Volume 23, Number 6—June 2017
Distribution and Quantitative Estimates of Variant Creutzfeldt-Jakob Disease Prions in Tissues of Clinical and Asymptomatic Patients
|Amplification round||Reference vCJD 10% brain homogenate dilution series, no. of positive PMCA reactions/no. reactions conducted
*A 10% (wt/vol) homogenate was prepared by using frontal cortex from a symptomatic patient with vCJD (same homogenate as in Table 1). Samples were serially diluted 10-fold (10−2–10−10) before being used to seed PMCA reactions. Six individual replicates of each sample dilution were tested. PMCA substrate was prepared by using brains from transgenic mice overexpressing the ARQ variant of sheep prion protein. PMCAs were subjected to 6 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 volume) were mixed with fresh substrate (9 volumes) to seed the following round. Part of the same product was analyzed by Western blotting for abnormal PrPres by using Sha31 antibody epitope YEDRYYRE. Values are number of PrPres Western Blot–positive replicates corresponding to each round and each dilution. PMCA, protein misfolding cyclic amplification; PrPres, proteinase K–resistant prion; vCJD, variant Creutzfeldt-Jakob disease.