NR AXSG
AU Martin,J.; McCutcheon,S.; Hunter,N.; Houston,F.
TI Can PrPsc be Detected in Blood - A Western Blot Approach?
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Pathology and Pathogenesis P03.112
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Background: Transmissible spongiform encephalopathies (TSEs) are a group of fatal infectious neurodegenerative diseases affecting both humans and animals. We have previously demonstrated transmission of bovine spongiform encephalopathy and scrapie between sheep via blood transfusion. In humans, four variant Creutzfeldt-Jakob disease (vCJD) cases have been reported which probably resulted from infected blood transfusions, highlighting the concern of TSE transmission via blood.
Aims/Objectives: The focus of this study is to investigate which blood components in scrapie-infected sheep carry infection using PrPsc as a marker for TSE agents. Although many standard methods for PrPsc detection have been applied to blood, PrPsc has only been detected by protein misfolding cyclic amplification (PMCA) technology in 263K infected hamster blood, so initially optimisation of a sensitive immunoassay for PrPsc detection was required.
Methods: The sensitivity of a standard Western blot assay for detection of proteinase K (PK) resistant PrPsc was increased by use of phosphotungstic acid (NaPTA) precipitation, combined with screening a panel of novel monoclonal antibodies against sheep PrP. To establish sensitivity limits for comparison with cellular components of blood SMB (scrapie-infected mouse brain) cells were also tested.
Results: The use of NaPTA combined with novel monoclonal antibodies resulted in an overall increase of assay sensitivity of approximately 10-fold. The monoclonal antibodies tested have a broad specificity and two can detect PrPsc in the equivalent of 2 µg wet brain tissue and in approximately 6 x 104 SMB cells. The optimized Western blot assay has been applied to buffy coat (predominantly leukocyte) fractions from animals clinically affected with scrapie. In each case 50 ml of blood was obtained yielding approximately 1 x 108 buffy coat cells to test. Preliminary results have revealed PK resistant protein bands and this is currently being investigated further.
Conclusion: If PK resistant PrPsc is not confirmed in leukocytes, this indicates that the proportion of infected cells is less than 0.01 %, highlighting the need for extremely sensitive assays to detect PrPsc in blood. An alternative explanation is that abnormal PrP in blood is PK sensitive, and therefore cannot be detected by methods relying on PK digestion to distinguish it from PrPc.
AD J. Martin, S. McCutcheon, N. Hunter, F. Houston, Roslin Institute, Neuropathogenesis Unit, UK
SP englisch
PO Schottland
EA pdf-Datei und Poster (Postertitel: Can surrogate markers be identified in sheep scrapie buffy coat proteomes?)