NR AWJS
AU Martin,J.; McCutcheon,S.; Hunter,N.; Houston,F.
TI Can PrPsc be detected in blood - a western blot approach?
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 PA-36
PT Konferenz-Poster
AB We have shown that TSE agents can be transmitted via blood using sheep models and in humans vCJD cases have been reported that were probably the result of infected blood transfusions. However, it is not known which blood components carry infection and using various approaches, PrPsc has not yet been conclusively detected in TSE-infected blood. It may be that PrPsc levels in TSE infected blood are low and the sensitivity of current detection methods insufficient. We have developed a sensitive immunoassay in an attempt to detect PrPsc in blood. NaPTA precipitation and a panel of novel monoclonal antibodies have been used to increase sensitivity. The method has been optimised using natural scrapie and experimental murine brain homogenates. To establish sensitivity limits for comparison with cellular components of blood SMB (scrapie-infected mouse brain) cells have also been tested. Assay sensitivity has been confirmed using scrapie-infected brain and SMB cell spikes. The methodology has been applied to buffy coat blood fractions from clinical scrapie-infected sheep. It was found that the overall sensitivity of the assay using novel antibodies combined with NaPTA concentration increased approximately 10-fold. The monoclonal antibodies tested have a broad specificity and two can detect PrPsc in the equivalent of 2 µg wet brain tissue and in approximately 6 x 104 SMB cells. This level of sensitivity was confirmed using SMB cells spiked into peripheral blood mononuclear cells (PBMC). We are currently applying the method to clinical buffy coat and plasma fractions from scrapie infected sheep. Although preliminary our results highlight the need for further assay development to achieve the required sensitivity for PrPsc detection in blood. By identifying the blood fractions that carry PrPsc we ultimately aim to improve our understanding of TSE transmission and pathogenesis. This work may also aid in the development of safer methods of blood transfusion in humans and also blood diagnostic tests.
AD J. Martin, S. McCutcheon, F. Houston: Institute for Animal Health, TSE Division, Compton Laboratory, Compton, Newbury, Berkshire, RG20 7NN, UK; N. Hunter: Institute for Animal Health, Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh, EH9 3JF, UK. E-mail: jo.martin@bbsrc.ac.uk
SP englisch
PO Italien