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Articles |
Spectral Diagnostics Inc., 135-2 The West Mall, Toronto, Ontario, Canada M9C 1C2.
a Author for correspondence. Fax 416 626-7383; e-mail qshi{at}ica.net
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We used four recombinantly engineered cTnI fragments to study the regions of cTnI recognized by the Stratus®, Opus®, and ACCESS® immunoassays. The stability of these regions in serum was analyzed with Western blot.
Results: The measurement of several control materials and different forms of purified cTnI using selected commercial assays demonstrated five- to ninefold variation. Both the Stratus and Opus assays recognized the N-terminal portion (NTP) of cTnI, whereas the ACCESS assay recognized the C-terminal portion (CTP) of cTnI. Incubation of recombinant cTnI in normal human serum produced a marked decrease in cTnI concentration as determined with the ACCESS, but not the Stratus, immunoassay. Western blot analysis of the same samples using cTnI NTP- and CTP-specific antibodies demonstrated preferential degradation of the CTP of cTnI.
Conclusions: The availability of serum cTnI epitopes is markedly affected by the extent of ligand degradation. The N-terminal half of the cTnI molecule was found to be the most stable region in human serum. Differential degradation of cTnI is a key factor in assay-to-assay variation.© 1999 American Association for Clinical Chemistry
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In patients diagnosed with acute myocardial infarction, serum cTnI concentrations are increased within 4 to 6 h, peak at 12 to 18 h, and remain increased for 5–9 days after the onset of chest pain (4). Recent reports further demonstrated the high sensitivity (97%), specificity (98%), and predictive value (99.8%) of increased cTnI concentration for the diagnosis of acute myocardial infarction (5)(6). The cTnI assay is also useful in the risk assessment of patients with acute coronary syndromes (7).
Several commercial immunoassays (8)(9)(10) are available for the quantitative measurement of serum cTnI. Ten- to 20-fold variations in cTnI concentrations were reported when a given patient sample was measured with several different assays (9)(11). Currently, all of the factors responsible for this observed variation are not fully understood. Although the lack of a primary reference method or standard material to calibrate these assays may partially account for these variations, the different serum forms of cTnI recognized by the antibodies used in these immunoassays may be responsible for the remaining discrepancies. Recently, Wu et al. (11) tested different forms of cTnI, such as free cTnI, cTnC-cTnI complex, cTnI-TnT complex, and cTnC-cTnI-cTnT complex, using different cTnI assays. Generally, the observed variation between assays was <10-fold. However, a maximum variation of 20-fold was observed when a patient sample was tested (11). It is known that cTnI degradation may occur in both ischemic myocardium (12)(13)(14) and the circulation. Thus, we hypothesized that each assay may recognize different regions of cTnI and that the rate of degradation of these regions in myocardium or serum may be different. To test this hypothesis, recombinant peptides corresponding to different regions of cTnI were engineered and analyzed using the major commercial assays.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After fermentation and induction with isopropyl-ß-D-thiogalactopyranoside, bacterial host cells expressing the desired proteins were disintegrated by a combination of lysozyme digestion and sonication. Inclusion bodies containing cTnI were sedimented from the homogenate by centrifugation and were solubilized with a Tris-HCl buffer containing 8 mol/L urea. The solubilized cTnI was further purified using CM-Sephadex ion-exchange chromatography and refolded by the removal of urea. Both cTnT and cTnC were found predominantly in the soluble fractions of the bacterial lysate. Recombinant cTnT was purified by DEAE-Bio-gel ion-exchange chromatography from the bacterial lysate and further polished by precipitation with 326 g/L ammonium sulfate. Recombinant cTnC was isolated from the bacterial lysate by precipitating bacterial proteins with 436 g/L ammonium sulfate. The resulting supernatant containing cTnC was further purified by DEAE-Bio-gel ion-exchange chromatography.
