NR AXPT
AU Keevil,B.; Collin,R.; Herve,R.
TI A Rapid, Sensitive Dual Stain Procedure to Discriminate between PrPsc and Other Proteins in Tissue Contaminating Surgical Stainless Steel Surface
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Epidemiology, Risk Assessment and Transmission P04.88
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB We have previously developed a rapid, sensitive staining procedure for in situ detection of prion amyloid material on surgical stainless steel surfaces using fluorescent thiazole reagents. To increase the resolution of the amyloid material in tissues and on contaminated surfaces, and discriminate between PrPsc and other proteins, we now report the development of a Thioflavine T / SYPRO Ruby dual staining procedure. This is used in combination with our episcopic differential interference contrast/epifluorescence (EDIC/EF) microscope for rapid scanning of instrument surfaces. Double-blind studies using various dilutions of ME7 infected brain in normal brain homogenate produced a dose-related signal on contaminated surgical stainless steel surfaces, as assessed by image analysis. The sensitivity of our method proved over 2-fold better compared to the classic Western blot procedure with the same prepared samples. It is believed that the sensitivity improvement is actually greater since testing instrument surfaces by Western blot relies on swabbing the contaminated surface, and we have shown previously that PrPsc binds to surfaces more strongly than other proteins. Moreover, if the instrument is serrated or pitted then it is more difficult to elute biological deposits from the surface for analysis. The detection limit for the new procedure was demonstrated to be <100fg PrPsc; approx. 2 attomoles. Therefore, this new sensitive microscopy procedure is rapid and can be applied directly to instrument surfaces to check for amyloid contamination.
AD B. Keevil, R. Collin, R. Hervé, University of Southampton, Environmental Healthcare Unit, UK
SP englisch
PO Schottland