NR AOSW
AU Gassner,D.; Golla,R.
TI In vitro PrPsc amplification in a human blood buffy coat model system
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - DG-25
PT Konferenz-Poster
AB
We attempted to enhance the efficiency of the PMCA technique in the cell-free conversion of PrPc to PrPsc. First, we optimized the conditions in a hamster model with regard to temperature, sonication intensity and cycle number. Secondly, we applied these conditions to human(h) buffy coat preparations of lymphocytes and other mononuclear cells with high PrPc content (by Accuspin method) which were spiked (1:100) with homogenate of vCJD brain tissue RU 98/148 (CJD Resource Centre),UK). Near 'physiological' conditions were applied preserving PrPc/Sc rafts. No SDS was used in the conversion buffer and the number of sonication / incubation cycles were reduced to a minimum due to the generation of insoluble superaggregates with increasing cycle numbers. Added aggregated vCJD PrPsc acted as a seed in the conversion of h buffy coat PrPc to protease-resistant isoform when checked in a sensitive western blot assay.
Without PMCA added PrPsc was undetectable.
AD Dieter Gassner, Ruth Golla, Roche Diagnostics GmbH, Centralized Diagnostics, Germany
SP englisch
PO Deutschland