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1
Sanofi Diagnostics Pasteur, Z.A. Leopha Rue d'Italie, 69780 Mions, France.
2
Centre Hospitalio Universitaire Nîmes,
Service de Cardiologie, 30006 Nîmes, France.
3
Centre National de la Recherche Scientifique,
Unité Mixte de Recherche, 9921, Faculté de
Pharmacie, 34060 Montpellier, France.
4
Sanofi Recherche, 34084 Montpellier, France.
5
Hôpital St. Jacques, 25030 Besançon, France.
6
Sanofi Diagnostics Pasteur, 92230 Marnes-la-Coquette,
France.
a Author for correspondence. Fax 33 4 78 21 75 06; e-mail sylvie.trinquier{at}sanofi.com.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cTnI has been used as a marker of myocardial injury for almost 10 years, and several assays are available commercially (13)(14)(15). At present, cTnI is considered to be the most reliable marker of acute myocardial infarction (AMI), particularly because of its specificity and sensitivity (13)(14)(15)(16). cTnI has also been proven to be a specific marker of perioperative myocardial infarction (17)(18), cardiac contusion (19), and cardiac ischemia during cardiac surgery; as such cTnI is used to evaluate and compare different cardioprotective procedures in routine cardiac operations and in heart transplantations (20)(21). Several commercial cTnI assays are currently available; however, the predominant form in which cTnI is released into the bloodstream after myocardial damage has not been determined. Furthermore, cTnI values can differ by a factor 10 or more from one commercial assay to another (22). This can be explained in part by the different monoclonal antibodies (mAbs) used in the assays, which lead to differences in the recognition of the various cTnI forms. Differences in the reference materials used in assay calibration can also explain this divergence. Identification of the circulating forms of cTnI in different cardiac injuries is essential for choosing mAbs adapted to the detection of the prevalent form, which consequently increases the clinical sensitivity of the assay. This knowledge would also be very helpful for establishing a common calibrator, thus enabling harmonization of cTnI values determined with the different commercial assays.
Katrukha et al. (23) used mAbs specific for human cTnI and were able to determine the concentration of the free form and a complexed form. They suggested that the largest part of cTnI is liberated into the bloodstream in the form of a complex, probably with TnC, and that only a small part circulates in the free form. In this study, we developed three two-site immunoassays that allowed us to identify the free form of cTnI as well as each complexed form: the binary complex IC, the binary complex IT, and the ternary complex cTnT-cTnI-TnC (TIC). Using these three immunoassays, we analyzed 35 serum samples from patients with AMI and patients receiving a cardioplegia during heart surgery with the objective of identifying the predominant cTnI form(s) released into the bloodstream after myocardial damage.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blood samples from 20 patients receiving either crystalloid cardioplegia (10 patients) or cold blood cardioplegia followed by warm reperfusion (10 patients) were collected just before cardiopulmonary bypass and after aortic unclamping at 6, 9, 12, and 24 h and daily thereafter for 5 days. Samples were centrifuged after collection, and serum was stored immediately at -20 °C until use (24). Patients receiving cardioplegia had first undergone elective coronary artery bypass grafting. Not included in this group were patients requiring only one distal anastomosis, patients with an ejection fraction <0.30, patients undergoing a repeat operation, and patients with concomitant heart valve disease or unstable angina.
Blood samples from 12 healthy donors with no history of heart disease were centrifuged after collection, and the serum was stored immediately at -20 °C until use. These samples were confirmed cTnI negative by the ACCESS® immunoassay system (Sanofi Diagnostics Pasteur-Beckman Instruments) and cTnT negative by the ELECSYS® immunoassay system (Boehringer Mannheim).
Because leftover blood from routine collections was used for all samples and no patient identifiers were used, informed consent was deemed unnecessary.
mAbs
Different mAb combinations were tested in a sandwich enzyme
immunoassay in an attempt to find the optimal mAb pairs for the
measurement of the various cTnI forms. Four antibodies were selected to
perform the immunoassays: mAb 1, monoclonal mouse anti-cTnI (Sanofi
Recherche) (25); mAb 2, monoclonal mouse anti-cTnI (Sanofi
Recherche) (25); mAb 2I-11, monoclonal mouse anti-TnC
(Spectral Diagnostics); mAb 7G-7, monoclonal mouse anti-cTnT (Hytest),
which recognizes fragment 60–71 on the cTnT molecule. mAbs 1 and 7G-7
were used as detection antibodies labeled with horseradish peroxidase
(HRP). mAbs 2 and 2I-11 were used as capture antibodies immobilized
onto 96-well microtitration plates.
