质粒DNA抗原酶联免疫吸附试验检测抗双链DNA抗体的研究

An ELISA for detection of anti-dsDNA by using plasmid DNA as antigen

目的 建立以质粒DNA为抗原的检测血清抗双链DNA (ds DNA)抗体的酶联免疫吸附试验 (ELISA)方法及探讨其临床应用价值。方法 将原核表达载体质粒 pBV 2 2 0用碱裂解法提取纯化DNA后 ,按 1∶150稀释包被在多聚 L 赖氨酸处理的微孔板上 ,以辣根过氧化物酶标记的葡萄球菌A蛋白 (HRP SPA)为酶标记物 ,建立检测血清抗双链DNA抗体的ELISA方法。并以短膜虫为底物的间接免疫荧光法 (IIF)为参比方法 ,对 64例系统性红斑狼疮 (SLE)、8例混合性结缔组织病、17例类风湿性关节炎患者的血清抗ds DNA抗体进行对比检测。结果 紫外分光法于波长 2 60nm测DNA浓度为 1 54g/L。ELISA抗ds DNA法和IIF法检测 3组患者阳性率分别为 2 3 4%和 17 2 %、12 5%和 12 5%、11 8%和 5 9% ,符合率分别为 93 8%、10 0 %、94 1%。以经典IIF法为金标准 ,ELISA检测抗ds DNA抗体的特异性为 93 4% ,敏感性为 10 0 %。结论 用质粒DNA作抗原建立的检测血清抗ds DNA抗体具有良好的精密度、敏感性和特异性 ,检出率高于IIF法 。

Objective To develop an ELISA for detection of anti-dsDNA antibody by using plasmid DNA as antigen.Methods DNA in plasmid pBV220 for prokaryotic expression vector was purified by base-cleavage. The microtiterplates were pretreated by poly-L-lysine and coated by the plasmid DNA in a dilution of 1∶50 as antigen. An ELISA method for detection of serum anti-dsDNA antibodies was developed with HRP-SPA as enzyme-labeled marker.The IIF using crithidia lucilia as substrate was performed simultaneously for comparison. The serum samples from 64 patients with SLE, 8 with MCTD and 17 with RA were detected. Results The concentration of DNA was 1 54 g/L by UV spectrophotometer at wavelength of 260 nm. The positive percentage of ELISA for anti-dsDNA was higher than that of IIF. By comparison with IIF the positive percentages in SLE, MCTD and RA groups were 23 4% vs 17 2%, 12 5% vs 12 5% and 11 8% vs 5 9%, respectively, and the coincident rates between the 2 methods were 93 8%, 100% and 94 1% respectively. The sensitivity and specificity of the developed ELISA for detection of anti-dsDNA were 100% and 93 4% when IIF was as gold standard.Conclusion The ELISA by using plasmid DNA as antigen to detect anti-dsDNA has fine precision, sensitivity and specificity. Its positive rate is higher than that of IIF thus it will contribute to monitor the activities for SLE patients′ condition.