WO2016149537A1

WO2016149537A1 - Bank vole prion protein as a broad-spectrum substrate for rt-quic-based detection and discrimination of prion strains

Info

Publication number: WO2016149537A1
Application number: PCT/US2016/022945
Authority: WO
Inventors: Byron Winslow CAUGHEY; Christina Doriana ORRU; Bradley Richard GROVEMAN; Lynne DePuma RAYMOND; Romolo NONNO; Andrew Hughson; Kentaro Masujin
Original assignee: The United States Of America, As Represented By The Secretary, Department Of Health And Human Services; Istituto Superiore Di Sanita
Priority date: 2015-03-17
Filing date: 2016-03-17
Publication date: 2016-09-22

Abstract

In some embodiments, a method is disclosed for detecting a transmissible spongiform encephalopathy in a subject. In some embodiments, the method includes performing a prion protein amyloid seeding assay on a biological sample from the subject to detect PrP^D. In additional embodiments, a method is disclosed for discriminating whether a bovine is affected by classical or atypical L-type or H-type bovine spongiform encephalopathy, an ovine is affected by classical or atypical Nor98 scrapie, or a human is affected by sporadic or variant Creutzfeldt- Jakob disease.

Description

BANK VOLE PRION PROTEIN AS A BROAD-SPECTRUM SUBSTRATE FOR RT- QUIC-BASED DETECTION AND DISCRIMINATION OF PRION STRAINS

CROSS REFERENCE TO RELATED APPLICATION

This claims the benefit of U.S. Provisional Application No. 62/134,476, filed March 17,

2015, and U.S. Provisional Application No. 62/148,679, filed April 16, 2015. The prior applications are incorporated by reference in their entirety.

FIELD OF THE DISCLOSURE

This relates to the field of detection, specifically to a universal substrate, bank vole prion protein, that can be used for the detection of prions in a biological sample, and can be used to discriminate between classical and atypical L-type bovine spongiform encephalopathy in cattle, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt- Jakob disease in humans.

BACKGROUND

Prion diseases, or transmissible spongiform encephalopathies, are neurodegenerative disorders that include Creutzfeldt- Jakob disease (CJD), Gerstmann-Straussler-Scheincker syndrome (GSS), fatal familial insomnia (FFI) and sporadic fatal insomnia (sFI) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in cervids. The origin of prion diseases can be infectious, genetic or sporadic. Many prion diseases also have subtypes or strains that can be distinguished based on the PRNP (prion protein) genotype, transmission characteristics, clinical manifestations, neuropathological lesion profiles and/or biochemical properties of the disease- associated forms of prion protein (PrP^D). PrP^D, or a subset thereof, is the predominant molecular component of the infectious agent, or prion, which propagates itself by inducing misfolding of the hosts' normal protease- sensitive prion protein, PrP^c or PrP^Sen, into additional PrP^D. This propagation mechanism appears to involve seeded, or templated, polymerization in which the given PrP^D conformation is imposed upon normally monomeric PrP^Sen molecules as they are recruited into growing PrP^D multimers that can take the form of amyloids (Bessen RA et al. (1995) Nature 375: 698-700; Telling GC et al, Science 21 A: 2079-2082).

PrP^D usually includes forms called PrP^Res that, unlike the normal PrP^Sen, are partially resistant to digestion by proteinase K (PK). The banding pattern of PrP^Res in immunoblots can vary distinctively depending on the prion strain, host species and/or PRNP genotype. With most prion diseases the predominant 21-32-kDa variably glycosylated PrP^Res fragments observed on immunoblots extend from ragged N-termini between residues -80-96 to the GPI-anchored C- terminus (e.g., at residue 231 in humans). In contrast, the PrP^Res associated with sheep Nor98 scrapie and human GSS linked to the P102L, F198S, A117V and H187R PRNP mutations include much smaller 6-14 kDa bands (Monaco S, et al. (2012) PLoS One 7: e32382; Piccardo P, et al. (2001) Am. J. Pathol. 158: 2201-2207; Gotte DR, et al. (2011) PLoS One 6: e27510). These bands are internal fragments with ragged N-and C-termini within residues -80—160 (Pirisinu L et al. (2013) PLoS One 8: e66405). In cases of P102L GSS, brain tissue from some individuals can also give 21-32 kDa PrP^Res bands with the 7-8 kDa bands, while others give the 21-32 kDa PrP^Res bands but lack the 7-8 kDa bands. The former cases are referred to as GSS P102L* and the latter as GSS P102L.

A major challenge for the prion disease field is the development of sufficiently practical and sensitive tests for routine prion disease detection and strain discrimination in medicine, agriculture, wildlife management and research. The Real Time Quaking Induced Conversion (RT-QuIC) assay, which is based on PrP^D-seeded amyloid fibrillization of recombinant prion protein (rPrP^Sen), is highly specific and ultra-sensitive for detection of multiple human and animal prion diseases. Moreover, like the initially described prion protein "amyloid seeding assay" (Colby DW et al. (2007) Proc Natl Acad Sci USA 104: 20914-20919), RT-QuIC is more practical than comparably ultra- sensitive assays by being relatively rapid and based on a 96-well plate format with fluorescence readout (Atarashi R et al. (2011) Nature Medicine 17: 175-178). RT-QuIC assays improved on the initial amyloid seeding assay of Colby et al. (op. cit.) by the selection of reaction conditions that markedly retard potentially confounding non-specific, PrP^D- independent amyloid formation by rPrP^Sen (Wilham JM et al. (2010) PLoS Pathogens 6:

el001217; Atarashi R et al. (2011) Nature Medicine 17: 175-178). Appropriate combinations of prion strain and rPrP^Sen substrate have been important in the performance of various RT-QuIC assays (Wilham JM et al. (2010) PLoS Pathogens 6: el001217; McGuire LI et al. (2012) Annals of Neurology 72: 278-285; Orru et al., (2011) mBio 2(3):e00078-l l ; Cramm M et al. (2015) Mol Neurobiol 51: 396-405; Cramm M et al. (2015) Mol Neurobiol. (Epub ahead of print)). For several types of prion disease, however, no effective rPrP^Sen substrate has been identified; these types include human GSS arising from P102L*, F198S, Al 17V and H187R PRNP mutations and the atypical sheep scrapie strain Nor98. Moreover, no single substrate has yet been shown to detect all of the different prion variants of humans, cattle, sheep, cervids and rodents. SUMMARY OF THE DISCLOSURE

The most demanding and costly requirement for detection of prions in various prion protein amyloid seeding assays, for example RT-QuIC assays, is the availability of suitable recombination protease K sensitive (rPrP^Sen) substrates. Testing facilities have to produce or procure multiple rPrP^Sen sequences to be able to test for multiple prion types. It is disclosed herein that all of the prion diseases tested to date, from humans and other mammals, can be detected sensitively by using bank vole rPrP^Sen. Thus, bank vole rPrP^Sen provides a unique and rapid platform for broad-based prion detection and strain discrimination. The use of bank vole _rp_rpSen _aj_{so ena}bi_es discrimination of different disease causing prions (PrP^D), so that it can be determined which type of PrP^D is responsible for a diease in a specific subject of interest.

In some embodiments, a method is disclosed for detecting a transmissible spongiform encephalopathy in a subject. The method can include performing a prion protein amyloid seeding reaction on a biological sample from the subject to detect PrP^D. A biological sample from the subject is contacted with a recombinant bank vole protease sensitive prion protein (rPrP^Sen) to form a reaction mixture. The reaction mixture is incubated to permit coaggregation of PrP^D with the recombinant bank vole protease sensitive prion protein (rPrP^Sen). Incubation conditions are maintained that promote coaggregation of the recombinant bank vole protease sensitive prion protein (rPrP^Sen) with the PrP^D to result in a conversion of the recombinant bank vole sensitive prion protein (rPrP^Sen) to recombinant protease resistant and/or amyloid prion protein (rPrP-res^(Sc)), while inhibiting development of spontaneously forming (PrP^D- independent) recombinant protease resistant and/or amyloid prion protein (rPrP-res^(spon)). Any aggregates formed in the reaction mixture are agitated by shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking. In some embodiments, agitating aggregates comprises agitation in the absence of sonication. rPrP- res^(Sc) is the detected in the reaction mixture. The detection of rPrP-res^(Sc) in the reaction mixture indicates that the subject has the transmissible spongiform encephalopathy.

In additional embodiments, a method is disclosed for discriminating whether cattle are affected by classical or atypical L-type or H-type bovine spongiform encephalopathy, a sheep is affected by classical or atypical Nor98 scrapie in sheep, or a human is affected by sporadic or variant Creutzfeldt-Jakob disease.

In further embodiments, the method includes performing a prion protein amyloid seeding assay on a first biological sample from the host. The first assay includes contacting the first biological sample with a recombinant bank vole protease sensitive prion protein (BV rPrP^Sen) to form a first reaction mixture, and incubating the first reaction mixture to permit coaggregation of p_rp^D p_{resenl m 1η6} biological sample with the recombinant bank vole protease sensitive prion protein (BV rPrP^Sen). Incubation conditions are maintained that promote coaggregation of the recombinant bank vole protease sensitive prion protein (BV rPrP^Sen) with any PrP^D present in the biological sample, and result in a conversion of the recombinant bank vole protease sensitive prion protein to recombinant protease resistant and/or amyloid prion protein (rPrP-res^(Sc)) while inhibiting development of spontaneously forming (PrP^D-independent) recombinant protease resistant and/or amyloid prion protein (rPrP-res^(spon)). Aggregates formed during this incubation are agitated, optionally without sonication, wherein the reaction conditions comprise shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking. rPrP-res^(Sc) is detected in the first reaction mixture.

In some embodiments, the method also includes performing a second prion protein amyloid seeding reaction on a second biological sample from the subject. The method includes contacting the second biological sample with: 1) a recombinant Syrian golden Hamster protease sensitive prion protein (Hamster 90-231 rPrP^Sen) for cattle; 2) a chimeric hamster-sheep protease sensitive prion protein (Ha-S rPrP^Sen) for sheep; 3) a Syrian golden Hamster protease sensitive prion protein (Hamster 23-231 rPrP^Sen) for human 4) a sheep protease sensitive prion protein (sheep rPrP^Sen) to form a second reaction mixture, and incubating the second reaction mixture to permit coaggregation of PrP^D present in the second sample with the recombinant protease sensitive prion protein Hamster 90-231 rPrP^Sen (for bovine test samples) or with the Ha-S rPrP^Sen (ovine samples) or with the Hamster 23-231 rPrP^Sen (human samples) or with the Sheep rPrP^Sen (bovine samples). Incubation conditions are maintained that promote coaggregation of the above mentioned rPrP^Sens with the PrP^D to result in a conversion of these recombinant protease- sensitive prion protein prion proteins to recombinant protease resistant or thioflavin T-positive amyloid prion proteins (Hamster 90-231, Ha-S, Hamster 23-231 or Sheep rPrP-res^(Sc)) while inhibiting development of spontaneously forming recombinant protease resistant or thioflavin T- positive prion protein (rPrP-res^(spon)). Aggregates formed are agitated, optionally without sonication, wherein the reaction conditions include shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking. Detection of rPrP-res^(Sc) in the first reaction mixture but not the second reaction mixture indicates that the subject has atypical C-Type BSE (bovine samples), or atypical Nor98 scrapie (ovine samples) or variant CJD (human samples). Detection of rPrP-res^(Sc) in the first reaction mixture and the second reaction mixture indicates that the subject has BSE (bovine samples), or classical scrapie (ovine samples) or sporadic CJD (human samples). In some embodiments, the methods can be used to determine if a cow, sheep or a goat has C-BSE, H-BSE or L-BSE. The foregoing and other features and advantages of the invention will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. RT-QuIC detection of GSS P102L and lack of detection for GSS F198S, P102L* and sheep atypical Nor98 scrapie using hamster rPrP^Sen 90-231. Serial dilutions (10^" ⁶ to lO ⁹) of GSS P102L brain tissue dilutions were used to seed quadruplicate RT-QuIC reactions with hamster 90-231 _rPrP^Sen as the substrate, 300mM NaCl, 0.002% SDS. The same _rp_rpSen _an(j RT_Q_UIC conditions listed above were used in reactions seeded with the designated brain tissue dilutions of GSS human patients with F198S or P102L* PRNP point mutations, Alzheimer's disease (AD), sheep without prion disease or with Nor98 scrapie. Average ThT fluorescence readings from replicate wells for each type of sample were plotted as a function of time. Similar results were observed in at least 10 independent experiments using other human, hamster or hamster-sheep chimeric substrates (not shown).

Figures 2A-2D. Detection of GSS P102L, F198S and A117V PrP^D types by RT- QuIC using BV rPrP^Sen, 300mM NaCl and 0.001% SDS. Quadruplicate RT-QuIC reactions were seeded with 10^~4 dilutions of human frontal cortex brain tissue from GSS patients with the P102L, F198S, or Al 17V PRNP mutation. Negative control reaction were seeded with 10^"4 dilutions of frontal cortex brain tissue from a cerebral ischemia patient. A final SDS

concentration of 0.002% (A and B) or 0.001% (C and D) in combination with 130 mM (A and C) or 300 mM (B and D) NaCl were used with BV rPrP^Sen. Similar results were seen in three independent experiments. Traces from representative RT-QuIC experiments are the average of four replicate wells.

Figures 3A-3E. RT-QuIC sensitivity for detection of human GSS P102L, P102L*, A117V, F198S, and H187R seeding activity using BV rPrP^Sen. The designated dilutions of frontal cortex brain tissue from the designated GSS P102L (A), P102L* (B), A117V (C), F198S (D), and H187R (E) patients were used to seed RT-QuIC reactions with 0.001% SDS and 300mM NaCl. Negative control reactions were seeded with Alzheimer's disease (AD) brain tissue (A-E). Representative data from one of three independent experiments is shown as the averages of fluorescence values from four replicate wells.

Figure 4. RT-QuIC detection of 28 types of prion seeds from 5 different species using new BV rPrP^Sen substrate. RT-QuIC reactions were seeded with lO ⁴ brain tissue dilutions of the indicated human and animal prion types in the presence of 300mM NaCl and 0.001% SDS. Equivalent dilutions of species- and brain region-matched samples from uninfected individuals were used as specificity controls. Prion types that have been detected previously by RT-QuIC using other substrate are indicated in black, whereas those that have only been detectable using our selected set of conditions and BV PrP^Sen are indicated in red. The traces show the average fluorescence from four replicate wells. Similar data were obtained from a minimum of three independent experiments with each prion type.

Figure 5. Western blot of BV rPrP^Res products from RT-QuIC reactions seeded with various human prion types. Reaction products were digested with 10μg/mL of PK. Immunoblots were probed with the C-terminal antibody R20 (hamster PrP epitope residues 218- 231). Numbers on left indicate molecular mass in kilodaltons. Immunoblots are representitive of one biological replicate (n=total biological replicates tested). Each biological replicate was analyzed at least twice. ^(#) Samples for which cerebellum, frontal cortex, basal ganglia and thalamus from the same patient were analyzed. A minimum of two sets of RT-QuIC bank vole _rp_rpRes roducts were generated from a given prion type and independently subjected to immunoblotting analysis.

Figures 6A-6D. Western blots of BV rPrP^Res from RT-QuIC reactions seeded with sheep, bovine, cervine and rodent prion types. PK-treated RT-QuIC products from mouse (A), hamster (B), bovine (C), cervine (C) and ovine (D) prion seeds were probed with R20 (hamster PrP epitope residues 218-231). In (D), the classical scrapie-seeded reactions include those seeded with samples from PRNP VRQ/VRQ and ARQ/ARQ sheep (not designated). The Nor98-seeded reactions were seeded with samples from ARR/ARR, ARQ/AHQ and ARQ/ARQ sheep. RT-QuIC reactions and immunoblotting analysis for each of these types of prions were done twice with similar results. Immunoblots are representative of biological replicates (n) independently tested. Each biological replicate was assayed at least twice.

Figures 7A-7D. Detection and discrimination of Classical (C-BSE) and Atypical (L- type BSE). RT-QuIC reactions were seeded with 10^"4 brain tissue dilutions of brain stem (blue, C-BSE) or frontal cortex (L type-BSE) from Italian cattle. Negative control reactions (NBH) were seeded with 10^"4 dilutions of frontal cortex or brain stem from uninfected cattle. Ha (90- 231) _rPrP^Sen (300mM NaCl and 0.002% SDS; (A)-(B), Ha (23-231) or Hu (23-231) _rPrP^Sen

(300mM NaCl and 0.002% SDS; B), or BV (23-230) _rPrP^Sen (300mM NaCl and 0.001% SDS; (C) were used as substrates. RT-QuIC analysis was performed at least twice for each sample with similar results. Results are plotted as the averages from four replicate wells. (D) PK-treated RT-QuIC products from C were probed with R20 (hamster PrP epitope residues 218-231). Figures 8A-8F. Detection and discrimination of classical and Nor98 sheep scrapie with BV and Ha-S rPrP^Sen substrates. RT-QuIC reactions were seeded with dilutions of cerebellum or cerebral cortex from uninfected, classical or Nor98 atypical scrapie positive sheep. The Nor98 (ARR/AHQ, ARQ/ARQ, ARQ/AHQ and ARR/ARR PRNP genotypes) reactions were seeded with 10^"4 (light grey) brain tissue dilutions. Additional 10^"3 (dark grey) brain tissue dilutions are also shown for weaker samples. Classical sheep scrapie brain tissue from eight animals (ARQ/ARQ, VRQ/VRQ PRNP genotypes) was diluted 10^"4 (A and D). Equivalent dilutions of cerebellum or frontal cortex brain tissue dilutions (ARQ/ARQ and ARQ/ARR PRNP genotypes) were used as specificity controls (C and F). Either Ha-S rPrP^Sen (300mM NaCl and 0.002% SDS; A-C) or BV _rPrP^Sen (300mM NaCl and 0.001% SDS; D-F) were used as substrates. RT-QuIC analysis was performed at least twice for each sample with similar results. Results are plotted as the averages from four replicate wells.