Labeling of antibodies with HRP.
The HRP used to label
detection antibodies was obtained from Boehringer Mannheim
Biochemicals. We labeled mAbs 1 and 7G-7 by mixing 3 mg of each mAb
with 2.25 mg of oxidized HRP and incubating the mixture for 2 h in
the dark under gentle shaking at room temperature. Labeled mAbs were
stabilized with 4 g/L sodium borohydride (Aldrich Chemical) and
incubated for 2 h at 4 °C in the dark under gentle shaking.
After dialysis overnight at 4 °C in phosphate-buffered saline (PBS
Dulbecco; Seromed), the conjugates were diluted in 550 mL/L
glycerol and stored at -20 °C.
Coating of the microtiter plates.
The capture mAbs 2 and 2I-11
were immobilized onto 96-well microtitration plates (Nunc Maxisorp) by
physical adsorption. Wells were coated with 250 µL of the mAb diluted
to 5 mg/L in PBS Dulbecco. After incubation overnight at 4 °C, the
microplates were washed three times with 300 µL of PBS Dulbecco
containing 1 mL/L Tween 20 (Sigma Chemical). They were then saturated
with 250 µL of Tris-buffered saline (TBS) containing 10 g/L bovine
serum albumin (BSA; Sigma), incubated for 1 h at 37 °C, and
washed three times with 300 µL of PBS Dulbecco containing 1 mL/L
Tween 20.
immunoassays
Assay description.
Four two-site immunoassays were performed
in coated 96-well microtitration plates. The first assay, the total
cTnI assay for detection of free and complexed cTnI, was performed with
two anti-cTnI mAbs: mAb 2 coated onto the microplates was used as the
capture mAb, and the mAb 1-HRP conjugate diluted 1/5000 in PBS Dulbecco
containing 10 g/L BSA and 1 mL/L Tween 20 was used as the detection
mAb. The second assay, the IC-TIC assay, was performed
with an anti-cTnI mAb associated with an anti-TnC mAb. mAb 2I-11 coated
onto the microplates was used as the capture antibody, and the mAb
1-HRP conjugate diluted 1/5000 in TBS containing 10 g/L BSA and 1 mL/L
Tween 20 was used as the detection antibody. The third assay, the
IT-TIC assay, used an anti-cTnI mAb and an anti-cTnT
mAb. mAb 2 coated onto the microplates was used as the capture
antibody, and the mAb 7G-7-HRP conjugate diluted 1/200 in TBS
containing 10 g/L BSA and 1 mL/L Tween 20 was used as the detection
antibody. The fourth assay, the cTnT-TnC binary complex (TC)-TIC assay,
used an anti-TnC mAb and an anti-cTnT mAb. mAb 2I-11 coated onto the
microplates was used as the capture antibody, and the mAb 7G-7-HRP
conjugate diluted 1/200 in TBS containing 10 g/L BSA and 1 mL/L Tween
20 was used as the detection antibody.
Assay protocol.
One hundred microliters of serum sample or
calibrator and 100 µL of the appropriate mAb-HRP conjugate were added
to each coated well and incubated for 6 h at 4 °C under gentle
shaking for the total cTnI assay, the IT-TIC assay, and the TC-TIC
assay, and for 1 h 30 min at 4 °C for the IC-TIC assay. After the
plates were washed five times with PBS Dulbecco containing 1 mL/L Tween
20, 200 µL of the substrate
o-phenylenediamine/hydrogen peroxide (Sanofi Diagnostics
Pasteur) was added to each well and incubated for 15 min in the
dark at room temperature. The reaction was stopped by the addition of
50 µL 2 mol/L H2SO4. The absorbance
was measured at 492 nm with the LP400 microplate reader (Sanofi
Diagnostics Pasteur). Samples with an absorbance >3.0, the limit of
the microplate reader, were diluted in normal human serum (Scantibodies
Laboratory), which was confirmed cTnI negative by the ACCESS
immunoassay system and cTnT negative by the ELECSYS immunoassay system.
calibrator solution preparations
Calibrator solutions were prepared for each cTnI form: free cTnI,
the TIC ternary complex, the IC binary complex, and the IT binary
complex.
TIC complex calibrator solutions.
