Figure 9. Analytical sensitivity of endogenous vCJD agent PMCA detection in the blood of affected patients.

1/10 dilution series (in PMCA buffer) were prepared using (A) WBC homogenate from one French vCJD affected patient and (B) buffy coat homogenates from two UK vCJD affected patients. These dilutions series were used to seed serial PMCA amplifications (up to 6 rounds) using brain homogenate from ovine PrP transgenic mouse (ARQ variant) as substrate. After each round, PMCA products were analyzed by Western Blot (WB) for the presence of abnormal PK resistant PrP (PrP^res - antibody Sha31 epitope YEDRYYRE). On each gel a classical scrapie isolate (PK digested) was used as positive control (WB control). (A) WBC homogenate from the French vCJD affected patient (Hu vCJD WBC) and healthy patients (H-WBC) were submitted to three amplification rounds. Each dilution was tested in duplicate. The equivalent whole blood amount used to seed the reactions is indicated in the figure. (B) PrP^res WB detection after the first (white circle), the second (grey circle), the third (black circle) and the sixth (white triangle) round of PMCA. Reactions were unseeded (no seed) or seeded with serial 1/10 dilution of the nine BC samples provided by the MRC unit (London, UK). (C) WBC from the French vCJD affected patient was tested either alone or after pooling with WBC from 11 (p12), 23 (p24), 47 (p48) or 95 (p96) healthy controls. The WBC homogenates used to prepare pools were equivalent to 50 µL of starting whole blood. Reactions seeded with WBC from healthy controls (H-WBC) were included as controls.