NR AOWZ
AU Morel,N.; Simon,S.; Frobert,Y.; Volland,H.; Creminon,C.; Grassi,J.
TI Specific determination of PrPsc by a combined use of plasminogen and monoclonal antibodies. Application to the diagnosis of prion diseases.
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - DG-28
PT Konferenz-Poster
AB
The establishment of methods allowing preclinical and ante mortem diagnosis of TSEs is considered as a high priority. From a practical point of view, this implies to detect PrPsc (the only unequivocal marker of TSEs) in body fluids as blood and urine. This constitutes an actual challenge because the concentration of PrPsc in these fluids is usually estimated to be 1000 to 10000 folds lower than in central nervous system tissues. In addition, the biochemical features of brain PrPsc (aggregation, PK resistance) are not obviously conserved in these media.
Here we describe a new approach based on a combined use of plasminogen binding and antibody recognition which allows to concentrate and detect very efficiently PrPsc from large volume samples (up to 50 ml). In this method, PrPsc is first specifically captured by plasminogen immobilised on magnetic beads as previously described (1). After a washing step, PrPsc is denatured in controlled conditions allowing its recognition by monoclonal anti-PrP antibodies while the association with plasminogen is maintained. This direct assay is achieved in less than 5 hours and is much more easy and rapid than previously described techniques which involve elution of plasminogen from solid-phase immobilised plasminogen before western-blot determination (1). Specific capture is obtained in a 2% (w/v) tissue homogenate using an optimised buffer containing a mixture of detergent, salt and protein and does not require any PK treatment. When applied to serial dilutions of TSE infected brains this test proved to be as sensitive as the BioRad/CEA test (2). Its capacity to concentrate PrPsc diluted in large volumes of samples is illustrated by its performances in media as different as water and plasma. This new approach could prove to be very useful for detecting low levels of PrPsc in body fluids.
References:
1- Fischer M.B. et al., (2000) Nature, 408, 479-483.
2- Moynagh and Schimmel (1999) Nature, 400, 105.
AD Nathalie Morel, Stéphanie Simon, Yveline Frobert, Hervé Volland, Christophe Créminon, Jacques Grassi, CEA, service de Pharmacologie et d'Immunologie, CEA/Saclay, 91190 Gif sur Yvette, France
SP englisch
PO Deutschland