Figures 9A-9D. RT-QuIC sensitivities for detection of classical and Nor98 scrapie using BV or Ha-S rPrP^Sen substrates. Brain homogenates from classical scrapie positive sheep (A and C, ARQ/ARQ) and atypical Nor98 scrapie positive sheep (B and D, VRQ/VRQ) were serially diluted (10 ⁴ to lO ⁸) for RT-QuIC analysis using Ha-S _rPrP^Sen with 300mM NaCl and 0.002% SDS (A and B) or BV _rPrP^Sen with 300mM NaCl and 0.001% SDS (C and D) substrates. RT-QuIC testing was performed independently twice with similar results. Traces show averages of quadruplicate wells.

Figure 10. Detection and discrimination of human sCJD and vCJD with hamster

23-231 and BV rPrP^Sen substrates. Dilutions (10^~4) of frontal cortex brain tissue from two confirmed sCJD (patient a and b) and two vCJD cases (patient c and d) were used to seed RT- QuIC reactions. Testing was performed using either hamster (23-231) in the presence of 300mM NaCl and 0.002% SDS (top panel) or BV PrP^Sen in the presence of 300mM NaCl and

0.001 %SDS (bottom panel). These samples were tested in three independent experiments with similar results. Traces represent the averages of four replicate wells.

Figures 11A-11D. RT-QuIC sensitivities for detecting sCJD and vCJD with hamster 23-231 and BV rPrP^Sen. Brain homogenates from one sCJD and one vCJD patient were serially diluted (10 ⁵ to 10^"8) for RT-QuIC analysis. Hamster 23-231 (A and B) and BV _rPrP^Sen (C and D) were used as a substrate. Data from reactions seeded with tissue from sCJD (patient a) and vCJD (patient c) are shown as the average fluorescence of quadruplicate replicate wells. Such testing was performed in three independent experiments with similar results.

Figure 12. PMCA will detect classical but not atypical Nor98 sheep scrapie seeding activity using bank vole brain homogenate as the substrate. No amplification of Nor98- seeded tubes was observed after 2 rounds of PMCA with 10% brain homogenates of bank vole 1091 or 109M, while classical scrapie was amplified after only one round as usual with bank vole 109M. The experiment was repeated several times with different Nor98 seeds and with similarly negative outcomes. Immunoblots were probed with mAb 9A2 (sheep PrP epitope residues 102-104).

Figure 13. RT-QuIC detection of seeding activity from PK-digested purified PrP^Res using bank vole _rPrP^Sen 23-230. Isolated PK-digested PrP^Res from E200K-, F198S- and Al 17V-GSS infected brain tissue as well as a mock purification (blue) were diluted 10⁶ fold and were used to seed quadruplicate RT-QuIC reactions with bank vole (23-230) rPrP^Sen as the substrate, 300mM NaCl, 0.001% SDS. Average ThT fluorescence readings from replicate wells for each type of sample were plotted as a function of time.

Figure 14 is Table 1. Diagnosis, genotype and brain regions for human samples. In this figure, the following annotations are used: ¹ Sporadic Creutzfeldt-Jakob disease; ² Variant Creutzfeldt- Jakob disease; ³ Iatrogenic Creutzfeldt-Jakob disease; ⁴ Genetic Creutzfeldt-Jakob disease; ⁵ Gerstmann-Straussler-Scheinker syndrome; ⁶ Fatal familial insomnia; ⁷ Sporadic fatal insomnia; ⁸ Chronic Wasting Disease, φ All French cases for which the source of prion contamination were growth hormone (7 patients) and dura matter graft (1 patient). ^#Samples from the same patient

Figure 15 is Table 2. Prion disease, Prnp genotype and brain region for animal samples† Sheep Prnp genotype at codons 136/154/171.

Figures 16A-16C. Detection of human (16A &16B), ovine and bovine (16C) prion seeding activity from brain. Prion seeding activity was detected from 10^~3 brain tissue dilutions from GSS (16A), variant and sporadic CJD (16B), classical and atypical sheep scrapie (16C), and classical and atypical BSE (16C) using bank vole 90-230 rPrP^sen substrate.

Figures 17A-17B. Sensitivity for detection for classical BSE and variant CJD. Ten fold serial dilutions of brain tissue (10^~3-10^~8) from a C-BSE infected cattle (16A) and a variant CJD infected human (16B) were tested by RT-QuIC using bank vole 90-230 rPrP^sen substrate.

Figures 18A-18C. Use of bank vole 90-231 for detection of human prion seeding activity from CSF. Prion seeding activity was detected from 20μΙ_^ of undiluted human CSF from patients with variant (18A), sporadic (18B), or genetic (18C) CJD or Fatal Familial Insomnia (18C) using bank vole 90-230 rPrP^sen substrate.

Figure 19. Western blot analysis of PrP^res in brain homogenate samples from cattle affected by C-BSE, atypical L- or H-BSE. The immunoblot was probed with an HRP- conjugated mAb T2. Lanes 1 and 2: uninfected cattle; Lanes 3 and 4: C-BSE (n=2); Lanes 5 to 7: L-BSE (n=3); Lanes 8 to 10: H-BSE (n=3). All the samples were digested with 40 μ^ηιΐ PK at 37 °C for 1 h. Molecular markers are shown on the left in kilodaltons (kDa).

Figure 20. RT-QuIC detection of C-BSE and atypical L- and H-type BSE prion

Sen

seeding activity using bank vole rPrP 23-230, M₁₀₉. Quadruplicate RT-QuIC reactions were seeded with 10 ⁵ dilutions of brain tissues from uninfected (n=2), C-BSE (n=2), L-BSE (n=3) and H-BSE (n=3) affected-cattle. A final SDS concentration of 0.001% in combination with

Sen

300mM NaCl was used with BV rPrP 23-230, M_log substrate. Similar results were observed in three independent experiments; representative RT-QuIC data is shown. Thioflavin T fluorescence measurements (the average of four replicate wells; y-axis) are plotted as a function of time (hours; x-axis).

Figure 21. RT-QuIC end-point dilution analyses of brain tissues from H-BSE-

Sen

affected cattle using bank vole rPrP 23-230, M₁₀₉. The designated dilution of brain tissue from H-BSE cattle #'s 1, 2 or 3 and uninfected cattle (n=2) were used to seed RT-QuIC reactions

Sen

in the presence of 0.001% SDS and 300mM NaCl using BV rPrP 23-230, M109 as the substrate. Each trace indicates the average fluorescence (y-axes) from four replicate wells seeded with the same brain homogenate dilution. Similar results were observed in two independent experiments. The calculated 50% seeding dose (SD₅₀) per mg of brain tissue is indicated for each brain samples tested.

Figures 22A-22L. RT-QuIC detection of C-, L- and H-type BSE prion seeding activity in brain samples using multiple rPrP^Sen substrates. Quadruplicate RT-QuIC reactions were seeded with 10^~4 brain tissue dilutions from uninfected, C-BSE, L-BSE and H-BSE affected-cattle in the presence of 0.001% SDS. A final concentration of either 300mM NaCl [BV _rPrPsen 23-230, Mio9 (A), BV _rPrP^Sen 90-230, M109 (B), BV _rPrP^Sen 23-230, 1109 (C), Hu-BV _rPrPsen 23-230, M109 (D) , Ha _rPrP^Sen 23-231 (G), Ha _rPrP^Sen 90-231 (H), Sh _rPrP^Sen ARQ 25- 234 (I), VRQ 25-234 (J), ARR 25-234 (K) and Ha-S _rPrP^Sen 23-234 (L)] or 130mM NaCl [Mo _rPrPsen 23-231 (E), Hu _rPrP^Sen 23-231 (F) ] was used. Traces from representative RT-QuIC experiments are shown as the average of ThT fluorescence (y-axes) from four replicate wells.

Figures 23A and 23B. Schematic for RT-QuIC based discrimination for C-, L- and H-type BSE. Decision tree illustrating the steps for RT-QuIC discrimination of C-, L- and H- type BSE strains. The table summarizes the possible outcome s of RT-QuIC testing using both BV 23-230, M109 and Sh ARR 25-234 _rPrP^Sen in the same plate. The graphs represent three examples of RT-QuIC kinetics observed using BV _rPrP^Sen 23-230, M109 and Sh ARR 25-234 _rPrPsen _{arrows m} table and the graphs indicate the shift in lag phase using Sh ARR relative to BV rPrP^Sen.

Figure 24. Detection and discrimination of C-, L- and H-type BSE prion seeding activity by RT-QuIC using BV rPrP^Sen 23-230, M109 and Sh rPrP^Sen ARR 25-234. Serial dilutions of brain homogenates (10^~3, 10^"4 and 10^"5) from three C-BSE-, three L-BSE-, and three H-BSE-affected cattle and one uninfected animal were tested by RT-QuIC. The same brain homogenate dilutions (circles, triangles and squares indicate individual animals) were used to seed reactions with either BV _rPrP^Sen 23-230, M109 (dark grey) or Sh _rPrP^Sen ARR 25-234 (light shade of grey). Representative data from at least two independent experiments are shown. Traces are the average fluorescence (y-axes) of four replicate wells.

Figures 25A and 25B. Western blot analysis of BV _rPrP^Res products from RT-QuIC reactions seeded with C-, L- and H-type BSE strains. Reaction products were digested with 10 μg/ml PK at 37°C for 1 h. BV rPrP^Res conversion products were detected using C-terminal antiserum R20 (hamster PrP epitope residues 218-231). Panel A: BV rPrP^Res conversion products from reactions seeded with uninfected (lanes 1 and 2, n=2), C-BSE (lanes 3 to 8, n=6), L-BSE (lanes 9 to 11, n=3), or H-BSE brain tissue dilutions (lanes 12 to 14, n=3). RT-QuIC and immunoblotting analyses was performed at least twice for each brain sample with similar results. Molecular markers are shown on the left in kilodaltons (kDa). Panel B: ImageQuant TL software densitometry quantification of the relative intensity of the lower (10 kDa: open bar) and the upper (12 kDa: solid black bar) bands of BV rPrP^Res products generated by seeding with C-, L- or H-type BSE brain homogenate dilutions. Results are represented as the mean + standard deviation (SD) from two independent experiments in which seeding activity from each of the three BSE strains was detected. The asterisks indicate statistically significant differences in the signal intensity of the 12kDa band between L-BSE and other BSE strains (Student's t test: *p< 0.001).

Figures 26A-26C. RT-QuIC analysis of CSF from C-, L- and H-type positive cattle using BV _rPrP^Sen 23-230, M109, BV _rPrP^Sen 90-230, M109 and Hamster _rPrP^Sen 90-231. CSF samples from two C-BSE, four L-BSE, two H-BSE-affected cattle and two uninfected cattle were tested by RT-QuIC. BV _rPrP^Sen 23-230, M109 (A), BV _rPrP^Sen 90-230; M109 (B) and Ha _rp_rps^en 90-231 were used as substrates in the presence of 0.002% SDS and 300mM NaCl.

Reactions were incubated at 42°C (BV _rPrP^Sen 23-230, M10 ) or 55°C (BV _rPrP^Sen 90-230, M109 and Ha rPrP^Sen 90-231). Traces are the average fluorescence (y-axes) of replicate wells. * Fractions indicate the number of positive wells out of the total number of replicate reactions for each sample. SEQUENCE LISTING

The nucleic and amino acid sequences listed are shown using standard letter

abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. For nucleic acid sequences, only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file [Sequence_Listing, March 17, 2016, 53,248 bytes], which is incorporated by reference herein. In the accompanying sequence listing:

SEQ ID NO: 1 is an amino acid sequence of amino acids 23 to 230 of bank vole prion protein (residue 109M), see also GENBANK® Accession No. AF367624.1, as available on July 31, 2008, which is incorporated herein by reference.

KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGTWGQPHGGGWGQPHGGGWGQPH GGGWGQPHGGGWGQGGGTHNQWNKPSKPKTNMKHVAGAAAAGAVVGGLGGYML GSAMSRPMIHFGNDWEDRYYRENMNRYPNQVYYRPVDQYNNQNNFVHDCVNITIKQ HTVTTTTKGENFTETDVKMMERVVEQMCVTQYQKESQAYYEGRS

This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below.

SEQ ID NO: 2 is an amino acid sequence of a recombinant Syrian golden hamster prion protein (leader sequence in bold).

MANLSYWLLALFVAMWTDVGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGG TWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHNQWNKPS KPKTN MKHMAGAAAAGAVVGGLGGYMLGSAMSRPMMHFGNDWEDRYYRENMNRYPNQV YYRPVDQYNNQNNFVHDCVNITIKQHTVTTTTKGENFTETDIKIMERVVEQMCTTQYQ KESQAYYDGRRS

SEQ ID NO: 3 is an amino acid sequence of a recombinant human (129M) prion protein, starting at residue 23 (23-231).

KKRPKPGG WNTGGSRYPG QGSPGGNRYP PQGGGGWGQP HGGGWGQPHG GGWGQPHGGG WGQPHGGGWG QGGGTHSQWN KPSKPKTNMK HMAGAAAAGA VVGGLGGYML GSAMSRPIIH FGSDYEDRYY RENMHRYPNQ VYYRPMDEYS NQNNFVHDCV NITIKQHTVT TTTKGENFTE TDVKMMERVV EQMCITQYER ESQAYYQRGS S

See also GENBANK® Accession No. M13899.1, January 8, 1995, which is incorporated herein by reference.

This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below. SEQ ID NO: 4 is an amino acid sequence of a recombinant human (129V) prion protein, starting at reside 23 (23-231).

KKRPKPGG WNTGGSRYPG QGSPGGNRYP PQGGGGWGQP HGGGWGQPHG GGWGQPHGGG WGQPHGGGWG QGGGTHSQWN KPSKPKTNMK HMAGAAAAGA VVGGLGGYVL GSAMSRPIIH FGSDYEDRYY RENMHRYPNQ VYYRPMDEYS NQNNFVHDCV NITIKQHTVT TTTKGENFTE TDVKMMERVV EQMCITQYER ESQAYYQRGS S

SEQ ID NO: 5 is an amino acid sequence of a full-length chimeric Hamster-Sheep (H-S) prion protein wherein residues 23-137 are of the Syrian hamster sequence and the remaining residues 138-231 were homologous to sheep residues 141-234 (R154,Q171 polymorph).

KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGTWGQPHGGGWGQPHGGGWGQPH GGGWGQPHGGGWGQGGGTHNQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYML GS AMSRPLIHFGND YEDRYYRENMYRYPNQVYYRPVDQYSNQNNFVHDCVNITVKQH TVTTTTKGENFTETDIKIMERVVEQMCITQYQRESQAYYQRGAS

SEQ ID NO: 6 is amino acid sequence of amino acids 23-231 of Hamster prion protein. KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGTWGQPHGGGWGQPHGGGWGQPH GGGWGQPHGGGWGQGGGTHNQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYML GSAMSRPMMHFGNDWEDRYYRENMNRYPNQVYYRPVDQYNNQNNFVHDCVNITIKQ HTVTTTTKGENFTETDIKIMERVVEQMCTTQYQKESQAYYDGRRS

SEQ ID NO: 7 is an amino acid sequence of amino acids 90-231 of Hamster prion protein.

GQGGGTHNQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPMMHFGN DWEDRYYRENMNRYPNQVYYRPVDQYNNQNNFVHDCVNITIKQHTVTTTTKGENFTE TDIKIMERVVEQMCTTQYQKESQAYYDGRRS

SEQ ID NO: 8 is an amino acid sequence 23-230 of Bank Vole prion protein (residue

1091): KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGTWGQPHGGGWGQPHGGGWGQPH GGGWGQPHGGGWGQGGGTHNQWNKPSKPKTNIKHVAGAAAAGAVVGGLGGYMLG SAMSRPMIHFGNDWEDRYYRENMNRYPNQVYYRPVDQYNNQNNFVHDCVNITIKQHT VTTTTKGENFTETDVKMMERVVEQMCVTQYQKESQAYYEGRSSRAVLLFSSPPVILLIS F

SEQ ID NO: 9 is an amino acid sequence of a recombinant ovine residues 25-234 (136A 154R 171Q) prion protein, residues 25-234; see GENBANK® Accession No. AJ567988, as available on April 15, 2015, incorporated herein by reference.

KKRPKP GGGWNTGGSR YPGQGSPGGN RYPPQGGGGW GQPHGGGWGQ PHGGGWGQPH GGGWGQPHGG GGWGQGGSHS QWNKPSKPKT NMKHVAGAAA AGAVVGGLGG YMLGSAMSRP LIHFGNDYED RYYRENMYRY PNQVYYRPVD QYSNQNNFVH DCVNITVKQH TVTTTTKGEN FTETDIKIME RVVEQMCITQ YQRESQAYYQ RGAS

SEQ ID NO: 10 is an amino acid sequence of a recombinant bovine (6-octarepeat) prion protein.

KKRPKP GGGWNTGGSR YPGQGSPGGN RYPPQGGGGW GQPHGGGWGQ

PHGGGWGQPH GGGWGQPHGG GWGQPHGGGG WGQGGTHGQW NKPSKPKTNM KHVAGAAAAG AVVGGLGGYM LGSAMSRPLI HFGSDYEDRY YRENMHRYPN QVYYRPVDQY SNQNNFVHDC VNITVKEHTV TTTTKGENFT ETDIKMMERV VEQMCITQYQ RESQAYYQRG AS

SEQ ID NO: 11 is the amino acid sequence of the mouse prion protein 23-231.

KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGTWGQPHGGGWGQPHGGSWGQPHG GSWGQPHGGGWGQGGGTHNQWNKPSKPKTNLKHVAGAAAAGAVVGGLGGYMLGS AMSRPMIHFGNDWEDRYYRENMYRYPNQVYYRPVDQYSNQNNFVHDCVNITIKQHTV TTTTKGENFTETDVKMMERVVEQMCVTQYQKESQAYYDGRRS

SEQ ID NO: 12 is the amino acid sequence of bank vole 90-230 109M GQGGGTHNQWNKPSKPKTNMKHVAGAAAAGAVVGGLGGYMLGSAMSRPMIHFGND WEDRYYRENMNRYPNQVYYRPVDQYNNQNNFVHDCVNITIKQHTVTTTTKGENFTET DVKMMERVVEQMCVTQYQKESQAYYEGRS

SEQ ID NO: 13 is the amino acid sequence of Bank vole 90-230 1091

GQGGGTHNQWNKPSKPKTNIKHVAGAAAAGAVVGGLGGYMLGSAMSRPMIHFGND WEDRYYRENMNRYPNQVYYRPVDQYNNQNNFVHDCVNITIKQHTVTTTTKGENFTET DVKMMERVVEQMCVTQYQKESQAYYEGRS

SEQ ID NO: 14 is the amino acid sequence of Human-BV Chimera (Human 23- 165[129M]/Bank vole 166-230)

KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPH GGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYML GSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPVDQYNNQNNFVHDCVNITIKQHT VTTTTKGENFTETDVKMMERVVEQMCVTQYQKESQAYYEGRS

SEQ ID NO: 15 is the amino acid sequence of BV-Human Chimera 109M (Bank vole

23-165/Human 166-231)

KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGTWGQPHGGGWGQPHGGGWGQPH GGGWGQPHGGGWGQGGGTHNQWNKPSKPKTNMKHVAGAAAAGAVVGGLGGYML GSAMSRPMIHFGNDWEDRYYRENMNRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQH TVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSS

SEQ ID NO: 16 is the amino acid sequence of BV-Human Chimera 1091 (Bank vole 23- 165/Human 166-231)

KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGTWGQPHGGGWGQPHGGGWGQPH GGGWGQPHGGGWGQGGGTHNQWNKPSKPKTNIKHVAGAAAAGAVVGGLGGYMLG SAMSRPMIHFGNDWEDRYYRENMNRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHT VTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSS

SEQ ID NO: 17 is the amino acid sequence of bank vole 1-230 (109M)

MANLSYWLLAFFVTTWTDVGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGG TWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHNQWNKPSKPKTN MKHVAGAAAAGAVVGGLGGYMLGSAMSRPMIHFGNDWEDRYYRENMNRYPNQVY YRPVDQYNNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCVTQYQ KES Q A YYEGRS

SEQ ID NO: 18 is the amino acid sequence of Human (129M) 1-231:

MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGG GGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTN MKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYR PMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERES QAYYQRGS

SEQ ID NO: 19 is the amino acid sequence of Sheep (ARQ) 1-234:

MVKSHIGSWILVLFVAMWSDVGLCKKRPKPGGGWNTGGSRYPGQGSPGGNRYPPQ GGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGGWGQGGSHSQWNKPSKP KTNMKHVAGAAAAGAVVGGLGGYMLGSAMSRPLIHFGNDYEDRYYRENMYRYPNQ VYYRPVDQYSNQNNFVHDCVNITVKQHTVTTTTKGENFTETDIKIMERVVEQMCITQY QRESQAYYQRGASVILFS SEQ ID NO: 20 is the amino acid sequence of mouse 1-231:

MANLGYWLLALFVTMWTDVGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGG TWGQPHGGGWGQPHGGSWGQPHGGSWGQPHGGGWGQGGGTHNQWNKPSKPKTNL KHVAGAAAAGAVVGGLGGYMLGSAMSRPMIHFGNDWEDRYYRENMYRYPNQVYYR PVDQYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCVTQYQKE SQAYYDGRRSS

SEQ ID NO: 21 is the amino acid sequence 25-234 of a recombinant ovine (136A 154R 171R) prion protein.

KKRPKPGGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQP HGGGWGQPHGGGGWGQGGSHSQWNKPSKPKTNMKHVAGAAAAGAVVGGLGGYML GSAMSRPLIHFGNDYEDRYYRENMYRYPNQVYYRPVDRYSNQNNFVHDCVNITVKQH TVTTTTKGENFTETDIKIMERVVEQMCITQYQRESQAYYQRGAS

This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below. SEQ ID NO: 22 is the amino acid sequence of Human-BV Chimera (Human 23-165 [129V]/Bank vole 166-230)

KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPH GGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYVLG SAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPVDQYNNQNNFVHDCVNITIKQHTV TTTTKGENFTETDVKMMERVVEQMCVTQYQKESQAYYEGRS

SEQ ID NO: 23 is the amino acid sequence of Human (129V) 1-231:

MANLGCWML VLF VAT WSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGG GGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTN MKHMAGAAAAGAVVGGLGGYVLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYR PMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERES QAYYQRGS SEQ ID NO: 24 is the amino acid sequence 1-234 of a recombinant ovine (136A 154R

171R) prion protein.

MVKSHIGSWILVLFVAMWSDVGLCKKRPKPGGGWNTGGSRYPGQGSPGGNR YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGGWGQGGSHSQWNK PSKPKTNMKHVAGAAAAGAVVGGLGGYMLGSAMSRPLIHFGNDYEDRYYRENMYRY PNQVYYRPVDRYSNQNNFVHDCVNITVKQHTVTTTTKGENFTETDIKIMERVVEQMCI TQ YQRES Q A Y YQRG AS

SEQ ID NO: 25 is an amino acid sequence 1-230 of Bank Vole prion protein (residue

1091):

MANLSYWLLAFFVTTWTDVGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGG TWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHNQWNKPS KPKTNI

KHVAGAAAAGAVVGGLGGYMLGSAMSRPMIHFGNDWEDRYYRENMNRYPNQVYYR

PVDQYNNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCVTQYQKE

SQAYYEGRSSRAVLLFSSPPVILLISF

SEQ ID NO: 26 is an amino acid sequence of a full-length chimeric Hamster-Sheep (H- S) prion protein wherein residues 23-137 are of the Syrian hamster sequence and the remaining residues 138-231 were homologous to sheep residues 141-234 (R154, R171 polymorph).

KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGTWGQPHGGGWGQPHGGGWGQPH

GGGWGQPHGGGWGQGGGTHNQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYML GSAMSRPLIHFGNDYEDRYYRENMYRYPNQVYYRPVDRYSNQNNFVHDCVNITVKQH

TVTTTTKGENFTETDIKIMERVVEQMCITQYQRESQAYYQRGAS

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

The Real Time Quaking Induced Conversion (RT-QuIC) assay is based on prion-seeded fibrillization of recombinant prion protein (rPrP^Sen). This type of assay provides highly specific and ultra- sensitive for detection of multiple human and animal prion diseases. Moreover, RT- QuIC is more practical than comparably ultra-sensitive assays because it is relatively rapid and utilizes a 96-well plate format with fluorescence readout. In RT-QuIC reactions prion- associated seeds induce amyloid fibril formation of bacterially expressed recombinant PrP^Sen (rPrP^Sen). The resulting rPrP^Res amyloid fibrils are then detected by the enhanced fluorescence of the amyloid-sensitive dye, thioflavin T (ThT). The most demanding and costly requirement for RT-QuIC testing is often the availability of suitable rPrP^Sen substrates. Testing facilities have to produce or procure multiple rPrP^Sen sequences to be able to test for multiple prion types.

However, using the methods disclosed herein, all mammalian prion disease types tested so far (n=28) can be detected sensitively by a single substrate, bank vole rPrP^Sen. The use of bank vole rPrP^Sen as a universal RT-QuIC substrate improves the practicality, efficiency and cost-effectiveness of ultra-sensitive prion detection and strain discrimination.

In addition, currently there are no rPrP^Sen substrates that can be used for the detection of several human prion disease types, for example Gerstmann-Straussler-Scheincker syndrome (GSS) types P102L*, F198S, A117V and H187R, nor is there a substrate that can be used to detect the atypical Nor98 scrapie strain in sheep. It is disclosed herein that a single substrate, bank vole PrP^Sen (BV rPrP^Sen), can be used to detect all of these diseases. In addition, the disclosed methods can be used to detect many different prion variants of humans, cattle, sheep, cervids and rodents. It was determined that recombinant bank vole rPrP^Sen, when expressed in E. coli and purified, can be used as a universally sensitive substrate for multiple prion strains from multiple species, and, most notably, for prions for which no effective substrate has been available. Furthermore, bank vole rPrP^Sen -based RT-QuIC reactions can be used for prion strain discrimination of classical and atypical L-type and H-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt- Jakob disease in humans (when no genetic prion disease is indicated by PRNP genotyping). Terms

Unless otherwise noted, technical terms are used according to conventional usage.

Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology , published by Blackwell Science Ltd., 1994 (ISBN 0-632- 02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

Aggregate: More than one molecule in association, such as dimers, multimers, and polymers of prion proteins, for instance aggregates, dimers, multimers, polymers and amyloid fibrils of protease resistant prion protein (e.g. PrP^D, PrP^Res, rPrP-res^(Sc), or rPrP-res^(spon)).

Agitation: Introducing any type of turbulence or motion into a mixture or reaction mix, for examples by sonication, stirring, or shaking. In some embodiments, agitation includes the use of force sufficient to fragment rPrP-res^(Sc) aggregates or amyloids, which disperses rPrP- _res(Sc) _agg_reg_ates and/or polymers to facilitate further amplification. In some examples fragmentation includes complete fragmentation, whereas in other examples, fragmentation is only partial, for instance, a population of aggregates can be about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% fragmented by agitation. Exemplary agitation methods are described in the Examples section below.

Amyloid: Fibrillar ultrastructure of protein aggregates that contains cross-beta structure and typically stains in characteristic ways with certain dyes such as thioflavin T (ThT). In the latter case, the fluorescence yield of the dye is enhanced by binding to amyloids. Many different proteins can form amyloids in association with a wide variety of diseases. However, the amyloids associated with mammalian prion diseases are formed strictly from the hosts' prion protein. Multiple amyloid forms of prion protein, such as rPrP-res^(Sc) or rPrP-res^(spon) can also be formed in vitro, and such forms are often distinguishable by conformation or other features from known forms of PrP^D.

Antibody: A polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen or a fragment thereof. An antibody can specifically bind PrP-res/PrP^Sc. Antibodies can be composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody.

The term antibody includes intact immunoglobulins and the variants and portions of them well known in the art, such as Fab' fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (for example, humanized antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3^rd Ed., W.H. Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (λ) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains"). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs". The extent of the framework region and CDRs have been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDRl is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds an antigen of interest has a specific VH region and the VL region sequence, and thus specific CDR sequences. Antibodies with different specificities (due to different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).

References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab. References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A "monoclonal antibody" is an antibody produced by a single clone of B -lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected, or a progeny thereof. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

Antibody binding affinity: Affinity of an antibody for an antigen, such as PrP-res. In one embodiment, affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol, 16:101-106, 1979. In another embodiment, binding affinity is measured by an antigen/antibody dissociation rate. In yet another embodiment, a high binding affinity is measured by a competition radioimmunoassay. In several examples, a high binding affinity is at least about 1 x 10^"8 M. In other embodiments, a high binding affinity is at least about 1.5 x 10^"8 M, at least about 2.0 x 10^"8 M, at least about 2.5 x 10^"8 M, at least about 3.0 x 10^" ⁸ M, at least about 3.5 x lO ⁸ M, at least about 4.0 x lO ⁸ M, at least about 4.5 x lO ⁸ M, or at least about 5.0 x 10^"8 M.

Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term "antigen" includes all related antigenic epitopes. "Epitope" or "antigenic determinant" refers to a site on an antigen to which B and/or T-cells respond. In one embodiment, T-cells respond to the epitope, when the epitope is presented in conjunction with an MHC molecule. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. An antigen can be a tissue-specific antigen, or a disease-specific antigen, such as PrP-res. These terms are not exclusive, as a tissue-specific antigen can also be a disease specific antigen.

Amyloid: Fibrillar ultrastructures of protein aggregates that, among several common characteristics, typically interact with the dye thioflavin T (ThT) to enhance the dye's fluorescence. Although many different proteins can form amyloids in association with a wide variety of diseases, the amyloids associated with mammalian prion diseases are formed strictly from the hosts' prion protein.

Bovine Spongiform Encephalopathy: A fatal neurodegenerative disease in cattle that causes a spongy degeneration in the brain and spinal cord. This disease has a long incubation period, about 30 months to 8 years, and all breeds of cattle are susceptible. The disease can be transmitted to human beings by eating food contaminated with the brain, spinal cord or digestive tract of infected carcasses. Cows affected by bovine spongiform encephalopathy will move apart from the herd and show progressively deteriorating behavioral and neurological signs, and often exhibit an increase in aggression. Affected animals react excessively to noise or touch and slowly become ataxic.

The incidence of classical BSE (C-BSE) has decreased as a result of disease-control programs such as the ruminant feed ban (Ducrot et al., 2008, Vet Res. 39:15). However, phenotypically atypical forms of BSE have been identified in several countries (Jacobs et al., 2007, J Clin Microbiol. 45: 1821-1829). These emerging bovine prion strains are H-type atypical BSE (H-BSE) or atypical L-type BSE (L-BSE). The atypical BSE strains can be differentiated biochemically by the electrophoretic mobility and glycoform pattern of PrP^Res after Proteinase K (PK) digestion (Casalone et al., 2004, Proc Natl Acad Sci U S A. 101:3065- 3070). The atypical bovine prion strains mainly affect older animals (Windl and Dawson, 2012, Subcell Biochem. 65:497-516.), are believed to be sporadic forms of bovine prion diseases (Brown et al., 2006, Emerg Infect Dis. 12:1816-1821). Classical and L-type BSE forms have been detected and discriminated by RT-QuIC using rPrP^Sen substrates that are different than the bank vole rPrP^Sen described herein (see also Orru et al., J Clin Microbiol. 2015 Apr;53(4):1115- 20). In some embodiments, the presently disclosed methods provide detection of C-BSE, L- BSE and H-BSE. In a Western blot analysis, the prions from the L-type form of BSE have a lower molecular mass than the prions from C-BSE, and the prions from the H-type form of BSE have a higher molecule mass then the prions from C-BSE. In some non-limiting examples, C- BSE prions have molecular masses of 27.560.5, 21.860.7 and 17.660.6 kDa for the di-, mono- and unglycosylated bands, respectively. The L-Type estimated molecular masses of the di-, mono- and unglycosylated moieties are all slightly lower (by ~1 kDa) than those of the C-type BSE. Thus, these molecular masses are approximately 26.360.6, 20.760.6 and 17.360.6 kDa. In the case of H-type the molecular masses are higher (by -1.5 kDa) for all three bands. Thus, the di-, mono- and unglycosylated forms have molecular weights of approximately 28.761.5, 23.361.2 and 19.361.2 kDa, respectively. (See Dudas et al., PLoS One 5: el0638, 2010, incorporated herein by reference).

Chronic Wasting Disease (CWD): A transmissible spongiform encephalopathy (TSE) of cervids, such as deer, elk, and moose. Most cases of CWD occur in adult animals. The disease is progressive and always fatal. The most obvious and consistent clinical sign of CWD is weight loss over time. Behavioral changes also occur in the majority of cases, including decreased interactions with other animals, listlessness, lowering of the head, lethargy, repetitive walking in set patterns, and a smell like meat starting to rot. In elk, behavioral changes may also include hyperexcitability and nervousness. Affected animals continue to eat grain, but may show decreased interest in hay. Excessive salivation and grinding of the teeth also are observed. Most deer also exhibit increased drinking and urination.

Creutzfeldt- Jakob disease: A transmissible spongiform encephalopathy of humans. The disease leads to rapid neurodegeneration, causing the brain tissue to develop microscopic holes and take a more sponge-like texture. The first symptom of CJD is rapidly progressive dementia, leading to memory loss, personality changes and hallucinations. Other frequently occurring features include anxiety, depression, paranoia, obsessive-compulsive symptoms, and psychosis. Physical problems occur, such as speech impairment, jerky movements (myoclonus), balance and coordination dysfunction (ataxia), changes in gait, rigid posture, and seizures.

There are several forms, including variant CJD (vCJC), sporadic CJD (sCJD), iatrogenic CJD, and familial CJD (fCJD). Iatrogenic CJD can be transmitted by contaminated harvested human brain products, immunoglobulins (IVIG), corneal grafts, dural grafts or electrode implants (acquired or iatrogenic form (iCJD). Genetic mutations in the PRNP gene causes familial

(fCJD), while sCJD appears subjects without germline PRNP mutations. Ten to fifteen percent of CJD cases are familial. It is believed that vCJD is caused by consumption of animal products including bovine prions; vCJD can also be transmitted by blood transfusions. There is no treatment for CJD.

Conservative variant: In the context of a prion protein, refers to a peptide or amino acid sequence that deviates from another amino acid sequence only in the substitution of one or several amino acids for amino acids having similar biochemical properties (so-called conservative substitutions). Conservative amino acid substitutions are likely to have minimal impact on the activity of the resultant protein. Further information about conservative substitutions can be found, for instance, in Ben Bassat et al. (J. Bacteriol , 169:751-757 ^', 1987), O'Regan et al. (Gene, 77:237-251, 1989), Sahin-Toth et al. (Protein Set , 3:240-247, 1994), Hochuli et al. (Bio/Technology, 6: 1321-1325, 1988) and in widely used textbooks of genetics and molecular biology. In some examples, prion protein variants can have no more than 1, 2, 3, 4, 5, 10, 15, 30, 45 conservative amino acid changes.

In one example, a conservative variant prion protein is one that functionally performs substantially like a similar base component, for instance, a prion protein having variations in the sequence as compared to a reference prion protein. For example, a prion protein or a conservative variant of that prion protein, will aggregate with PrP^Res (or PrP^Sc), for instance, and will convert rPrP^Sen to rPrP-res^(Sc) (or will be converted to rPrP-res^(Sc)). In this example, the prion protein and the conservative variant prion protein do not have the same amino acid sequences. The conservative variant can have, for instance, one variation, two variations, three variations, four variations, or five or more variations in sequence, as long as the conservative variant is still complementary to the corresponding prion protein.

