Figure 2. Protein misfolding cyclic amplification (PMCA) of peripheral tissues from patients with variant Creutzfeldt-Jakob disease (vCJD). PMCA reactions were seeded with a 10-fold dilution series of vCJD tissue homogenates (10⁻²–10⁻⁹) obtained postmortem from CJD-infected patients. At least 4 replicates of each sample dilution were tested in 2 independent PMCA experiments. Patients vCJD-1 (A), vCJD-2 (B), vCJD-3 (C), and vCJD-4 (D) died of clinical vCJD. These 4 patients were infected with prions containing methionine at codon 129 of the PRNP gene (homozygote). Patient vCJD-A (E) died during an asymptomatic or preclinical stage of the disease. This patient was infected with a prion containing methionine/valine at codon 129 of the PRNP gene (heterozygote). PMCA substrate was prepared by using brains from transgenic mice overexpressing the ARQ variant of sheep prion protein. Unseeded reactions and a reaction seeded with tissues from 2 non–vCJD-infected control patients (NC-1 and NC-2; Table 3) were included as specificity controls. PMCAs were subjected to 4 rounds of amplification, each composed of 96 cycles (sonication for 10 s and incubation for 14.5 min at 39.5°C) in a Qsonica700 Sonicator (Qsonica LLC, Newtown, CT, USA). After each round, reaction products (1 vol) were mixed with fresh substrate (9 vol) to seed the following round. Part of the same product was analyzed by Western blotting for abnormal proteinase K–resistant prions (PrP^res) (Sha31 antibody epitope YEDRYYRE). For each round, the highest dilution showing a positive Western blot result in at least half of the replicates tested is indicated. Circles, round 1; ∇ round 2; Δ, round 3; squares, round 4. Neg, negative.