Development of an Assay for the Detection of PrPres in Blood and Urine Based on PMCA Assay and ELISA Methods
Abstract
The focus of this program is the development of a pre-clinical blood-based TSE diagnostic. The assay is been developed with plasma from hamsters infected with the 263K strain of scrapie. The assay has shown high sensitivity and specificity and good reproducibility. In this funding period we addressed two critical issues: Proteinase K (PK) digestion to discriminate PrPc, the normal form of the protein and PrPres the infection-specific form of the protein and denaturation of PrPres in plasma. The first issue was partially resolved when we discovered that PK digestion of plasma PrPc can be conducted without addition of SDS. We can now titer plasma and determine which PK condition is appropriate to digests all PrPc and no infectivity. We also identified the condition for plasma PrPres denaturation. We are currently developing the reagents and protocols for a proof of principle titration in plasma after PK digestion. This is the last step in the development of a complete plasma diagnostic assay. In a large, long term, limiting dilution titration of untreated, whole urine from scrapie infected hamsters, we have now conclusively shown that urine from TSE infected animals contains significant levels of infectivity and we will have a precise titer in another 6 months. Urine could thereby be an alternative substrate for disease detection. Bladder and kidney are also infectious.
Document Details
- Document Type
- Technical Report
- Publication Date
- Sep 01, 2006
- Accession Number
- ADA462905
Entities
People
- Luisa L. Gregori
- Robert G. Rohwer