Figure 4. PMCA optimization for detection of macaque-adapted vCJD (m-vCJD) prions in preclinical blood samples. A) Tenfold serial dilutions of m-vCJD BH were amplified by the regular PMCA substrate (right panel) and substrate supplemented with 100 μg/mL heparin (left panel). After completion of 2 PMCA rounds, samples were digested with 50 μg/mL of PK and analyzed by Western blot. B) PL and BC (500 μL) samples collected 1 month before disease onset from 3 m-vCJD infected macaques (M1, M2, and M3) were sarkosyl precipitated and analyzed by 4 PMCA rounds using hep-substrate. Samples from noninfected macaques were analyzed as negative controls (–). N refers to transgenic mouse normal BH without proteinase K treatment, which was used as a migration control. BH, brain homogenate; PMCA, protein misfolding cyclic amplification; vCJD, variant Creutzfeldt-Jakob disease.