Figure 2. A) Semiquantitative densitometric analysis (optical density × area in mm2) of Western blot data (Figure 1, panel A, top panel), showing the amplification factors (+PMCA/−PMCA) obtained for all 4 seeds (bovine BSE, ovine scrapie, ovine BSE, and human vCJD in the PRNP-129MM substrate. B) Amplification of PrPd associated with ovine BSE (left) and ovine scrapie (right) from each of 3 different sheep in PRNP-129MM substrate as determined by Western blotting using MAb 3F4 to detect PrPres after limited proteinase K digestion. Substrate was seeded with brain homogenates prepared from sheep with confirmed scrapie and BSE such that each PMCA reaction mix contained an equivalent amount of PrPd according to detection of PrPres by Western blot titration after limited proteinase K digestion. PRNP-129MM substrate seeded with vCJD brain homogenate was included as a positive control in each experiment. C) Amplification of PrPd associated with ovine scrapie and BSE in substrates prepared from PRNP-129 methionine homozygous humanized transgenic mouse brain tissue (MM substrate) and NSB substrate. Substrates were prepared as 10% (wt/vol) homogenates in PMCA conversion buffer (10). Each substrate was seeded with brain homogenates prepared from sheep with confirmed scrapie and BSE so that each PMCA reaction mix contained an equivalent amount of PrPd as determined by detection of PrPres by Western blot titration after limited proteinase K digestion. Reaction mixes were divided into 2 lots: 1 was stored immediately at –80°C (−PMCA) and the other was subjected to 48 cycles of PMCA (+PMCA) by using standard conditions (10). After limited proteinase K digestion, PrPres in samples −/+PMCA was detected by Western blotting using MAb 6H4. PMCA, protein misfolding cyclic amplification; BSE, bovine spongiform encephalopathy; vCJD, variant Creutzfeldt-Jakob disease; MM, methionine homozygous; PrPd, disease-associated prion protein; MAb, monoclonal antibody; PrPres, protease-resistant prion protein; NSB, normal ARQ/ARQ sheep brain tissue. Values on the left in panels B and C are in kilodaltons.