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. 2017 Dec 8;7:17241. doi: 10.1038/s41598-017-17090-x

Figure 1.

Figure 1

PMCA of CWD prions. (A) To optimize CWD PrPSc amplification by PMCA and determine the limit of detection, brain extracts from CWD sick animals was serially diluted (10−4 to 10−13) in buffer and subjected to various consecutive rounds of 144 PMCA cycles. As negative controls, 5 tubes (C1 to C5) in which PMCA was done without CWD brain homogenate were used to control for possible cross-contamination. After each PMCA round, an aliquot of 10 µL was taken to analyze for PrPSc signal by western blot using the 6H4 anti-PrP antibody. All samples, except the normal brain homogenate (NBH), were treated with 10 µg/mL of PK for 1 h at 37 °C, before western blotting to differentiate PrPSc from PrPC. (B) Whole blood from a healthy deer was spiked with CWD brain homogenate at distinct final dilutions (10−6 to 10−11). The same dilutions were spiked in buffer (PBS) as control (right panel). After processing by high-speed centrifugation in the presence of sarkosyl (as described in Methods), samples were subjected to three consecutive rounds of PMCA. The PrPSc signal was assessed by Western blot analysis after PK digestion. NBH refers to the transgenic normal (healthy) brain homogenate, used as migration control marker. Dashed lines in some of the blots indicate splicing, done to remove unrelated lanes. Numbers in the right indicate the position of molecular weight markers.