In some embodiments, a conservative variant prion protein includes one or more conservative amino acid substitutions compared to the prion protein from which it was derived, and yet retains prion protein biological activity. For example, a conservative variant prion protein can retain at least 10% of the biological activity of the parent prion protein molecule from which it was derived, or alternatively, at least 20%, at least 30%, or at least 40%. In some preferred embodiments, a conservative variant prion protein retains at least 50% of the biological activity of the parent prion protein molecule from which it was derived. The conservative amino acid substitutions of a conservative variant prion protein can occur in any domain of the prion protein.

Contacting: "Contacting" includes in solution and solid phase, for example contacting a sample with a specific binding agent, such as an antibody that specifically binds PrP-res.

Conditions sufficient to detect: Any environment that permits the desired activity, for example, that permits an antibody to bind an antigen, such as PrP-res, and the interaction to be detected, or conditions that allow ThT to be detected. For example, such conditions include appropriate temperatures, buffer solutions, and detection means such as and digital imaging equipment.

Detect: To determine if an agent (such as a signal or protein, for example PrP-res) is present or absent. In some examples, this can further include quantification, for example the quantification of the amount of PrP^D in a sample, such as a nasal brushing, blood sample, serum sample, tissue sample, or a fraction of a sample. Diagnostic: Identifying the presence or nature of a pathologic condition, such as, but not limited to, identifying the presence of PrP^D or PrP^Res. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of true positives). The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the false positive rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis. "Prognostic" is the probability of development (for example severity) of a pathologic condition.

Disaggregate: To partially or complete disrupt an aggregate, such as an aggregate of p_rpRes _{or r}p_rp__res(Sc)

Encode: Any process whereby the information in a polymeric macromolecule or sequence is used to direct the production of a second molecule or sequence that is different from the first molecule or sequence. As used herein, the term is construed broadly, and can have a variety of applications. In some aspects, the term "encode" describes the process of semi- conservative DNA replication, wherein one strand of a double-stranded DNA molecule is used as a template to encode a newly synthesized complementary sister strand by a DNA-dependent DNA polymerase.

In another aspect, the term "encode" refers to any process whereby the information in one molecule is used to direct the production of a second molecule that has a different chemical nature from the first molecule. For example, a DNA molecule can encode an RNA molecule (for instance, by the process of transcription incorporating a DNA-dependent RNA polymerase enzyme). Also, an RNA molecule can encode a peptide, as in the process of translation. When used to describe the process of translation, the term "encode" also extends to the triplet codon that encodes an amino acid. In some aspects, an RNA molecule can encode a DNA molecule, for instance, by the process of reverse transcription incorporating an RNA-dependent DNA polymerase. In another aspect, a DNA molecule can encode a peptide, where it is understood that "encode" as used in that case incorporates both the processes of transcription and translation.

Fatal familial insomnia (FFI): A very rare autosomal dominant inherited prion disease.

It is almost always caused by a mutation to the protein PrP^c, but can also develop spontaneously in patients with a non-inherited mutation variant called sporadic fatal insomnia (sFI). FFI has no known cure and involves progressively worsening insomnia, which leads to hallucinations, delirium, and confusional states like that of dementia. The average survival span for patients diagnosed with FFI after the onset of symptoms is 18 months, with a range of 7 to 36 months. The disease has been found in 40 families worldwide, affecting about 100 people. If only one parent has the gene, the offspring have a 50% risk of inheriting it and developing the disease. The age of onset is variable, ranging from 18 to 60, with an average of 50. Genetic testing is advisable to avoid passing the mutation to offspring. The disease has four stages, taking 7 to 18 months to run its course:

Stage 1: The person has increasing insomnia, resulting in panic attacks, paranoia, and phobias. This stage lasts for about four months.

Stage 2: Hallucinations and panic attacks become noticeable, continuing for about five months.

Stage 3: Complete inability to sleep, followed by rapid weight loss. This lasts for about three months.

Stage 4: Dementia, during which the patient becomes unresponsive or mute over the course of six months. Death usually follows.

Other symptoms include profuse sweating, pinpoint pupils, sudden entrance into menopause (women), impotence (men), neck stiffness, constipation, elevated blood pressure and elevanted heart rate. Atrophy of the thalamus is one of the most common signs of fatal familial insomnia.

In humans, the gene encoding prion protein (PrP^c) is located on the short (p) arm of chromosome 20 at position pi 3. FFI patients and those with familial Creutzfeldt-Jakob disease carry a mutation at codon 178 of the PRNP gene. FFI is invariably linked to the presence of the methionine codon at position 129 of the mutant allele, whereas fCJD is linked to the presence of the valine codon at position 129. FFI (but not sFI) is also linked to a change of the amino acid at position 178 when an asparagine (N) is found instead of the normal aspartic acid (D)

(accompanied by the methionine at position 129).

Fluorophore: A chemical compound, which when excited by exposure to a particular stimulus, such as a defined wavelength of light, emits light (fluoresces), for example at a different wavelength (such as a longer wavelength of light). Fluorophores are part of the larger class of luminescent compounds. Luminescent compounds include chemiluminescent molecules, which do not require a particular wavelength of light to luminesce, but rather use a chemical source of energy. Therefore, the use of chemiluminescent molecules (such as aequorin) can eliminate the need for an external source of electromagnetic radiation, such as a laser. Thioflavin T is a fluorophore of use for the detection of amyloid and prions.

Examples of particular fluorophores that can attached to antibodies that specifically binds PrP^Sc are provided in U.S. Patent No. 5,866,366 to Nazarenko et al, such as 4-acetamido- 4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS), 4- amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4- anilino-l-naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumaran 151); cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'- isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid; 5-[dimethylamino]naphthalene-l-sulfonyl chloride (DNS, dansyl chloride); 4- dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5- (4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6- carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararos aniline; Phenol Red; B-phycoerythrin; o- phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1- pyrene butyrate; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X

isothiocyanate, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives; LightCycler Red 640; Cy5.5; and Cy56-carboxyfluorescein; 5- carboxyfluorescein (5-FAM); boron dipyrromethene difluoride (BODIPY); Ν,Ν,Ν',Ν'- tetramethyl-6-carboxyrhodamine (TAMRA); acridine, stilbene, -6-carboxy-fluorescein (HEX), TET (Tetramethyl fluorescein), 6-carboxy-X-rhodamine (ROX), Texas Red, 2',7'-dimethoxy- 4',5'-dichloro-6-carboxyfluorescein (JOE), Cy3, Cy5, VIC® (Applied Biosystems), LC Red 640, LC Red 705, Yakima yellow amongst others.

Other suitable fluorophores include those known to those skilled in the art, for example those available from Molecular Probes (Eugene, OR). In particular examples, a fluorophore is used as a donor fluorophore or as an acceptor fluorophore. In some examples, a fluorophore is detectable label, such as a detectable label attached to an antibody. Gerstmann-Straussler-Scheinker syndrome (GSS): A very rare, usually familial, fatal neurodegenerative disease caused by prions that affects patients from 20 to 60 years in age. Many symptoms are associated with GSS, such as progressive ataxia, pyramidal signs, and even adult-onset dementia; the symptoms progress as the disease progresses. GSS can be caused by a substitution at codon 102 from proline to leucine (P102L) in the prion protein gene (PRNP), encoded on chromosome 20, although other mutations are associated with GSS. However, it can also be caused by F198S, A117V and H187R mutations, and other point PRNP mutations. The trait is an autosomal-dominant trait. There is no cure for GSS, nor is there any known treatment to slow the progression of the disease. GSS is the slowest to progress among human prion diseases. The duration of GSS ranges from 3 months to 13 years, with an average duration of 5 or 6 years.

Immunoassay: A biochemical test that measures the presence or concentration of a substance in a sample, such as a biological sample, for example a nasal brushing or a blood sample, or a serum sample obtained from a subject, using the reaction of an antibody to its cognate antigen, for example the specific binding of an antibody to a protein, such PrP-res. Both the presence of antigen or the amount of antigen present can be measured. In some examples, the amount of PrP^Res is measured.

Immunoprecipitation (IP): The technique of precipitating a protein antigen out of solution using an antibody or peptides that specifically binds to that particular protein. These solutions will often be in the form of a crude lysate of an animal tissue. Other sample types could be body fluids or other samples of biological origin. Generally, in IP the antibody or peptides are coupled to a solid substrate at some point in the procedure.

Isolated: An "isolated" biological component, such as a peptide or assembly of polypeptides (for example PrP^Sc), cell, nucleic acid, or serum samples has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been "isolated" thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a cell as well as chemically synthesized peptide and nucleic acids. The term

"isolated" or "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, an isolated peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell. Preferably, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or even at least 99% of the peptide or protein

concentration.

Nucleic acid molecule: A polymeric form of nucleotides, which can include both sense and anti sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. A "nucleic acid molecule" as used herein is synonymous with "nucleic acid" and "polynucleotide." A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single and double stranded forms of DNA. A nucleic acid molecule can include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.

Prion: A type of infectious agent composed mainly of protein. All known prion diseases affect the structure of the brain or other neural tissue, and all are untreatable and fatal. The "transmissible spongiform encephalopathies (TSEs)" or prion diseases are fatal neurodegenerative disorders that include human Creutzfeldt-Jakob disease (CJD), Gerstmann- Straussler-Scheincker syndrome (GSS), fatal familial insomnia (FFI) and sporadic fatal insomnia (sFI), bovine spongiform encephalopathy (BSE), sheep scrapie, cervid chronic wasting disease (CWD), and transmissible mink encephalopathy (TME).

Prions are believed to infect and propagate by refolding abnormally into a structure that is able to convert normal molecules of the protein into the abnormally structured forms (PrP^D for disease-associated forms, also called PrP^Sc), PrP^BSE (for bovine spongiform encephalopathy) or PrP^vCJD (for variant CJD), which are usually partially resistant to proteinase K (PK) digestion, and hence will be designated generically herein as PrP-res or PrP^Res for PK-resistant. Most, if not all, known prions can polymerize into amyloid fibrils rich in tightly packed beta sheets. This altered structure often renders them unusually resistant to denaturation by chemical and physical agents, making disposal and containment of these particles difficult.

In prion diseases, the disease-associated or pathological form(s) of prion protein PrP^D or PrP^Sc, which is typically, but not always, partially protease-resistant (i.e., PrP^Res), appears to propagate itself in infected hosts by inducing the conversion of its normal host-encoded protease- sensitive precursor, called PrP-sen, PrP^Sen or PrP^c, which is sensitive to proteinase K digestion, into PrP^D. PrP^c (also called PrP^Sen or PrP-sen) is a monomeric

glycophosphatidylinositol-linked glycoprotein that is low in β-sheet content, and highly protease-sensitive. Conversely, PrP^D (and PrP^Sc) aggregates, such as PrP^Res aggregates, are high in β-sheet content and often partially protease-resistant. Mechanistic details of the conversion are not well understood, but involve direct interaction between PrP^D and PrP^c, resulting in conformational changes in PrP^c as the latter is recruited into the growing PrP^D multimer (reviewed in Caughey & Baron, Nature 443, 803-810, 2006). Accordingly, the conversion mechanism has been tentatively described as autocatalytic seeded (or nucleated) polymerization.

In the amyloid seeding assays disclosed herein, addition of a biological sample comprising PrP^D, PrP^Res, or prions results in the conversion of recombinant PrP^Sen (rPrP^Sen) to a recombinant protease-resistant and/or amyloid form of rPrP-res^(Sc) in a reaction mixture which can then be detected, such as using thioflavin T. Unlike natural PrP^c expressed in mammalian cells, bacterially expressed rPrP^c (rPrP^Sen) lacks glycans and the glycophosphatidylinositol anchor. rPrP-res^(Sc) is a generic term for the PrP^D-induced rPrP conversion product, regardless of the species and strain of origin of the prions. The recombinant protein aggregate, rPrP-res^(Sc), is usually not infectious, unlike naturally occurring PrP^Sc or PrP^D.

PMCA or Protein Misfolding Cyclic Amplification: A method for amplifying PrP^Res in a sample by mixing PrP^c or rPrP^Sen with the sample, incubating the reaction mix to permit p_rpRes _to ijjjjjate t e conversion of PrP^c or rPrP^Sen to aggregates of PrP-res or rPrP-res^(Sc), fragmenting any aggregates formed during the incubation step by sonication, and repeating one or more cycles of the incubation and fragmentation steps.

Polypeptide: A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L- optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The terms "polypeptide" or "protein" as used herein is intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term "polypeptide" is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.

The term "polypeptide fragment" refers to a portion of a polypeptide which exhibits at least one useful epitope. The term "functional fragments of a polypeptide" refers to all fragments of a polypeptide that retain an activity of the polypeptide. Biologically functional fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell.

QuIC or Quaking Induced Conversion: A particular type of PrP^D seed detection assay, in which shaking of the reaction vessels is performed instead of sonication to agitate the reaction. A "prion protein amyloid seeding assay" is an assay for PrP^D (such as PrP^Res) seeds that induce protease-resistant and/or amyloid rPrP-res^(Sc) formation from rPrP^Sen.

Real Time (RT)-QuIC: A type of QuIC assay that includes intermittent shaking without sonication to agitate the reaction and includes the use of a fluorescent readout, such as the fluorescent dye thioflavin T (ThT) to detect amyloid produced by a prion protein amyloid seeding assay. Exemplary protocols are disclosed, for example, in Wilham et al., PLOS Pathog. 6(12): el001217, pages 1-15. Generally, this assay uses rPrP^Sen as a substrate, intermittently shaken reactions, predominantly detergent- free (such as < 0.003% of SDS) or detergent-free, and chaotrope-free reactions conditions, and ThT-based fluorescent detections of prion-seeded recombinant PrP amyloid fibrils. The chaotrope-free reaction conditions distinguish RT-QuIC from the initially described amyloid seeding assay of Colby DW et al. (2007) Proc Natl Acad Sci USA 104: 20914-20919 and markedly retard potentially confounding non-specific, PrP^D- independent amyloid formation by rPrP^Sen [Wilham JM et al. (2010) PLoS Pathogens 6:

el001217; Atarashi R et al. (2011) Nature Medicine 17: 175-178]. Both QuIC and RT-QuIC can be used to detect PrP^D with amyloid seeding activity. PrP^D is detected by the production of ThT- reactive amyloid in this assay.

Sample: A biological sample obtained from a subject, such as a human or veterinary subject, which contains for example nucleic acids and/or proteins. As used herein, biological samples include all clinical samples useful for detection of PrP-res/prions in subjects, including, but not limited to, nasal brushings, saliva, cells, tissues, and bodily fluids, such as: blood;

derivatives and fractions of blood, such as serum; extracted galls; biopsied or surgically removed tissue, including tissues that are, for example, unfixed, frozen, fixed in formalin and/or embedded in paraffin; tears; milk; skin scrapes; surface washings; urine; sputum; cerebrospinal fluid; prostate fluid; pus; muscle; lymphoid tissues; olfactory mucosa; urine; feces; or bone marrow aspirates. The tissue can be any tissue of interest. In specific non-limiting examples, the tissue can be skin tissue or brain tissue. In particular embodiments, the biological sample is obtained from a cow, sheep or a goat, such as in the form of a blood sample. The sample can be a serum sample or nasal brushing. Samples also include environmental samples, such as soil or water samples.

Scrapie: A fatal, degenerative transmissible spongiform encephalopathy that affects the nervous systems of sheep and goats, but is not transmissible to humans. Changes are mild at first; slight behavioral changes and an increase in chewing movements may occur. Ataxia and neurological signs then develop, and affected sheep struggle to keep up with the flock. Some sheep scratch excessively and show patches of wool loss and lesions on the skin. Scratching sheep over the rump area may lead to a nibbling reflex, which is characteristic for the condition. Signs of a chronic systemic disease appear later, with weight loss, anorexia, lethargy, and possibly death. There are no treatments (or cures) for this disease. Thus, one of the most common ways to contain scrapie is to quarantine and destroy those affected. However, scrapie tends to persist in flocks and can also arise apparently spontaneously in flocks that have not previously had cases of the disease. Thus, diagnosis is critical.

Nor98 is also called "atypical scrapie" or "non-classical scrapie." This disease differs from classical scrapie in that it is not transmitted (or is very poorly transmitted) under natural conditions. Nor98 is rarely found in more than one animal in a flock. Nor98 is widely distributed in both sheep and goats, while classical scrapie is usually found in clusters and is primarily found in sheep. In Nor98, clinical signs are only rarely documented in younger animals; animals are usually diagnosed at slaughter at greater than 5 years of age. Some sheep genotypes are resistant to scrapie; all genotypes are susceptible to Nor98. Generally, depopulation and movement restriction is not required for Nor98 infected sheep and goats. (See the USDA website, such as

aphis.usda.gov/animal ealth/animal_diseases/scrapie/downloads/nor98-like_information.pdf).

Sequence identity: The similarity between two nucleic acid sequences or between two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences. Methods for aligning sequences for comparison are described in detail below, in section IV E of the Detailed Description.

Single Round: Performing a method wherein serial amplification is not performed. For example, rPrP-res^(Sc) can be amplified in a sample, by mixing the sample with purified rPrP^Sen to make a reaction mix; performing an amplification reaction that includes (i) incubating the reaction mix to permit coaggregation of the rPrP^Sen with the PrP^D that may be present in the reaction mix, and maintaining incubation conditions that promote coaggregation of the rPrP^Sen with the PrP^D and results in a conversion of the rPrP^Sen to rPrP-res^(Sc) while inhibiting development of rPrP-res^(spon) (protease-resistant rPrP products that are generated spontaneously in the absence of prions or PrP^D) (ii) agitating aggregates formed during step (i); (iii) optionally repeating steps (i) and (ii) one or more times. rPrP-res^(Sc) is detected in the reaction mix, wherein detection of rPrP-res^(Sc) in the reaction mix indicates that PrP^D was present in the sample. Additional substrate (rPrP^Sen) can be added during the reaction, such as during the lag phase (between the addition of the sample and the formation of detectable of rPrP-res^(Sc)). However, a portion of the reaction mix is not removed and incubated with additional rPrP^Sen in a separate reaction mixture. Sonication: The process of disrupting or dispersing biological materials using sound wave energy.

