NR AXXM
AU Tattum,M.H.; Jones,S.; Pal,S.; Collinge,J.; Jackson,G.S.
TI Discrimination between Prion-infected and Normal Blood Samples by PMCA
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Protein Misfolding P01.59
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Background: Diagnosis of prion disease from blood samples will require the detection of minute quantities of PrPsc. Protein Misfolding Cyclic Amplification (PMCA) is a technique which can amplify small amounts of seed PrPsc to a level detectable by conventional methods. Application of PMCA to the testing of blood samples may enhance the ability to detect PrPsc in blood and allow ante-mortem detection of prion infection.
Aims: The PMCA methodology was used to the amplification of PrPsc present in blood samples from RML infected mice culled at various time point throughout the incubation period for disease. Also, the coupling of PMCA to the amplification of PrPsc captured by immunoprecipitation from large volumes of blood and subsequent detection by high sensitivity ELISA.
Methods: Blood samples were diluted into substrate brain homogenate and subjected to PMCA. Serial PMCA (sPMCA) involved repeated rounds of amplification and redilution into fresh substrate. PrPsc recovered from immunoprecipitation of RML brain homogenate spiked into whole blood was also subjected to sPMCA. All samples were analysed by Western blotting and ELISA.
Results: After sPMCA, blood samples from RML infected mice showed amplification of PrPsc to levels readily detectable by western blotting and standard ELISA. Detectable levels of PK resistant material were detected following PMCA of material recovered by immunoprecipitation of RML spiked into 8ml of whole blood.
Discussion: Serial PMCA performed on small volumes of whole blood samples (1µl) gave amplification of PK resistant material to a level detectable by standard immunoassays. Amplification of PrPsc immunoprecipitated from a large volume of whole blood suggest that PMCA can provide a valuable amplification step for the determination of prion infection in combination with immunoprecipitation and high sensitivity ELISA.
AD Tattum,M.H.; Jones,S.; Pal,S.; Collinge,J.; Jackson,G.S., Institute of Neurology, MRC Prion Unit, UK
SP englisch
PO Schottland
EA pdf-Datei und Poster (Titel ergänzt: Discrimination between prion-infected and normal blodd samples by PMCA at a pre-clinical stage)