Bank vole prion protein extends the use of RT-QuIC assays to detect prions in a range of inherited prion diseases

Abstract

The cerebrospinal fluid (CSF) real-time quaking-induced conversion assay (RT-QuIC) is an ultrasensitive prion amyloid seeding assay for diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) but several prion strains remain unexplored or resistant to conversion with commonly used recombinant prion protein (rPrP) substrates. Here, bank vole (BV) rPrP was used to study seeding by a wide range of archived post-mortem human CSF samples from cases of sporadic, acquired and various inherited prion diseases in high throughput 384-well format. BV rPrP substrate yielded positive reactions in 70/79 cases of sporadic CJD [Sensitivity 88.6% (95% CI 79.5-94.7%)], 1/2 variant CJD samples, and 9/20 samples from various inherited prion diseases; 5/57 non-prion disease control CSFs had positive reactions, yielding an overall specificity of 91.2% (95% CI 80.1-97.1%). Despite limitations of using post-mortem samples and our results' discrepancy with other studies, we demonstrated for the first time that BV rPrP is susceptible to conversion by human CSF samples containing certain prion strains not previously responsive in conventional rPrPs, thus justifying further optimisation for wider diagnostic and prognostic use.

Figure 1

Comparisons between RT-QuIC reactions seeded by CSF samples from sCJD and vCJD cases, using either Hu rPrP or BV rPrP. Each curve is the mean with standard deviation of 4 replicate wells. (A) RT-QuIC reactions using BV rPrP (blue) and Hu rPrP (red) in 96-well format at 45 °C, seeded by vCJD CSF; BV rPrP showed a positive result (2/4) but with a long lag phase while Hu rPrP was negative (0/4). (B) Comparison of RT-QuIC reactions using BV rPrP, seeded by CSFs from sCJD (blue) and vCJD (red) cases at 45 °C; reactions seeded by sCJD CSFs showed the classic positive curves while that seeded by vCJD CSF replicated the delayed positive curve in (A).

Figure 2

Comparisons of reaction volumes in RT-QuIC reactions using BV rPrP in 384-well format. Reactions seeded by CSF samples from sCJD and vCJD at (A) total volume of 50  μL at 45 °C, (B) total volume of 75 μL at 45 °C and (C) total volume of 50 μL at 50 °C. Each curve is the mean with standard deviation of 4 replicate wells. This series of experiments showed that positive results can be replicated at lower reaction and analyte volumes; raising the temperature to 50 °C improved the efficiency of the reaction by reducing the lag phase and by producing a higher maximal rfu.

Figure 3

Sample traces of RT-QuIC reactions using BV rPrP in 384-well format. Reaction volumes of 50 μL at 50 °C seeded by CSF from (A) Alzheimer’s Disease (AD), (B) sCJD 129MM, (C) sCJD 129MV, (D) sCJD 129VV, (E) vCJD 129MV, (F) E200K, (G) E196K, (H) 6-OPRI and (I) P102L. Positive results were seen in sCJD with different PRNP codon 129 genotypes, vCJD and a range of IPD mutations, while the reaction seeded by AD CSF remained negative throughout. Individual coloured signal curves in each panel represent the signal curves of each well (of which there are 4) of the corresponding prion species/type indicated.

Figure 4

Estimation of CSF prion seeding doses. RT-QuIC reactions seeded by serial dilutions of sCJD 129MM brain homogenates at 50 °C ; each curve representing the average rfu readings across replicate wells (A), and dose response curve for estimation of prion seeding doses based on time to threshold lag phases (B). Estimation of CSF prion seeding doses were derived from the standard calibration curve (B) constructed from the lag phases (time to threshold rfu) of RT-QuIC reactions with BV rPrP seeded by serial dilutions of sCJD BH in (A). Data points in (B) represent the mean of 4 wells with standard errors of the mean.

Figure 5

Correlation of CSF prion seeding doses with functional decline and duration of illness. Correlation of the MRC Scale slope (percentage decline in function per day) with prion seeding dose by RT-QuIC (A), and Correlation of the clinical duration from symptom onset with prion seeding dose by RT-QuIC (B). No correlation was seen with rate of functional decline (A) but a positive correlation was found (r = 0.44, p = 0.0004) with total duration of illness. However the latter positive correlation is dependent on 2 extreme outliers with long durations of illness.