Development of sensitive diagnostic methods for transmissible spongiform encephalopathies.

为了提高朊病毒蛋白检测的灵敏度，对免疫印迹法的组织样品制备方法进行了改进。在小鼠瘙痒症模型中，腹腔感染后1周，用新方法制备的脾样品中可检测到朊病毒蛋白。希望有比蛋白质印迹法更方便的方法来检测许多样品中朊病毒蛋白的存在。我们建立了一种ELISA，其灵敏度比小鼠模型的Western blotting高约两倍。本研究首先确认了该ELISA方法可用于羊瘙痒病样品的检测，然后将该方法应用于北海道屠宰场采集的近300份羊样品的检测，并开发了检测污染胶原蛋白中朊蛋白的样品制备方法。本研究探索了用酶降低胶原蛋白溶液粘度的方法和从酶处理的胶原蛋白溶液中浓缩朊病毒的方法，并对绵羊朊病毒基因多态性与羊瘙痒病的关系、朊病毒蛋白在绵羊各器官中的表达以及绵羊载脂蛋白E基因多态性作为羊瘙痒病的危险因素进行了研究。

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.