NR AXLI
AU Edgeworth,J.A.; Weissman,C.; Clarke,A.R.; Jackson,G.S.; Collinge,J.
TI A Rapid, Quantitative Assay for the Presence of Steel-bound Prion Infectivity in Cultured Cells
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Epidemiology, Risk Assessment and Transmission P04.16
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Background: Although in many cases PrPsc is an accurate surrogate marker for the presence of prion infectivity in some circumstances it can be dissociated from biological activity. The standard method for direct assay of infectivity has historically been rodent bioassay. However, recent advances have allowed the use of highly sensitive cell-culture assays to detect the presence of prions. Such assays have proved extremely useful for the investigation of many aspects of prion biology but have not been appropriate for use in prion decontamination studies due to the need to study the behaviour of infectivity bound to steel surfaces.
Aims & Objectives: To develop, optimise and validate a cell based bioassay that can sensitively and quantitatively detect RML prions bound to stainless steel wires with sensitivity equivalent to or exceeding conventional whole animal bioassay.
Methods: Dilutions of RML-infected homogenates of known titre were adsorbed to surgical steel surfaces pre-treated in various ways to give optimal transfer of infectivity. The susceptible subclone of murine neuroblastoma cells (N2a-PK1) were grown in contact with the wires and harvested. The cells were cultured and assayed for the presence of propagating PrPsc and from this the number of infectious units bound to the wires quantified.
Results: The new assay has improved sensitivity and is capable of detecting infectivity in a 1010 fold dilution of RML infected brain. Application of this assay facilitates the rapid study of how prion infectivity behaves when bound to surgical steel. This has confirmed that prions adsorbed to steel surfaces have an altered behaviour to those in solution, including enhanced resistance to thermal inactivation.
Discussion or Conclusion: This novel assay offers a new methodology for the evaluation and validation of prion decontamination methodologies and we have demonstrated that an enzyme detergent method for prion decontamination can reduce infectious titre by over 109 fold.
AD J.A. Edgeworth, A.R. Clarke, G.S. Jackson, J. Collinge, Institute of Neurology, MRC Prion Unit, UK; C. Weissman, Scripps Florida, USA
SP englisch
PO Schottland