A 10% normal hamster brain homogenate (NBH) was prepared in phosphate buffered saline (PBS) containing 1% Triton X-100, 4mM EDTA and 1% protease inhibitor cocktail (Abcam). PrPspecific Mabs were generated against recombinant (murine or hamster) PrP or brain-derived proteinase K (PK)-resistant purified PrP Sc [35] from brains of clinical mice infected with the ME7 mouse-adapted scrapie strain or clinical hamsters infected with the 263K hamster-adapted scrapie strain. The Mabs used in this study were purified (Montage Antibody Purification kit; Millipore, Billerica, CA), isotyped (ELISA Mouse Antibody Isotyping kit; Thermo Fisher, Rockford, IL), and epitope mapped ( Table 1) . The immunoreactivity of all the Mabs were analyzed on western blots against denatured, PK-digested and undigested PrP derived from uninfected and infected brain homogenates as well as by ELISA against recombinant PrP. With the exception of Mab 3F4, each of the individual Mabs had equivalent immunoreactivity against murine and hamster PrP Sc on an immunoglobulin concentration basis. All of the Mabs were highly reactive against both hamster PrP C and PrP Sc isoforms and, for the PMCA studies, were individually added to the 10% NBH at a final concentrations of 50 mg/ml. A 10% 263K brain homogenate was prepared in PBS only and diluted to a final concentration of 10 24 . A 100 ml aliquot of this homogenate was initially combined with 10 ml of 10% NBH (with or without added Mab). Each sample was sonicated (QSONIC at 480W power, 60 Amplitude, 40,000 J energy, 90 sec process time, 3 sec pulse on21 sec pulse off), then incubated at 37uC for 1 hr. This was defined as one cycle of serial PMCA (sPMCA). At the completion of each cycle, an additional 10 ml of 10% NBH (with or without Mab) was added. At the end of every five cycles, 100 ml of the total volume was transferred to a new tube containing an equal volume of 10% NBH (with or without Mab) and the cycling reactions continued. At the completion of 40 cycles (sPMCA 40 ), 500 ml from each sample was PK-treated (100 mg/ml final concentration, 50uC, 30 min), followed by the addition of protease inhibitor cocktail. The sample was heated (100uC, 10 min) and then centrifuged at 16,0006g for 2 min at room temperature. The supernatant was combined with 6X Laemmli sample buffer, and 50 ml was electrophoresed in a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to nitrocellulose membrane. The membrane was blocked for 1 hr in PBS containing 0.1% Tween 20 (PBST) with 5% non-fat dry milk and incubated with 2 mg/ml biotinylated Mab 08-6/2F11. The membrane was washed 3 times (10 min each) with PBST, incubated for 60 min in HRP-conjugated streptavidin (Invitrogen) (1:5000 in PBST containing 5% non-fat dry milk) followed by 3 additional PBST washes and detection of proteins with ECL Supersignal West Dura kit (Thermo Fisher). Quantification of PrP Sc was performed by densitometric analysis using NIH Image J software.