Fig. 4
Amplification of MM2-sCJD prions by PMCA, using ovine PrPC as substrate. (a, b) Endpoint titration of tg338-passaged MM2-sCJD prions by a single round of PMCA. Brain (a) or spleen (b) homogenates from tg338 mice infected with MM2-sCJD prions were serially diluted in tg338 healthy brain lysate, as indicated, and submitted to one round of PMCA. Each amplified dilution was analyzed by immunoblotting (Sha31 antibody) for the presence of PrPres and characterization of electrophoretic pattern. Amplification of 127S prion seeds (21 kDa-PrPres) has been done in parallel for comparison [41]. (c) PrPres banding pattern in the brain and spleen tissue of tg338 mice inoculated with PMCA amplified products from tg338-passaged MM2-sCJD prions (10−8 dilution seed) and on subpassage (MM2 → PMCA). On 2nd passage, mice were challenged with either brain (Br) or spleen (Sp) extracts. The equivalent of 1 and 2 mg of brain and spleen tissue was loaded on the SDS-PAGE gels (Sha31 antibody). 127S PrPres is shown as control. (d) Endpoint titration by PMCA of uncloned (MM2) and cloned (Cl.MM2) tg338-passaged MM2-sCJD prions. The limiting dilution achieved was compared to that observed after PMCA reactions seeded with brain homogenates from tg338 mice inoculated with PMCA-amplified MM2-sCJD prions (2nd passage; MM2 → PMCA → tg338). (e) Amplification of human MM2-sCJD prions by PMCA, using tg338 healthy brain lysate as substrate. PMCA reactions were seeded with 103-diluted brain material from human PrP tg650 mice infected with uncloned (3rd passage; MM2-tg650) or cloned (4th passage; Cl.MM2-tg650) Three amplification rounds were performed. After each round, the amplified products were analyzed for PrPres content and electrophoretic pattern by immunoblot (Sha31 antibody) or diluted 1:10 in fresh substrate to seed the following round