NR AXUX
AU Ramsay,A.; Gonzalez,L.; Horton,R.; Everest,S.; Toomik,R.; Tonelli,Q.; Leathers,V.; Terry,L.
TI Development of an EIA to Detect PrPd in Rectal Mucosa Lymphoid Tissue from Sheep with Scrapie
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Pathology and Pathogenesis P03.125
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Disease-associated PrP (PrPd) accumulates in the lymphoid tissue of the rectal mucosa in a high proportion of scrapie infected sheep at clinical and preclinical stages of disease. PrPd in biopsy samples was detected by immunohistochemistry (IHC), and Western blot (WB) analysis with a consistency comparable to tissues collected from the central nervous system (CNS), and other lymphoreticular system (LRS) tissues (Gonzalez et al., 2006). The aim of the present work was to investigate the suitability of the rectal mucosa lymphoid tissue for use in a rapid EIA detection system for diagnosis of scrapie in live animals.
We have optimised a rapid test, based on the chronic wasting disease (CWD) antigen test kit EIA (IDEXX laboratories?), in order to detect PrPd in necropsy and biopsy samples. This assay system was originally designed for the detection of abnormal prion protein in retropharyngeal lymph nodes from white tail and mule deer and is based on a PrPd-specific ligand for antigen capture.
Initial optimisation studies demonstrated that there was no significant loss of signal when biopsy samples of approximately 120mg were used, compared to the standard kit weight of 300mg. Release of PrPd from the tissue matrix was optimised by the addition of a large ceramic bead to the ribolysation step and by increasing the number of cycles. The addition of digestive enzymes was investigated but was found to have no significant effect on signal strength. Finally, an increase in time during the primary incubation step along with the addition of a gentle motion was used to optimise sensitivity.
In conclusion, we have developed a protocol for use on rectal mucosa lymphoid tissue using the IDEXX herdchek EIA test kit that, once optimised, provided specificity and sensitivity figures of 99.2% and 93.5%, respectively, compared to IHC results in the same samples; sensitivity values reached 100% for sheep subjected to two sequential biopsies four months apart.
References: GONZµLEZ, L. DAGLEISH, M.P., BELLWORTHY, S.J., SISą, S., STACK, M.J., CHAPLIN, M.J., DAVIS, L.A., HAWKINS, S.A.C., HUGHES, J. & JEFFREY, M. (2006) Postmortem diagnosis of preclinical and clinical scrapie in sheep by the detection of disease-associated PrP in their rectal mucosa. Veterinary Record 158, 325-331.
AD A. Ramsay, R. Horton, S. Everest, L. Terry, Veterinary Laboratories Agency, Molecular Pathogenesis and Genetics, UK; L. Gonzalez, VLA Lasswade, Pathology, UK; R. Toomik, Q. Tonelli, V. Leathers, IDEXX Laboratories inc., USA
SP englisch
PO Schottland
EA pdf-Datei und Poster (Posterautoren: A. Ramsay, L. Terry, R. Horton, R. Toomik, V. Leathers, Q. Tonelli, M. Dalgliesh, M. Jeffrey und L. Gonzalez; Postertitel: Adaptation and evaluation of a rapid test for the diagnosis of sheep scrapie in samples of rectal mucosa)