Specific binding agent: An agent that binds substantially only to a defined target. In some embodiments, a specific binding agent is an antibody that specifically binds PrPr^es but not PrP^c.

The term "specifically binds" refers to the preferential association of an antibody or other ligand, in whole or part, with an antigen. Specific binding may be distinguished as mediated through specific recognition of the antigen. Although selectively reactive antibodies bind antigen, they may do so with low affinity. On the other hand, specific binding results in a much stronger association between the antibody (or other ligand) and antigen (or cells bearing the antigen) than between the bound antibody (or other ligand) and another protein (or cells lacking the antigen). Specific binding typically results in greater than 2-fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody or other ligand (per unit time) to a cell or tissue expressing the target epitope as compared to a cell or tissue lacking this epitope. A variety of immunoassay formats are appropriate for selecting antibodies or other ligands specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Suitable methods and materials for the practice or testing of the disclosure are described below. However, the provided materials, methods, and examples are illustrative only and are not intended to be limiting. Accordingly, except as otherwise noted, the methods and techniques of the present disclosure can be performed according to methods and materials similar or equivalent to those described and/or according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification (see, for instance, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al, Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al , Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al, Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999).

I. Overview of Several Embodiments

Method of determining whether a subject has a transmissible spongiform

encephalopathy. These methods use bank vole rPrP^Sen as a substrate for an amplification reaction performed on a biological sample from the subject. In specific non-limiting examples, the Bank Vole PrP comprises the amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17 or SEQ ID NO: 25. In other non-limiting exmaples, a chimeric bank vole rPrP comprises the amino acid sequence set forth as SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 22. In some embodiments, the disclosed methods utilize the amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 22, optionally with an N-terminal methionine In other embodiments, the disclosed methods utilize the amino acid sequence set forth as SEQ ID NO: 17 or SEQ ID NO: 25, optionally without an N-terminal methionine.

The subject can be any subject of interest, including human and veterinary subjects. The subject can be, for example, a human, sheep, cow, goat, sheep, or cervid (such as deer, moose, elk and reindeer). In some embodiments, the subject is a human, and the transmissible spongiform encephalopathy is Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-

Scheincker syndrome (GSS), fatal familial insomnia (FFI) or sporadic fatal insomnia (sFI). In some example, these methods utilize a tissue sample, such as a skin sample.

In other embodiments, the subject is a cow, and the transmissible spongiform

encephalopathy is bovine spongiform encephalopathy (BSE). In further embodiments, the subject is a cervid, and wherein the transmissible spongiform encephalopathy is chronic wasting disease (CWD). In further embodiments, the subject is a sheep and the transmissible spongiform encephalopathy is classical or atypical scrapie, including Nor98 scrapie. In other embodiments, the subject is a human, and has, or is suspected to have Gerstmann-Straussler- Scheincker (GSS) P102L, F198S, Al 17V and/or H187R. In yet other embodiments, the subject is suspected of having CJD, such as sCJD, vCJD, or gCJD. The methods can be used to test biological samples, such as blood, plasma or serum samples, such as to determine they do not contain prions.

The subject can be suspected of having the transmissible spongiform encephalopathy. Alternatively, the subject can be diagnosed with the transmissible spongiform encephalopathy, and the methods disclosed herein can be used to confirm the diagnosis. In some examples, the subject is bovine, or ovine subject and the transmissible spongiform encephalopathy is classical or atypical bovine spongiform encephalopathy (BSE) or classical or atypical (e.g. Nor98) scrapie. In other examples, the subject is a human, and the transmissible spongiform

encephalopathy is GSS P102L, P102L*, F198S, Al 17V or H187R. Alternatively the subject is a human, and the transmissible spongiform encephalopathy is genetic CJD (gCJD), such as E200K, V210I or six octarepeat insertion gCJD. In other embodiments, the subject is a human, and the transmissible spongiform encephalopathy is FFI, such as caused by the D178N PRNP mutation, or sFI. In other examples, the subject is a human, and the transmissible spongiform encephalopathy is sporadic, variant, iatrogenic or genetic CJD. In further example, the subject is a cervid and the transmissible spongiform encephalopathy is chronic wasting disease. In yet other examples, the subject is a bovine, sheep or goat, and the transmissible spongiform encephalopathy is bovine spongiform encephalopathy.

In some embodiments, other assays are preformed, such as QuIC, RT-QuIC, PMCA, an electrophoretic mobility assay, or any other assay that indicates that the subject has the disease. In further embodiments, clinical symptoms are assessed.

In some embodiments, the method includes performing a prion protein amyloid seeding assay on a biological sample from the subject to detect PrP^D. A biological sample from the subject is contacted with a recombinant bank vole protease sensitive prion protein (rPrP^Sen) to form a reaction mixture. The reaction mixture is incubated to permit coaggregation of any disease-associated form of prion protein (called PrP^D when present in the biological sample) with the recombinant bank vole protease sensitive prion protein (rPrP^Sen). Incubation conditions are maintained that promote coaggregation of the recombinant bank vole protease sensitive prion protein (rPrP^Sen) with the PrP^D to result in a conversion of the recombinant bank vole sensitive prion protein to recombinant protease resistant and/or amyloid form of prion protein (rPrP- res^(Sc)) while inhibiting development of spontaneously forming recombinant protease resistant and/or amyloid form of prion protein (rPrP-res^(spon)).

Any aggregates formed in the reaction mixture are agitated by shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking. The period of rest and the period of shaking can be equal or unequal. In some embodiments, agitating aggregates comprises agitating aggregates in the absence of sonication. rPrP-res^(Sc) is then detected in the reaction mixture. The detection of rPrP-res^(Sc) in the reaction mixture indicates that the subject has the transmissible spongiform encephalopathy.

The method can include adding additional recombinant bank vole rPrP^Sen to the reaction mixture without removing rPrP-res^(Sc) prior to detecting the presence of rPrP-res^(Sc). In some embodiments, the additional recombinant bank vole rPrP^Sen is added to the reaction mixture without serial rounds of amplification. rPrP-res^(Sc) is then detected. In additional embodiments, the period of rest and the period of shaking are substantially equal. In other embodiments, the period of rest and the period of shaking are unequal.

Methods are also disclosed herein for discriminating whether a subject is affected with classical or atypical L-type bovine spongiform encephalopathy, a subject is affected with classical or atypical Nor98 scrapie, or a human subject is affected with sporadic or variant Creutzfeldt- Jakob disease.

A method of discriminating whether a sheep subject has atypical (Nor98) scrapie or classical scrapie. A first prion protein amyloid seeding assay is performed that includes contacting the first biological sample with a recombinant bank vole protease sensitive prion protein (rPrP^Sen) to form a first reaction mixture, and incubating the first reaction mixture to permit coaggregation of PrP^D present in the biological sample with the recombinant bank vole protease sensitive prion protein (rPrP^Sen). Incubation conditions are maintained that promote coaggregation of the recombinant bank vole protease sensitive prion protein (rPrP^Sen) with any p_rp^D p_{resenl m 1η6} biological sample, and result in a conversion of the recombinant bank vole protease sensitive prion protein to recombinant protease resistant and/or amyloid form of prion protein (rPrP-res^(Sc)) while inhibiting development of spontaneously forming recombinant protease resistant and/or amyloid form of prion protein (rPrP-res^(spon)). Aggregates formed during this incubation are agitated, wherein the reaction conditions comprise shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking. In some embodiments, the period of rest and the period of shaking are substantially equal. In other embodiments, the period of rest and the period of shaking are unequal. rPrP-res^(Sc) is detected the first reaction mixture.

The method also includes performing a second prion protein amyloid seeding assay on a second biological sample from the subject. The second assay includes contacting the second biological sample with a second recombinant protease sensitive prion protein (rPrP^Sen), wherein the second protease sensitive prion protein is a chimeric hamster-sheep, human, mouse, bovine or sheep protease sensitive prion protein to form a second reaction mixture, and incubating the second reaction mixture to permit coaggregation of PrP^D present in the second sample with the second recombinant protease sensitive prion protein (rPrP^Sen). Incubation conditions are maintained that promote coaggregation of the second recombinant protease sensitive prion protein (rPrP^Sen) with the PrP^D to result in a conversion of the second recombinant protease sensitive prion protein to a second recombinant protease resistant and/or amyloid form of prion protein (rPrP-res^(Sc)) while inhibiting development of spontaneously forming recombinant protease resistant and/or amyloid form of prion protein (rPrP-res^(spon)). Aggregates formed during step (viii) are agitated, wherein the reaction conditions comprise shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking. In some embodiments, the period of rest and the period of shaking are substantially equal. rPrP-res^(Sc) is detected in the second reaction mixture, wherein detection of rPrP-res^(Sc) in the first reaction mixture but not the second reaction mixture indicates that the subject has atypical (Nor98) scrapie, and wherein detection of rPrP-res^(Sc) in the first reaction mixture and the second reaction mixture indicates that the subject has classical scrapie. In some specific non-limiting examples the second recombinant protease sensitive prion protein is the recombinant hamster sheep protease sensitive prion protein, and the hamster sheep protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 5 or SEQ ID NO: 26, optionally with an N-terminal methionine. In other non-limiting examples, the second recombinant sensitive prion protein is the human protease sensitive prion protein, and the human protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 3, SEQ ID NO: 4, optioinally with an N-terminal methionine, or SEQ ID NO: 18 or SEQ ID NO: 23, optionally without the N-terminal methionine. In additional non-limiting examples, the second recombinant protease sensitive prion protein is the mouse protease sensitive prion protein, and the mouse protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 11, optionally with the N-terminal methionine, or the amino acid sequence set forth as SEQ ID NO: 20, optionally without the N-termainl methionine. In yet other non- limiting examples, the second recombinant protease sensitive prion protein is the sheep protease sensitive prion protein, and the sheep protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 9 or 21, optionally with an N-terminal methionine, or ii) the amino acid sequence set forth as SEQ ID NO: 19 or SEQ ID NO: 24, optionally without an N- terminal methionine. In further non-limiting examples, the second recombinant protease sensitive prion protein is the bovine protease sensitive prion protein and the bovine protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 10, optionally with an N-terminal methionine.

A method of discriminating whether a human subject, such as a subject that does not have a genetic PRNP disease mutation, is affected with variant Creutzfeldt-Jakob disease or sporadic Creutzfeldt-Jakob disease. A first prion protein amyloid seeding assay is performed that includes contacting the first biological sample with a recombinant bank vole protease sensitive prion protein (rPrP^Sen) to form a first reaction mixture, and incubating the first reaction mixture to permit coaggregation of PrP^D present in the biological sample with the recombinant bank vole protease sensitive prion protein (rPrP^Sen). Incubation conditions are maintained that promote coaggregation of the recombinant bank vole protease sensitive prion protein (rPrP^Sen) with any PrP^D present in the biological sample, and result in a conversion of the recombinant bank vole protease sensitive prion protein to recombinant protease resistant (or amyloid form of) prion protein (rPrP-res^(Sc)) while inhibiting development of spontaneously forming recombinant protease resistant and/or amyloid prion protein (rPrP-res^(spon)). Aggregates formed during this incubation are agitated, wherein the reaction conditions comprise shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking. In some embodiments, the period of rest and the period of shaking are substantially equal. In other embodiments, the period of rest and the period of shaking are unequal. rPrP-res^(Sc) is detected the first reaction mixture.

A second prion protein amyloid seeding assay is also performed on a second biological sample from the subject. The second biological sample is contacted with a second recombinant protease sensitive prion protein (rPrP^Sen), wherein the second protease sensitive prion protein comprises amino acids 23-213 of a hamster protease sensitive prion protein, to form a second reaction mixture. Incubation conditions are maintained that promote coaggregation of the second recombinant protease sensitive prion protein (rPrP^Sen) with the PrP^D to result in a conversion of the second recombinant protease sensitive prion protein to a second recombinant protease resistant and/or amyloid form of prion protein (rPrP-res^(Sc)) while inhibiting development of spontaneously forming recombinant protease resistant prion protein (rPrP- res^(spon)). Aggregates formed during step (viii) are agitated, wherein the reaction conditions comprise shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking. In some embodiments, the period of rest and the period of shaking are substantially equal. rPrP-res^(Sc) is detected in the second reaction mixture. Detection of rPrP-res^(Sc) in the first reaction mixture but not the second reaction mixture indicates that the subject has variant Creutzfeldt-Jakob disease, and detection of rPrP- res^(Sc) in the first reaction mixture and the second reaction mixture indicates that the subject has sporadic Creutzfeldt-Jakob disease. In specific, non-limiting examples, the second

recombinant protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 6 or SEQ ID NO: 7, optionally with an N-terminal methionine. In another embodiment, the second recombinant protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 2, optionally without an N-terminal methionine.

The C-BSE epidemic in cattle, and its causation of the vCJD in humans, have had serious impacts on both the cattle industry and human health. The incidence C-BSE has been virtually eliminated by effective, albeit costly, measures to minimize its propagation within cattle, and zoonotic transmission from cattle into humans. However, the recent appendix-based evidence for widespread subclinical prion infections of humans (Gill ON, Spencer Y, Richard-Loendt A, et al. BMJ 347:f5675) raises the specter of longer-term consequences of the C-BSE epidemic. At the same time, the likelihood that atypical forms of BSE can arise spontaneously in cattle, as apparently occurs with sporadic CJD in humans, suggests that continuous surveillance for H- and L-BSE, coupled with appropriate containment measures, will be required to prevent outbreaks in cattle and mitigate risks to humans and other species. The current commercially available "rapid tests" that are approved for postmortem BSE surveillance are indeed practical and effective at identifying the many problematic cases of classical or atypical BSE-infected cattle that might indicate a focus of bovine prion disease or threaten the human food supply. However, these tests are not sensitive enough to detect all potential sources of BSE infection because they are orders of magnitude less sensitive than bioassays for BSE infectivity (Gray JG, Dudas S, Graham C, et al. J Vet Diagn Invest 24:976-980; Meloni D, Davidse A, Langeveld JP, et al. PLoS One 7:e43133; Safar JG, Scott M, Monaghan J, et al. Nat. Biotechnol. 20:1147- 1150; and Buschmann A, Groschup MH. J. Infect. Dis. 192:934-942). Thus, highly sensitive and practical tests are needed for the detection and discrimination of each of the established strains of BSE in cattle.

In some embodiments, methods are disclosed herein for distinguishing whether a subject has atypical L-type bovine spongiform encephalopathy or classical bovine spongiform encephalopathy. The subject can be a cow, sheep or a goat. In some embodiments, the method includes performing a first prion protein amyloid seeding assay on a first biological sample from the subject. The first assay includes contacting the first biological sample with a chimeric recombinant bank vole protease sensitive prion protein to form a first reaction mixture. The first reaction mixture is incubated to permit coaggregation of BSE-associated prion protein (e.g. p_rp^BSE) p_{resenl m 1η6} biological sample with the recombinant bank vole sensitive prion protein (rPrP^Sen), and incubation conditions are maintained that promote coaggregation of the recombinant bank vole rPrP^Sen with the PrP^BSE to result in a conversion of the recombinant bank vole rPrP^Sen to recombinant protease resistant (or amyloid prion protein) (rPrP-res^(Sc)) while inhibiting development of spontaneously forming recombinant protease resistant (or amyloid prion protein) (rPrP-res^(spon)). Aggregates formed in the first reaction mixture are agitated by shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking. rPrP-res^(Sc) is then detected in the first reaction mixture. In some embodiments, the period of rest and the period of shaking are substantially equal.

The methods also include performing a second prion protein amyloid seeding assay on a second biological sample from the subject. The second assay includes: contacting a second biological sample from the subject with a second recombinant protease sensitive protein to form a second reaction mixture, wherein the second protease sensitive prion protein (rPrP^Sen) second protease sensitive prion protein is a hamster or a human protease sensitive prion protein. The second reaction mixture is incubated to permit coaggregation of PrP^BSE present in the second sample with the second rPrP^Sen. Incubation conditions are maintained that promote

coaggregation of the second rPrP^Sen with the PrP-res to result in a conversion of the second _rp_rpSen _to recombinant second protease resistant (or amyloid prion protein) (rPrP-res^(Sc)) while inhibiting development of spontaneously forming recombinant protease resistant prion protein (rPrP-res^(spon)). Aggregates formed in the second reaction mixture are agitated, wherein the reaction conditions include shaking the second reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking. In some embodiments, the period of rest and the period of shaking are substantially equal. rPrP-res^(Sc) is detected in the second reaction mixture.

The detection of rPrP-res^(Sc) in the first reaction mixture indicates that the subject has bovine spongiform encephalopathy. The detection of rPrP-res^(Sc) in the second reaction mixture indicates that the subject has atypical L-type bovine spongiform encephalopathy, and the absence of rPrP-res^(Sc) in the second reaction mixture indicates that the subject has classical bovine spongiform encephalopathy. In specific non-limiting examples, the second recombinant protease sensitive prion protein is the human protease sensitive prion protein, and the human protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 3, SEQ ID NO: 4, optionally with an N-terminal methionine, or the amino acid sequence set forth as SEQ ID NO: 18 or SEQ ID NO: 23, optionally without the N-termainl methionine. In other specific non- limiting examples, the second recombinant protease sensitive prion protein is the hamster protease sensitive prion protein, and the hamster protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 2, optionally without the N terminal methionine, orthe amino acid sequence set forth as SEQ ID NO: 6 or SEQ ID NO: 7, optionally with an N-terminal methionine.

