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Letters |
Department of Thyroid, Metabolic,, and Cardiovascular Diagnostics R/D, Abbott Laboratories, Abbott Park, IL 60064
a Author for correspondence. Fax 847-938-7920; e-mail David.Wilson{at}add.ssw.com
To the Editor:
A recent report by Apple et al. (1) presented the performance characteristics of the AxSYM Troponin I (cTnI) assay, which included method comparison data with the Dade-Behring Stratus, Behring Opus, and Beckman Access cTnI assays. In commenting on the absolute differences in measured cTnI between methods (2- to 100-fold), the authors spoke of the need for unified rationale for antibody selection in assays for cTnI. Indeed, this has been a topic of substantial discussion in the recent literature in view of the large differences observed between cTnI methods [e.g., see Ref. (2)]. Most recently, Shi et al. (3) added to earlier results of Katrukha et al. (4) on cTnI degradation. In both reports, degradation of cTnI and differential epitope specificities between commercial methods were cited as significant contributors to between-method variation. Both studies also indicated that the C-terminal portion of the molecule is labile to proteolytic degradation, whereas the N-terminal half of the molecule is relatively stable. Katrukha et al. (4) identified a region between amino acid residues 30 and 110 that appeared to be the most stable. Shi et al. (3) noted that the N-terminal portion appeared to be as stable as the 30–110 fragment, probably a result of the primary structure rather than protective association with troponin C. Shi et al. (3) further suggested that of the large differences noted between methods, it is likely that differences in calibration are smaller contributors than between-method differences in sensitivity to cTnI degradation attributable to antibody selection. To facilitate understanding in this complex area, we describe the antibody selection for the AxSYM cTnI assay.
The AxSYM assay uses a two-step sandwich format, whereby sample and anti-cTnI-coated microparticles are combined to form an antigen complex on the microparticles. After an incubation, the reaction mixture is transferred to the glass fiber matrix of the AxSYM Matrix Cell, where the microparticles are captured, washed, and probed for cTnI with a conjugate composed of a second anti-cTnI antibody and alkaline phosphatase.
The two antibodies used in the AxSYM assay were chosen before the current knowledge about cTnI degradation. Antibody selection was based on extensive antibody screening with a goal of identifying an efficacious pair that would exhibit similar sensitivity to both free and complexed troponin. The screening was performed using the BIAcore system (Pharmacia Biosensor). The BIAcore system is a biosensor that utilizes the quantum mechanical phenomenon of surface plasmon resonance (SPR) to detect and analyze the interactions of biomolecules. Antibodies were evaluated on the basis of their affinity for cTnI [both free form and complexed with troponin T (TnT) and troponin C (TnC), as purchased from HyTest Ltd], as well as their ability to form an efficacious antigen sandwich between solid-phase antibody (immobilized on the surface of a gold/glass sensor chip interface), cTnI, and a secondary antibody. Interactions between these molecules alter the refractive index of the sensor chip, which is detected as a change in the SPR angle. Angle shifts are recorded in real time as resonance units and are related to the association/disassociation of the interacting molecules. From this analysis, association constants were determined, and relative epitope positioning was qualitatively mapped.
We evaluated 342 potential monoclonal, polyclonal, and mono-polyclonal antibody pairs. Of these, eight pairs with the highest binding potential were further evaluated as the solid-phase capture antibody or the signal generator antibody in the AxSYM assay format. Although exhibiting good dose–response characteristics in the AxSYM for free troponin, most of these pairs exhibited distinctly different relative affinities for free and complexed cTnI. The two antibodies that were chosen were a monoclonal antibody specific for an epitope in the mid portion of the cTnI primary structure (residues 87–91, cell line 8E10; HyTest), and a peptide affinity-purified goat polyclonal antibody specific for residues 20–39 (Bio Pacific). Residues 1–31 represent a cardiac-troponin-specific region (5). These antibodies exhibited similar apparent association constants for free and complexed cTnI based on the BIAcore analysis (for the monoclonal, Ka = 1.10 x 105 and 1.45 x 105; for the polyclonal, Ka = 1.06 x 105 and 1.17 x 105 for free and complexed troponin, respectively), as well as good dose–response characteristics in the AxSYM.
As it turned out, the antibodies selected for the AxSYM are specific for the more stable mid and N-terminal portions of the cTnI molecule. According to Shi et al. (3), the Stratus assay also exhibits a specificity for the N-terminal rather than the C-terminal portion of cTnI. This would be consistent with the similar clinical performance noted between these two methods by Apple et al. (1). The 3.5-fold difference in the slope of the linear regression between the AxSYM and Stratus (1) would thus appear to be attributable more to differences in calibration than to antibody selection.
References
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