生物素-亲合素系统-时间分辨荧光免疫分析法检测抗核抗体方法学的建立和临床应用

Establishment and application of biotin-avidin-system-time-resolved fluoroimmunoassay for the quanti- tive detection of antinuclear antibody-Ig

目的建立高灵敏度、宽量程的定量检测患者血清中抗核抗体的生物素．亲合素系统．时间分辨荧光免疫分析（BAS，TRFIA）方法。方法以hela细胞核和抗人免疫球蛋白（GAM）分别作为固相抗原和生物素化抗体，Eu3＋标记亲合素；标准品或样品中抗核抗体与固相核抗原、生物素GAM、Eu3＋标记亲合素形成固相核抗原．抗核抗体．生物素化抗体-Eu3＋标记亲合素的多层夹心免疫反应复合物；加入荧光增强液解离Eu3＋，形成强荧光螯合物，荧光强度与样品中待测抗核抗体的含量呈正比，建立BAS-TRFIA方法检测抗核抗体；对建立的BAS-TRFIA法检测抗抗核抗体的精密度、检测范围、稳定性等进行分析。收集50名健康献血者、105例系统性红斑狼疮、109例类风湿关节炎、25例原发性胆汁性肝硬化、29例干燥综合征、23例硬皮病、25例混合性结缔组织病患者血清标本，采用建立的BAS-TRFIA法检测这些患者血清中的抗核抗体，计算阳性率。结果BAS—TRFIA法检测抗核抗体高、中、低3种浓度混合血清的批内（n=20）精密度分别为3．13％、3．74％和5．76％，批间（n=8）精密度分别为5．31％、6．25％和7．46％，均优于酶联免疫吸附试验（ELISA）法；方法的灵敏度为2．24U／ml；平均回收率为100．74％；该方法测定值与进口ELISA法所得结果高度相关（Rz=0．989）；BAS．TRFIA法检测健康献血者、系统性红斑狼疮、类风湿关节炎、原发性胆汁性肝硬化、干燥综合征、硬皮病、混合性结缔组织病患者血清标本抗核抗体，阳性率分别为0、100％、23％、96％、38％、91％、92％；一强阳性标本，BAS．TRFIA法测抗核抗体的曲线良好线性范围在稀释浓度1：12．5～1：409600的范围内；ELISA法测抗核抗体的曲线良好线性范围在稀释浓度1：100～1：51200的范围内，BAS-TRFIA法与ELISA法相比，抗核抗体可测量范围明显提高；试剂盒置于37℃水浴箱7d后，BAS—TRFIA法结合率仅下降15．9％，而ELISA法结合率下降58．4％，BAS—TRFIA法试剂盒稳定性较好。结论首次建立稳定的高灵敏度和宽检测范围的BAS-TRFIA法检测人血清中的抗核抗体，对早期诊断自身免疫病及监测疗效具有重要意义，有望在各检验科室得以普遍应用。

Objective To establish an analytical method with high sensitivity and wide range in biotin-avidin and time-resolved fluoroimmunoassay system for the quantitative detection of antinuclear antibody （ANA）-Ig （GAM）. Methods ANA in standard preparation or sample was combined with solid nuclear antigen, biotinylation antibody and the europium（Eu3＋）-labelled avidin to form the compounds of solid nuclear antigen-antibody-biotinylation antibody-SA-Eu3＋. The fluorescence enhancement fluid was added to dis-sociate the Eu3＋, the content of ANA was directly proportional to fluorescence intensity, the BAS-TRFLA was esta- blished for the quantitative detection of ANA-lg （GAM）. The sensitivity, specificity, reliability and range of detection were evaluated. Serum of 50 blood donor, 105 patients with systemic lupus erythematosus （SLE）, 109 patients with rheumatoid arthritis （RA）, 25 patients with PBC, 29 patients with Sjogren＇s syndrome（SS）, 23 patients with Sjogren＇s cleroderme 25 patients with MCTD were included in this study. The positive rate was calculated. Results The in ner-grotIwdifference between high, medium and low densities mixture serum was 3.13%, 3.74% and 5j.76%and the inter-group precision rate was 5.31%, 6.25% and 7.46% in BAS-TRFLA. The sensitivity of TRFIA was better than that of the ELISA method. The low detection limit was 2.24 U/ml. The mean recovery rate w as 100.74%. The results measured by the TRFIA and ELISA methods were closely correlated （R2=0.978）. The＇positive rate of blood donor, and patient with SLE, RA, PBC, SS, scleroderma and MCTD were 0, 100%, 23%, 96%, 38%; 91%, 92% respectively. When compared with ELISA, the detection range of TRFIA was wider,and stability was better. Conclusion BAS-TRFIA is a stable method for detection of ANA with high sensitive and wide range of detection. It is important for the early diagnosis of autoimmune disease and monitoring the treatment efficacy of autoimmune disease. And this method may be widely used in clinical laboratories in the future.