NR APAJ
AU Weissmann,C.; Klöhn,P.C.; Stoltze,L.; Enari,M.; Flechsig,E.
TI SSCA and SCEPA: Quantitative, Highly Sensitive Cell-based Infectivity Assays for Mouse Scrapie Prions
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Oral sessions OS-09
PT Konferenz-Vortrag
AB PrPsc is a surrogate marker for prion disease, but its level does not necessarily correlate with the infectivity titer. Infectivity is usually quantified by injecting samples i.c. into indicator animals and determining the time to appearance of disease (incubation time method) or by injecting serial dilutions of the sample and determining the dilution at which 50% of the animals acquire scrapie (endpoint method). Several cell lines can be infected by prions, as evidenced by the accumulation of PrPsc and/or infectivity after multiple passages. Murine neuroblastoma-derived N2a cells are susceptible to certain strains of mouse prions, however only a small proportion of cells accumulate detectable levels of PrPsc. We have isolated N2a sublines highly susceptible to RML mouse scrapie prions (but not to prions of other species) as well as such that are relatively resistant. We observed that infected monolayers, when blotted onto membranes and assayed for PrPsc, gave a speckled pattern, suggesting that micro-colonies of infected cells, embedded in a lawn of uninfected cells, were being displayed. We then succeeded in visualising individual PrPsc-positive cells filtered onto membranes of an Elispot plate and established conditions under which the proportion of PrPsc-positive cells was a function of the prion titer. Using the Standard Scrapie Cell Assay (SSCA) and the N2aPK1 cell line we could quantify RML prion concentrations as low as those that can be determined in the mouse bioassay. The SSCA is carried out in a 96-well format, allows the processing of hundreds of samples in one run, can be completed in 2, as compared to 20 weeks in the most rapid mouse bioassay, and is two orders of magnitude cheaper. We also developed the Scrapie Cell End Point Assay (SCEPA), which, whilst more time consuming, is more sensitive and robust, capable of detecting infectivity at a 10-9 dilution of RML-infected mouse brain, which is beyond the range of the standard mouse bioassay.
AD Charles Weissmann, Peter-Christian Klöhn, Lars Stoltze, Masato Enari, Eckhard Flechsig, MRC Prion Unit, University College London, UK
SP englisch
PO Deutschland