NR AXYK
AU Waldron,S.; Rogers,M.
TI Assessing Acute infection in Prion Diseases
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Natural and Experimental Strains P02.02
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Routine detection of PrPsc is based on the different sensitivities of PrPc and PrPsc to proteinase K (PK). PrPc is sensitive to digestion by PK while PrPsc shows variable resistance to digestion resulting in an N-terminally truncated protein of ~27-30kDa (PrP27-30). The majority of antibodies used for detection of PrP are directed against linear epitopes that preferentially bind to native PrPc and not native PrPsc. The epitopes within PrPsc are revealed by denaturation. Cell culture models of infection are available that potentially allow the acute or non-persistent phases of the infection to be investigated. However, this requires a sensitive assay that detects PrPsc in its native state.
To establish a sensitive assay for the detection of prion infection in cell culture we have used a combination of trypsin in conjunction with N-terminal-specific anti-PrP antibodies allowing detection of PrPsc in its native state. We have shown that trypsin leaves PrPsc intact after digestion but digests PrPc. We have developed an ELISA that detects native PrPsc using this approach and are further transferring this system into flow cytometry. Using flow cytometry allows sensitive detection of acute infection and simultaneous detection of other cellular markers following infection with different strains and species of infectious prion.
In addition, detection with N-terminal antibodies allows discrimination between the inoculum used (PK treated) and newly synthesised PrPsc. Therefore we can distinguish the new infection from the infectious material by the presence of the N-terminal.
AD S. Waldron, M. Rogers, University College Dublin, School of Biology and Environmental Science, Ireland
SP englisch
PO Schottland