NR AOXP
AU Oliver,J.; Lane,A.; Thorne,L.; Jackman,R.; Quarterman,E.; Stanley,C.; Wilson,S.
TI Detection of PrPsc in scrapie-infected ovine blood
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - DG-34
PT Konferenz-Poster
AB
Background. We have developed the PrPsc-specific ligand, Seprion, into a very simple microplate immunoassay which removes the need for sample preparation, including proteinase K, in post-mortem brain assays. This Seprion Assay has 100% sensitivity and specificity compared to current gold standards (1) and can be applied to CJD, BSE, Scrapie and CWD. We present here our progress in developing the assay for detection of TSEs in blood.
Method. Animals were asymptomatic, from an experimentally infected flock. Blood from a scrapie-free flock was used as negative controls. 20 ml of water was added to 5 ml of citrated blood. The white cell fraction was collected by centrifugation and resuspended in Seprion Capture Buffer. PrPsc was assayed as described in (1). Briefly, PrPsc from the lysed cells was captured to immobilised Seprion ligand in a microtiter well and detected with an anti-prion HRP conjugate. In addition, PrPsc was captured from the lysed white cell fraction from 80 ml pooled positive blood using Seprion-coated magnetic beads. Captured PrPsc was eluted and analysed by PAGE and Western blotting.
Results. All 3 negative animals and 6/9 animals from the experimental flock were negative by the Seprion assay, with signals of less than 0.11 at OD450nm. Three animals were positive with signals of 0.33, 1.28 and 4.00 at OD450nm.Western blotting of positive blood showed a triplet of antibody reactive bands at the same molecular weight as the recombinant PrP marker.
Discussion. PrPsc was detected in 3/9 experimentally infected sheep and this correlated with an appropriate PrP-specific band upon Western blot analysis. We are currently analysing successive blood samples from these animals and from the other animals in the exposed flock. The Seprion Assay will be correlated with Immunocapillary Electrophoresis and brain pathology upon culling. Assay of a large scrapie-free population is underway.
Reference.1. Stanley et al, Clin. Chem., to be published September 2003.
AD Josephine Oliver, Amin Lane, Emma Quarterman, Christopher Stanley, Stuart Wilson, Microsens Biotechnologies, UK; Leigh Thorne, Roy Jackman, Veterinary Laboratories Agency, UK
SP englisch
PO Deutschland