Generation and characterization of D-dimer specific monoclonal antibodies for use in latex agglutination test

The commercially available D-dimer assays used in the clinical practice often show differences in the results, and their specificity and sensitivity are rather unsatisfactory. Our aim was to develop a new monoclonal antibody against D-dimer with a proper specificity, and estimating its suitability using in a latex agglutination diagnostic test. Monoclonal antibodies were generated using hybridoma technology. Their titer was determined by a self-developed ELISA method. The cross-reactions of the antibodies were tested. Characterization of the epitope specificity of a selected antibody was performed through digestion of D-dimer followed by Western blotting. The amino acid sequences of the active antigen fragments were determined. According to the ELISA results, 38 cell groups were constated as antibody-producing hybridomas, among them 7 gave raised titer of antibody and were cloned. Based on the cross-reaction analysis, none of the antibodies gave cross-reaction with fibrin-E and fibrinogen-E fragments but reacted with fibrin D and fibrinogen D fragments. A low cross-reaction was showed with fibrinogen and fibrin X and Y. Contrary to the others, antibody 2B9 gave no cross-reaction with fibrinogen and reacted weakly with fibrin X and Y fragments. According to the epitope analysis the antibody 2B9 binds to amino acids 94-99 and to amino acids 140-147 on the beta chain and it recognizes the amino acids 23-32 and 93-98 on the gamma chain of D-dimer. Considering the characteristics of the above mentioned monoclonal antibody 2B9, we found that it is suitable to be a basis for a D-dimer diagnostic test with proper specificity.