STANDARDIZATION OF THE PROTEIN CALIBRATORS ISOLATION METHODOLOGY FOR THROMBOPHILIA MARKERS DETECTING IMMUNODIAGNOSTIC TEST SYSTEMS

Abstract

The most accurate laboratory methods for thrombophilia diagnostics are based on the quantitative determination of the blood plasma specific markers that appear as a result of the coagulation cascade activation. Soluble fibrin and D-dimer belong to the main of the last ones. An alteration in the concentration of such markers can indicate thrombin concentration growth and the formation of soluble oligomeric fibrin. It should be pointed out that simultaneous detection of these markers can establish the correlation between the accumulation of soluble fibrin and fibrinolysis and nowadays is provided only by enzyme-linked immunoassay. Thus, the usage of immunodiagnostic test systems for the detection of thrombophilia markers is highly relevant today. The important components of immunodiagnostic test system are protein calibrators, the isolation standardization of which plays a key role for accurate construction of a calibration curve and obtaining objective results as a consequence. Aim. The objective of this study was to develop the soluble fibrin and D-dimer isolation methodology and its standardization for their further use as the protein calibrators for thrombophilia markers detecting immunodiagnostic test systems. Materials and Methods. Soluble fibrin and D-dimer were isolated from collected human blood by fibrinogen salting out with further fibrin polymerization with thrombin and hydrolysis with plasmin. Quality control of the obtained proteins was carried out using SDS-PAGE and turbidimetric measurements with further checking of the proteins as calibrators for the thrombophilia markers detecting immunoassay. Results. Obtained proteins meet the necessary specifications and can be used as calibrators for immunodiagnostic test systems. Soluble fibrin and D-dimer were checked by SDS-PAGE for the absence of impurities. Turbidimetric measurements showed the polymerization capability of the soluble fibrin and the inhibition of the polymerization by D-dimer. Conclusion. The standardized isolation methodology of soluble fibrin and D-dimer can be used to obtain protein calibrators for appropriate immunodiagnostic test systems.