基于斑点免疫金渗滤法的蛋白微矩阵

Protein microarray based on dot immunogold filtration assay

目的 :开发一种低成本、简单易用的基于免疫金渗滤法的蛋白微矩阵用于平行检测分析血清中多种靶分子 .方法 :用点样仪将人IgG和多种抗原点到硝酸素纤维膜上 ,经小牛血清白蛋白封闭后垫以高分子吸水材料 ,随后滴加血清样本 ,免疫金显色 ,CCD阅读仪分析相对灰度值 ,并与ELISA法的检测结果进行对比 .结果 :微矩阵的IgG检测范围在 1～5 0ng之间 ;按照 4 0 0份阴性血清相对灰度值的均数加上 3倍标准差制定临界值 .在 186份随机血清试验中 ,与ELISA试剂盒比较 ,检测结果无显著性差异 ,总体灵敏度和特异度均大于90 % .结论 :蛋白微矩阵与ELISA方法相比 ,检测效果相当 ,且具有操作步骤简单 ,易于使用 ,检测成本低 ,耗时少的优点 ,适合于在临床检验上的应用 .

AIM: To develop a cheap and easy-to-use protein microarray based on dot immunogold filtration assay for parallel analysis of several targets in serum. METHODS: The human IgG and several antigens were printed on activated nitrocellulose membrane with robotics. After blocked with PBS, the membrane printed was underlaid by macromolecular bibulous material. The patients’ sera samples and immunogold was instilled in the membrane respectively, the relative gray level of human IgG bound to the printed antigens were analysed by CCD reader. The results were compared with ELISA kits. RESULTS: The range of detection for IgG was from 1-50 ng in our microarray. The detection limits was set by the mean of 200 negative serumal samples plus triplicate standard deviation.We assayed 186 random sera, compared with ELISA kit, there was no significance between the two methods, and >90% sensitivity and specificity was obtained. CONCLUSION: Compared with ELISA kits, the microassay performs well equivalently. Furthermore, the microassay is easy-to-use and have important advantage in cost and time. It is suitable for clinical diagnosis.