Figure 3. Western blots of proteinase K–resistant prions (PrPres) in PMCA reactions seeded with peripheral tissues. PMCA reactions were seeded with a 10-fold dilution series (10−2–10−9) of variant Creutzfeldt-Jakob disease (vCJD) tissue homogenates that had been collected postmortem from vCJD patients during the clinical stage (symptomatic vCJD patient 1–vCJD patient 4 [P1–P4]) or at an asymptomatic or preclinical stage of the disease (vCJD asymp) (Table 2). Reactions seeded with tissues from 2 non-vCJD patients (Table 2) were used as controls, and an unseeded (lane U) reaction was included as a specificity control. Reactions were then subjected to 4 amplification rounds, 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). PMCA reactions were analyzed by using Western blotting for abnormal PrPres (Sha31 antibody epitope YEDRYYRE). A sheep scrapie sample and a vCJD reference isolate were used as controls. For the 7 tissues tested, the dilution of tissue homogenates used to seed the PMCA reactions is indicated below the immunoblots. Cont, control; WB, Western blot.