In additinal embodiments, methods are also disclosed for determining whether a subject that has bovine spongiform encephalopathy has atypical L-type bovine spongiform

encephalopathy (L-BSE), atypical H-type bovine spongiform encephalogpathy, or classical bovine spongiform encephalopathy (C-BSE) comprising. The subject can be a cow, sheep or a goat. These methods include contacting a first biological sample with a recombinant bank vole protease sensitive prion protein (rPrP^Sen) to form a first reaction mixture, and incubating the first reaction mixture to permit coaggregation of disease-associated prion protein (PrP^D) present in the biological sample with the recombinant bank vole protease sensitive prion protein (rPrP^Sen). Incubation conditions are maintained that promote coaggregation of the recombinant bank vole protease sensitive prion protein (rPrP^Sen) with the PrP^D present in the sample to result in a conversion of the recombinant bank vole protease sensitive prion protein to recombinant protease resistant and/or amyloid prion protein (rPrP-res^(Sc)) while inhibiting development of spontaneously forming recombinant protease resistant and/or amyloid prion protein (rPrP- res^(spon)). Aggregates formed during this incubation are agitated, optionally without sonication, wherein the reaction conditions comprise shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking. In some embodiments, the period of rest and the period of shaking are substantially equal. rPrP-res^(Sc) is detected in the first reaction mixture. Any rPrP-res^(Sc) formed in the first reaction mixture is detected.

These methods also include performing a second prion protein amyloid seeding assay on a second biological sample from the subject. The second prion proten amyloid seeding assay includes contacting the second biological sample with a second recombinant protease sensitive prion protein (rPrP^Sen), to form a second reaction mixture, and incubating the second reaction mixture to permit coaggregation of any PrP^D present in the second sample with the second recombinant protease sensitive prion protein (rPrP^Sen). Incubation conditions are maintained that promote coaggregation of the second recombinant protease sensitive prion protein (rPrP^Sen) with the PrP^D to result in a conversion of the second recombinant protease sensitive prion protein to a second recombinant protease resistant and/or amyloid prion protein (rPrP-res^(Sc)) while inhibiting development of spontaneously forming recombinant protease resistant and/or amyloid prion protein (rPrP-res^(spon)). Aggregates formed during this incubation are agitated, optionally without sonication, wherein the reaction conditions comprise shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking. In some embodiments, the period of rest and the period of shaking are substantially equal. rPrP-res^(Sc) is detected in the second reaction mixture. rPrP-res^(Sc) formed in the second reaction mixture is detected. Detection of rPrP-res^(Sc) in the first reaction mixture but not the second reaction mixture indicates that the subject has classical BSE. Detection of rPrP-res^(Sc) in the first reaction mixture and the second reaction mixture indicates that the subject has either L- type BSE or H-type BSE.

The method can also include quantitating the rPrP-res^(Sc) in the first reaction mixture and quantitating the rPrP-res^(Sc) in the second reaction mixture. In some embodiments, detecting the presence of rPrP-res^(Sc) in the first sample and the second biological sample includes the use of thioflavin T (ThT). In specific non-limiting examples, the first reaction mixture has a first lag phase to the conversion of the bank vole rPrP^Sen to the first rPrP-res^(Sc), and wherein the second reaction mixture has a second lag phase to the conversion of the second rPrP^Sen to the second rPrP-res^(Sc). A shorter length of the second lag phase as compared to the first lag phase indicates that the subject has L-BSE, and a longer length of the second lag phase as compared to the first lag phase indicates that the subject has H-BSE. In other specific non-limting examples, the second rPrP^Sen is sheep rPrP^Sen, such as the amino acid sequence set forth as one of SEQ ID NO: 9 or SEQ ID NO: 21, optionaly with an N-terminal methionine, or the amino acid sequence set forth as SEQ ID NO: 19 or SEQ ID NO: 24, optionally without the N-terminal methionine.

In some embodiments, for discriminating between two different conditions, the subject has been diagnosed as having a transmissible spongiform encephalopathy. This can include evaluating clinical symptoms of the veterinary or human subject, such as behavioral and neurological symptoms. The disease can have been detected by any other diagnostic test for the presence of prions, such as, but not limited to, PMCA, RT-QuIC, or an assay based on electrophoretic mobility of proteins.

In some embodiments, detecting the presence of rPrP-res^(Sc) in a sample, such as a first sample and/or a second sample, comprises the use of thioflavin T (ThT). In additional embodiments, the biological sample, such as the first biological sample and/or the second biological sample, are an olfactory mucosal (for example, nasal) brushing, saliva, blood, serum, cerebral spinal fluid sample, muscle, lymphoid tissue, urine, feces, tissue, or bone marrow sample. The tissue can be any tissue of interest. The tissue can be fresh tissue or fixed tissue, such as formalin-fixed tissue. In specific non-limiting examples, the tissue can be skin tissue or brain tissue. In other embodiments, rPrP-res^(Sc), such as the rPrP-res^(Sc) in the first reaction mixture and in the second reaction mixture, is quantitated.

In further embodiments, the shaking cycles include a period of rest that precedes the period of shaking. In any of the disclosed methods, the shaking cycle can be, for example, 60 to 180 seconds in total length. The period of rest and the period of shaking can be equal. In a specific non-limiting example, the period of rest and the period of shaking are each about 60 seconds in length, and the shaking cycle is about 120 seconds in length. In other embodiments, the period of rest and shaking can be of different durations (unequal). For example, about 50 to about 300 seconds of shaking to about 10 to about 100 seconds of rest. In one specification non- limiting example, the shaking cycle is about 120 seconds in length, and includes 100 seconds of shaking and 20 seconds for rest. In some non-limiting examples, the shaking cycles are repeated 1 to about 300 times, such as about 1 to abou 200 times. The period of rest and the period of shaking can be substantially equal.

In some embodiments, PrP^D (e.g., PrP^CJD, PrP^BSE or PrP^Res) can be captured from a biological sample, such as a olfactory mucosal (for example, nasal) brushing, saliva, blood, serum, cerebral spinal fluid sample, muscle; lymphoid tissues; olfactory mucosa; urine; feces; tissue sample, or bone marrow sample, prior to performing the PrP amyloid seeding assay(s). The tissue can be fresh tissue or fixed tissue, such as formalin-fixed tissue. In specific non- limiting examples, the tissue can be skin tissue or brain tissue. Thus, the method can include contacting a sample from the subject, such as the sheep, human, cow, goat, or cervid, with an effective amount of an antibody that specifically binds prions, or PrP^Res for sufficient time to form an immune complex and separating the immune complex to form the biological sample used in the disclosed methods. Methods for immunoprecipitation are disclosed below. These methods can be used to prepare the biological sample.

Disclosed below are rPrP^Sen substrates, PrP amyloid seeding methods, methods for immunopreciptiation, and methods for detection that can be used in any of the embodiments herein disclosed.

II. PrP Substrates

As disclosed herein, prion protein amyloid seeding assays, such as RT-QuIC and QuIC assays (see below), performed using recombinant bank vole rPrP^Sen, can be used to detect any type of transmissible spongiform encephalopathy. In some embodiments, the subject is a human, and the transmissible spongiform encephalopathy is Creutzfeldt- Jakob disease (CJD), Gerstmann-Straussler-Scheincker syndrome (GSS), fatal familial insomnia (FFI) or sporadic fatal insomnia (sFI). In other embodiments, the subject is a cow, sheep or a goat, and the transmissible spongiform encephalopathy is bovine spongiform encephalopathy (BSE). In other embodiments, the subject is a cervid, and the transmissible spongiform encephalopathy is chronic wasting disease (CWD). In some embodiments, the subject is a sheep, and the transmissible encephalopathy is scrapie. Exemplary rPrP^Sen of use in the disclosed methods are provided in SEQ ID NOs: 1-26. An rPrP^Sen of use in the disclosed methods can include an N- terminal methionine. However, the N-terminal methionine can also be not present in a rPrP^Sen of use in the disclosed methods. Thus, an N-terminal methionine can be added to SEQ ID NOs: 1-16, 21-22 and 26, and these rPrP^Sen can be used in any of the methods disclosed herein. When referring to these sequences, the N-terminal methionine is "optional," indicating that a methionine can be added at the N-terminus of these amino acid sequences. Similiarly, the N- terminal methionine can be deleted from SEQ ID NOs: 17-20 and 23-25, and these rPrP^Sen can be used in the any of the methods disclosed herein. When referring to these sequences, the N- terminal methionine is "optional," indicating that the N-terminal methioine can be removed from these amino acid seuqences.

Bank vole rPrP^Sen can be used to detect PrP^D (such as PrP^Res) in biological samples from subjects, such as a human, cow, sheep, cervid or goat subjects. Hence bank vole rPrP can be used to detect target PrP^D (such as PrP^Res) in a sample taken from a subject of interest, such as a sheep showing symptoms of scrapie, a human, or a cow, sheep or a goat with clinical symptoms of a bovine BSE infection. If rPrP-res^(SC) is seeded by a biological sample from a subject suspected of having a prion disease, then the subject has the transmissible spongiform encephalopathy. If rPrP-res^(SC) is not seeded by a biological sample from the subject, then the subject does not have the transmissible spongiform encephalopathy. In certain embodiments, the bank vole PrP^sen comprises or consists of the amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 13, optionally including an N-terminal methionine. In other embodimetns, the bank vole bank vole PrP^sen comprises or consists of the amino acid sequence set forth as SEQ ID NO: 17 or SEQ ID NO: 25, optionally without the N- terminal methionine. The bank vole bank vole PrP^sen can be chimeric and can comprise or consists of the amino acid sequence set forth as SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 22, optionally with an N-temrinal methionine. The bank vole PrP^Sen can include the amino acid sequence set forth as SEQ ID NO: 17 or SEQ ID NO: 25, amino acids 21-230 of SEQ ID NO: 17 or SEQ ID NO: 25, 22-230 of SEQ ID NO: 17 or SEQ ID NO: 25, 23-230 of SEQ ID NO: 17 or SEQ ID NO: 25 of 24-230 of SEQ ID NO: 17 or SEQ ID NO: 25, optionally including an N-terminal methionine. In some embodiments, at most 1, 2, 3, 4, or 5 amino acids is deleted from the N- or C-terminus of the polypeptide. In some embodiments, the p_rpsen dQgg _not i_nci_u(j_e amino acids 1-22 (the leader sequence). In additional embodiments, an N-terminal methionine is present.

In a chimeric rPrP^Sen, a portion of the protein is from one species, and a portion of the protein is from another species. In one example about 10 to about 90%, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% about 80% or about 90% of the rPrP^Sen is from one species, and, correspondingly, about 90%, about 80%, about70%, about 60%, about 50%, about 40%, about 30%, about 20% or about 10% is from another species. Chimeric proteins can include, for example, hamster rPrP^Sen and rPrP^Sen from another species, such as sheep rPrP^Sen. Thus, the chimeric protein includes hamster rPrP^Sen and sheep _rp_rpSen Additional chimeric proteins include bank vole rPrP^Sen and rPrP^Sen from another species, such as human. In certain embodiments, the chimeric bank vole PrP^sen comprises or consists of the amino acid sequence set forth as SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 22, optionally including an N-terminal methionine.

In some embodiments, chimeric hamster (Ha) sheep (S) rPrP^Sen is used in QuIC and/or RT-QuIC reactions, such as to distinguish between scrapie and Nor98. A chimeric hamster- sheep rPrP^Sen construct can be used, such as, but not limited to, to distinguish between scrapie and Nor98. In some embodiments, a chimeric rPrP^Sen (designated Ha-S rPrP^Sen) is used, wherein the chimeric molecule includes residues 23-137 were of the Syrian hamster sequence (see SEQ ID NO: 2) and the remaining residues 138-231 were homologous to sheep residues 141-234 (R154,Q171 polymorph), see SEQ ID NO: 9, chimeric protein shown in SEQ ID NO: 5. In further embodiments, the Ha-S PrP^c comprises or consists of the amino acid sequence set forth as SEQ ID NO: 5 or SEQ ID NO: 26. An N-terminal methionine can be added to the chimeric sequence.

In further embodiments, sheep rPrP^Sen is used in QuIC and/or RT-QuIC reactions, such as to distinguish between C-BSE, L-BSE and H-BSE. In certain embodiments, the sheep rPrP^Sen includes amino acids 1-234, or amino acids 25-234 of a sheep rPrP^Sen. Exemplary amino acid sequences are shown in SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 24, wherein the N-terminal methionine is optional.

rp_rpSen _use(j _{m me reac}ti_on can be any recombinant prion protein of interest, for example prion protein from cells, for example bacterial cells or eukaryotic cells engineered to over express the protein. Any bank vole, hamster, or sheep prion protein sequence can be used to generate the rPrP^Sen.

Full length bank vole, hamster, human, sheep or mouse rPrP^Sen polypeptide can be used in the assays disclosed herein. However, a partial prion protein sequence expressed as rPrP^Sen can correspond to the polypeptide sequences of the natural mature full-length PrP^c molecule, meaning that the rPrP^Sen polypeptide lacks both the amino-terminal signal sequence and carboxy-terminal glycophosphatidylinositol-anchor attachment sequence. These proteins are also of use. Exemplary sequences are provided above.

In another example, amino acids 23-230, 30-230, 40-230, 50-230, 60-230, 70-230, 80- 230, or 90-230 of bank vole prion protein can be utilized in the assays disclosed herein. In other examples, amino acids 23-231, 30-231, 40-231, 50-231, 60-231, 70-231, 80-231 or 90-231 of hamster prion protein is utilized. In yet other embodiments, amino acids 23-231, 30-231, 40- 231, 50-231, 60-231, 70-231, 80-231 or 90-231 of human prion protein is utilized. In further embodiments, amino acids 23-230, 30-230, 40-230, 50-230, 60-230, 70-230, 80-230, or 90-230 of mouse prion protein can be utilized. In yet other examples, amino acids 1-234, 25-234, 30- 234, 40-234, 50-234, 60-234, 70-234, 80-234 or 90-234 of sheep prion protein is utilized. One or two amino acids can also be deleted from the C terminus of sheep prion protein (e.g., so that that amino acid 233 or 232 of the sheep prion protein is utilized, such that 1-233, 25-233, etc., or 1-232, 25-232, etc., are utilized).

Other prion proteins can also be utilized, such as those that have 1, 2, 3, 4, or 5 amino acids deleted from the carboxy terminus. In some embodiments, the prion protein includes the leader sequences (amino acids 1-22), or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids of the leader sequence. In other embodiments, the prion protein does not include the leader sequence (amino acid 1-22). Exemplary leader sequences are shown in bold in the "Sequence Listing" section provided above. An N-terminal methionine is optional, as discussed above.

One of skill in the art can readily produce these polypeptides using the sequence information provided herein, or using information available in GENBANK®. SEQ ID NOs: 1, 8, 12, 13 and 17 are exemplary bank vole amino acid sequences that can be utilized in some embodiments. SEQ ID NOs: 14-16 and 22 are chimeric bank vole amino acid sequences that can be utilized in some embodiments.

In some embodiments, to produce rPrP^Sen, host cells are transformed with a nucleic acid vector that expresses the rPrP^Sen, for example bank vole rPrP^Sen, or a fragment thereof, or a chimeric hamster-sheep rPrP^Sen. These cells can be mammalian cells, bacterial cells, yeast cells, insect cells, or whole organisms, such as transgenic mice. Other cells also can serve as sources of the rPrP^Sen. In particular examples the cell is a bacterial cell, such as an E. coli cell. Purified _rp_rpSen f_{rom r}p_rpSen expressing _cerj_{s ΟΓϊ m} some cases, raw cell lysates, can be used as the source of the non-pathogenic protein.

In some embodiments the recombinant protein is fused with an additional amino acid sequence. For example, over expressed protein can be tagged for purification or to facilitate detection of the protein in a sample. Some possible fusion proteins that can be generated include histidine tags, Glutathione S-transferase (GST), Maltose binding protein (MBP), green fluorescent protein (GFP), and Flag and myc-tagged rPrP These additional sequences can be used to aid in purification and/or detection of the recombinant protein, and in some cases are subsequently removed by protease cleavage. For example, coding sequence for a specific protease cleavage site can be inserted between the PrP^c coding sequence and the purification tag coding sequence. One example for such a sequence is the cleavage site for thrombin. Thus, fusion proteins can be cleaved with the protease to free the PrP^c from the purification tag.

Any of the wide variety of vectors known to those of skill in the art can be used to over- express rPrP^Sen. For example, plasmids or viral vectors can be used. These vectors can be introduced into cells by a variety of methods including, but not limited to, transfection (for instance, by liposome, calcium phosphate, electroporation, particle bombardment, and the like), transformation, and viral transduction.

Recombinant PrP^Sen also can include proteins that have amino sequences containing substitutions, insertions, deletions, and stop codons as compared to wild type sequences. In certain embodiments, a protease cleavage sequence is added to allow inactivation of protein after it is converted into prion form. For example, cleavage sequences recognized by Thrombin, Tobacco Etch Virus (Life Technologies, Gaithersburg, Md.) or Factor Xa (New England Biolabs, Beverley, Mass.) proteases can be inserted into the sequence. In some embodiments, inactivation of protein after it is converted into the PrP^Res seeded form is unnecessary because the rPrP-res^(Sc) resulting from the reaction has little or no infectivity.

Changes also can be made in the rPrP^Sen protein coding sequence. For example, mutations can be made to match a variety of mutations and polymorphisms known for various mammalian prion protein genes in any rPrP^Sen protein coding sequence, such as at most 1, 2, 3, 4, 5, 6, 7, 8, 8 or 10 substitutions. Cells expressing these altered prion protein genes can be used as a source of the rPrP^Sen, and these cells can include cells that endogenously express the mutant rPrP gene, or cells that have been made to express a mutant rPrP protein by the introduction of an expression vector. Use of a mutated rPrP^Sen can be advantageous, because some of these proteins are more easily or specifically converted to protease-resistant forms, or are less prone to spontaneous (prion-independent) conversion, and thus can further enhance the sensitivity of the method.

