结核分枝杆菌可视化抗体检测蛋白芯片的制备

目的：利用克隆表达的7种结核分枝杆菌优势表位抗原，建立可视化抗体检测蛋白芯片，用于结核病辅助诊断。方法：将7种结核分枝杆菌优势表位抗原，即38kD、ESAT-6、CFP10、MPT64、Mtb8、Mtb8.4和Mtb16.3点于修饰的基片上，制备可检测7种结核抗体的多靶点蛋白微阵列，建立免疫金银染色检测系统；使用该芯片对48例临床结核病患者血液样品进行检测，并与“金标准”痰涂片（48例）和痰培养（其中的29例）检测结果进行比较，分析其敏感性；对30名献血员血液样品进行检测，分析其特异性。结果：可视化抗体检测蛋白芯片的敏感性分别为98.5％和96.6％，而痰涂片和痰培养检测方法的敏感性分别为35.4％和48.3％；可视化抗体检测蛋白芯片的特异性为93.3％。结论：建立的结核分枝杆菌可视化抗体检测蛋白芯片检测敏感性显著高于痰涂片和痰培养方法，可用于结核病的临床辅助诊断，提高痰涂片和痰培养假阴性的检出率。

Preparation of the Visual Protein Chip Based on Epitope-Dominant Antigens of Mycobacterium tuberculosis

Objective： The visual protein chip was prepared for tubercle baeillus（TB） auxiliary diagnosis, in which the seven epitope-dominant antigens of Mycobacterium tuberculosis were used as detecting proteins. Methods： Seven epitope-dominant antigens of M.tuberculosis, i.e. 38kD, ESAT-6, CFP10, MPT64, Mtb8, Mtb8.4 and Mtb16.3, were expressed in E. coli, and they were purified and dotted on the chemically modified glass slides. Immunogold-silver staining system was chosen to make the positive results visual. 48.samples of TB patient＇s sera and 30 samples of donor＇s sera were tested by the multi-target protein microarray. The sensitivity and specificity of the visual protein chip were analyzed and compared with those of the sputum smear and the sputum culture respectively. Results： The sensitivities of the visual protein chip obtained in two assays were 98.5% and 96.6% respectively, which were remarkably higher than the values reached by the sputum smear and the sputum culture. The specificity of the visual protein chip was 93.3%. Conclusion： The sensitivity of the visual protein chip based on epitope-dominant antigens of M.tuberculosis was higher than that of the sputum smear and the sputum culture significantly. It can be the clinical auxiliary diagnosis of TB to detect the false negative cases of sputum smear and/or culture.