Comparison of Immunoreactivity of Five Human Cardiac Troponin I Assays toward Free and Complexed Forms of the Antigen: Implications for Assay Discordance

Troponin, consisting of three components, troponin C (TnC), troponin I (TnI), and troponin T (TnT), is a major component of the structural proteins involved in striated and cardiac muscle contraction (1)(2). TnI and TnC bind tightly to each other in the presence of Ca2+ with an association constant, Ka, of ∼108-109 L/mol (1)(2)(3). TnT binds to both TnC and TnI, although less weakly than the binding between TnC and TnI. The cardiac isoforms of TnI (cTnI) and TnT are structurally different from the corresponding skeletal isoforms, and therefore they have recently established themselves as biochemical markers of myocardial damage (4)(5)(6)(7). The currently available cTnI assays produce differing results (8)(9)(10). One important reason is that the assays may differ in their responses to the various isoforms of cTnI present in circulation or in biochemical preparations. In addition to “free” cTnI (I) and its binary and ternary complexes with TnC and TnT (IC or ICT) (8)(11)(12), cTnI may exist in phosphorylated (13), oxidized (14), and proteolytically degraded (8)(11)(12) forms. The latter modified forms of cTnI also may exist as binary or ternary complexes. All of these forms may have different recognition patterns in different immunoassays. The predominant form of cTnI in acute myocardial infarction (AMI) patients is the binary complex IC (8)(11). Samples treated with a Ca2+ chelator such as EDTA would contain mostly free cTnI because chelation of Ca2+ disrupts IC and ICT complexes. We explored the possibility that the sera from patients with different pathological conditions may contain different isoform distributions of cTnI, thus generating discordant results among assays that recognize the isoforms differently. Here we report the immunoreactivity of five commonly used commercial cTnI immunoassays (Bayer ACS:180®, Dade Stratus®, Beckman Access®, Behring OPUS®, and Abbott AxSYM®) toward the two major forms of the analyte: free cTnI and its binary complex, IC. Free cTnI (I) and its binary complex (IC), both isolated from human heart, were obtained from Scripps Laboratories. Their concentrations, determined by protein assay (Bio-Rad), were provided by the vendor and then converted to molar cTnI concentrations, using molecular weights of 21 000 and 35 000 for I and IC, respectively. Normal human serum, obtained from Scantibodies, tested negative for cTnI in all five cTnI assays. Buffered stock solutions of both I and IC, as obtained from the vendor, and the serum were mixed to produce 0, 5, 10, and 20 μg/L solutions of cTnI, which were analyzed by five cTnI immunoassays: ACS:180, Stratus, AxSYM, OPUS, and ACCESS. Manufacturer-suggested assay protocols and platforms were used for all assays. All five assays use a “sandwich” method, where the analyte is sandwiched between capture and label antibodies, and thus the generated signal is directly proportional to the concentration of TnI in the sample. Details of the assays are in listed in Table 1. Comparison of the five cTnI immunoassays used in this study. The capture antibody is different from the label antibody, recognizing different sites on the cTnI molecule. b–d From the package inserts for the kits; ACS:180; AxSYM; and d ACCESS. Dasgupta et al. (15). Comparison of the five cTnI immunoassays used in this study. The capture antibody is different from the label antibody, recognizing different sites on the cTnI molecule. b–d From the package inserts for the kits; ACS:180; AxSYM; and d ACCESS. Dasgupta et al. (15). When the results of the methods listed in Table 1(y) were compared with the Stratus (x), the linear correlation slopes were 0.14 for ACCESS, 1.13 for ACS:180, 1.43 for OPUS, and 3.42–5.14 for AxSYM. We calculated the OPUS (y)/Stratus (x) slope as the ratio of slopes for (ACS:180/Stratus) and (ACS:180/OPUS). The two different slopes of AxSYM (y) vs Stratus (x) were obtained from different studies: 3.42 from the package insert of the AxSYM kit, and 5.14, from our own studies (15). The AMI cutoff values varied in slightly different order: ACCESS (0.15 μg/L) < ACS:180 ∼ Stratus (1.5 μg/L) < AxSYM ∼ OPUS (2.0 μg/L). The ACCESS and AxSYM assays differed by a factor of as much as 36-fold (5.14/0.14). The other