Preparation Of Anti-D-Dimer Monoclonal Antibody And Application In Fluorescence Latex Immunoassay

D-dimer is a unique marker of fibrin degradations. D-dimer level indicates whether thereis fibrinogen formation and degradation or not. So the D-dimer test is useful to determin whatextent fibrin formation has been initiated, or to learn whether there is any change in the courseof a specific therapy and disease process. In practice, D-dimer measurement has been mostcomprehensively validated in the exclusion of venous thromboembolism（VTE）in certainpatient populations; and the monitoring of coagulation activation in disseminatedintravascular coagulation(DIC). All the D-dimer tests use antibody and antigen reactionprinciple. Recently,Most of the anti-D-dimer antibodies produced by hybridoma technique orgene engineering,and they are used in all kinds of D-dimer assays includes ELISA（Microplate,VIDAS,Membrane) and agglutination（Dimertest latex,SimpliRED,TinaQuant/Liatest）.In this experment,D-dimer which was collected from fibrin degration was used asimmunogen,and immunized the BALB/c mouse. By the improved hybridoma techniquewhich used methylcellulose semisolid media substitite for traditional fluid medium,10hybridoma cells secreting monoclonal antibody were established and named1-3C,2-1B,2-1D,2-4B,2-6D,4-1D,4-3C,4-3D,4-4C,5-3B respctively. By indirect ELISA,the antibodytiters of them reached10~6. The antibody type test showed that2-1D were belonging to IgG1;1-3C,2-1B,2-4B,4-3C,4-3D were belonging to IgG2a;2-6D,4-1D,4-4C,5-3B werebelonging to IgG2b; and the light chains of the antibodies were all κ chains,which wereanalyzed by using a commercial ELISA kit. Western-blot demonstrated that4-1D,5-3B havereaction with fibrinogen; C1, C2have reaction with fibrinogen degration;1-3C,2-1B,2-6D,4-3C,4-3D,4-4C have special reaction with D-dimer. The sandwich ELISAshowed that15antibody pairs gave positive results when detective D-dimer. Consequently,apair of monoclonal antibodies2-6D and C2had been screened by the methods ofELISA,colloidal gold immunoassay and immunofluorometric.Fluorescence latex immnochromatographic assay is a fast immunity method which hasthe benefits of convenience, accuracy and specificity. The experiment used D-dimermonoclonal antibodies to manufacture fluorescence latex immnochromatographic strip and itcan test D-dimer quantity via instrument. One D-dimer antibody labelled with fluorescencelatex, another D-dimer and goat-anti-mouse IgG were coated to nitrocellulose membrane at adose of2.0μg/cm and3.4μg/cm respectively as the test line and the control line. Assembled them and incised into lateral test strips. Sensitivity test showed that the fluorescence lateximmunochromatographic strip could detect100μg/L D-dimer. The test showed highspecificity and have no cross reaction with fibrinogen and its degrations.137specimens fromhospital were detected by the fluorescence latex immunochromatographic strip and the resulthad a high coincidence (positive92.1%，negative98%) compared with the ELISA test kit.The result of experiment showed that the fluorescence latex immunochromatographicstrip which developed with monoclonal antibody against D-dimer has good sensitivity andspecificity, and had high coincidence with the ELISA test kit. The D-dimer pair used well insandwich ELISA and colloidal gold immunoassay can also be applied in development offluorescence latex immunochromatographic strip which provides a rapid, time-saving andconvenient means to detect the D-dimer. Therefore, the preparation of anti-D-dimerantibodies and the producing of fluorescence latex immunochromatographic strip had greatcontribution on the detectiing, monitoring and curing of thrombus diseases.