ИСТИНА

D-dimer is an acknowledged marker of blood clotting. It has been shown that D-dimer concentration is elevated (higher than 0.5 μg/ml) in plasma of patients with pulmonary thromboembolism, deep vein thrombosis and disseminated intravascular coagulation of different etiology. Despite the long history of D-dimer use in clinical practice, there are some problems concerning its quantitative determination. D-dimer as well as its precursor - fibrin degradation products (FDPs) are present in blood of patients to varying ratios. Different monoclonal antibodies (Mabs) utilized in different D-dimer assays recognize D-dimer and FDPs with unequal efficiency and that causes significant discrepancy in results obtained by such assays. In addition, differences in Mabs specificities make it impossible to use D-dimer as a standard or a calibrator in current generation of D-dimer assays. The aim of this work was to obtain monoclonal antibodies with the same specificity to D-dimer and FDPs and to design an assay prototype that equally recognizes D-dimer and FDPs in patient plasma. Hybridomas producing D-dimer-specific MAbs were generated using standard techniques. Mabs that did not show cross-reactivity with fibrinogen were tested with D-dimer and FDPs in different two-site combinations. To obtain preparations of D-dimer and FDPs with equal concentrations, a fibrin clot was initially partially and then completely digested by plasmin. The partially digested fibrin was used as a source of FDPs, whereas the completely digested product was utilized as a source of D-dimer. Thus, the FDPs and D-dimer preparations contained the same amount of cross-linked fibrin-derived materials. Two-site antibody combination with capture antibody DD189 and detection antibody DD255 (labeled with stable Eu3+-chelate) gave an equal response with FDPs and D-dimer in the range of the antigen concentrations from 20 to 1000 ng/ml and was selected for the further analysis. DD189-DD255 assay was used to analyze plasma protein profiles obtained by gel filtration on Superdex 200 column. It was shown that plasma samples from different patient groups contained different ratios of D-dimer and FDPs. D-Dimer levels in blood of three patients who had gone through a surgical operation were comparable with the level of FDPs or exceeded it. In contrast, FDPs were the main products of fibrin degradation in samples from two thrombotic patients. In two septic patients' blood different ratios of D-dimer and FDP were observed. Our results show that the ratio of D-dimer and FDPs varies between patients with different diseases. This fact means that a precise determination of fibrin-derived cross-linked products in patients’ blood requires the use of antibodies with equal specificity to D-dimer and FDPs. Use of antibodies with the same reactivity to D-Dimer and FDPs would also allow the use of D-Dimer as a calibrator. This would have a significant impact on standardization of different D-Dimer assays and would benefit number of practicing clinicians who currently need to deal with the variability of different D-Dimer assays.