NR AWPA
AU Toomik,R.; Ramsay,A.; Gonzalez,L.; Dagleish,M.P.; Horton,R.; Hawthorn,J.; Everest,S.; Terry,L.; Estey,L.; Leathers,V.
TI Detection of ovine scrapie in ramalt tissue using an ELISA
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 DIA-54
PT Konferenz-Poster
AB Earlier studies of recto-anal mucosa-associated lymphoid tissue (RAMALT), obtained from animals at various stages of disease and subjected to immunohistochemistry (IHC), and Western blot (WB) analysis have shown that PrPsc could be detected with a consistency comparable to central nervous system, and other lymphoreticular system tissues. The aim of the present work was to investigate the suitability of using an IDEXX post mortem PrPsc ELISA for the detection of scrapie in rectal biopsy tissue as a potential diagnostic tool for live animal testing. An initial small scale evaluation was conducted with the IDEXX Herdchek Chronic Wasting Disease Antigen Test Kit (CWD-EIA). The test was conducted according to the package insert with ovine RAMALT tissue replacing the cervid retropharyngeal tissue normally evaluated in the test. This preliminary evaluation resulted in a specificity of 100% (n=38) and diagnostic sensitivity of 75% (56/75 positive). Based on this promising data, the decision was made to further optimize the IDEXX CWD-EIA for RAMALT applications. A small panel of IHC positive and negative tissue was made available for optimization purposes. The key steps evaluated were tissue disruption, PrPsc sample capture and PrP detection conditions. Release of PrPsc from the tissue matrix was optimised by the addition of a large ceramic bead to the disruption step and increasing the number of cycles. The addition of digestive enzymes was investigated but was found to have no significant effect on signal strength. An increase in capture time, during the sample incubation step along with the addition of a gentle motion was used to optimize sensitivity. Experiments also demonstrated that there was no significant loss of signal when biopsy sized tissue (approximately 120mg) was used, compared to the standard kit weight of 300mg. Using the optimized protocol, a population of 256 scrapie negative RAMALT samples and 213 scrapie positive RAMALT samples (IHC positive) were evaluated on the IDEXX CWD-EIA Test Kit using the modified protocol. The specificity of the optimized protocol was 100% and sensitivity was 94%. In conclusion, we were able to modify the IDEXX CWD Antigen Test Kit protocol for optimal detection of PrPsc in ovine rectal mucosal tissue.
AD R. Toomik, L. Estey, V. Leathers: IDEXX Laboratories Inc., One IDEXX Drive, Westbrook, Maine 04092, USA; A. Ramsay, R. Horton, J. Hawthorn, S. Everest, L. Terry: TSE Molecular Biology, Veterinary Laboratories Agency, Weybridge, KT15 3NB, UK; L. González: Pathology, Veterinary Laboratories Agency, Lasswade, Midlothian EH26 OPZ, UK; M.P. Dagleish: Moredun Research Institute, Penicuik, Midlothian, EH26 0PZ, UK
SP englisch
PO Italien