NR AOQM
AU Birkmann,E.; Schäfer,O.; Weinmann,N.; Wilm,A.; Riesner,D.
TI Detection of BSE and Scrapie without PK-digestion via Dual-Colour
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - DG-11
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. 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 FCS, which detects and quantifies aggregated proteins in solution. The protein aggregates are fluorescence labelled with specific antibodies and can be detected as single particles by FCS. The principle was already successfully applied to the detection of Aß-petides in cerebrospinal fluid of AD-patients and prion aggregates in the CSF of CJD-patients.
The sensitivity of our test was enhanced by improving the experimental procedure and the sample processing. To detect more prion particles a new scanning mechanism was introduced to the FCS setup. The specificity was increased by applying FCS in the dual colour mode with two different antibodies. The sample preparation was modified so that PK-digestion could be avoided therefore not only PK-resistant but also PK-sensitive, i.e. probably early states of the pathogenic PrP could be detected. The system was applied to nervous tissue of Scrapie infected hamsters and BSE infected cattle. Further experiments determining and enhancing the sensitivity in earlier steps of the disease are in progress. Therefore we try to enhance the sensitivity by amplification of the prion particles.
AD E. Birkmann, O. Schäfer, N. Weinmann, A. Wilm, D. Riesner, Universität Düsseldorf, Germany
SP englisch
PO Deutschland