Fig. 7
rSDS-PrPSc oligomers are detected in patients with new variant CJD (vCJD) when tested with RCA. a-b Frozen, homogenized brain samples from two patients with vCJD, one patient with sCJD (codon 129 M/M genotype) (positive control) and one healthy control (NBH) were from NIBSC. Each sample was identified by the number attributed by the NIBSC. The RCA assay was carried out as before (see legend to Fig. 5) but adapted to human samples: 50 μL of 10 % brain homogenates in 100 μL PBS/2 % Sarkosyl were incubated with 2 mM MR100 for 2 h. Before centrifugation, 30 μL was collected and mixed with 30 μL of 2X loading buffer for immunoblotting analysis (a). The rest of the sample was centrifuged at 11,000 g for 5 min, and supernatants (S) and pellets (P) were immunoblotted with the SAFmix (b) [16]. c-d For comparison, the same brain homogenates (50 μL of 10 % brain homogenates in 100 μL PBS/2 % Sarkosyl) were processed with the classical proteinase K digestion assay without MR100. Samples were analyzed before (c), and after proteinase K digestion (125 μg/mL PK at 37 °C for 1 h) (d). The reaction was stopped by addition of a protease inhibitor cocktail, before analysis of rPrPSc by western blotting with the SAF mix. Molecular masses (20–75 kDa) are on the left side of the panels