sPMCA analysis of ruminant prions. Serial 10-fold dilutions of 10% (w/v) brain homogenate from clinically affected sheep with either scrapie or BSE (A and B as indicated), or cattle with clinical BSE (A and B as indicated) were seeded into sPMCA reactions (seeds contained 1,000 to 0.0001 nl of 10% brain homogenate per gel lane as indicated; reactions containing no PrP^Sc seed are also shown). Serial PMCA substrates were 10% (w/v) brain homogenates from TSE-free bovine or ovine sources. The latter were animals with either VRQ/VRQ (V/V) or ARQ/ARQ (A/A) PRNP genotypes (as indicated). Each sample underwent either four (A) or five (B) rounds of sPMCA and products were digested with proteinase K before analysis of 10 µl of each sample on western blots. PrP was detected with monoclonal antibodies SHA31 and P4 and molecular weight markers of 30 and 20 kDa are indicated. Brain homogenate seed were added directly into sPMCA reactions (A) or were first spiked 1 in 10 into unpasteurised whole milk (B). Milk has obtained from TSE-free animals and processed for sPMCA as described by Maddison and co-workers;1 all sPMCA reactions were also carried out as described previously.1 Bovine BSE was amplified and detected from 1 nl of brain homogenate seed and ovine BSE from 0.01 nl (lanes marked with an asterisk, A). The direct western blot analysis of 1 µl and 3 µl of the bovine BSE and ovine BSE 10% brain homogenates respectively produced detectable levels of PrP^Sc (data not shown) indicating amplifications of 1,000-fold and 300,000-fold for bovine BSE and ovine BSE respectively by sPMCA. Bovine BSE amplified by sPMCA in bovine brain homogenate substrate (C, BSE-b) and ovine BSE amplified by sPMCA in ovine brain homogenate substrate (C, BSE-o) both produced PK-resistant PrP^Sc fragments that displayed lower molecular weights than analogous fragments produced with ovine scrapie amplified by sPMCA in ovine brain homogenate substrate (C, Sc-o).