Wang Guoqiang, Gao Xueqin, Han Jinxiang, Pan Jihong, Huang Hainan
Key Laboratory for Biotech-drugs of the Ministry of Health, Shandong Medicinal and Biotechnology Center, Jinan 250062, China.
Sheng Wu Gong Cheng Xue Bao. 2008 Aug;24(8):1496-504.
We selected 12 antigens corresponding to commonly used autoantibodies in clinical practice to prepare antigen microarray. We chose NBT/BCIP color reaction as the end detection strategy to develop a new autoantibody protein chip detection system. Using this system, we optimized the best spotting solution, spotting concentration of the 12 antigens and the dilution of serum. We prepared a protein chip that could detect 12 autoantibodies simultaneously using the optimized antigen concentration. We established a new method to determine the cutoff of each autoantibodies by evaluation of 678 positive and 120 negative serum of clinical sample. We also evaluated the sensitivity and specificity of our new detection system. The optimal spotting solution was 0.1% TBST, the dilution of serum was 1:4 and the best spotting concentration of the 12 antigens were ANA 520 microg/mL, Ro-60/SSa 465 microg/mL, La/SSb 530 microg/mL, Jo-1 530 microg/mL, Scl-70 525 microg/mL, Sm 520 microg/mL, Ro-52/SSa 615 microg/mL, RF 340 microg/mL, CCP 465 microg/mL, ulRNP 410 microg/mL, CENP-B 490 microg/mL and dsDNA 580 microg/mL respectively. It had higher coincidence rate compared to current clinical used methods. We have developed a 12 antigens protein chip for the detection of autoantibodies based on the NBT/BCIP color reaction system. This system was fast, convenient, efficient, and cost-effective.
我们选择了12种与临床实践中常用自身抗体相对应的抗原，用于制备抗原微阵列。我们选择NBT/BCIP显色反应作为最终检测策略，开发一种新的自身抗体蛋白芯片检测系统。使用该系统，我们优化了最佳点样溶液、12种抗原的点样浓度以及血清的稀释度。我们使用优化后的抗原浓度制备了一种能够同时检测12种自身抗体的蛋白芯片。通过对678份临床样本阳性血清和120份阴性血清的评估，我们建立了一种确定每种自身抗体临界值的新方法。我们还评估了新检测系统的敏感性和特异性。最佳点样溶液为0.1%TBST，血清稀释度为1:4，12种抗原的最佳点样浓度分别为ANA 520μg/mL、Ro-60/SSa 465μg/mL、La/SSb 530μg/mL、Jo-1 530μg/mL、Scl-70 525μg/mL、Sm 520μg/mL、Ro-52/SSa 615μg/mL、RF 340μg/mL、CCP 465μg/mL、ulRNP 410μg/mL、CENP-B 490μg/mL和dsDNA 580μg/mL。与目前临床使用的方法相比，它具有更高的符合率。我们基于NBT/BCIP显色反应系统开发了一种用于检测自身抗体的12抗原蛋白芯片。该系统快速、便捷、高效且具有成本效益。