In certain embodiments, cysteine residues are placed at positions 94 and 95 of the hamster prion protein sequence in order to be able to selectively label the rPrP at those sites using sulfhydryl-reactive labels, such as pyrene and fluorescein linked to maleimide -based functional groups. In certain embodiments, these tags do not interfere with conversion but allow much more rapid, fluorescence-based detection of the reaction product. In one example, pyrenes in adjacent molecules of rPrP-res^(Sc) are held in close enough proximity to allow eximer formation, which shifts the fluorescence emission spectrum in a distinct and detectable manner. Free pyrenes released from, or on, unconverted rPrP^Sen molecules are unlikely to form excimer pairs. Thus, the reaction can be run in a multiwell plate, digested with proteinase K, and then excimer fluorescence can be measured to rapidly test for the presence of rPrP-res^(Sc). Sites 94 and 95 were chosen for the labels because the PK-resistance in this region of PrP^Res distinguishes rPrP-res^(Sc) from rPrP-res^(spon), giving rise to the 17 kDa rPrP-res^(Sc) band. Other positions in the PK-resistant region(s) that distinguish the 17-kDa rHa PrP-res^(Sc) fragment from all rHaPrP-res^(spon) fragments also can work for this purpose.

Recombinant prion proteins (rPrP^Sen) can be produced by any methods known to those of skill in the art. In one example, in vitro nucleic acid amplification (such as polymerase chain reaction (PCR)) can be utilized as a method for producing nucleic acid sequences encoding prion proteins. PCR is a standard technique that is described, for instance, in PCR Protocols: A Guide to Methods and Applications (Innis et al., San Diego, CA: Academic Press, 1990), or PCR Protocols, Second Edition (Methods in Molecular Biology, Vol. 22, ed. by Bartlett and Stirling, Humana Press, 2003).

A representative technique for producing a nucleic acid sequence encoding a recombinant prion protein by PCR involves preparing a sample containing a target nucleic acid molecule that includes the prion protein-encoding sequence. For example, DNA or RNA (such as mRNA or total RNA) can serve as a suitable target nucleic acid molecule for PCR reactions. Optionally, the target nucleic acid molecule can be extracted from cells by any one of a variety of methods well known to those of ordinary skill in the art (for instance, Sambrook et al. ,

Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel et al. , Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992). Prion proteins are expressed in a variety of mammalian cells. In examples where RNA is the initial target, the RNA is reverse transcribed (using one of a myriad of reverse transcriptases commonly known in the art) to produce a double- stranded template molecule for subsequent amplification. This particular method is known as reverse transcriptase (RT)-PCR. Representative methods and conditions for RT-PCR are described, for example, in Kawasaki et al. (In PCR Protocols, A Guide to Methods and Applications, Innis et al. (eds.), 21-27, Academic Press, Inc., San Diego, California, 1990).

The selection of amplification primers will be made according to the portion(s) of the target nucleic acid molecule that is to be amplified. In various embodiments, primers (typically, at least 10 consecutive nucleotides of prion-encoding nucleic acid sequence) can be chosen to amplify all or part of a prion-encoding sequence. Variations in amplification conditions may be required to accommodate primers and amplicons of differing lengths and composition; such considerations are well known in the art and are discussed for instance in Innis et al. (PCR Protocols, A Guide to Methods and Applications, San Diego, CA: Academic Press, 1990). From a provided prion protein-encoding nucleic acid sequence, one skilled in the art can easily design many different primers that can successfully amplify all or part of a prion

protein-encoding sequence.

As described herein, a number of prion protein-encoding nucleic acid sequences are known. Though particular nucleic acid sequences are disclosed, one of skill in the art will appreciate that also provided are many related sequences with the functions described herein, for instance, nucleic acid molecules encoding conservative variants of a prion protein. One indication that two nucleic acid molecules are closely related (for instance, are variants of one another) is sequence identity, a measure of similarity between two nucleic acid sequences or between two amino acid sequences expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences.

Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981 ; Needleman and Wunsch, /. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Set USA 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5: 151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988; Huang, et al., Computer Applications in the Biosciences 8: 155-165, 1992; Pearson et al.,

Methods in Molecular Biology 24:307-331, 1994; Tatiana et al., (1999), FEMS Microbiol. Lett., 174:247-250, 1999. Altschul et al. present a detailed consideration of sequence-alignment methods and homology calculations (/. Mol. Biol. 215:403-410, 1990). The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™, Altschul et al, J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence-analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the internet under the help section for BLAST™.

For comparisons of amino acid sequences of greater than about 30 amino acids, the "Blast 2 sequences" function of the BLAST™ (Blastp) program is employed using the default BLOSUM62 matrix set to default parameters (cost to open a gap [default = 5]; cost to extend a gap [default = 2]; penalty for a mismatch [default = -3]; reward for a match [default = 1];

expectation value (E) [default = 10.0]; word size [default = 3]; number of one-line descriptions (V) [default = 100]; number of alignments to show (B) [default = 100]). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the sequence of interest.

For comparisons of nucleic acid sequences, the "Blast 2 sequences" function of the BLAST™ (Blastn) program is employed using the default BLOSUM62 matrix set to default parameters (cost to open a gap [default = 11]; cost to extend a gap [default = 1]; expectation value (E) [default = 10.0]; word size [default = 11]; number of one-line descriptions (V) [default = 100]; number of alignments to show (B) [default = 100]). Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, r at least 98%, or at least 99% sequence identity to the prion sequence of interest.

Another indication of sequence identity is nucleic acid hybridization. In certain embodiments, prion protein-encoding nucleic acid variants hybridize to a disclosed (or otherwise known) prion protein-encoding nucleic acid sequence, for example, under low stringency, high stringency, or very high stringency conditions. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na⁺ concentration) of the hybridization buffer will determine the stringency of hybridization, although wash times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, chapters 9 and 11.

The following are representative hybridization conditions and are not meant to be limiting.

Very High Stringency (detects sequences that share at least 90% sequence identity)

Hybridization: 5x SSC at 65 °C for 16 hours

Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65 °C for 20 minutes each

High Stringency (detects sequences that share at least 80% sequence identity)

Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours

Wash twice: 2x SSC at RT for 5-20 minutes each

Wash twice: lx SSC at 55 °C-70°C for 30 minutes each

Low Stringency (detects sequences that share at least 50% sequence identity)

Hybridization: 6x SSC at RT to 55°C for 16-20 hours

Wash at least twice: 2x-3x SSC at RT to 55°C for 20-30 minutes each.

Prion protein variants that include the substitution of one or several amino acids for amino acids having similar biochemical properties (so-called conservative substitutions), can also be used in the presently described methods. Conservative amino acid substitutions are likely to have minimal impact on the activity of the resultant protein, such as its ability to convert to PrP-res. Further information about conservative substitutions can be found, for instance, in Ben Bassat et al. (J. Bacteriol , 169:751-757 ^', 1987), O'Regan et al. (Gene, 77:237-251, 1989), Sahin-Toth et al. (Protein Set , 3:240-247, 1994), Hochuli et al.

(Bio/Technology, 6: 1321-1325, 1988) and in widely used textbooks of genetics and molecular biology. In some examples, prion protein variants can have no more than 3, 5, 10, 15, 20, 25, 30, 40, or 50 conservative amino acid changes. The following table shows exemplary conservative amino acid substitutions that can be made to a prion protein, for example the prion proteins shown in SEQ ID NOs: 1-26, such that they can still be used in the presently claimed assays. Table A:

As noted above, in some embodiments, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids is substituted in one of SEQ ID NOs: 1-26, wherein the rPrP^Sen retains the ability to amplify prions in an assay such as RT-QuIC.

To purify PrP^Sen from recombinant sources (or PrP^Sen from natural sources), the composition is subjected to fractionation to remove various other components from the composition. Various techniques suitable for use in protein purification are well known. These include, for example, precipitation with ammonium sulfate, PTA, PEG, antibodies and the like, or by heat denaturation followed by centrifugation; chromatography steps such as metal chelate, ion exchange, gel filtration, reverse phase, hydroxylapatite, lectin affinity, and other affinity chromatography steps; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. III. Standard QuIC (SQ) and RT-QuIC

Disclosed below are reactions conditions that can be used with recombinant hamster 90- 231 and 23-231 chimeric hamster sheep protease sensitive prion protein, human protease sensitive prion protein, mouse protein sensitive prion protein, bovine protein sensitive prion protein, sheep protease sensitive prion protein, bank vole protease sensitive prion protein, and recombinant forms thereof. These methods are of use with any sample of interest. The methods disclosed herein include the use of a sample from a subject, such as, but not limited to, a nasal brushing, saliva, cerebral spinal fluid, blood, fecal, tissue, urine, or serum sample. The tissue can be any tissue of interest. The tissue can be fresh tissue or fixed tissue, such as formalin- fixed tissue. In specific non-limiting examples, the tissue can be skin tissue or brain tissue.

The prion detection method termed protein misfolding cyclic amplification (PMCA) is based on the ability of prions to replicate in vitro in tissue homogenates containing protease sensitive prion protein (PrP^Sen), see, for instance, PCT Publication No. WO0204954). PMCA involves amplification of protease resistant prion protein (PrP-res), the pathologic form, through incubation with a suitable prion protein substrate derived from brain tissue, serial amplification of the PrP^c, for instance by alternating incubation and sonication steps, and detection of the resulting PrP-res. In some instances, incubation and sonication are alternated over a period of approximately three weeks, and intermittently a portion of the reaction mix is removed and incubated with additional PrP^c in order to serially amplify the PrP^Res in the sample. Following the repeated incubation/sonication/dilution steps, the resulting PrP^Res is detected in the reaction mix. Although brain extract-based PMCA is a very sensitive assay for detecting PrP-res, it has practical drawbacks, notably the time required to achieve optimal sensitivity (several days to weeks) and the use of brain-derived PrP^c as the amplification substrate. This method also uses sonication.

In contrast, in the QuIC methods (standard QuIC (also called SQ or QuIC) and real time (also called RTQ or RT-QuIC)), agitation is performed by shaking and not by sonication. These assays use bacterially-expressed rPrP^Sen as a substrate Atarashi et al. , (2008) Nat Methods, 5, 211-212, incorporated herein by refernece), which can be obtained rapidly in high purity and in large amounts, whereas purification of naturally occurring PrP^c from brain tissue is difficult and gives much lower yields (Deleault et al. (2005) /. Biol. Chem. 280, 26873-26879; Pan et al. (1993) Proc. Natl. Acad. Set USA 90, 10962-10966; Hornemann et al., (2004) EMBO Rep. 5, 1159-1164). Furthermore, unlike PrP^c in brain homogenates or purified from brain, rPrP^Sen can be easily mutated or strategically labeled with probes to simplify and accelerate the detection of relevant products.

Thus, there are two types of PrP^D- or PrP^Res-associated amyloid seeding activity detection methods that utilize rPrP^Sen, one that uses sonication (rPrP-PMCA) (Atarashi et al , (2007) Nat Methods, 4, 645-650) and one that utilizes shaking (QuIC) (Atarashi et al. , (2008) Nat Methods, 5, 211-212) in the absence of sonication. These methods facilitate fundamental studies of the structure and conversion mechanism of PrP^D. Site-directed mutations can allow precise labeling of rPrP^Sen with a variety of probes that can report on conformational changes, and both inter-molecular and intra-molecular distances within rPrP-res ^(Sc) aggregates, which are formed on conversion of rPrP^Sen to the protease resistant and/or amyloid form, rPrP-res^(Sc). Furthermore, RT-QuIC allows detection of the amyloid RT-QuIC product using thioflavin T (ThT). In enhanced RT-QuIC, rPrP^Sen can be preemptively replenished before much detectable (ThT-positive) polymerization has occurred (such as before 24 hours of incubation), while retaining the existing rPrP-res^(Sc).

The QuIC and RT-QuIC methods generally involve mixing a sample (for example a olfactory mucosal sample (such as nasal brushing), saliva sample, blood sample, tissue sample (fixed or fresh), CSF sample, urine sample, fecal sample, or plasma sample that is suspected of containing prions or PrP^D) with purified rPrP^Sen to make a reaction mix, and performing a primary reaction to form and amplify specific forms of rPrP-res^(Sc) in the mixture. This primary reaction includes incubating the reaction mix to permit the PrP^D to initiate the conversion of _rp_rpSen _{to S ec}ifi_c aggregates or polymers of rPrP-res^(Sc); fragmenting any aggregates or polymers formed during the incubation step; and repeating the incubation and fragmentation steps one or more times, for instance from about 1 to 2 times, 1 to 4 times, 1 to 10 times, 1 to 20 times, 1 to 30 times, 1 to 40 times, 1 to 50 times, 10 to 20 times, 10 to 30 times, 10 to 40 times, or 10 to about 50 times. In some embodiments of the method, serial amplification of rPrP-res^(Sc) is carried out by removing a portion of the reaction mix and incubating it with additional _rp_rpSen j_n othej- embodiments, additional rPrP^Sen is added to the reaction, such as during the lag phase (prior to the formation of detectable rPrP-res^(Sc), such as prior to 24 hours of the reaction), and the incubation and fragmentation steps are repeated.

In further embodiments, the method is performed without serial amplification, such that substrate bound prions are retained in a reaction vessel, and that substrate is replenished without removing potential rPrP-res^(Sc) seeds. For example, rPrP-res^(Sc) can be amplified in a sample, by mixing the sample with purified rPrP^Sen to make a reaction mix; performing a prion protein amyloid seeding assay that includes (i) incubating the reaction mix to permit coaggregation of the rPrP^Sen with the PrP^D that may be present in the reaction mix, and maintaining incubation conditions that promote coaggregation of the rPrP^Sen with the PrP^D and results in a conversion of the rPrP^Sen to rPrP-res^(Sc) while inhibiting development of rPrP-res^(spon); (ii) agitating aggregates formed during step (i); (iii) optionally repeating steps (i) and (ii) one or more times. rPrP-res^(Sc) is detected in the reaction mix, wherein detection of rPrP-res^(Sc) in the reaction mix indicates that PrP^D was present in the sample. Additional substrate (rPrP^Sen) can be added during the reaction (such as bank vole rPrP^Sen or hamster or chimeric hamster sheep rPrP^Sen to their respective reaction), such as during the lag phase between the addition of the sample and the detection of rPrP-res^(Sc) formation. However, when a single round of rPrP-res^(Sc) amplification is used, a portion of the reaction mix is not removed and incubated with additional rPrP^Sen. In some embodiments, the rPrP^Sen can be replenished by adding additional rPrP^Sen substrate to the reaction mix.

Generally, with either QuIC or RT-QuIC (also called SQ or RTQ, respectively), the reaction includes the use of shaking in the absence of sonication (the QuIC reaction), and the use of cycles of shaking/rest that are about 1:1 in duration. In one non-limiting example, the reaction alternates 60 seconds of shaking and 60 seconds of no shaking (rest). In another non- limiting example, the reaction alternates 30 seconds of shaking and 30 seconds of no shaking (rest). However, the times can be varied, such as 45 seconds of shaking and 45 seconds of no shaking or 70 seconds of shaking and 70 seconds of no shaking. The shaking cycle can be, for example, about 20 to about 180 seconds in length, such as about 30 to about 180 seconds in length, about 40 to about 180- seconds in length, about 50 to about 180 seconds in length, or about 60 to about 180 seconds in length, with equal periods of rest and shaking. Thus, in some embodiments the period of rest and the period of shaking are equal. However, in other embodiments, the period of rest and the period of shaking are unequal.

In some embodiments, the period of rest and the period of shaking are about 120 seconds in length for the total cycle. In other embodiments the total cycle time is about 60 to 180 seconds in length, such as, but not limited to 60, 70, 80, 90, 100, 110, 120, 130, 140 , 150, 160, 170, or 180 seconds in length. The period of shaking and rest in each cycle can be equal, as discussed above.

In other embodiments, the period of rest and the period of shaking are unequal. For example, the reaction includes 90 seconds of shaking and 30 seconds of no shaking, or 100 seconds of shaking and 20 seconds of no shaking, or 80 seconds of shaking and 40 seconds of rest. In additional embodiments, the total cycle time is about 60, 70, 80, 90, 100, 110 or 120 seconds in length and includes at least 30 seconds, at least 40, or at least 50, or at least 60 seconds of shaking. In specific non-limiting examples, the total cycle time is 60 to 180 seconds in length, such as, but not limited to 60, 70, 80, 90, 100, 110, 120, 130, 140 , 150, 160, 170, or 180 seconds in length. Reactions have also been found to be particularly efficient at 37-60° C, for example 45-55° C, such as about 55° C, or at about 42°C to 46°C or 42°C to 55°C, such as 42°C to about 50° C or at about 42°C. These conditions are particularly effective at promoting the formation of rPrP-res^(Sc), while inhibiting rPrP-res^(spon) formation of unseeded reactions.

The reaction can be performed for 3 to 12 hours, such as 6 to 12 hours, such as 8 to 10 hours. However, longer amplification reactions of 14 hours, 16 hours, 20 hours, 24 hours, such as at least 45 hours, 48 hours, 55 hours or even 65 or 96 hours, can also provide excellent results, depending on the reaction temperature. In some embodiments, the reaction is performed for 3 to 96 hours. The reaction can be performed, for example, for 5 to 55 hours, 12 to 55 hours, 24 to 55 hours, 36 to 55 hours, 48 to 55 hours or 50 to 55 hours. In other examples, the reaction can be performed for no more than 12 hours, no more than 24 hours, no more than 36 hours, no more than 48 hours, no more than 55 hours, no more than 72 hours, no more than 96 hours or no more than 120 hours. In some examples the reaction is performed from about 5 hours to about 120 hours. In several specific non-limiting examples, the reaction is performed for 24, 48, or 55 hours. The reaction can be performed for 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours. In some specific examples, the reaction is performed for 55 hours. In some embodiments, the reaction is performed using sodium chloride (NaCl) at a concentration of about 100 mM to about 900 mM, such as about 100 to about 700, or about 100 mM to about 500 mM NaCl. In additional embodiments, about 100 mM, 200 mM, 300 mM, 400 mM NaCl. In other embodiments, the reaction is performed using 200 to 400 mM NaCl, usch as using 300 mM NaCl. In methods wherein RT-QuIC and/or a combination of immunoprecipitation and real time QuIC is used (IP-RTQ reactions or eQuIC), ThT can be used to detect rPrP-res^(Sc).