The troponin complex was reconstituted by mixing equal amounts of purified troponin subunits in the presence of urea, ß-mercaptoethanol, CaCl2, and MgCl2 essentially as described by Tao et al. (18). Crude cTnI fragments were used for qualitative assessment without further purification.
characterization of recombinant proteins
Protein concentrations were determined with the Bradford assay
(19). Recombinant proteins or their fragments were analyzed
with SDS-PAGE as described by Laemmli (20). Western blot
analyses of recombinant cTnI fragments and serum cTnI degradation
products were performed according to the original method of Towbin et
al. (21), using a goat polyclonal antibody (BiosPacific)
that recognizes residues 27–39 of cTnI and a monoclonal antibody
(8I-18; Spectral Diagnostics) that recognizes the C-terminal portion
(150–210) of cTnI. The final concentration of the goat polyclonal
antibody was 15 mg/L, whereas the final concentration of the 8I-18
antibody was 20 mg/L.
degradation of cTnI IN SERUM
To investigate the degradation pattern of cTnI in serum, purified
free recombinant cTnI or cTnI complexes were added to 100 mL/L normal
human serum (NHS) to a final concentration of 160 mg/L and incubated at
37 °C for 2, 4, 6, 24, and 48 h. cTnI concentrations were
measured after 1250- to 2500-fold dilution in NHS. Thirty microliters
of sample from each time point was used for Western blot analysis.
immunoassays for cTnI
Three commercially available assay systems, the
Stratus® II (Dade International), the
ACCESS® (Beckman Instruments), and the
Opus® (Behring Diagnostics), were used to
measure various forms of cTnI, following the manufacturers'
instructions. All values reported here are the mean values of duplicate
determinations.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The recombinant cTnI used in these studies was compared with
cTnI purified from human heart for the characterization of
immunoreactivity and its ability to complex with other troponin
subunits. Western blot, ELISA, and BIAcore were used to compare
their immunoreactivity. No significant differences were found between
the recombinant and the isolated cTnI (data not shown).
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measurement of different cTnI FORMS
To investigate method variation, the following materials were
measured for apparent cTnI mass concentration, using the Stratus,
ACCESS, and Opus assay systems: (a) Stratus control
material, (b) ACCESS control material, (c) Opus
control material, (d) purified recombinant cTnI (25 µg/L)
added to NHS, (e) purified recombinant cTnI-cTnC complex (25
µg/L) added to NHS, and (f) purified recombinant
cTnI-cTnC-cTnT complex (25 µg/L) added to NHS (Table 1
). All values were derived from the means of duplicate
determinations. Because of the reversible nature of the equilibrium
between complexed and free troponin subunits, both the cTnI-cTnC and
the cTnI-cTnC-cTnT preparations contained a percentage of free cTnI. In
general, the maximum observed variation was less than fivefold, with
the exception that no values were obtained for the Stratus control
material when analyzed with the ACCESS and Opus assays.
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segment mapping of selected immunoassays
We have observed that when clinical samples stored for 12 to 36
months at -20 °C are retested for their cTnI concentration, the
Stratus system often yields a relatively unchanged or even an increased
value when compared with the initial values for the samples (Table 2
). In contrast, cTnI concentrations measured with the ACCESS
assay consistently demonstrate a marked decrease when compared with
their initial values. To identify which region of the cTnI molecule is
recognized by each assay, four overlapping cTnI fragments corresponding
to amino acid residues 1–148 (cTnIf1), 55–210 (cTnIf2), 1–99
(cTnIf3), and 104–210 (cTnIf4) were engineered (Table 3
) and expressed in E. coli. The primary sequences of
the fragments were verified by DNA sequencing, and the fragment sizes
(17.5, 18.2, 11.7, and 12.6 kDa, respectively, by prediction) were
confirmed with SDS-PAGE. Western blot analyses of the four cTnI
fragments (Fig. 2
) showed that cTnIf1 and cTnIf3 reacted with an N-terminal
portion-specific polyclonal antibody (Fig. 2A
, lanes 1 and 3,
respectively), but not a C-terminal portion-specific monoclonal
antibody (Fig. 2B
, lanes 1 and 3, respectively), whereas cTnIf2 and
cTnIf4 reacted with a C-terminal portion-specific monoclonal antibody
(Fig. 2B
, lanes 2 and 4, respectively), but not the N-terminal
portion-specific antibody (Fig. 2A
, lanes 2 and 4, respectively).
