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目的优化制备蛋白质微阵列的几个关键因素,初步建立自身免疫性疾病抗体检测微阵列,以期用于自身免疫性疾病的诊断。方法选择与制备蛋白质微阵列有关的4个因素,即温育时间、封闭剂、封闭时间和与二抗结合时间,进行正交优化,得出制备蛋白质微阵列的最优化条件,并根据该条件,进一步确定6种自身抗原适合的点样浓度及血清的最佳稀释度。根据优化条件,制备出同时检测6种自身抗体的微阵列,并初步应用,与间接免疫荧光(IIF)法和酶联免疫吸附试验(ELISA)法加以比较,对结果进行评价。结果制备蛋白质微阵列的最佳优化条件是温育2h,封闭剂采用含有1%小牛血清白蛋白和2．5%蔗糖的PBST,封闭时间为30min,与二抗反应30min,6种自身抗原适合点样浓度从100μg/ml到300μg/ml不等,最佳血清稀释浓度是1∶2。微阵列法对抗核抗体(ANA)的检测与IIF法相比,灵敏度是88．6%,特异度是83．3%,微阵列法对其他5种自身抗体的检测与ELISA法相比,灵敏度在80．0%～88．2%之间,特异度在90．2%～98．6%之间。结论上述优化条件适合于制备自身免疫性疾病抗体检测微阵列,微阵列检测结果与现有常规方法检测结果比较一致,值得推广应用。
Objective To optimize several key factors in the preparation of protein microarrays and to establish an antibody detection microarray for autoimmune diseases in order to diagnose autoimmune diseases. Methods Four factors related to the preparation of protein microarray were selected as incubation time, blocking agent, blocking time and the time of binding with secondary antibody. The optimal conditions for preparing protein microarray were obtained. According to this condition , To further determine the appropriate spotting concentration of 6 kinds of self-antigens and the optimal dilution of serum. According to the optimized conditions, six kinds of autoantibodies were detected simultaneously. The microarray was initially applied and compared with indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA) to evaluate the results. Results The optimal conditions for the preparation of protein microarray were incubation for 2h, blocking agent containing 1% bovine serum albumin and 2.5% sucrose in PBST, blocking time 30min, reaction with secondary antibody 30min, 6 autoantigen Appropriate spotting concentration ranging from 100μg / ml to 300μg / ml, the best serum dilution is 1: 2. Compared with the method of IIF, the sensitivity of microarray detection of anti-nuclear antibody (ANA) was 88.6% and the specificity was 83.3%. Compared with the ELISA method, the sensitivity of microarray detection method of other five kinds of autoantibodies was 80 .0% ~ 88.2%, the specificity was between 90.2% ~ 98.6%. Conclusion The above optimization conditions are suitable for the preparation of antibody detection microarray for autoimmune diseases. The results of microarray detection are consistent with those of conventional methods and are worth popularizing.