Properties of C-BSE, CWD, and vCJD amplification products in a second round of PMCA. Hu-C-BSE, hu-vCJD, and hu-CWD (from a previous round of PMCA) were supplemented with fresh human brain homogenate and subjected to a second round of PMCA. The reactions were normalized by PrPres level and the product diluted (1:3, 1:6, 1:12, 1:24) in fresh human brain homogenate (PRNP codon 129MM) before PMCA. Odd numbers correspond to samples without PMCA; even numbers correspond to the reactions after PMCA (A, B, and C). The PrP detection antibody was 3F4. C-BSE, C-type bovine spongiform encephalopathy; CWD, chronic wasting disease; vCJD variant Creutzfeldt-Jakob disease; PMCA, protein misfolding cyclic amplification; hu, human; PrPres, protease-resistant prion protein; M, molecular marker.