three assays agreed more closely (± 25%). Various survey data for cTnI also indicate similar differences among the assay results (16). Many of the difference are probably contributed by standardization. As noted by other researchers (8)(9)(10), such interassay differences underscore the urgent need for universal standardization for this important analyte. Fig. 1 presents the comparison of immunoreactivity of the five assays to IC (Fig. 1A) and I (Fig. 1B). The assays have different orders of immunoreactivity toward free cTnI and IC complex: ACCESS < Stratus < ACS:180 < OPUS < AxSYM for IC; and Stratus < ACCESS < OPUS < ACS:180 < AxSYM for I. The order of immunoreactivity for IC was similar to the order of their responses in the method comparison study (Table 1). However, whereas the ACS:180, ACCESS, and AxSYM cTnI assay responses to I remained similar to their responses to IC, the Stratus and OPUS assays behaved quite differently to I than to IC, producing responses to I that were 6.41 and 3.19 times less than IC (as determined the ratio of slopes, IC/I; Fig. 1). The ACS:180 cTnI assay showed the best “equimolarity” (i.e., the ability to recognize I and IC equally well), with a IC/I slope ratio of 1.07. The slope ratios for the ACCESS and AxSYM assays were 1.23 and 1.4, respectively. Results of five different cTnI assays compared for serum samples supplemented with binary IC complex (A) or free cTnI (B). In A and B, the solid line is the line of identity. (A), the ACS:180 assay is closest to the line of identity, AxSYM and OPUS are above the line of identity, and ACCESS and Stratus are below the line of identity. The linear correlation slopes of the five assay results with respect to the added cTnI concentrations are: ACS:180, 0.92 (r = 0.999); Stratus, 0.53 (r = 0.998); AxSYM, 1.68 (r = 0.978); OPUS, 1.37 (r = 0.999); and ACCESS, 0.26 (r = 0.999). (B), the ACS:180 assay is closest to the line of identity, AxSYM is above the line, and the other assays are below the line of identity. The linear correlation slopes of the five assay results with respect to the added cTnI concentrations are: ACS:180, 0.86 (r = 0.992); Stratus, 0.08 (r = 0.978); AxSYM, 1.20 (r = 0.999); OPUS, 0.43 (r = 0.968); and ACCESS, 0.21 (r = 0.990). Our observations agree well with those of Wu et al. (8) who compared nine different immunoassays for cTnI toward synthetically prepared oxidized or reduced forms of I or IC. When they compared the more common oxidized forms of IC and I, six assays in their study were approximately equimolar, two others responded to IC better than to I by ∼1.5-fold, and one assay responded to IC ∼3.2-fold more than to I. Because the binary IC complexed form of cTnI is believed to account for ≥90% of cTnI in the serum of most AMI patients (8)(11)(12), the ACS:180 and Stratus assays show close agreement among most samples (Table 1). However, if the complexed form of cTnI is released into the circulation only after extensive damage to myocytesand the subsequent necrosis of cardiac muscle, the appearance of IC or ICT complexes in the circulation could take several hours to days, thus delaying the confirmation of AMI diagnosis. On the other hand, 5–6% of the total cardiac cTnI exists as a free form in the cytoplasm (17). If the free cTnI is released faster than IC complex from damaged myocytes, initial cTnI concentrations may contain a higher percentage of free cTnI. These samples may show a higher frequency of discordance between the equimolar and nonequimolar cTnI assays. We think that the differential immunoreactivity of cTnI assays to its isoforms may explain some cases of assay discordance. A possible example can be found in a method comparison study with samples from many categories of cardiac patients (18). When the Stratus, OPUS, and ACCESS assay results for 138 such samples were compared, a poorer linear regression coefficient (r = 0.774) was found between Stratus and ACCESS than between Stratus and OPUS (r = 0.92) or OPUS and ACCESS (r = 0.90) (18). Our finding that the order of equimolarity between these three assays is ACCESS (most equimolar) > OPUS > Stratus (least equimolar) may explain those data. An assay that detects both forms of cTnI equally well could be an advantage over a method that detects only one form of cTnI because the presence of any form of cTnI in serum is indicative of cardiac damage.