疯牛病和羊痒病WESTERN BLOT检测方法的建立
evelopment of Western Blot for Detecting BSE and Scrapie
以朊蛋白单抗AH6和碱性磷酸酶标记的酶标马抗鼠二抗建立了疯牛病和羊痒病的Western Blot检测方法。对Western Blot 各种反应条件进行摸索，并确定了最佳工作条件，结果表明：当匀浆缓冲液为RIPA时最佳反应条件为浓缩胶内电泳电压为恒压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100V 1.5小时；封闭液为3%BSA时，封闭15分钟，封闭效果最好；AH6的最佳稀释浓度为1：4000，4℃下孵育过夜，马抗鼠二抗的最佳稀释浓度1：1000，室温下孵育30分钟。采用已确立的反应条件对样品进行检测并与Prionics®-Check WESTERN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%，而进口试剂盒分别为100%，100%，与进口试剂盒无显著差异，这为在该基础上建立国产试剂盒提供了条件。
estern Blot 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 affected factors and conditions of Western Blot were explored, and the optimal reaction conditions of Western Blot were determined. It was shown that when used the homogenization buffer RIPA, the optimal voltage of resolving gel electrophoresis was 90Vand in stacking gel was up to 160V, the optimal voltage and time of blotting was 100V for 1.5h, the optimal PVDF blocking buffer was 3%BSA in TBST, the optimal dilution of monoclonal antibody (mAb) AH6 was 1:4000 and incubated for 12-18 h at 4 °C, the optimal dilution of horse anti-mouse IgG antibodies coupled to alkaline phosphates (AP) was 1:1000 and incubated 30 min at room temperature. Following the determination of conditions of Western Blot, Samples were detected and the result of it was compared with that of Prionics®-Check WESTERN kit. It show t
孟丽平、白玉、宁章勇、赵德明、王辉暖、吴常德、郝俊峰、王传武、杨建民
生物科学研究方法、生物科学研究技术基础医学
疯牛病羊痒病Western Blot
BSE scrapie Western Blot
孟丽平,白玉,宁章勇,赵德明,王辉暖,吴常德,郝俊峰,王传武,杨建民.疯牛病和羊痒病WESTERN BLOT检测方法的建立[EB/OL].(2005-02-28)[2025-05-10].http://www.paper.edu.cn/releasepaper/content/200502-82.点此复制
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