抗-dsDNA抗体吖啶酯化学发光定量检测方法的性能验证

Establishment of acridinium-labeled chemiluminescence quantitative detection method for anti-dsDNA antibody

目的建立抗-dsDNA抗体吖啶酯化学发光定量检测方法并评价其性能。方法以链酶亲和素通用型磁珠为固相,通过生物素-链酶亲和素系统捕获免疫复合物,采用间接法原理建立抗-dsDNA抗体吖啶酯化学发光定量检测方法。对该方法的线性范围、灵敏度、精密度、抗干扰能力进行评价,确定该方法的正常参考值,并进行系统性红斑狼疮(systemic lupus erythematosus,SLE)临床样本检测。结果该方法抗体浓度为50~3000IU/ml时线性良好,最低检测限为1.878~2.535IU/ml(<10IU/ml),3次实验回收率均为95%~100%,准确性好;试剂检测精密度高,批内变异<5%且总变异<10%;干扰实验结果表明样本中含有1200mg/dl、150mg/dl、30mg/dl、30ng/ml、150IU/ml的三酰甘油、血红蛋白、胆红素、生物素、类风湿因子时不影响检测结果。通过检测185例正常人血清,确定正常参考值为100IU/ml,特异性达99%;检测54例SLE患者样本,其阳性率为42.6%,与ELISA参比试剂阳性率(46.3%)相似。结论此吖啶酯化学发光方法检测抗-dsDNA抗体的灵敏度高、精密性好,性能稳定,线性范围可达3000IU/ml,其检测SLE疾病的敏感性与ELISA相当,特异性为99%。检测过程仪器自动化操作,简便、快速、无污染,适用于临床。

Objective To develop an acridinium-labeled chemiluminescence quantitative detection method for anti-dsDNA antibody and evaluate its performance.Methods The anti-dsDNA antibody assay was an indirect immunoassay.dsDNA was labeled with biotin and murine anti-human IgG monoclonal antibody was labeled with acridinium.Magnetic beads coated with streptavidin were used as the solid phase to capture the antigen-antibody-secondary antibody complex via biotin-streptavidin interaction.The linearity range,sensitivity,precision and interference rejection were evaluated,the reference value was established,and clinical samples of SLE patients were detected at the same time.Results The linearity was good within the scope of 50 - 3 000 IU/ml and the lowest detection limit was 1.878 - 2.535 IU/ml (< 10 IU/ml).The percentage of recovery ranged from 95% to 99%,which indicating a good accuracy.In addition,the precision of assay was really high,the coefficient of variation of intra-assay was less than 5% and total variation was less than 10%.Interference experiment results showed that there was no influence in detection when triacylglycerol, hemoglobin,bilirubin,biotin and rheumatoid factor existed in samples with the concentration of 1 200 mg/dl,150 mg/dl,30 mg/dl, 30 ng/ml and 150 IU/ml,respectively.The normal reference value (100 IU/ml) was established,with specificity of 99%.Totally 54 clinical SLE patient specimens were detected,and the sensitivity of this method was 42.6%,similar to 46.3% of ELISA.Conclusion The acridinium-labeled chemiluminescence quantitative detection method for anti-dsDNA antibody has a high sensitivity,good precision and stable performance.The linearity range can reach 3 000 IU/ml.It has a similar sensitivity to ELISA method for SLE specimen detection (99% specificity).The assay is simple and automatic,which is suitable for clinical application.