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E. coli lysates expressing the four cTnI fragments were
tested for their immunoreactivity with the different assays. Because
any value above the assay cutoff was considered positive, lysates
containing high concentrations of each fragment were used to determine
whether the specific fragments were recognized by the assays. E.
coli lysate not expressing any recombinant protein was used as the
negative control, and purified full-length recombinant cTnI was used as
the positive control. Our results (Table 3
) demonstrate that both the
Stratus and Opus methods recognized the N-terminal portion of cTnI,
whereas the ACCESS assay recognized the C-terminal portion of cTnI.
degradation of cTnI IN HUMAN SERUM
To further investigate whether the instability of the C-terminal
portion of cTnI was attributable to the preferential degradation of
this region, purified recombinant cTnI was added to 100 mL/L NHS and
incubated at 37 °C for 2, 4, 6, 24, and 48 h. The cTnI
concentrations at different time points measured with the Stratus assay
showed an initial increase and then a slow decrease of the apparent
concentration. In contrast, when the same set of samples were analyzed
with the ACCESS assay, a steady decrease of the apparent concentration
was observed (Fig. 3
). Western blot analysis of these samples (30 µL) after
electrophoresis in a 10–20% SDS-gradient gel, using an N-terminal
segment-specific antibody, demonstrated the accumulation of two
low-molecular mass polypeptides (~10–12 kDa) over the incubation
period (Fig. 4
A), indicating that a portion of the N-terminal region was
strongly resistant to proteolytic cleavage. When a C-terminal
segment-specific antibody was used, no accumulation of low-molecular
mass polypeptide was observed, and the overall intensity of all of the
detected bands decreased (Fig. 4B
). To determine whether the troponin
complexes are degraded in the same way as free cTnI, both the
recombinant cTnI-cTnC and the cTnI-cTnC-cTnT complexes were incubated
in NHS and their proteolytic patterns compared with Western-blot
analysis (Fig. 5
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Our results also suggest
that the C-terminal portion of serum cTnI is preferentially degraded.
Consequently, assays recognizing the C-terminal portion of the cTnI
molecule tend to yield much lower apparent concentrations when compared
with assays recognizing the N-terminal portion. We hypothesize that
both the differences in cTnI epitopes recognized by these assays and
the differences in the serum stability of cTnI degradation products
account for a large part of the variation commonly observed with
patient samples. Factors that may cause variation between methods include (a) the use of different calibrators for assay calibration; (b) different sample requirements (serum, plasma, and/or whole blood) and the resulting matrix effects (e.g., fibrin and anticoagulants); (c) differential recognition of various epitopes that may not be equally accessible or have a different conformation when cTnI is complexed with TnC and/or TnT; (d) the requirement that different regions (between epitopes) of cTnI remain intact (Serum proteolytic fragments of cTnI containing these regions may have very different half-lives because of differential clearance or preferential degradation.); (e) assay-specific false positives; and (f) the existence of cTnI polymorphisms (22) that may affect certain epitopes, but not others.
The method variation between cTnI assays, which for a given patient
sample may reach as high as 20-fold (11), is not entirely
attributable to the fact that each assay is calibrated using different
calibrators. As demonstrated in Table 1
, assay-specific calibration has
been shown to account for at most a fourfold difference among assays.
Thus, there appear to be other important factors that affect the
measurement of cTnI. Because all of the methods are sandwich-based
immunoassays, signal generation requires that all targeted epitopes are
accessible and have the correct confirmation, and that the regions
between these epitopes remain intact. Because each assay uses a unique
set of antibodies, different regions of cTnI are required to be intact.
Therefore differential degradation of selected regions of cTnI likely
contributes to the observed differences between cTnI methods.
cTnI, when incubated at 37 °C in NHS, showed a steady decline in
apparent TnI concentration when monitored with the ACCESS assay.
Interestingly, an initial increase of up to twofold was seen when these
same samples were measured for cTnI concentration with the Stratus.
This is in agreement with the data in Table 2
. It is likely that the
cleavage of the C-terminal portion of cTnI facilitates the binding of
the antibodies used in the Stratus assay to the molecule and leads to
an increase in the observed concentration. We hypothesized that this
variation may be caused by the different serum stabilities of the
target cTnI segments recognized by each assay. Further analysis of
these experimental samples with Western blots using terminal-specific
antibodies revealed that the N-terminal portion of the cTnI molecule
was highly resistant to further proteolytic degradation even in the
absence of TnC. Consequently, assays measuring this portion of the cTnI
molecule may not show a dramatic decrease in the presence of
substantial cTnI degradation. Furthermore, the finding of accumulated
stable N-terminal proteolytic fragments agrees with our Stratus assay
results (Fig. 3
). In contrast, no accumulation of C-terminal fragments
was observed, suggesting that this portion of cTnI was rapidly
degraded. This observation agrees with the results obtained with the
ACCESS assay (Fig. 3
), a C-terminal segment-specific method.
