自身免疫性疾病抗体检测微阵列制备条件的优化和初步应用  

Preparation conditions optimization and preliminary application of the microarray for detecting autoantibodies

作　　者：徐华[1] 韩金祥[1] 高雪芹[1] 黄海燕[1] 李钿[2] 潘继红[1] 刘灿兰[3] 

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,济南250062 [2]山东省立医院风湿免疫实验室 [3]山东省泰安市中心医院感染管理科

出　　处：《中华风湿病学杂志》2006年第6期349-352,i0003,共5页Chinese Journal of Rheumatology

基　　金：山东省科技厅重点基金(003100104)

摘　　要：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 preparing protein microarray, and establish microarrays to detect autoantibodies. Methods Four factors, including incubation time of the microarray after spotting, the blocking reagent, the length of blocking time, the length of time conjugating with the second-antibody, were selected to carry out orthogonal optimization, then we determined the optimal spotting concentration of antigen and the best dilution of serum with the optimized conditions obtained from the above. On the basis of the optimized conditions, we prepared microarray to detect six autoantibodies simultaneously, which were compared with IIF and ELISA. Results The best conditions we determined from the experiment were： incubating the spot microarray for 120 min, PBST containing 1% BSA and 2.5% saccharose as the blocking reagent and blocking for 30 min, reacting with the second-antibody for 30 min, the appropriate spotting concentrations of six antigens differed from 100 μg/ml to 300 μg/ml and the best serum dilution was 1：2. Compared with IIF for ANA detection, the sensitivity of the microarray was 88.6%, the specificity was 83.3%, and compared with ELISA in detecting the other five autoantibodies, the sensitivity of the microarray was between 80.0% and 88.2%, and the specificity was between 90.2% and 98.6%. Conclusion The optimization conditions we obtained are suitable for preparing protein microarray, and the detection sensitivity and specificity of autoantibodies by microarray are consistent with the conventional methods in general. The microarray for detecting autoantibodies is applicable in the clinical diagnosis of autoimmune disease.

关 键 词：蛋白质阵列分析 正交试验 免疫系统疾病 自身抗体 

分 类 号：R593.2[医药卫生—内科学] R446.62[医药卫生—临床医学]