Development of sensitive diagnostic methods for transmissible spongiform encephalopathies.

为了提高蛋白质检测的灵敏度，对蛋白质印迹检测组织样品的方法进行了改进。在小鼠瘙痒病模型中，用新方法可在腹腔感染1周后的脾组织标本中检测到PrP蛋白。人们希望有一种比蛋白质印迹法更方便的方法来检测许多样本中是否存在普恩蛋白。我们用小鼠模型建立了一种敏感性比免疫印迹法高约两倍的ELISA。我们首先确定了该方法可用于羊瘙痒病样本，然后将该方法应用于北海道屠宰场近300个绵羊样本，并正在开发检测污染胶原蛋白中Pron蛋白的样品制备方法。我们找到了用酶降低胶原液粘度和从大量酶处理胶原液中浓缩普鲁恩的方法。我们还检测了绵羊普鲁恩基因的多态性与绵羊瘙痒病的关系，普鲁恩蛋白在绵羊不同器官中的表达，以及绵羊载脂蛋白E基因的多态性作为瘙痒病的危险因素。

In order to increase the sensitivity for detection of prion protein, the method to prepare samples from tissues for Western blotting was reformed. In mouse scrapie model, prion protein could be detected in the samples prepared from the spleen 1 week after intraperitoneal infection by the new method. It is to be desirable that more convenient method than Western blotting to examine many samples for the presence of prion protein. We developed an ELISA whose sensitivity was about two times higher than that of Western blotting using mouse model. We first confirmed that this ELISA method could be used for sheep scrapie samples, then applied the ELISA method for near 300 sheep samples obtained from slaughter house in Hokkaido.We are developing the method for preparing samples to detect prion protein in contaminated collagen. We found the ways to reduce viscosity of collagen solution by enzymes and to concentrate prion from large volume of the enzyme treated collagen solution.We also examined polymorphisms of sheep prion gene in relation to sheep with scrapie, expression of prion protein in various sheep organs, and polymorphisms of sheep apolipoprotein E gene as a risk factor of scrapie.