FIG 6.

Specific probing of the V2^CJD strain in sCJD isolates by protein misfolding cyclic amplification. PMCA reactions were seeded with a 1/10 dilution series (10⁻¹ to 10⁻⁸ dilutions of 10% brain homogenate in PMCA buffer) corresponding to different sCJD patients and/or different brain areas from a single patient (Tables 1 and 3). PMCA reactions were subjected to 3 rounds of amplification, each comprising 96 cycles (10-s sonication for 14 min and 50-s incubation at 39.5°C) in a Qsonica700. This PMCA protocol allows amplification of the V2^CJD strain, but not the M1^CJD strain (Fig. 5). After three PMCA rounds, products were analyzed by Western blotting (WB) for the presence of abnormal PK-resistant PrP (PrP^res antibody Sha31 epitope YEDRYYRE). A type 1 (case 1) and type 2 (case 6) PrP^res were included as controls on each WB gel. For each sample, the WB shows only PrP^res presence/absence at dilutions bracketing the endpoint dilutions (Table 5).