Our results are in agreement with those obtained for the degradation of
the C-terminal portion of cTnI in rat and human cardiac tissue reported
by Van Eyk et al. (14), Ardelt et al. (23), and
Katrukha et al. (24), and in human serum reported by Morjana
(25) and Katrukha et al. (24). Katrukha et al.
(24) suggested that cTnI fragment located between amino acid
residues 30 and 110 is the most stable region, possibly because of its
protection by TnC. Our results indicate that the proteolytic resistance
of N-terminal portion, which is similar or identical to the stable
fragment mentioned above, is not attributable to the complex with TnC,
but to the nature of that portion's primary amino acid sequence. The
complexed forms of cTnI are degraded in a similar pattern to, but at a
much slower rate than, free cTnI, indicating that the C-terminal
portion of cTnI is protected by other troponin subunits from
proteolytic cleavage. We also noticed partial cleavage of a small
fragment from the NH2 terminus of cTnI. Using an
in-house monoclonal antibody, 3I-265, which recognizes residues 27–36,
we could detect only a limited number of N-terminal portion proteolytic
fragments (data not shown). These results strongly suggest that the
differential degradation of different regions of cTnI in human serum or
in cardiac tissue is a major reason for the variation between
commercial cTnI methods. Our results also suggest that all methods
studied preferentially recognize the cTnI complex over free cTnI (Table 1
). It is also apparent that none of the materials in Table 1
is
completely representative of the serum form of cTnI.
It has been reported by Adams et al. (26) that 97% of cTnI was found in the myofibril fraction and that <3% of cTnI was found in the cytosolic fraction of cardiac tissue. Their results suggest that >97% of total cellular cTnI exists in a complexed form. The existence of a high percentage of complexed cTnI is probably attributable to the relatively high intracellular concentrations of both TnI and the other troponin subunits and to the high affinity constant between these subunits. Because the half-life of free cTnI in circulation is only 5 min (27), the major forms of cTnI in the bloodstream of patients are more likely to be troponin complexes. This has been suggested by the results of Wu et al. (11) and Katrukha et al. (28). Recently, Morjana (25) reported that affinity-purified cTnI from a myocardial infarction patient is associated with both TnC and TnT. However, Giuliani et al. (29) reported that using three immunoenzymatic sandwich assays specific for free cTnI, cTnI-TnC, and cTnI-TnC-cTnT, respectively, the predominant serum form of cTnI is the cTnI-TnC binary complex.
We, as well as Katrukha et al. (24), have demonstrated the degradation of the C-terminal portion of cTnI in serum. Other investigators have reported the degradation of cTnI in ischemic tissue (12)(13)(14), resulting from the removal of a C-terminus fragment. It is therefore reasonable to assume that cTnI exists in serum mainly in a complexed and C-terminal degraded form, although a small portion (1–30) of N-terminal fragment may be also partially degraded. Saijo et al. (30) observed that skeletal muscle TnC and amino acids 1–47 of skeletal muscle TnI can form a core that is resistant to proteolytic digestion. Krudy et al. (31) reported that truncated cTnI containing only amino acids 33–80 was sufficient to form a stable complex with TnC. These findings support the notion that troponin complexes containing a C-terminal truncated cTnI can be relatively stable in serum. It should be noted that because the release of cTnI from the myocardium, the clearance of cTnI from the circulation, and the dissociation of troponin complexes are continuous processes, the extent of cTnI degradation in patient samples is highly variable.
To help reduce this method variation, all assays should use the same calibrator (an international reference material). The ultimate goal of assay standardization is to minimize the variation between methods. This standardization protocol, however, requires that the ideal reference standard display the same molecular characteristics as the substance being measured. Because of both the heterogeneous nature and biochemical complexity of the serum forms of cTnI, the standardization of cardiac troponin assays is proving to be a serious challenge. Although one could prepare a material comparable to the cTnI serum forms, this alternative is not reasonable because of the variable nature of the troponin complexes and the difficulty in reproducing the serum degradation of these complexes. Alternatively, utilization of a calibrator containing equimolar concentrations of all of the epitopes recognized by these assays in conjunction with antibodies that recognize epitopes in the most stable common segment of the serum cTnI will greatly assist the standardization process.
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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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