NR AWOU
AU Thackray,A.M.; Hopkins,L.; Bujdoso,R.
TI PK-sensitive ovine PrPsc revealed by conformation-dependent immunoassay
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions PR-34
PT Konferenz-Poster
AB Detergent extraction and limited proteolysis of PrPsc results in fibrillar aggregates of a protease-resistant core (PrP27-30) that polymerise spontaneously into amyloid. Aggregated forms of PrP copurify with prion infectivity, although infectivity does not always correlate with the presence of PrP27 - 30. Limited digestion with PK to discriminate between PrPc and PrPsc may underestimate the repertoire of conformations of PrPsc and their quantity within prion-infected tissues. We have developed a conformation-dependent immunoassay (CDI) utilising time-resolved fluorescence to study the conformers of ovine disease-associated PrP in the absence of PK-treatment to discriminate between PrPc and PrPsc. The CDI utilises a panel of either N- or C-terminal-specific anti-PrP monoclonal antibodies that recognise regions of the prion protein that are differentially buried or exposed. Epitopes buried in PrPsc show an increase in immunoreactivity after denaturation of the PrP molecule, whereas those that were solvent exposed do not change in their reactivity. PrPsc for analysis was precipitated from brain tissue of scrapie-positive sheep by detergent extraction, with or without the use of sodium phosphotungstic acid. Native and denatured PrPsc was subsequently subjected to a capture-detector CDI. PrPsc was readily detectable in homozygous VRQ and ARQ scrapie-infected sheep brains. The highest levels of PrPsc were found in homozygous VRQ scrapie-infected brains. The quantity of PrPsc was significantly reduced when samples were treated with PK prior to the CDI. These data show that the level of PrPsc in brain samples from cases of natural scrapie display genotypic differences and that a significant amount of this material is PK-sensitive.
AD Centre for Veterinary Science, Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 OES, UK
SP englisch
PO Italien