Development of sensitive diagnostic methods for transmissible spongiform encephalopathies.

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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.