免疫法检测D-二聚体的研究
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摘要
D-二聚体是交朕纤维蛋白特异的降解产物,它的生成或增高反映了血浆中凝血系统和纤溶系统的激活,是唯一反映凝血和纤溶的理想指标,在临床上已视为体内高凝状态和纤溶亢进的分子标志物。D-二聚体的定量检测可定量反映药物的溶栓效果,及可用于诊断、筛选新形成的血栓。血浆D-二聚体抗原的测定方法主要有胶乳凝集法、酶联免疫吸附(ELISA)法、光电散射比浊法和胶体金免疫渗透法(GIA ),但是到目前为止,商品的D-二聚体检测手段都尚存在一定局限性。胶体金标记技术和免疫荧光技术是近些年来发展迅速的检测技术,具有简便、快速、灵敏度高等优点。因此,本研究拟开发用胶体金和荧光染料为标记物的D-二聚体检测试剂盒,并考察试剂盒对实际样品的检测性能。
首先,选用样品纸、吸收纸、硝酸纤维素膜(NC膜)及被衬垫组装成试纸条。NC膜上分别固定D-Dimer抗体A1和兔抗鼠IgG抗体,作为检测线和控制线,浓度均为2.2 mg·mL-1。若样品中含有待测抗原则与标记物包被的抗体结合,形成抗原-标记抗体复合物,当复合物移动到检测线时,检测线上固定的D-Dimer抗体与样品中的抗原-标记抗体复合物结合,形成抗体-抗原-标记抗体交联物。其余的标记抗体继续移动到质控线与质控抗体结合。由于检测线上的小分子蛋白交联物与质控线上的抗体在硝酸纤维素膜都是固定的,结合在两处的胶体金或荧光素就会显现一定的颜色或荧光。
其次,采用柠檬酸三钠法制备胶体金,根据颜色、波长扫描曲线,选择粒径为18 nm的胶体金颗粒标记抗体,即100 mL氯金酸溶液(0.01%)中需加入还原剂柠檬酸三钠(1%)的量为6 mL。通过实验,胶体金标记D-Dimer抗体的pH为7.4,每毫升最佳包被量为25.6μg。本实验测定了胶体金法与生化分析仪的比对试验,回归方程为y=-0.119+1.045c,相关系数r=0.993。用胶体金法测定了灵敏度、重复性、稳定性、生物性物质干扰等性能。结果显示,该胶体金法制备的试剂盒灵敏度高(0.473μg·mL-1),并有良好的重复性(STDEV=0.093～0.115)和抗干扰性,4℃条件下可以保存很长时间。
最后,用荧光染料Cy5对DD2抗体进行包被,包被完的蛋白在PBS缓冲液(pH 7.2,10 mM)中透析24小时,以除去多余的荧光染料。荧光染料-抗体蛋白稀释至500倍时有良好的检测效果,此时可以降低背景信号,还可避免荧光猝灭的发生。本实验测定了荧光素法与生化分析仪的比对试验,回归方程为y=0.00984+0.1962c,相关系数r=0.975。用荧光素法测定了灵敏度、重复性、稳定性、生物性物质干扰等性能。结果显示,该荧光素法制备的试剂盒灵敏度高(0.278μg·mL-1),并有良好的重复性(STDEV=0.029～0.093)和抗干扰性,4℃条件下可以保存很长时间。
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℃.
首先,选用样品纸、吸收纸、硝酸纤维素膜(NC膜)及被衬垫组装成试纸条。NC膜上分别固定D-Dimer抗体A1和兔抗鼠IgG抗体,作为检测线和控制线,浓度均为2.2 mg·mL-1。若样品中含有待测抗原则与标记物包被的抗体结合,形成抗原-标记抗体复合物,当复合物移动到检测线时,检测线上固定的D-Dimer抗体与样品中的抗原-标记抗体复合物结合,形成抗体-抗原-标记抗体交联物。其余的标记抗体继续移动到质控线与质控抗体结合。由于检测线上的小分子蛋白交联物与质控线上的抗体在硝酸纤维素膜都是固定的,结合在两处的胶体金或荧光素就会显现一定的颜色或荧光。
其次,采用柠檬酸三钠法制备胶体金,根据颜色、波长扫描曲线,选择粒径为18 nm的胶体金颗粒标记抗体,即100 mL氯金酸溶液(0.01%)中需加入还原剂柠檬酸三钠(1%)的量为6 mL。通过实验,胶体金标记D-Dimer抗体的pH为7.4,每毫升最佳包被量为25.6μg。本实验测定了胶体金法与生化分析仪的比对试验,回归方程为y=-0.119+1.045c,相关系数r=0.993。用胶体金法测定了灵敏度、重复性、稳定性、生物性物质干扰等性能。结果显示,该胶体金法制备的试剂盒灵敏度高(0.473μg·mL-1),并有良好的重复性(STDEV=0.093～0.115)和抗干扰性,4℃条件下可以保存很长时间。
最后,用荧光染料Cy5对DD2抗体进行包被,包被完的蛋白在PBS缓冲液(pH 7.2,10 mM)中透析24小时,以除去多余的荧光染料。荧光染料-抗体蛋白稀释至500倍时有良好的检测效果,此时可以降低背景信号,还可避免荧光猝灭的发生。本实验测定了荧光素法与生化分析仪的比对试验,回归方程为y=0.00984+0.1962c,相关系数r=0.975。用荧光素法测定了灵敏度、重复性、稳定性、生物性物质干扰等性能。结果显示,该荧光素法制备的试剂盒灵敏度高(0.278μg·mL-1),并有良好的重复性(STDEV=0.029～0.093)和抗干扰性,4℃条件下可以保存很长时间。
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℃.
引文
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[2]郭雪梅.快速D-二聚体检测在诊断静脉血栓形成中的应用进展[J].中国实验诊断学,2000,4(1):41-43
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[4]黄蘩嫱,刘丽,杨莉等.一种新的纤溶活性测定法及在心肌梗死中的应用[J].上海医学检验杂志,1999,14(2):83-85
[5]邢晓光,杨晨.胶体金免疫渗透试验定量检测D-dimer评价[J].天津医科大学学报,2004,10(3):436-438
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