Tesee western blot

The ear canals of culled sheep were checked for the presence of cerumen. The postmortem TSE test protocol varied depending on the study and tests were one or a combination of tests, such as immunohistochemistry as described for ovine BSE, classical and atypical scrapie (28 (link)–30 (link)), and rapid postmortem test [TeSeE ELISA or TeSeE Western blot (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK)] (31 (link)), according to the manufacturer’s instructions. Clinically healthy sheep from the CS-free flock were only tested by the ELISA; the TSE diagnostic test in the other sheep always included immunohistochemistry.
Lymphoreticular tissues were also examined by immunohistochemistry from all sheep except for sheep from the CS-free flock and reported field suspects.

Brain samples were homogenized at room temperature in a buffer containing Tris-HCl pH 7.4, 150 mM NaCl, 0.5% (w/v) Triton X-100, 0.5% sodium deoxycholate, and treated with Proteinase K (4 μg/ml) for 1 h at 37 °C, or with PNGase F (New England Biolabs) for 2 h at 37 °C for deglycosylation. Samples were boiled in SDS sample buffer and run on 12% pre-cast polyacrylamide gels (Bio-Rad) and transferred to polyvinylidene fluoride membranes by a semi-dry blotter. Membranes were blocked for 1 h in 5% fat-free dried milk, and incubated with primary antibody Bar224 (Spi Bio, France) overnight at 4 °C. Secondary antibody (goat anti-mouse) labeled with alkaline phosphatase (ALP: GE Healthcare, UK) was used to visualize bands with a fluorescence imager (Typhoon 9200, GE Healthcare, UK) after incubation with the ALP substrate (ECFTM, Western blotting reagent pack, GE Healthcare). In addition, an EU and OIE approved test, (TeSeE WESTERN BLOT, Bio-Rad) was used according to the recommendations of the manufacturers. The immunodetection was performed using the monoclonal antibody SHa31 that recognizes the amino acids (YEDRYYRE), corresponding to codons 145–152 in human PrP. Peroxidase activity was revealed using enhanced chemiluminescence (ECL, GE Healthcare, UK) substrate.