时间分辨荧光免疫分析法测定抗环瓜氨酸肽抗体方法学的建立

Detection of anti-cyclic citrullinated peptide antibodies by time-resolved fluoroimmunoassay

目的建立高灵敏度、宽量程的定量检测患者血清中的抗环瓜氨酸肽（CCP）抗体的时间分辨荧光免疫分析方法。方法以CCP重组抗原和鼠抗人IgG抗体分别作为固相抗原和Eun标记抗体，如果样本中存在抗CCP抗体，则形成CCP重组抗原-抗CCP抗体-Eu^3＋标记鼠抗人IgG抗体复合物，加人解离增强液解离铕离子，检测荧光强度，样本中抗CCP抗体含量与荧光强度成正比；对建立的时间分辨荧光免疫（TRFIA）法检测抗CCP抗体的线性范同、精密变、检测范同进行分析；对32例阳性血清标本分别利用TRFIA及酶联免疫吸附试验（ELISA）法检测抗CCP抗体，分析其相关性；收集50名健康献血者，32例系统性红斑狼疮，26例干燥综合征，10例硬皮病，20洲混合性结缔组织病，18例多发性硬化症，利用TRFIA检测其血清中的抗CCP抗体，计算临床特异度．TRFIA与ELISA2种方法检测结果的一致性检验采用Mc、Nenw检验。结果TRFIA法检测高、中、低3种浓度混合血清的批内（20份）精密度分别为2％、3％和4％，批问（8份）精密度分别为3％、4％和7％；平均回收率为101％；方法的灵敏度为1．0U／ml；TRFIA法检测健康献血者、系统性红斑狼疮、干燥综合征、硬皮病、混合性结缔组织病及多发性硬化症患者血清中抗CCP抗体,临床特异度分别为98％、97％、96％、100％、95qc、100％；2种方法检测的相关系数为0．989；TRFIA比ELISA的榆洲范同更宽．稳定性能好。结论首次建立稳定的高灵敏度和宽检测范同的TRFIA法检测人血清中的抗CCP抗体，对早期诊断RA硬临测疗效具有蘑要意义，该方法以其优势有望在各检验科室得以普遍应用．

Objective To develop a method for detecting anti-cyclic citrullinated peptide （anti-CCP） in the serum of rheumatoid arthritis patients. Methods The range of lineage correlation, precision and detection range of time-resolved fluoroimmunoassay （TRFIA） was analyzed. Thirty-two positive serum of antiCCP, and the sera from 50 heahh blood donors, 32 SLE patients, 26 patients with SS, 10 patients with seleroderma, 20 patients with MCTD and 18 patients with MS were tested in this study. The clinical specificity was assessed. The consistency between TRFIA and ELISA was analyzed by Mc Nemar test. Results Analyzed by SPSS 13.0 statistical software, the intra-batch precision （n=20） rate was 2%, 3% and 4% respectively, and the inter-batch precision （n=8） was 3%, 4% and 7% respectively for 3 different concentrations. The elinieal specificily was 98%, 97cA, 96%, 100%, 95% and 100% in the sera of healthy blood donors, SLE, SS, scleroderma, MCTD and MS patients respectively. The correlation coefficient was 0.989.The average recover＇ rate was 101%, and the sensitivity.was 1 U/ml. When compared with ELISA, the detection range of TRFIA was wider and also with betterstability. Conclusion TRFIA is a stable method with high sensitivity and wide detection range. It can be used to detect anti-CCP antibody. It is important for early diagnosis of RA and monitor the curative effect of RA. And this method has potential to be used in clinical diagnosis.