We used troponin
complex TIC from human heart tissue (Hytest). The concentration of the
TIC complex stock solution was 0.31 g/L in cTnI, as determined by gel
scanning by the manufacturer. We prepared the TIC complex calibrator
solutions by diluting the TIC complex stock solution in the
Scantibodies normal human serum described above to give 0, 0.1, 0.5, 1,
2, 5, 10, 30, and 50 µg cTnI/L.
Free cTnI calibrator solutions.
Free cTnI calibrator solutions
were prepared with cTnI (purity >95%) from human heart tissue
(Hytest). The concentration of the cTnI stock solution, determined
spectrophotometrically, was 0.05 g/L. The cTnI stock solution was
diluted with the Scantibodies normal human serum described above to
give 0, 0.1, 0.5, 1, 2, 5, 10, 30, and 50 µg/L.
IC complex formation and calibrator solution preparation.
TnC
(purity >98%) and cTnI from human heart tissue were used to
form the IC binary complex. A sixfold molar excess of TnC was added to
cTnI diluted in the Scantibodies normal human serum containing 2 mmol/L
CaCl2 (23). The mixture was incubated for 30
min at 4 °C under gentle shaking. Using the stock solution of IC
complex at 500 µg/L in cTnI, we made the following dilutions in the
Scantibodies normal human serum containing 2 mmol/L CaCl2,:
0, 0.1, 0.5, 1, 2, 5, 10, 30, and 50 µg cTnI/L.
IT complex formation and calibrator solution preparation.
cTnT
(purity >98%) from human heart tissue (Scripps) and cTnI purified
from human heart tissue (Hytest) were used to form the binary complex
IT. A sixfold molar excess of cTnT was added to cTnI diluted in the
Scantibodies normal human serum containing 2 mmol/L CaCl2.
This mixture was incubated for 30 min at 4 °C under gentle shaking.
Using the stock solution of IT complex at 500 µg/L in
cTnI, we made the following dilutions in the
Scantibodies normal human serum containing 2 mmol/L CaCl2:
0, 0.1, 0.5, 1, 2, 5, 10, 30, and 50 µg cTnI/L.
sensitivity of the immunoassays
To illustrate the sensitivity of our immunoassays for each
calibrator, we calculated the response curve slope for each calibration
curve (26)(27).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The calibration curves for the total cTnI assay is shown in Fig. 1
A. With this assay, we detected all forms of cTnI: free cTnI,
the IC binary complex, the IT binary complex, and the TIC ternary
complex. Free cTnI and the binary complexes IC and IT were detected
with the same sensitivity [free cTnI, slope (A · L/µg)
= 1.055; complex IC, slope (A · L/µg) = 1.130; complex
IT, slope (A · L/µg) = 1.186, where A is the
absorbance at 492 nm]. The sensitivity for the complex TIC was
slightly lower: slope (A · L/µg) = 0.853.
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The calibration curves for the IC-TIC assay are shown in Fig. 1B
. With
this assay, we detected two cTnI forms: the IC binary complex, and the
TIC ternary complex. The slope (A · L/µg) obtained for
the complex IC was 0.099. The sensitivity for the complex TIC was
lower: slope (A · L/µg) = 0.054. Free cTnI diluted in
normal human serum was detected only faintly at high concentrations. A
test with free cTnI diluted either in serum or in succinate buffer at
100 and 1000 µg/L showed that the signal was increased only in serum,
which suggested that the normal human serum we used probably contained
TnC from skeletal muscle, which formed an IC complex when cTnI was
present at rather high concentrations (
30 µg/L). When the IT
complex was tested with this assay, the signal was the same as that for
the nonspecific binding.
The calibration curves for the IT-TIC assay are shown in Fig. 1C
. This
assay also detected two forms of cTnI: the IT binary complex and the
TIC ternary complex. The sensitivity (A · L/µg) of this
assay for the TIC complex was 0.579, whereas the sensitivity of the
assay for the IT complex was lower: slope (A · L/µg) =
0.092. For free cTnI and IC complex, the signal detected was the same
as that for the nonspecific binding.
The total cTnI assay was more sensitive than the IC-TIC assay for detection of the IC and the TIC complexes and more sensitive than the IT-TIC assay for the detection of the IT complex. The total cTnI assay and the IT-TIC assay detected the TIC complex with approximately the same sensitivity.
