Sensitivity of moRK13 cell assay for the detection of RML mouse prions.

<p>Serial 10-fold dilutions (from 10⁻⁴ to 10⁻⁸) of RML 10% brain homogenate were inoculated in duplicate to moRK13 cells and the inoculated cultures were proceeded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020563#pone-0020563-g001" target="_blank">figure 1D</a>. PrP^res was analyzed by western blot after one (A) or two rounds (B) of cell assay. M are standard molecular mass marker proteins in kDa.</p

Sensitivity of ovRK13 cell assay for the detection of PG127 ovine prion.

<p>A) Morphology of inoculated ovRK13 cultures kept in the same wells during the whole cell assay procedure (d0 is the time of inoculation and d28 is 4 weeks later). B) Sensitivity of ovRK13 cell assay as assessed by immunoblotting. <i>Right panel:</i> Serial 10-fold dilutions (from 10⁻⁴ to 10⁻⁶) of infectious PG127 10% brain homogenate were inoculated to single wells of ovRK13 cells. Four weeks later, inoculated cultures were analyzed for PrP^res by immunoblotting. Positive transmission was detected for dilutions up to 10⁻⁵. No PrP^res was observed when inoculated ovRK13 did not express the ovine PrP (dox-). <i>Left panel:</i> total PrP from infected cells was analyzed before (−) or after (+) PK digestion to illustrate band shift upon PK proteolysis. M are standard molecular mass marker proteins (20, 30 and 40 kDa). C) Sensitivity of ovRK13 cell assay as assessed by Elispot. Replicate wells from the same experiment shown in Fig. 1B were analyzed. Left: representative wells of an Elispot plate showing spots given by ovRK13 cells exposed to the indicated dilutions of PG127. Right: double-logarithmic plot of spot number versus PG127 dilution shown for inoculations in the presence (triangle) or in the absence (square) of dox. For each dilution, the mean value ± SD of 8 measurements is shown. Background values for ovRK13 cells inoculated in the absence of dox are less than 4 spots per 50,000 cells. D) Sensitivity is strongly improved by 2 successive rounds of cell assay. Serial 10-fold dilutions (from 10⁻⁵ to 10⁻⁷) of infectious PG127 10% brain homogenate were inoculated to duplicate wells of ovRK13 cells. Four weeks later, PrP^res in a 1^st set of inoculated cultures was isolated (1^st round) while cultures of the 2^nd set were homogenized to inoculate new ovRK13 cells. Four weeks later, PrP^res was isolated (2^nd round) and all the samples were analyzed by immunoblotting. M are standard molecular mass marker proteins (20, 30 and 40 kDa).</p

Cell assay detection of plastic-bound prions.

<p>Infectivity from 10% PG127 brain homogenate was diluted either in culture medium (prion in culture medium) or in Triton-DOC lysis buffer (prion in detergents) and incubated for 2 h into plastic wells. Samples were removed, wells were thoroughly rinsed and air-dried. OvRK13 cells were then seeded in the presence (+) or in the absence (−) of dox. PrP^res in the cultures was analyzed 4 weeks later by immunoblotting and compared to PrP^res levels in ovRK13 cultures subjected to the standard cell assay (no coating). M are standard molecular mass marker proteins (20, 30 and 40 kDa).</p

PMCA analysis of white blood cells and platelets samples.

<p>Platelets (A) and white blood cells (WBC) (B) from sheep D2 collected at the indicated time points (dpi) were subjected to two successive rounds of PMCA. Unseeded reactions were run in parallel. Samples were processed for PrP^res isolation and analyzed by immunoblotting. A western-blotting positive control (cont) is included in each gel.</p

Evaluation of the infectivity present in platelets prepared from scrapie infected sheep by two different methods: PMCA and inoculation into tg338 mice (bioassay).

<p>Five susceptible VRQ/VRQ sheep were orally challenged with 2 g of brain homogenate (10^6.6 ID₅₀/g IC in tg<i>338</i> mice) between 6 and 10 months of age. The five VRQ/VRQ sheep respectively died at 198, 193, 198, 194 and 191days post inoculation (dpi). Classical scrapie was confirmed by histopathology (vacuolar change in central nervous system) and detection of abnormal PrP deposit in central nervous system and lymphoid tissues. At different time points, whole blood was collected from each donor and aliquots of platelets corresponding to 15 mL of plasma were prepared. Platelet homogenates (in 200μL) were inoculated in groups of six tg338 mice. Mice were monitored up to occurrence of TSE compatible clinical sign onset or killed at 250 days post inoculation. All mice were tested for presence of abnormal PrP deposition in brain. Incubation period in mice are presented in days (+/−SD). When less than 3 mice were positive, individual incubation period are given. In parallel the same homogenates were tested by PMCA (using tg338 mice brain homogenate as substrate). Each sample was run in 5 replicates for 2 successive rounds and the number of positive reactions is presented.</p

Cell-based assay of white blood cells infectivity from asymptomatic scrapie sheep.

<p>White blood cells from 5 infected sheep (D1 to D5) were isolated 80 days and 130 days post inoculation (dpi) when sheep were still asymptomatic. White blood cell homogenates (4×10⁷ cells) were inoculated to recipient ovRK13 cells. After 2 successive rounds of cell assay, the cultures were assayed for PrP^res by immunoblotting. PrP^res level is higher in cells infected with D3 130 dpi sample but its banding pattern is similar to that in cells infected with the other samples. M are molecular mass marker proteins (20, 30 and 40 kDa).</p

Evaluation of the infectivity present in white blood cells prepared from scrapie infected sheep by Cerebellar Organotypic Slice Culture Assay.

<p>Immunoblots of PK-treated slice culture homogenates probed with anti-PrP antibody Sha31, showing PrP^res accumulation in slice culture. (A) Cerebellar organotypic slices were prepared from tg338 pups and maintained in culture during 42 days <i>in vitro</i> after exposure to white blood cells prepared from blood collected from five scrapie infected sheep (D1, D2, D3, D4 and D5) at different times: 50 days post inoculation (dpi), 80 dpi, 130 dpi and at the terminal stage (180 dpi). For quantification purposes, slice cultures were also exposed to serial dilutions of PG127 scrapie-infected brain stock prepared from terminally ill tg338 mice, previously used <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104287#pone.0104287-ArellanoAnaya1" target="_blank">[31]</a>. To visualize low levels of PrP^res, membranes were exposed over-night (B).</p