1. Defining and Assessing Analytical Performance Criteria for Transmissible Spongiform Encephalopathy–Detecting Amyloid Seeding Assays 
John G. Gray, Catherine Graham, Sandor Dudas, Eric Paxman, Ben Vuong, Stefanie Czub 
The Journal of Molecular Diagnostics 
Volume 18, Issue 3, Pages (May 2016)
DOI: /j.jmoldx
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2. Figure 1
ThT signal times from PrP conversion reactions seeded by CWD+ and CWD− elk brain tissue homogenate. Each data point represents a ThT signal time generated by a PrP conversion reaction when the reaction's fluorescence (480 nm) exceeded a threshold of 500 units (squares represent CWD+ seeded times; circles represent CWD− seeded times). Data are categorically organized by seed homogenate dilution. ROC curve identified cutoff times and statistics are listed on the right axis for each dilution set (AUC; P < 0.001); cutoff times are marked by double-headed arrows for each seed homogenate dilution. Reported cutoff times maintain 100% specificity (no cutoff available for the 1.00 × 10−1 dilution set due to inhibition); cutoff times are 100% sensitive, except for the 4.45 × 10−5 dilution set (*92% sensitive). The shaded area illustrates the range of 100% specific cutoff times. Six replicates were conducted on each CWD+ and CWD− brain dilution, on two amyloid-seeding assay trials (n = 12). AUC, area under curve; CWD, chronic wasting disease; NA, not applicable; PrP, prion-related protein; ROC, receiver-operating characteristic; ThT, thioflavin T.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
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3. Figure 2
ThT signal times for an elk brain homogenate pool previously titrated for CWD infectivity by way of Tg(CerPrP-M132)1536+/−/Tg(CerPrP-E226)5037+/− mouse bioassay. Black squares represent ThT signal times seeded by the titred CWD+ elk brain homogenate pool; the median ThT signal time for each CWD+ dilution is shown as a blue line over the data. Open circles represent the median ThT signal time for each dilution of the CWD− control elk brain homogenate pool. Specific values for CWD+ median ThT signal times are also provided in Table 1, along with the proportion of replicates fulfilling the 30-hour CWD+ criterion. For reference purposes, double-headed arrows denote individual dilution ROC curve cutoff times when 100% sensitivity and specificity could be achieved. The shaded area between the 30-hour CWD+ cutoff criterion and 49 hours represents the time range when ThT signals exude high CWD+ likelihood values but cannot be statistically confirmed as CWD+ (Supplemental Figure S2). Data were compiled from four independent amyloid-seeding assay trials, using two FLUOstar plate readers. This particular elk brain homogenate pool had previously been titrated for elk CWD infectivity in Tg(CerPrP-M132)1536+/− and Tg(CerPrP-E226)5037+/− mice and is reported in Bian et al.41 Data are expressed as median with error bars representing range of ThT signal times. ∗n = 12, †n = 24, ‡n = 36, §n = 48. CWD, chronic wasting disease; PrP, prion-related protein; ROC, receiver-operating characteristic; Tg, transgenic; ThT, thioflavin T.
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4. Figure 3
A DR curve analysis fit to the transformed CWD+ ThT signal time data from the amyloid-seeding assay versus Tg mouse bioassay sensitivity comparison in Figure 2. The response value of a data point is calculated such that earlier ThT signals receive higher response values. Open circles denote means ± SD response value, whereas black circles represent the median response value interquartile range. Both mean and median response values are provided to emphasize their proximity, indicating ThT signal times within each dilution are more-or-less normally distributed. The DR curve shown is fit to the median response values (R2 = 0.903). The calculated EC50 dilution of the titred CWD+ homogenate was 2.22 × 10−6 (dashed vertical line), with a corresponding half-maximal response value of 7.14 (arrow), which translates to a ThT-signal time of approximately 14 hours. The solid horizontal line is a reference response value corresponding to the 30-hour CWD+ cutoff time. CWD, chronic wasting disease; DR, dose-response; Tg, transgenic; ThT, thioflavin T.