We decided to standardize our three assays with the only common form of cTnI detected, namely the TIC complex diluted in normal human serum. The detection limit (DL) was determined by testing 10 S0 samples (normal human serum without the TIC complex) and 10 S1 samples (complex TIC diluted in normal human serum to a concentration of 0.1 µg cTnI/L) in the three assays {DL = [(2 x SD S0)/(mean absorbance S1 - mean absorbance S0)] x [S1]}. The DLs for the total cTnI assay, the IC-TIC assay, and the IT-TIC assay were 0.01, 0.70, and 0.03 µg/L, respectively. The mean concentration of the cTnI forms in the 12 normal human sera was found to be below the DL for each assay.
With the TC-TIC assay, we were able to detect the TIC ternary complex analytically, but with poor sensitivity. When we reconstituted the TC binary complex in buffer or in serum, we were unable to detect it.
assay of cTnI forms in sera from 15 ami patients
Serum samples from 15 AMI patients collected at different times
after their arrival at the hospital for chest pain were assayed
simultaneously with the three assays calibrated with the TIC complex.
No matrix effects were introduced into the assay by dilution of the
samples. Fig. 2
shows the kinetics of the release of the cTnI forms in the sera
of AMI patients 1–4. These results are typical of those obtained for
the 15 AMI patients. The majority of samples were positive in the total
cTnI assay and the IC-TIC assay; only a few samples were positive in
the IT-TIC assay. Of the 15 patients, one had higher values in the
IT-TIC assay than in the IC-TIC assay. Fig. 3
illustrates the mean results for the 15 AMI patients,
determined by the three assays at each sampling time after their
arrival at the hospital. In the total cTnI assay, all patients were
positive between 3 and 18 h after their arrival at the hospital,
and 11 of 15 patients were positive between their arrival at the
hospital and 3 h later. We observed a peak at 12 h after
their arrival at the hospital, i.e., between 14 and 18 h after the
onset of chest pain, the corresponding concentration was 38.26 ±
26.02 µg/L (mean ± SD). The results followed a similar pattern
with the IC-TIC assay. The results for all patients were positive
between 6 and 18 h after their arrival at the hospital, and the
results for 6 of the 15 patients were positive for samples taken
between their arrival at the hospital and 3 h later . A peak was
observed 12 h after their arrival at the hospital; the
corresponding concentration was 42.15 ± 31.73 µg/L. The results
obtained with the IT-TIC assay showed that it was possible to detect
the IT complex and/or the TIC complex in serum samples from AMI
patients; however, the concentrations were very low compared with those
found by the total cTnI assay and the IC-TIC assay. Only 2 of 15
patients were positive between their arrival at the hospital and 3
h later. There was a peak at 12 h after the patients' arrival at
the hospital, but the corresponding concentration was low: 2.8 ±
18.43 µg/L.
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assay of cTnI forms in sera from 20 patients
receiving cardioplegia
We also tested serum samples collected from 10 patients
receiving crystalloid cardioplegia and from 10 patients receiving cold
blood cardioplegia. Serum samples were collected just before
cardiopulmonary bypass and after aortic unclamping at 6, 9, 12, and
24 h and daily thereafter for 5 days. Serum samples were tested
simultaneously by the three sandwich immunoassays calibrated with the
TIC complex. No matrix effects were introduced into the assay by
dilution of the samples. Fig. 4
shows the kinetics of the appearance of the cTnI forms in the
sera of six patients. Patients 5, 6, and 7 received crystalloid
cardioplegia, whereas patients 8, 9, and 10 received cold blood
cardioplegia. Fig. 5
illustrates the mean values at each sampling time of the cTnI
forms in serum samples from the patients receiving crystalloid
cardioplegia (Fig. 5A
) or cold blood cardioplegia (Fig. 5B
). As
expected, the concentrations were lower than those in the sera from AMI
patients. With the total cTnI assay and the IC-TIC assay,
maximum concentrations were observed for both populations at
6 h after aortic unclamping. For patients who received crystalloid
cardioplegia, the concentrations observed at the peak were 5.09 ±
4.36 µg/L in the total cTnI assay and 11.66 ± 10.38 µg/L in
the IC-TIC assay. For patients who received cold blood cardioplegia,
the concentrations observed at the peak were 6.02 ± 7.18 µg/L
in the total cTnI assay and 10.66 ± 14.11 µg/L in the IC-TIC
assay. The binary IT complex and the ternary TIC complex were not
present in these samples, as illustrated by the results with the IT-TIC
assay. In Fig. 5B
, there were two peaks at 6 and at 12 h after
aortic unclamping for the IC and/or TIC complexes measured using the
IC-TIC assay for the patients who had received cold blood cardioplegia.
