Troponin I TechNotes: Concept of precise immunoassay

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Troponin I TechNotes.

Cardiac isoform of Troponin I (cTnI) is a golden marker of cardiac muscle cell damage and death today. Different diagnostic platforms were designed for quantitative measurements of cTnI in human blood and are used extensively in big hospitals and small diagnostic laboratories. But still there is no between assay agreement and it often happens that one and the same blood sample gives different values when being analyzed by different cTnI assays.

The most common reason for the discrepancy in the assay measurements is difference in the epitope specificity of the antibodies used in different assays. Now we know that multiple factors are influencing cTnI measurements. Among them are posttranslational modifications (proteolytic degradation (1), phosphorylation (2)), complexing with other molecules (troponin C (3), heparin (2)) and cTnI-specific autoantibodies circulating in patients’ blood (4). Different mono- and polyclonal antibodies, utilized in assays, are sensitive to these factors in different degree.

HyTest specialists have been involved in cTnI studies almost for 15 years. We generated and tested several thousands of monoclonal antibodies specific to different regions of cTnI molecule; we tried dozens of hundreds of different two-site MAb combinations in order to find the best one for precise cTnI immunoassay. Summarizing results of our studies we can conclude that on this moment it is impossible to have one antibody pair (one capture and one detection antibody), which would be absolutely insensitive to all known cTnI modifications and interferences. According to our opinion MAb combinations, which could be used for the development of precise cTnI immunoassay, should utilize two monoclonal antibodies as capture (plate or particle coating) and two MAbs for detection (conjugated with the specific label). We call such an approach as “2+2 concept”. In these assays monoclonal antibodies should be selected in such a way, that if one of the MAbs (capture or detection) is sensitive to the presence of some factor in the sample, then the other MAb should be insensitive to the same factor. Thus the effect of negative or positive interference is minimized. Also one important parameter should be considered: antibodies utilized in the assay should be specific to the cardiac isoform of the protein and should not have crossreaction with the two skeletal isoforms.

Today HyTest can suggest several combinations of monoclonal antibodies useful for the development of cTnI assays according to 2+2 concept. Such assays would be cardiac specific and almost insensitive to all known factors that could affect cTnI measurements. Moreover, while selecting antibodies we also considered the fact that new generation of cTnI assays should display high sensitivity and antibody combinations could be used in point-of-care diagnostics platforms. So, assays described in Table 1 have good kinetics and they recognize standard preparation of antigen (cTnI in troponin complex) with sensitivity better than 50 pg/ml.

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