NR ATGU
AU Ostermann,J.; Lehto,M.; Gong,B.; Kim,J.; Cashman,N.R.
TI Epitope protection as a novel tool to characterize prion structures and for diagnosis of prion infections
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Oral sessions ORAL-51
PT Konferenz-Vortrag
AB
The principle problem in detecting prions is to distinguish between the normally folded prion protein, PrPc, that is present on most cells and in most body fluids, and its disease-causing conformation, PrPsc. Prions consist of oligomers of misfolded prion proteins that are tightly packed into particles. Little is known about the exact structure of these particles, and tools that provide new insights into their structure are much needed.
In most techniques to distinguish between PrPc and PrPsc, the former is removed by proteolysis. The tight packing of prion proteins in some aggregates prevents the proteolysis of all its subunits. After disaggregation, these subunits can then be detected by techniques such as ELISA or Western blotting. However, not all infectious prions are sufficiently tightly packed to include a protease inaccessible core. In order to provide a more powerful approach to distinguish between monomeric and oligomeric states of prion proteins, we have developed the epitope protection assay. Instead of proteolytically digesting the PrPc protein, we incubate samples containing PrPsc hidden in a large excess of PrPc with short lived and highly reactive chemicals in a proprietary process. The chemical modification masks more than 99% of selected epitopes of PrPc, leaving them unrecognizable to antibodies. Due to the short life time of the reactive chemical, only surface epitopes of PrPsc particles are modified, leaving the buried epitopes in the aggregate protected. These can then be detected after disaggregation of the particle, using standard detection methods, or ultrasensitive methods developed by us for use in blood screening. By establishing assays that rely on the intactness of 2 or more antibody binding sites on PrP, we can further increase the discriminatory power of this approach to detect prions in the presence of a large excess of PrPc. This feature is especially important to identify in samples such as blood.
Data will be presented that demonstrate the advantages of this method in comparison with other methods of prion detection.
IN Die Autoren machen die problematische Verdauung von PrPc überflüssig, indem sie mit hochreaktiven Chemikalien mehr als 99% aller Epitope auf Prionproteinen absättigen, bevor sie PrPsc-Aggregate auflösen. Maskiert werden so nur die einzelnen PrPc-Moleküle sowie die Oberflächen der Prionen. Die im Inneren der Prionen geschützten PrPsc werden nicht maskiert und können anschließend immunologisch nachgewiesen werden.
AD J.Ostermann, M.Lehto, B.Gong, J.Kim, N.Cashman, Amorfix Life Sciences, S-119, 2075 Bayview Avenue, Toronto, ON M4N 3M5, Canada
SP englisch
PO Deutschland