NR AUSL
AU Birkmann,E.; Weinmann,N.; Henke,F.; Stöhr,J.; Willbold,D.; Riesner,D.
TI Diagnostics of BSE and Scrapie without the use of PK-digestion
QU TSE-Forum, 6. Kongress - Nationale TSE-Forschungsplattform, Greifswald 26.6.-28.6.2006, Poster: Diagnostik DIA-01
PT Konferenz-Poster
AB
The infectious agents of prion diseases are composed primarily of the pathogenic isoform of the prion protein designated PrPsc, which is generated by a conformational change of the cellular isoform, PrPc. In contrast to its cellular isoform, the pathogenic isoform PrPsc forms insoluble aggregates. In commercial prion tests, the PK-resistance of PrPsc is accounted as a marker for the disease, but a portion of the disease related aggregated PrP is not PK-resistant (1). Therefore detection of prions, which does not rely on PK-digestion, would be favourable for a sensitive diagnosis. It would allow us detecting both PK-resistant as well as PK-sensitive PrPsc.
Our test system is based on Dual-Colour Fluorescence-Correlation-Spectroscopy (FCS). It is able to detect and quantify aggregated proteins in solution and distinguishes aggregated from monomeric states, irrespective of PK-resistance. We are able to distinguish Scrapie infected hamster as well as BSE infected cattle in the clinical stage from a control group (2). To increase the sensitivity, the prions were concentrated on a surface. The surface can be scanned systematically even for single prion particles.
For further enhancement of the sensitivity of the test system, we established a method to amplify the PrP aggregates that are found in infected animals. We present a system in which small amounts of purified PrPsc serve as seeds for the conversion, they convert recPrP into an aggregated, amyloid state as proven by ThioflavinT (ThT)-assay. The system is clearly different from that described by Soto (3), because only purified components in contrast to a cellular extract are used. In presence of the seed the conversion is specific, since it occurs within hours, whereas recPrP in the absence of seeds is converted into fibres only after several weeks (4). The system may be used as an amplification in a diagnostic test.
(1) Safar et al. (1998) Nature Med. 4, 1157-1160
(2) Birkmann et al. (2006) Biol. Chem., 387, 95-102
(3) Saborio et al. (2001) Nature, 411, 810-3
(4) Leffers et al. (2005) Biol. Chem., 386, 569-80
AD Eva Birkmann, Nicole Weinmann, Franziska Henke, Jan Stöhr, Dieter Willbold, Detlev Riesner, Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany
SP englisch
PO Deutschland
EA Photo des Posters, welches nach Auskunft von Frau Dr. Birkmann aktueller als der Tagungsband ist
OR Tagungsband