Tesee

We homogenized frozen brain tissues (175 + 20 mg) in 5% glucose in distilled water in grinding tubes (Bio-Rad, https://www.bio-rad.com) adjusted to 10% (wt/vol) by using a TeSeE Precess 48TM homogenizer (Bio-Rad). We determined the presence of PrP^res in transgenic mouse brains by using WB, using the reagents of the ELISA commercial test TeSeE (Bio-Rad). We prepared brain homogenates (10–100 µL of a 10% [wt/vol]) as previously described (18 (link)) and loaded samples into 12% Bis-Tris Gel (Criterion XT; Bio-Rad). We transferred proteins electrophoretically onto polyvinylidene fluoride membranes (Millipore, https://www.sigmaaldrich.com) and blocked overnight with 2% bovine serum albumin blocking buffer. We incubated membranes with Sha31 (25 (link)) mAb at a concentration of 1 µg/mL. Sha31 recognizes the 145-WEDRYYRE-152 epitope of the human-PrP^C sequence, which is conserved in sheep and bovine sequences. We detected immunocomplexes by incubating the membranes for 1 h with horseradish peroxidase conjugated antimouse IgG (GE Healthcare Amersham Biosciences, https://www.gelifesciences.com). We developed immunoblots with enhanced chemiluminescence in ECL Select (GE Healthcare Amersham Biosciences) and captured images by using the ChemiDoc WRS+ System and processed them by using Image Lab 5.2.1 software (both Bio-Rad).

Mouse prion bioassays were carried out in tg338 mice, which are transgenic for ovine VRQ PrP and are highly efficient for the detection of sheep scrapie infectivity (Le Dur et al., 2005 (link)). Mice (n = 6 per inocula) were injected intracerebrally with 20 µl of diluted fly head homogenate (to give approximately two fly-head equivalents per mouse) and monitored daily until the occurrence of clinical signs of mouse prion disease. Inoculated mice were euthanized when they started to show locomotor disorders and any impairment in their capacity to feed, or at a predefined end-point for the assay (either >250 days or in some cases >670 days post-inoculation) (Andreoletti et al., 2011 (link)). Brain tissue (cerebral cortex) was collected from euthanized mice and frozen for PrP^Sc analysis by western blot (TeSeE, Bio-Rad) or paraffin-embedded tissue (PET) blot analysis (Andreoletti et al., 2011 (link)).

Several combinations of heat plus PK treatment were studied on this work: Plain heat treatment, heat treatment plus PK digestion and finally PK digestion plus heat treatment.
Heat treatment (98 °C): Brain homogenates of Tga20 mice infected with BSE, RML or 22L strains were aliquoted (500 µl), placed in safe-lock tubes (Eppendorf) and heated at 98 °C for 2 hours in a thermocycler (Primus 96 Plus Thermal Cycler, MWG AG Biotech). Samples were removed, allowed to cool gradually to room temperature and harvested at −20 °C.
Heat treatment plus PK digestion (98 °C + PK): The heating process was done as stated above. After the gradually cooling of the samples, 250 µl were taken and aliquoted in 50 µl. Individual aliquots were mixed with 150 µl of healthy sheep brain and subjected to PK digestion with 40 µg/ml of PK using the reagents of the TeSeE (Bio-Rad) enzyme-linked immunosorbent assay at 37 °C for 15 min. The reaction was stopped by adding PMSF to a final concentration of 2 mM. Finally, samples were subjected to centrifugation (15,000 g, 7 min, 20 °C) and pellets were resuspended in 50 µl of PBS.
PK digestion plus heat treatment (PK + 98 °C): PK digestion was done as described above using as starting material 50 µl of unheated brain homogenates. After centrifugation, pellets were resuspended in 50 µl of PBS and subjected to heat treatment as described above.

Blood samples were taken from sixteen individual female reindeer (R. tarandus)—average age approximately 7 years (range 1.5–12 years old)—sampled in Iceland. The sampling was part of research dealing with general health of Icelandic reindeer with specific emphasis on chronic wasting disease (CWD) and presence of parasites. Sample collection was in accordance with Icelandic laws and regulations on sampling from wild animals (64/1994) and licenses of the Institute for Experimental Pathology at Keldur, University of Iceland (number #0001 kt-650269—4549), approved by the central animal ethics committee in Iceland (Icelandic Food Regulation Authority, MAST Matvælastofnun). The plasma was isolated according to standard procedures from EDTA blood samples. Brain samples from the same animals were screened for presence of prion disease by ELISA (TeSeE^®, Bio-Rad, UK) following standard procedures at the Institute for Experimental Pathology at Keldur, and the animals were confirmed to be disease free and healthy. Plasma was aliquoted at 250 µL and stored at −80 °C until further use for the individual experiments.

Samples from all positive animals from the primary passage were subject to four commercially available ELISA tests—IDEXX Herdchek™ with bovine conjugate, IDEXX Herdchek™ with ovine conjugate, BioRad TeSeE™ and BioRad S&G™. For each test, samples were extracted and the tests carried out in accordance with the manufacturers’ instructions.

PrP^Sc detection was carried out using two sandwich ELISA tests (TeSeE, Bio-Rad) following the manufacturer's instructions. The assay protocol includes a purification of PrP^Sc (TeSeE purification kit) consisting of (i) digestion of PrP^C with PK, (ii) precipitation of PrP^Sc by centrifugation and (iii) denaturation of PrP^Sc at 100°C, before immuno-enzymatic detection. In this ELISA, the capture antibody recognizes the octarepeat region of PrP [34] (link), while the detection antibody binds to the core part of the protein [32] (link).
PK resistance of the PrP^Sc portion recognized in the ELISA test was determined by measurement of the ELISA specific signal recovered after digestion with different concentrations of PK in ‘buffer A’ reagent (TeSeE purification kit, Bio-Rad). Each sample was first diluted in normal brain homogenate (between 100 and 10 000 fold) until a signal between 1.5 and 2 absorbance units was obtained after digestion with 50 µg/ml of PK. Triplicates of equilibrated samples were then submitted to a PK digestion with concentrations ranging from 50 to 300 µg/ml, before PrP^Sc precipitation and ELISA detection. Results were expressed as the percentage of residual signal as compared with the signal after 50 µg/ml PK digestion (lowest PK concentration). In each assay, two standardized controls (scrapie and BSE from sheep) were used as an internal standard.