质粒DNA抗原芯片检测抗双链DNA抗体的初步研究  

A Chip Technique for Detection of Anti-dsDNA by Using Plasmid DNA as Antigen

作　　者：徐华[1] 韩金祥[1] 高雪芹[1] 黄海燕[1] 潘继红[1] 王传玺[1] 

机构地区：[1]山东省医药生物技术研究中心,卫生部生物技术药物重点实验室,山东省现代医用药物与技术重点实验室,山东济南250062

出　　处：《生物技术通讯》2006年第6期895-897,共3页Letters in Biotechnology

基　　金：山东省科技厅重点项目(003100104)

摘　　要：目的:建立以质粒DNA作为抗原的检测血清中抗双链DNA(dsDNA)抗体的芯片方法,并与酶联免疫吸附实验比较,初步探讨用芯片法检测抗dsDNA抗体的临床价值。方法:将原核表达载体质粒pcDNAⅡ用质粒DNA快速抽提试剂盒提取纯化DNA后按1∶2稀释,用点样仪点在经3-氨丙基三乙氧基硅烷(APES)修饰的玻片上,温孵后用含有1%小牛血清白蛋白和2.5%蔗糖的PBST封闭,以Cy3标记的人IgG为二抗,建立检测dsDNA抗体的芯片方法,并与德国欧蒙公司生产的抗双链DNA检测ELISA试剂盒做比较,对包括58例系统性红斑狼疮(SLE)、25例干燥综合征(SS)、10例皮肌炎(DM)和7例类风湿关节炎(RA)在内的病人和60例健康人对照进行了抗dsDNA的对比检测。结果:对阳性标本的检测,与现用常规检测方法ELISA相比,芯片检测抗dsDNA的灵敏度为91.3%,特异度为90.7%,阳性预测值为89.3%,阴性预测值为92.5%;对健康对照的检测,2种方法均为阴性,符合率为100%。结论:与ELISA相比,用质粒DNA作为抗原建立的芯片方法的灵敏度和特异度较高,为今后建立同时检测多个自身抗体的芯片奠定了基础。Objective： To establish a chip technique to detect anti-dsDNA in the serum by using plasmid DNA as antigen, and compare it with ELISA. Methods： DNA in plasmid pcDNA Ⅱ for prokaryotic expression vector was purified by Plasmidfast extraction kit, then the plasmid DNA was spotted by the Cartisian spotting system in a dilution of 1：2 as antigen on the slides modified by APES. The slides were incubated then blocked by PBST containing 1% BSA and 2.5% saccharose, and the human IgG labeled with Cy3 was chosen as the second-antibody, to establish the chip method to detect anti-dsDNA antibody. ELISA in detecting anti-dsDNA antibody was performed simultaneously for comparion. The serm samples from 58 patients with SLE, 25-with SS, 10 with DM, 7 with RA and 60 health people were detected. Results： In comparison with ELISA in detecting anti-dsDNA, the sensitivity of the chip is 91.3%, the specificity is 90.7%, the positive predictive value is 89.3% and the negative predictive value is 92.5%; and the coincidence is 100% in detecting the health controls. Conclusion： The sensitivity and specificity of the chip using plasmid DNA as antigen are reasonable in comparison with ELISA in the detection of anti-dsDNA, and the foundation for developing the chip which can detect many kinds of autoantibodies simultaneously are established.

关 键 词：生物芯片 质粒DNA 抗双链DNA抗体 

分 类 号：Q789[生物学—分子生物学]