ИСТИНА

Cardiac isoform of human troponin I (hcTnI) is a reliable biomarker of the cardiomyocyte damage which accompanies such severe cardiovascular diseases as myocardial infarction. Monoclonal antibodies TnIMAB are able to recognize hcTnI with high affinity. Usage of recombinant antibodies and their fragments allows improving of existing immunodiagnostic systems and, therefore, is of a high value in fundamental and applied fields of modern biomedicine. Because of the technical difficulties of the whole molecule recombinant antibodies production their fragments are generally used for the implementation of different fundamental and applied tasks. Fab and scFv fragments are the most widespread among them. Fab fragments are characterized by higher affinity and stability. scFv fragments have an advantage of being of small size and having a single chain. In this study, we produced TnIMAB scFv and Fab fragments, compared their production yield, biochemical and immunochemical properties to each other and with the whole molecule antibodies. Light chain and heavy chain gene regions corresponding to TnIMAB scFv and Fab fragments were subcloned pET23a+ plasmids. Expressed fragments were found in the insoluble fraction and were subjected to renaturation followed by affinity chromatography (2-5 mg per 1L of cell suspension). TnIMAB affinity (KD) measured by surface plasmon resonance approach approximates 3 nM and was not significantly different from recombinant fragments and enzymatic Fab fragments. According to our studies recombinant Fab and scFv fragments specific to hcTnI possess immunochemical activity reaching in some applications the level of whole molecule antibodies and demonstrate all necessary immunochemical properties for being used in diagnostic systems.