Validation of Serum Cardiac Troponin I Reference Materials Using an Immunoprecipitation Method Coupled with Fluorescent Western Blot Analysis

Cardiac troponin I (cTnI) is adopted as the ‘gold standard’ cardiac biomarker following the universal definition of myocardial infarction (MI). Presently, the performance of commercial cTnI immunoassays varies significantly despite the recalibration using a troponin standard reference material, NIST SRM 2921. To standardize the cTnI immunoassays, a secondary reference material consisting of a panel of cTnI-positive human serum pools, is proposed by IFCC Working Group for Standardization of Troponin I (IFCC WG-TNI) for assay manufacturers to reference their assay calibrations and for improving intra-assay comparability and harmonization. The establishment of a reference measurement procedure for cTnI is required for the value assignment of the cTnI concentration of the proposed secondary reference material. We developed an immunoprecipitation method coupled with fluorescent western blot analysis, which is designed to reduce the interference from cTnI heterogeneity and cTn autoantibodies and determine the cTnI concentration. We used magnetic beads coupled with 6 different anti-cTnI monoclonal antibodies that bind specifically to different amino acid sequence regions of the cTnI molecule to immunoprecipitate the cTnI proteins from a cTnI positive serum pool sample followed by sensitive detection using a fluorescent western blot. The cTnI degradation in the positive pool sample was confirmed and a total concentration of cTnI was determined. Moreover, cTnI and cTn autoantibodies were found in the same pool sample by a slightly modified method. We demonstrated the utility of this method for supporting the development of the secondary cTnI-positive serum based reference material for the standardization of clinical cTnI immunoassays.