This can probably be explained by the fact that the concentrations of
the cTnI forms in the serum of one patient (Fig. 4
, patient 9) were so
high that the profiles of the curves were modified.
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results obtained for ami patients and patients receiving a
cardioplegia with the total cTnI assay and the ic-tic assay
calibrated with the ic complex
After analyzing the results obtained with our three immunoassays
calibrated with the TIC ternary complex, we decided to recalculate the
concentrations of the test samples measured by both the total cTnI
assay and the IC-TIC assay calibrated with the IC binary complex
(Fig. 1
, A and
B).
The results obtained with AMI patients and patients receiving either
cold blood or crystalloid cardioplegia are shown in Fig. 6
. The profiles of the troponin release observed with the
total cTnI assay and the IC-TIC assay in AMI patients (Fig. 6A
) are
perfectly superposed, with a peak at 12 h after their arrival at
the hospital. The corresponding concentrations were 26.68 ± 18.76
µg/L in the total cTnI assay and 26.57 ± 23.18 µg/L in the
IC-TIC assay. The same results were obtained in patients receiving cold
blood cardioplegia (Fig. 6B
). The profiles of the troponin release
obtained with the total cTnI assay and the IC-TIC assay are superposed,
with a peak at 6 h after aortic unclamping and corresponding
concentrations of 4.62 ± 5.73 µg/L in the total cTnI assay and
4.90 ± 7.23 µg/L in the IC-TIC assay. The profiles of the
troponin release observed with the total cTnI assay and the IC-TIC
assay in patients receiving crystalloid cardioplegia are not
superposed: between 6 and 24 h after aortic unclamping,
concentrations measured by the IC-TIC assay were higher than those
measured by the total cTnI assay. The maximum concentrations measured
were 2.89 ± 1.022 µg/L by the total cTnI assay and 3.94 ±
2.92 µg/L by the IC-TIC assay (Fig. 6C
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To calibrate the three immunoassays, we tested all the troponin forms: I, IC, IT, and TIC. Because the TIC complex form was the only common calibrator, we decided to use it to calibrate the assays.
Using the three immunoassays developed in this study, we measured the kinetics of the release of free cTnI, IC, IT, and TIC in serum samples from AMI patients at different times after their arrival at the hospital. Among the 15 AMI patients, all sera were found to be positive in the total cTnI assays and in the IC-TIC assay, whereas the sera from only 2 of the 15 patients were weakly positive when measured with the IT-TIC assay. Because the IT-TIC assay detects only the IT and/or TIC forms, we conclude that the IT and/or TIC forms exist only in a low proportion of AMI patients, and at a very low concentration. The presence of the IT and/or TIC forms in the serum samples of two AMI patients might be indicative of a poor prognostic; this must be confirmed in a larger population. The patterns of troponin release measured by the total cTnI assay and the IC-TIC assay were the same in patients receiving cardioplegia during heart surgery as those for AMI patients. Neither IT nor TIC was detected in the serum of the 20 patients with heart surgery by the IT-TIC assay. As suggested by the work of Etievent et al. (20), the increases in the concentrations of the troponin forms measured by the total cTnI assay and the IC-TIC assay were probably caused by cardiac ischemia during heart surgery.
Because TIC was not detected in the serum samples from most of the patients, even when the most sensitive assay was used, we concluded that the IT and TIC complexes were not predominant forms in either patient population studied. Therefore, the cTnI forms measured in the patients' sera by the total cTnI assay were probably free cTnI and/or the IC complex, and the IC-TIC assay probably only detected the IC complex. Using the TIC complex to calibrate the assays, we found a slightly higher mean concentration of cTnI forms in the serum samples when the IC-TIC assay was used than when the total cTnI assay was used. Moreover, the release kinetics of the cTnI forms observed for both patient populations studied showed the same profile when measured with the total cTnI assay as with the IC-TIC assay. In other words, the release patterns observed for free cTnI and/or the IC complex in the total cTnI assay and for IC complex in the IC-TIC assay were the same. On the basis of these findings, we hypothesized that the total cTnI assay and the IC-TIC assay were detecting the same molecule, namely the IC complex. Indeed, when we calibrated both the total cTnI assay and the IC-TIC assay with the IC complex, we found that the release profiles for the cTnI forms measured by the two assays in serum samples from AMI patients and from patients receiving cold blood cardioplegia were superposed. We therefore strongly suggest that the IC complex is the main circulating form for these pathologies. For patients receiving crystalloid cardioplegia, the release profiles were not well superposed. This discrepancy could be attributed to a component of the crystalloid cardioplegia solution, which might interfere with our immunoassays. This remains to be confirmed.
