardiac troponin T and cardiac troponin I: relative values in short-term risk stratification of patients with acute coronary syndromes Clin. Chem., March 1, 1998; 44(3): 494 - 501. [Abstract] [Full Text] [PDF]
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).
) and free cTnI measured by
7F4–414 assay (
) in the serum of four representative AMI
patients.