NR ARED
AU Weinmann,N.; Birkmann,E.; Schäfer,O.; Riesner,D.
TI Seeded Aggregation and Detection of PrPsc Aggregates
QU TSE-Forum, 4. Kongress - Nationale TSE-Forschungsplattform, Düsseldorf 28.10.-29.10.2004, Poster GL-12
PT Konferenz-Poster
AB
One of the characteristic steps of transmissible spongiform encephalopathies is the accumulation of a pathogenic isoform (PrPsc) of the host encoded prion protein. PrPsc forms aggregates and is partially resistant to protease digestion (PK). These properties distinguish PrPsc from PrPc as a disease-specific marker in the central nervous system and peripheral tissues of infected animals and are used in all tests commercially available so far.
We present a sensitive biophysical test system based on Dual-Colour Fluorescence-Correlation-Spectroscopy (FCS), which detects and quantifies aggregated proteins in solution and distinguishes aggregated from monomeric states. The advantage of FCS compared to conventional systems is that FCS allows analysis of aggregates irrespective of PK-resistance of the prion protein. Thus sensitivity of our detection system was enhanced by modifying the sample preparation so that PK-digestion could be avoided (1). Therefore not only PK-resistant but also PK-sensitive, i.e. probably early states of the pathogenic PrP could be detected.
According to the protein misfolding cyclic amplification (PMCA) (2) we established a system which enables us to use purified PrPsc as a seed for recPrP aggregation. That way the amount of insoluble aggregates in the sample could be increased remarkably.
Combination of our test system and seeded aggregation should result in an enhancement of sensitivity.
(1) Safar et al. (1998) Nature Med. 4, 1157-116
(2) Saborio et al. (2001) Nature 411, 811-813
AD Weinmann N., Birkmann E., Schäfer O. and Riesner D., Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany
SP englisch
PO Deutschland
OR Tagungsband