Katrukha Ag

Biochemical Factors Influencing Measurement of Cardiac Troponin I in Serum

Abstract Troponin I (cTnI), a sensitive and reliable marker of damaged cardiac tissue, is now widely used in clinics. But the existence of different cTnI assays with a wide variety of cut-off values and discrepancies between the results of measurements of one and the same sample by different assays is puzzling for clinicians. The most urgent issue at the moment is the development of the international standard, which can be used for the calibration of different assays, thus decreasing between assay biases. But another important item, which should be considered by manufacturers, is the standardisation of the epitopes of the antibodies used for the assay development. The importance of such standardisation originates from the complicated biochemical nature of cTnI. Here we briefly try to analyse the main factors that can influence antigen recognition by different antibodies and formulate principles of antibody selection for assay development.

Epitope mapping of anti‐troponin I monoclonal antibodies

Two groups of monoclonal antibodies (MAbs) specific to human cardiac troponin I (cTnI) were generated by immunization of mice by isolated cTnI (group I, 16 MAbs) or by the whole troponin complex (group II, 15 MAbs). Two sets of overlapping decapeptides covering the complete sequence of cTnI were prepared and used for epitope mapping by SPOT technique. Majority of MAbs (28 out of 31) interacts with synthetic peptides thus indicating that they recognize liner epitopes. MAbs raised against isolated cTnI preferentially recognize epitopes located at the N‐ or C‐terminal ends of cTnI. Nine out of fifteen MAbs raised against whole troponin complex interact with epitopes located in the N‐terminal part of cTnI. Generation of MAbs recognizing both isolated cTnI and cTnI inside of troponin complex and mapping their epitopes provides reliable detection of TnI in serum of patients with acute myocardial infarction.

New approach to standardisation of human cardiac Troponin I (cTnI)

Six different cTnI assays (5 commercial and one assay under development) were run with either their own calibrators or with standard dilutions of different forms of cTnI (free or complexed). Twenty-one serum samples from 7 AMI patients were analysed using these combinations of calibrators and assay constructs. The range of cTnI concentrations as measured in AMI serum samples by different assays was more than 10-fold using each assays own standards or when using free native or recombinant cTnI. With recombinant or in vitro formed ternary cTnI-cTnT-TnC complexes the differences were smaller (3-fold). The lowest between-manufacturer bias (1.4-fold) was obtained when the heart tissue derived native troponin complex was used for the calibration of the assays. For all assays, calibrated against this troponin I complex, the recalculated cut-off values were within 0.1-0.25 microgram/L. We conclude that to reduce assay-to-assay variation, native troponin complex is presently the preferred alternative for the standardisation of cTnI assays.

Isolation of human cardiac troponin T and localization of epitopes recognized by monoclonal antibodies to cardiac troponin T

Human cardiac troponin T has been isolated and its properties compared with those of rabbit skeletal and bovine cardiac troponin T. Seven monoclonal antibodies to troponin T have been obtained. Two antibodies cross‐reacted with both cardiac and skeletal troponin T and recognized epitopes located between residues 98–177 of bovine cardiac troponin T. Five other antibodies were specific for cardiac troponin T and recognized antigenic determinants located between residues 180–258 of bovine cardiac troponin T. Localization of antigenic determinants in the central part of troponin T seems to be due to the high hydrophilicity and flexibility of this part of the molecule. The monoclonal antibodies thus obtained may be used for diagnosing various types of human heart diseases.