Table 1.
Summary of patients with prion disease.
Fig 1.
Quantitation of seeding activity in brain tissue from a sporadic CJD patient using end-point RT-QUIC.
(a) BH (Pt 1) was diluted (5 × 10–5 to 5 × 10–9) and subjected to RT-QUIC reaction containing human recPrP substrate. The fluorescence of ThT was elevated at dilutions from 5 × 10–5 to 5 × 10–8. The 5×10–9 dilution yielded no reaction, as did a negative control consisting of A-CSF. (b) The percentage of positive reaction decreased in a sigmoidal curve within the dilution range when BH was used as the seed. The SD50 was calculated using the Spearman—Kärber method.
Fig 2.
End-point RT-QUIC analysis of 10 brain specimens from patients with prion diseases.
End-point RT-QUIC assay was performed three times. (a) Brain tissues from six patients with sCJD-MM1 were used to seed the RT-QUIC reaction. (b) Samples of GSS-P102L, sCJD-MV2, and sCJD-MM2 (cortical and thalamic forms) were used to seed RT-QUIC reaction.
Fig 3.
Correlation between SD50 and PrP-res in the brain.
(a) Human recPrP was serial diluted and tested by dot blotting. (b) A standard curve was constructed using diluted human recPrP. (c) Dot-blotting of BHs from patients with prion diseases. Pt 10 (MM2-thalamic form) had a very weak signal and fell below the limit of detection. (d) There was a linear correlation between SD50 and the level of PrPSc in nine patient’s brains (y = 1.281 × 10-10x, R2 = 0.7192). NC = Normal brain homogenate. y = value of PrP-res (μg/g brain). x = value of SD50/g brain
Fig 4.
Preparation of standard curves based on lag phase and aggregate area in RT-QUIC.
(a) Brain specimen from a patient with sCJD (patient 4) was subjected to serial three-fold dilution and RT-QUIC reaction, with four replicates for each dilution. (b) Standard curves (gray line) based on lag phase.
Fig 5.
Reduction of seeding activity by heat treatment.
(a) Brain from patient with sCJD-MM1 (patient 3) was treated at 121°C for 20 min or 60 min, and seeding activity was tested by end-point RT-QUIC. (b) Remaining SD50 after heat treatment. Black represents non-treated CJD-BH (patients 3–5 and 8–10). Horizontal stripes and white represents SD50 after heat treatment for 20 min and 60 min, respectively. Heat treatment caused reduction of SD50 (2.25 to 3.88 orders of magnitude). Data are presented as means ± standard deviation.