NR AXXN
AU Tattum,M.H.; Jones,S.; Pal,S.; Collinge,J.; Jackson,G.S.
TI A Highly Sensitive and Specific ELISA for the Determination of Prion Infection in Human Samples without the Use of Proteases
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Pathology and Pathogenesis P03.154
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Background: A highly sensitive assay for the detection of PrPsc is vital for the early diagnosis of Prion disease, as well for the screening of blood and organs. Currently the best diagnostic tool for prion disease is tonsil biopsy and Western Blotting. Proteinase K (PK) digestion is required to distinguish between PrPc and PrPsc, however, protease digestion can degrade up to 70% of the target antigen. Rapid detection of PrPsc in blood without protease digestion requires the development of a more sensitive assay.
Aims: Our aim was to increase the sensitivity and throughput of immunoassays for the detection of PrPsc in blood. In particular, identify conditions for the discrimination of PrPsc from PrPc without protease digestion. The assays have been optimised for the detection of vCJD brain homogenate spiked into whole blood from unaffected donors. In addition the ELISA assay has been coupled to the immunoprecipitation (IP) of vCJD tissue from large volumes of blood.
Methods: vCJD brain homogenate diluted into whole blood was analysed by enzymelinked immunosorbent assay (ELISA). PrPsc was selectively captured without PK digestion, and detected with a fluorogenic substrate. PrPsc was recovered by IP of vCJD brain homogenate spiked into up to 8ml of whole blood and analysed by ELISA.
Results: The optimised ELISA, which utilises unique antibodies, allows detection of PrPsc from vCJD brain homogenate serially diluted into whole blood down to pico gramme levels. Combining the ELISA with quantitative recovery of vCJD homogenate by IP, indicates detection of PrPsc at extremely low concentrations is possible from large volumes of whole blood.
Discussion: We have developed an assay which allows the rapid, sensitive and selective detection of PrPsc at biologically relevant levels. Coupled with selective capture of PrPsc from clinically practical volumes of blood allows detection at a level equivalent to a 106 fold dilution of brain.
AD M.H. Tattum, S. Jones, S. Pal, J. Collinge, G.S. Jackson, Institute of Neurology, MRC Prion Unit, UK
SP englisch
PO Schottland
EA pdf-Datei und Poster (Posterautoren ergänzt um A. Khalili)