NR ATKX
AU Weinmann,N.; Birkmann,E.; Riesner,D.
TI Detection by Fluorescence Correlation Spectroscopy and Amplification of PrPsc-aggregates without the use of PK-digestion
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Diagnosis DIA-35
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. It is generally agreed that PrPsc molecules are capable to induce the conversion of PrPc molecules into new PrPsc molecules. 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 a sample preparation without PK-digestion is badly required for a sensitive diagnosis, because this would lead to the possibility to detect both, PK-resistant as well as PK-sensitive PrPsc.
A test system based on Dual-Colour Fluorescence-Correlation-Spectroscopy (FCS) detects and quantifies aggregated proteins in solution and distinguishes aggregated from monomeric states, irrespective of PK-resistance of PrP. Thus sensitivity was enhanced by modifying the sample preparation so that PK-digestion could be avoided (1). Therefore, also PK-sensitive, i.e. probably early states of the pathogenic PrP can be detected. We are able to distinguish BSE infected cattle in the clinical stage of disease form a control group.
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, i.e. they are able to convert recPrP into an aggregated, amyloid state as proven by ThioflavinT (ThT)-assay. This conversion is specific for the seed, since it occurs within hours, whereas recPrP in the absence of seeds is converted into fibers only after several weeks (2). The system may be used as an amplification in a disgnostic test.
(1) Safar et al. (1998) Nature Med. 4, 1157-116
(2) Leffers et al. (2005) Biol. Chem., in press
IN Um einen empfindlicheren und vor allem zuverlässigeren TSE-Test zu entwickeln, amplifizieren die Autoren (vermutlich zyklisch) das PrPsc in vitro und verzichten auf die Inkubation des nicht immer proteaseresistenten PrPsc mit Proteinase K. Stattdessen weisen sie mittels Dual-Colour Fluorescence-Correlation-Spectroscopy spezifisch aggregiertes Prionprotein nach.
AD Nicole Weinmann, Eva Birkmann, Detlev Riesner, Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany
SP englisch
PO Deutschland