疯牛病和羊痒病Western blotting检测方法的建立

Development of Western blotting for detecting BSE and scrapie

以朊蛋白单抗AH6和碱性磷酸酶标记的马抗鼠酶标二抗建立了疯牛病和羊痒病的Western blotting检测方法。对Western blotting各种反应条件进行摸索,并确定了最佳工作条件,结果表明:匀浆缓冲液为RIPA时的最佳反应条件包括浓缩胶电泳电压为恒压90V,分离胶电泳电压为恒压160V,转印的最佳电压和时间为恒压100V1.5h;封闭液为3%BSA时,封闭15min,封闭效果最好;AH6的最佳稀释度为1∶4000,4℃下孵育过夜,马抗鼠二抗的最佳稀释度1∶1000,室温下孵育30min。采用已确立的反应条件对样品进行检测并与Prionics-Check WEST-ERN进口试剂盒的检测结果比较,发现其敏感性为100%,特异性为99.4%,与进口试剂盒(100%,100%)无显著差异,这为国产试剂盒研制提供了条件。

Western blotting for detecting BSE and scrapie was developed by using monoclonal antibody （mAb） AH6 and horse anti-mouse IgG antibodies coupled to alkaline phosphates （AP）. The various attected tactors and conditions of Western blotting were explored, and the optimal reaction conditions of Western blotting were determined. It was shown that when the homogenization buffer RIPA is used, the optimal voltage of resolving gel electrophoresis was 90 V and in stacking gel was up to 160 V, the optimal voltage and time of blotting was 100 V for 1.5 h, the optimal PVDF blocking buffer was 3% BSA in TBST, the optimal dilution of monoclonal antibody （mAb） AH6 was 1 ： 4 000 and incubated for 12-18 h at 4℃, the optimal dilution of horse anti-mouse IgG antibodies coupled to alkaline phosphates （AP） was 1 ： 1 000 and incubated 30 min at room temperature. Following ＂the determination of conditions of Western blotting, samples were detected and the result of it was compared with that of Prionics-Check WESTERN kit. It show that the sensitivity and the specificity of detection are 100% and 99. 4% respectively, and those of Prionics-Check WESTERN kit are 100% and 100% respectively. There was no significant difference in these two detection ways. The development of Western blotting will provide help for the development of Western blotting kit.