Study Of D-dimer By Immunity Method

D-Dimer in plasma is a specific degradation product of cross-linked fibrousprotein whose produce or increase reflects clotting system and fibrinolysis system's activation. D-Dimer is the only ideal indicator reflecting clotting and fibrinolysis, and it is regarded as molecule marker of hypercoagulation state and increased fibrinolytic activity in vivo. Quantitative detection can show medicine's thrombolysis ability,also can be used to diagnose new thrombus. Generally ,the D-Dimer antigen is detected by latex particle agglutination assay, enzyme-linked immunosorbent assay, immune turbidimetry and collid gold immunofiltration assay. However, these assays all have its own limitations. Colloidal gold labelling technique and immunofluorescent technique develop very quickly in recent years with advantages of simple, rapid and high sensitivity. Accordingly,this study was intended to design D-Dimer diagnostic kit using colloidal gold and fluorescent dye as makers, also to review the kit's performance in actual work.Firstly, sample pad, absorbing pad, nitrocellulose membrane were assembled to immunochromatographic test strip. Nitrocellulose membrane contains one line of immobilized monoclonal anti-dimer antibody A1, serving as the test line,and one line of immobilized rabbit anti-mouse IgG antibody,serving as the control line.Both immobilized antibodies are 2.2 mg·mL-1 concentration. If the sample contains D-Dimer antigen, the antigen will bind the gold-antibody conjugate(or fluorescent dye-antibody conjugate) to form a complex which then migrates down to the membrane and binds to the immobilized anti-ddimer antibody A1 of the test line. Hereafter, the unbound conjugate migrates further on the membrane and binds to the immobilized anti-IgG antibody of the control line. The control line can bind all the conjugate it is in contact with, regardless the sample contains ddimer or not. Because antibody A1 and anti-IgG antibody are immobilized on the membrane, the test line and control line will show a reddish color or fluorescence.In addition, colloid gold with different diameter were prepared by trisodim citrate reducion. According to the exosyndrome of color,scanning curve and scanning electron microscope, the colloid with 18nm in diameter was used to label antibody. The optimun pH for labeling was 7.4, and the amount of antibody was 25.6μg·mL-1. Paired comparison test between colloid gold method and chemistry analyzer was done, the linear regression equation was y=-0.119+1.045c, and correlation coefficient was 0.993. Sensitivity,repeatability, stability and specificity were tested. It shows that the kit by colloid gold method has a high sensitivity of 0.473μg·mL-1, good repeatability(STDEV=0.093～0.115) and specificity, can be stored for a long time at 4℃.The last, use fluorescent dye to label antibody, the labeled antibody was purified by dialyzing in PBS buffer for 24 hours. The kit has good detection result when fluorescent dye-antibody complex was diluted 500timers.Paired comparison test between fluorescent dye method and chemistry analyzer also was done, the linear regression equation was y=0.00984+0.19627c, and correlation coefficient was 0.975. Sensitivity,repeatability, stability and specificity were tested. It shows that the kit by fluorescent dye method has a high sensitivity of 0.278μg·mL-1, good repeatability(STDEV=0.029～0.093) and specificity, can be stored for a long time at 4℃.