Development of an Assay for the Detection of PrPres in Blood and Urine Based on PMCA Assay and ELISA Methods

摘要

The focus of this program is the development of a pre-clinical blood- based TSE diagnostic assay. The assay in the pilot form is developed with plasma from hamsters infected with the 263K strain of scrapie. The same assay can be adapted to human PrP test. In this funding period we completed the optimization of the conditions for proteinase K (PK) digestion of PrPres in scrapie infected hamster plasma. We have also started a large study to determine the PK concentration that preserves plasma infectivity while reducing the level of endogenous normal PrP to below the limit of detection of our PrP assay. The PK concentration that indicates no infectivity reduction will be used to digest infected plasma for detection of PrPres. We also found that urine excreted by infected hamsters harbors infectivity with infectivity titers similar to that of hamster plasma. More recently, we inoculated animals with urine from pre-clinical hamsters. This study is still on-going and will be completed by the end of the funding period. Urine could be a useful alternative to blood in a TSE diagnostic assay. We also completed the titration of bladder and kidneys with similar titers approximately 5 log10 ID50/g of tissue.