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5. Figure 4
A comparison of TSE rapid test results on a dilution series of elk CWD+ brain homogenate serially diluted in a CWD− brain homogenate diluent. Western blot analysis lanes and graph data points are labeled according to dilution number; CWD+ content decreases within each dilution, as the label number/(mg−/mg+) value increases. A: Results for the Prionics Check WESTERN (1°Ab 6H4 [dilution 1:5000], 2°Ab goat anti-mouse AP-conjugated). The far left lane contains 5 μL of 1:10 MagicMark XP molecular weight marker. Dilutions where results varied in their interpretability among the four blot trials are underscored (dashed white line). The white arrow indicates the plausible end point detection limit among repeated blots. B: Bio-Rad TeSeE ELISA results. C: The results from the IDEXX HerdChek CWD EIA. Data points represent the median OD value for each dilution (± OD range). Dashed horizontal lines in B and C represent the TSE+ cutoff OD threshold. Detection limits for the Bio-Rad TeSeE ELISA and IDEXX HerdChek CWD EIA are indicated by the inset text boxes. n = 4 (B and C). Ab, antibody; AP, alkaline phosphatase; CWD, chronic wasting disease; EIA, enzyme-linked immunoassay; ELISA, enzyme-linked immunosorbent assay; TSE, transmissible spongiform encephalopathy.
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6. Figure 5
A comparison of amyloid-seeding assay sensitivity for elk CWD versus TSE rapid tests. Data are categorically organized in rows, such that CWD+ content decreases from top to bottom. Dilution numbers (and tissue ratios) correspond to those in Figure 4. ThT signal times for the CWD+ dilution sets are shown as black squares (n = 14 per dilution number), whereas the CWD− control/diluent data are shown as open circles in the bottom row (n = 24). The shaded area represents the zone where ThT signals no longer fulfill the 30-hour CWD+ cutoff time. The vertical dashed line (at 47 hours) marks a retest time cutoff, because ThT signals occurring up to approximately 47 hours maintain a CWD+ post-test probability of ≥90%. Median ThT signal times for all CWD+ dilution and the CWD− control/diluent homogenate are marked by blue lines. For reference purposes, TSE rapid test detection limits are marked by name alongside the corresponding tissue ratio/dilution number. CWD, chronic wasting disease; EIA, enzyme-linked immunoassay; ThT, thioflavin T; TSE, transmissible spongiform encephalopathy.
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7. Figure 6
A dose-response curve analysis for the ThT signal time data seeded by CWD+ elk brain dilutions used to compare amyloid seeding assay and transmissible spongiform encephalopathy rapid test CWD sensitivities. CWD+ dilution data from Figure 5 were transformed such that ThT signals occurring earlier receive higher response values. Open circles represent means ± SD response values, whereas black circles represent median response values interquartile range. Data points are labeled respective to the CWD+ dilution numbers as they appear in Figures 4 and 5. The dashed gray vertical line marks the EC50 (545 mg−/mg+) calculated from mean response values; the dashed black vertical line marks the EC50 calculated from median response values (750 mg−/mg+). The upper 95% CI estimate for the EC50 is marked by the vertical line (1845 mg−/mg+). The horizontal line marks a reference response value for the 30-hour CWD+ classifier cutoff time. CWD, chronic wasting disease; ThT, thioflavin T.
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8. Supplemental Figure S1
Raw ThT fluorescence signal data from PrP conversion reactions seeded by CWD+ and CWD− elk brain tissue homogenate. Data point shading corresponds to the dilution of a 10% (w/v) elk brain homogenate; darkest to lightest reflects most to least concentrated, respectively. The dashed horizontal line indicates the fluorescence threshold (500 units), whereby the ThT signal time is recorded. ThT signal times are plotted in Figure 1. CWD, chronic wasting disease; PrP, prion-related protein; ThT, thioflavin T.
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9. Supplemental Figure S2
Changes in assay sensitivity versus specificity over assay duration. Curves were generated after amalgamating ThT signal times from all dilution sets shown in Figure 1; this is meant to represent a general sensitivity/specificity change, over assay duration, which also accounts for sample varying CWD+ content. The shaded region represents a time range where sensitivity and specificity are both ≥95% (dashed horizontal line). The dashed vertical line marks where sensitivity and specificity intersect at 98% each. The CWD+ likelihood ratio is plotted where assay specificity is <100%. CWD, chronic wasting disease; ThT, thioflavin T.
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