Figure 4. PrP^res detection in PMCA reactions seeded with white blood cells (WBC) from BSE infected and healthy sheep.

WBC from BSE orally challenged and TSE free control ARQ/ARQ sheep were homogenised and used to seed PMCA reactions. Brain homogenate from ovine PrP transgenic mouse (ARQ variant) was used as PMCA substrate. Each sample was submitted to up to 6 rounds of amplification. Resulting PMCA products were analyzed by Western Blot (WB) for the presence of abnormal PK resistant PrP (PrP^res -antibody Sha31 epitope YEDRYYRE). On each gel a classical scrapie isolate (PK digested) was used as positive control (WB control). (A) In BSE orally challenged sheep (ARQ/ARQ), WBC prepared from blood collected at different time points (indicated as months post inoculation: mpi) of the incubation period were tested. The first clinical signs developed at 20 mpi. (B) BSE affected sheep (3 different individuals-20 mpi) and TSE free controls sheep (breed, genotype and age matched) were submitted to up to 6 PMCA rounds to check the specificity of the amplification.