NR AWMH
AU Properzi,F.; Badhan,A.; Kloehn,P.C.; Wadsworth,J.; Weissmann,C.; Collinge,J.
TI Development of a high throughput automated system for screening of prion infectivity
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions CE-41
PT Konferenz-Poster
AB
Until recently, animal models or 'bioassays' have been used to study prion infectivity, which are long, costly experiments with inherent variability. In 2003 a novel approach to assaying infectivity was developed using cells rather than animals (1). The Scrapie Cell Assay (SCA) is 10 times faster and 100 times cheaper than bioassay. However, as originally developed, the SCA is a standard manual procedure and has an intrinsic technical complexity that does not allow the processing of a large number of samples and lacks good reproducibility. Therefore our aim was to automate the SCA for high throughput screening of RML prion infectivity and reduction of variability in readout. We used a Beckman Coulter Biomeck FX robot due to its suitability for 96 well-plate liquid handling. The SCA posed two crucial challenges to a robot. First, it is cell based and the results depend absolutely on the viability of the cells and the maintenance of their susceptibility to prion infection. Second, the quantification of infectivity requires complex technology - the 'Elispot' technique - for the visual readout of prion positive cells, the units by which infectivity is measured. We first taught the robot how to manipulate cells without increasing the death rate or lose their susceptibility to prions, and we and have now successfully overcome the main obstacles to automated Elispot analysis. The automated scrapie cell assay (ASCA) has been now available in the MRC Prion Unit for the last 10 months. We can run up to 600 samples per week at relatively low cost. We also have introduced a bar code system for sample tracking and data base analysis. The automation of the SCA is a technological breakthrough and will be critical in understanding fundamental unresolved issues of prion diseases. The automation of the technique will also provide a platform for high throughput diagnosis when cell lines susceptible to human prions become available.
1. Klohn, P. C., Stoltze, L., Flechsig, E., Enari, M., & Weissmann, C. A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions. Proc.Natl.Acad.Sci U.S.A 100, 11666-11671 (2003).
AD F. Properzi, A. Badhan, P.C. Kloehn, J. Wadsworth, J. Collinge: MRC Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, Queen Square, London WC1N 3BG, UK; C. Weissmann: Department of Infectology, Scripps Florida, USA f.properzi@prion.ucl.ac.uk
SP englisch
PO Italien