Taken together, our results suggest that troponin forms are not released in the order expected on the basis of their molecular weights, i.e., cTnI first, because of its low molecular weight, then IC, and then TIC. We would also have expected to find TIC released first, then IC, and finally cTnI, indicative of complex dissociation after the release into the bloodstream. In fact, an equilibrium probably exists between all the possible released forms, and the form that predominates, the IC complex, is the one with the highest association constant and, therefore, the most stable.
Katrukha et al. (23), using one pair of anti-cTnI mAbs that recognize only free cTnI and another pair of anti-cTnI mAbs that recognize free and complexed cTnI in the presence of EDTA, showed that the main part of cTnI is released as a complex, without specifying whether the isoform released is IC or TIC. The use of three immunoassays able to detect all of the cTnI complexes with mAbs directed against cTnI, cTnT, and TnC and the absence of detection of the IT and/or TIC forms in the sera of most patients allowed us to conclude that IC is the predominant complex circulating, and not TIC. These results corroborate the findings that cTnI is stabilized by TnC (28).
The findings reported here are of a great importance for the standardization of the commercially available cTnI assays. Because IC is the main circulating form and one of the most stable, it is likely to be the best form to use as a standard for the different assays. That would be the first step along the way to standardization. Because different mAbs are used in the different commercially available assays and because our results as well as those of Katrukha et al. (23) suggest that low amounts of other cTnI forms (free cTnI, TIC, and/or IT) are released, it seems important to use a representative panel of serum samples from patients in addition to a primary standard. This panel could be used by each manufacturer to determine the clinical sensitivity of the assay.
In conclusion, we have shown that cTnI circulates in blood mainly as the IC complex form in AMI and heart surgery patients shortly after the release of troponin. Free cTnI, IT, and/or TIC forms were found in the blood of a few AMI patients, but at low concentrations compared with IC. Therefore, a cTnI assay must have the following two features: it must be able to recognize the predominant form IC; and it must be able to recognize the cTnI forms released into the bloodstream under different clinical conditions, i.e., in patients with different types of myocardial injury. In this investigation, we analyzed the release of cTnI forms in only two patient populations: AMI patients and patients receiving cardioplegia during heart surgery. We are now planning a study on patients with unstable angina to see whether they show the same release pattern as AMI patients or, which is more likely, the same pattern as patients who have undergone heart surgery, our aim being to investigate whether we can associate the different cTnI forms with specific pathologies.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  D. C Gaze and P. O Collinson Multiple molecular forms of circulating cardiac troponin: analytical and clinical significance Ann Clin Biochem, July 1, 2008; 45(4): 349 - 355. [Abstract] [Full Text] [PDF]
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R. Labugger, J. A. Simpson, M. Quick, H. A. Brown, C. E. Collier, I. Neverova, and J. E. Van Eyk Strategy for Analysis of Cardiac Troponins in Biological Samples with a Combination of Affinity Chromatography and Mass Spectrometry Clin. Chem., June 1, 2003; 49(6): 873 - 879. [Abstract] [Full Text] [PDF]
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R. Labugger, L. Organ, C. Collier, D. Atar, and J. E. Van Eyk Extensive Troponin I and T Modification Detected in Serum From Patients With Acute Myocardial Infarction Circulation, September 12, 2000; 102(11): 1221 - 1226. [Abstract] [Full Text] [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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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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, calibration with
the TIC complex diluted in normal human serum (linear regression:
y = 0.853x + 0.027,
r = 0.99); 
, calibration with free cTnI diluted
in normal human serum (linear regression: y =
1.055x + 0.062, r = 0.99); 
,
calibration with the IC complex diluted in normal human serum
containing a sixfold molar excess of TnC (linear regression:
y = 1.130x + 0.021,
r = 0.99); X, calibration with the IT complex
diluted in normal human serum containing a sixfold molar excess of cTnT
(linear regression: y = 1.186x +
0.016, r = 0.99). (B), IC-TIC assay.
, IT-TIC assay.
