Fig 4. Optimisation of WB IOME RT-QuIC to determine cut-off time.

(A) Samples of whole blood from presymptomatic sheep (12–18 months post inoculation, mpi) that went on to develop clinical signs, and samples from symptomatic/clinical stage (terminal) BSE-infected (+ve) sheep typically tested positive by WB IOME RT-QuIC within 20 h, (B) including a dilution series of BSE-infected blood from one sheep (ID number: M200). The mean fluorescence from n = 10 negative controls (whole blood samples from mock-infected sheep) is plotted in black. Most negative controls did not display evidence of spontaneous fibrilisation until after 20 h (as indicated by vertical dotted lines). Each blood sample was tested in n = 4 replicate reactions. (C) Amyloid formation rates (1/t) were plotted for the aforementioned BSE-infected (+ve) and negative (-ve) control samples, over a period of 50 h. Rates for individual replicate reactions are plotted, including n = 32 prion positive reactions (plotted in red) and n = 40 negative control reactions (plotted in blue). Cut-off time was set at 20 h (equivalent to 1/t = 0.05, as indicated by horizontal dotted line) as positive replicates tended to amplify within this time frame (1/t ≥ 0.05), whereas most negative control reactions did not show evidence of spontaneous fibrilisation until after this time (1/t < 0.05). Although a small number of negative control reactions amplified before 20 h (3/40 reactions, in this example), this was only observed in 1/4 replicate reactions for a given blood sample. Thus, a positive sample was defined as one in which ≥ 50% (2/4) replicate reactions exceeded threshold fluorescence within a 20 h cut-off time.