In a RT-QuIC assay, the reaction product is detected in real time (RT). There is generally a lag phase in a QuIC reaction, wherein rPrP-res^(Sc) cannot be detected. The lag phase is considered to end when a statistically significant amount of rPrP-res^(Sc) can be detected, as compared to the background level of fluorescence. The length of the lag phase will vary when different substrates are used. In some embodiments, the length of the lag phase using two different substrates can be compared. In certain embodiments, the length of the lag phase can be used to discrimate between different forms of a disease, such as, but not limited to L-BSE, H- BSE, and C-BSE. The length of a lag phase can readily be determined by one of skill in the art. Exemplary methods are provided in the examples section.

In IP-RTQ, a solid substrate, such as a bead, such as magnetic beads can be used. The beads and any associated prions or prion-induced RTQ conversion products tend to cling to the bottom of reaction vessel, such as a well. Thus, the reaction fluid can easily be changed, and the substrate replenished in its pre-RTQ state, without removing many beads or bound reaction products from the well. The rPrP^Sen substrate can be replenished preemptively during the lag phase, such as before ThT positivity indicated much consumption by conversion to prion-seeded amyloid product. With replenishment, IP-RTQ is highly sensitive. In some embodiments, the overall sensitivity of the RTQ was increased by at least 1000-fold and overall reaction time is greatly reduced. In additional embodiments, the concentration of substrate can be 0.1 mg/ml, such as 0.05 to 0.2 mg/ml.

Thus, QuIC reaction can be an RT-QuIC reaction, and thus can include thioflavin T (ThT) which allows detection of the rPrP-res^(Sc). The RT-QuIC assay incorporates rPrP^Sen as a substrate, intermittent shaking of the reactions such as in 96-well plates, largely detergent- and chaotrope-free reaction conditions and ThT-based fluorescence detection of prion-seeded _rp_rpSen ajnyioid fibrils. One advantage of using ThT is that it can be included in the reaction mixture. Thioflavin T is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils (see Khurana et al., J. Structural Biol. 151: 229-238, 2005), and is commonly used to

Following amplification, the prion-initiated rPrP-res^(Sc) in the reaction mix is detected. If ThT is included in the reaction (RT-QuIC), then rPrP-res^(Sc) can be detected using fluorescence at 450 +/- 10 nM excitation and 480 +/- 10 nm emission (see for example, Wilham et al., PLOS Pathogens 6(12): 1-15, 2010, incorporated herein by reference.) ThT can be included directly in the amplification mixture. In some embodiments, if ThT is included, the reaction mix does not include chaotropes or detergents. In some embodiments, if ThT is included, the reaction mix does not include chaotropic agents or detergents that can alter the rPrP-res^(Sc)- sensitivity of ThT.

In one non-limiting example, in RT-QuIC reactions the final concentration of ThT in each reaction is 1 mM. In other examples, ThT is used at a final concentration of about 0.1 to 1 mM in the reaction.

The fluorescent emitted by ThT can be measured in real time (RT). There is usually a lag phase in a RT-QuIC reaction, wherein ThT fluorescence cannot be detected. At some point, a statistically significant amount fluorescence can be measured that is above background fluorescence. The time of initiation of the reaction to the time of appearance of a statistically significant amount of detectable fluorescence, which represents the presence of rPrP-res^(Sc), can be measure as the lag phase. The length of the lag phase can vary when different substrates are used. In some embodiments, the length of the lag phase using two different substrates in two different RT-QuIC assays can be compared. The length of the lag phase when a first substrate is used, as compared to the length of the lag phase when a second substrate is used, can be used to discrimate between different forms of a disease. In certain embodiments, the length of the lag phase can be used to discriminate L-BSE, H-BSE, and C-BSE. In specific non-limiting examples, the first substrate is bank vole rPrP^Sen, and the second substrate is a different rPrP^Sen. A variety of rPrP^Sen molecules can be used as the second substrate, see for example, SEQ ID NOs: 2-7, 9-11, 18-21 and 23-24. In some embodiments, the second substrate is a sheep rPrP^Sen, such as, but not limited to SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 24, optionally with an N-terminal methionine. In some specific non-limiting examples, there is a first lag phase for the first reaction, wherein bank vole rPrP^Sen is convereted rPrP- res^(Sc), and a second lag phase to the conversion of the second rPrP^Sen, such as sheep rPrP^Sen, to the second rPrP-res^(Sc). A shorter length of the second lag phase as compared to the first lag phase indicates that the subject has L-BSE, and wherein a longer length of the second lag phase as compared to the first lag phase indicates that the subject has H-BSE (see Figures 23A and 23B).

The sodium chloride (NaCl) concentration can be varied in the reaction. In some embodiments, a concentration of about 200-400 mM NaCl allows sensitive detection of PrP^D while reducing the incidence of spontaneous conversion of the substrate. In some embodiments, detergent at a concentration greater than 0.002% is not included in an RT-QuIC reaction. The detergent concentration with Bank vole rPrP^Sen can be, for example, not greater than 0.001% in combination with 300 mM NaCl. In specific non-limiting examples, the detergent is sodium dodecyl sulfate (SDS).

If standard QuIC is utilized, PrP^Res can be detected by means other than ThT

fluorescence, for example, using an antibody (see below). In some examples, the reaction mix is digested with proteinase K (which digests the remaining rPrP^Sen in the reaction mix) prior to detection of the rPrP-res^(Sc). Two types of refolded prion protein can be generated in QuIC reactions, one occurring spontaneously (rPrP-res^(spon)) and the other initiated by the presence of prions (rPrP-res^(Sc)) in the test sample. Thus, discrimination between the former and the latter can be done on the basis of differing protein fragment sizes generated upon exposure to proteinase K. An unexpectedly superior decrease in the amount of rPrP-res^(spon) formed is achieved with the QuIC assays. Thus, RT-QuIC (RTQ) (which includes thioflavin T) reactions need not be subjected to proteinase K treatment. Thus, this step is optional.

All of the methods disclosed herein, such as QuIC and RT-QuIC, will work under a variety of conditions. In several embodiments, optimal conditions that support specific prion/PrP-res-seeded QuIC or RT-QuIC include the use of a detergent, such as an ionic and/or a non-ionic detergent. The QuIC conditions can include the use of about 0.0001-0.1% of an ionic detergent, such as 0.001% to 0.002%, of an ionic detergent, such as sodium dodecyl sulfate (SDS). The QuIC conditions can include the use of about 0.0001-0.1% of a nonionic detergent, such as 0.001% to 0.002% of a nonionic detergent, such as TX-100 in the reaction mixture. In some embodiments, 0.001% of SDS is included in the reaction. In RT-QuIC reactions, lower final detergent concentration (such as 0-0.001% SDS) can be used. Without being bound by theory, higher concentrations can interfere with the thioflavin T fluorescence detection of rPrP- _res(Sc)_

IV. Methods for Detecting rPrP-res^(Sc) in Amplification Mixes in the Absence of ThT

Once rPrP-res^(Sc) has been generated by rPrP^Sen conversion, such as using rPrP-PMCA or a QUIC assay, rPrP-res^(Sc) can be detected in the reaction mixture. Direct and indirect methods can be used for detection of rPrP-res^(Sc) in a reaction mixture. Detection using ThT is described above. For methods in which rPrP-res^(Sc) is directly detected, separation of newly-formed rPrP- _res(Sc) f_rom remaining rPrP^Sen usually is required. This typically is accomplished based on the different natures of rPrP-res^(Sc) versus rPrP^Sen. For instance, rPrP-res^(Sc) typically is highly insoluble and resistant to protease treatment. Therefore, in the case of rPrP-res^(Sc) and rPrP^Sen, separation can be by, for instance, protease treatment. Lateral flow assays or SOPHIA can also be used.

A. Protease Treatment

When rPrP-res^(Sc) and rPrP^Sen are separated by protease treatment, reaction mixtures are incubated with, for example, Proteinase K (PK). An exemplary protease treatment includes digestion of the protein, for instance, rPrP^Sen, in the reaction mixture with 1-20 μg/ml of PK for about 1 hour at 37° C. Reactions with PK can be stopped prior to assessment of prion levels by addition of PMSF or electrophoresis sample buffer. Depending on the nature of the sample, incubation at 37° C with 1-50 μg/ml of PK generally is sufficient to remove rPrP^Sen.

rPrP-res^(Sc) also can be separated from the rPrP^Sen by the use of ligands that specifically bind and precipitate the misfolded form of the protein, including conformational antibodies, certain nucleic acids, plasminogen, PTA and/or various peptide fragments.

B. Western Blot

In some examples, reaction mixtures fractioned or treated with protease to remove _rp_rpSen _are ^_6η subjected to Western blot for detection of rPrP-res^(Sc) and the discrimination of rPrP-res^(Sc) from rPrP-res^(spon). Western blots can also be used to evaluate the molecule weight of rPrP-res^(Sc), such as for the detection of a prions casusing L-BSE, H.BSE, and C-BSE.

Typical Western blot procedures begin with fractionating proteins by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The proteins are then electroblotted onto a membrane, such as nitrocellulose or PVDF and probed, under conditions effective to allow immune complex (antigen/antibody) formation, with an anti-prion protein antibody. Exemplary antibodies for detection of prion protein include the 3F4 monoclonal antibody, monoclonal antibody D13 (directed against residues 96-106 (Peretz et al. (2001) Nature All, 739-743)), polyclonal antibodies R18 (directed against residues 142-154 ), and R20 (directed against C-terminal residues 218-232) (Caughey et al. (1991) /. Virol. 65, 6597-6603).

Following complex formation, the membrane is washed to remove non-complexed material. An exemplary washing procedure includes washing with a solution such as

PBS/Tween, or borate buffer. The immunoreactive bands are visualized by a variety of assays known to those in the art. For example, the enhanced chemoluminesence assay (Amersham, Piscataway, N.J.) can be used.

If desired, prion protein concentration can be estimated by Western blot followed by densitometric analysis, and comparison to Western blots of samples for which the concentration of prion protein is known. For example, this can be accomplished by scanning data into a computer followed by analysis with quantitation software. To obtain a reliable and robust quantification, several different dilutions of the sample generally are analyzed in the same gel.

C. ELISA, Immunochromato graphic Strip Assay, and Conformation Dependent Immunoassay

As described above, immunoassays in their most simple and direct sense are binding assays. Specific non-limiting immunoassays of use include various types of enzyme linked immunosorbent assays (ELISAs), immunochromatographic strip assays, radioimmunoassays (RIA), and specifically conformation-dependent immunoassays.

In one exemplary ELISA, anti-PrP antibodies are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a reaction mixture suspected of containing prion protein antigen is added to the wells. After binding and washing to remove non- specifically bound immune complexes, the bound prion protein can be detected. Detection generally is achieved by the addition of another anti-PrP antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection also can be achieved by the addition of a second anti-PrP antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label. In another exemplary ELISA, the reaction mixture suspected of containing the prion protein antigen is immobilized onto the well surface and then contacted with the anti-PrP antibodies. After binding and washing to remove non-specifically bound immune complexes, the bound anti-prion antibodies are detected. Where the initial anti-PrP antibodies are linked to a detectable label, the immune complexes can be detected directly. Again, the immune complexes can be detected using a second antibody that has binding affinity for the first anti-PrP antibody, with the second antibody being linked to a detectable label.

Another ELISA in which protein of the reaction mixture is immobilized involves the use of antibody competition in the detection. In this ELISA, labeled antibodies against prion protein are added to the wells, allowed to bind, and detected by means of their label. The amount of prion protein antigen in a given reaction mixture is then determined by mixing it with the labeled antibodies against prion before or during incubation with coated wells. The presence of prion protein in the sample acts to reduce the amount of antibody against prion available for binding to the well and thus reduces the ultimate signal. Thus, the amount of prion in the sample can be quantified.

Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.

In coating a plate with either antigen or antibody, one generally incubates the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate are then washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antibodies. These include bovine serum albumin, casein, and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface, and thus reduces the background caused by nonspecific binding of antibodies onto the surface.

It is customary to use a secondary or tertiary detection means rather than a direct procedure with ELISAs, though this is not always the case. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand. "Under conditions effective to allow immune complex (antigen/antibody) formation" means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin, milk proteins, and phosphate buffered saline

(PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background. "Suitable" conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours, at temperatures preferably on the order of 25° C to 27° C, or can be overnight at about 4° C or so.

Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. An exemplary washing procedure includes washing with a solution such as PBS/Tween or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes can be determined.

To provide a detecting means, the second or third antibody generally will have an associated label to allow detection. In some examples, this is an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, the first or second immune complex is contacted and incubated with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (for instance, incubation for two hours at room temperature in a PBS -containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, for instance, by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl- benzthiazoline-6-sulfonic acid) and H2O2, in the case of peroxidase as the enzyme label.

Quantification is then achieved by measuring the degree of color generation, for instance, using a visible spectra spectrophotometer.

D. _rp_rpS labeling

In certain embodiments, the recombinant rPrP^Sen substrate protein can be labeled to enable high sensitivity of detection of protein that is converted into rPrP-res^(Sc). For example, _rp_rpSen _can b_e radioactively labeled, epitope tagged, or fluorescently labeled. The label can be detected directly or indirectly. Radioactive labels include, but are not limited to ¹²⁵1, ³²P, ³³P, and ³⁵S. The mixture containing the labeled protein is subjected to a prion protein amyloid seeding assay, such as QuIC, and the product detected with high sensitivity by following conversion of the labeled protein after removal of the unconverted protein for example by proteolysis. Alternatively, the protein can be labeled in such a way that a signal can be detected upon the conformational changes induced during conversion. An example of this is the use of FRET technology, in which the protein is labeled by two appropriate fluorophores, which upon refolding become close enough to exchange fluorescence energy (see for example U.S. Pat. No. 6,855,503).

In certain embodiments, cysteine residues are placed at positions 94 and 95 of the hamster prion protein sequence in order to be able to selectively label the rPrP^Sen at those sites using sulfhydryl-reactive labels, such as pyrene and fluorescein linked to maleimide -based functional groups. In certain embodiments, these tags do not interfere with conversion but allow much more rapid, fluorescence-based detection of the reaction product. In one example, pyrenes in adjacent molecules of rPrP-res^(Sc) are held in close enough proximity to allow eximer formation, which shifts the fluorescence emission spectrum in a distinct and detectable manner. Free pyrenes released from, or on, unconverted rPrP^Sen molecules are unlikely to form eximer pairs. Thus, the rPrP-res^(Sc) prion protein amyloid seeding assay can be run in a multiwell plate, digested with proteinase K, and then eximer fluorescence can be measured to rapidly test for the presence of rPrP-res^(Sc). Sites 94 and 95 are chosen for the labels because the PK-resistance in this region of constituent PrP molecules distinguishes rPrP-res^(Sc) from rPrP-res^(spon), giving rise to the 17 kDa rPrP-res^(Sc)band. Other positions in the PK-resistant region(s) that distinguish the 17-kDa rPrP-res^(Sc) fragment from all rPrP-res^(spon) fragments also can work for this purpose.

In certain other embodiments, the use of a fluorescently-tagged rPrP^Sen substrate for the reaction is combined with the use an immunochromatographic strip test with an immobilized rPrP-res^(Sc) specific antibody (for example, from Prionics AG, Schlieren-Zurich, Switzerland). Binding of the rPrP-res^(Sc) to the antibody is then detected with a fluorescence detector.

V. Immunoprecipitation and Concentration

In some embodiments prions can be purified from a sample from a subject, such as a human, cervid, cow, sheep or goat, prior to performing a prion protein amyloid seeding assay to detect PrP^D (such as PrP^Res). The methods disclosed herein include the use of a sample from a subject, such as, but not limited to, a nasal brushing, saliva, cerebral spinal fluid, blood, fecal, tissue, urine, or serum sample. The tissue can be any tissue of interest. The tissue can be fresh tissue or fixed tissue, such as formalin-fixed tissue. In specific non-limiting examples, the tissue can be skin tissue or brain tissue. The sample is contacted with an antibody that specifically binds only the disease related conformation of a prion protein (e.g. PrP^D, also known as PrP^Res). Thus, a purified form of the PrP^Res that binds to the antibody, can be used as the biological sample tested in the disclosed assays. Any of the immunoprecipitation methods disclosed below can be used in the assays to detect a transmissible spongiform encepalopathy, or to distinguish between scrapie and Nor98.

In some embodiments, the sample, such as a biological sample from a subject (for example a human, cervid, cow, sheep, or goat) is contacted with a capture-monoclonal antibody (or epitope -binding fragment thereof), which can be immobilized on a solid substrate.

Monoclonal antibodies can be selected that specifically bind an epitope that is expressed on PrP^D or PrP^Res, but not on PrP^c.

The monoclonal antibodies that specifically bind PrP^D or PrP^Res can be from any species, such as murine antibodies. The monoclonal antibodies can be produced by known monoclonal antibody production techniques. Typically, monoclonal antibodies are prepared by recovering spleen cells from immunized animals with the protein of interest and immortalizing the cells in conventional fashion, for example, by fusion with myeloma cells or by Epstein-Barr virus transformation, and screening for clones expressing the desired antibody. See, for example, Kohler and Milstein Eur. J. Immunol. 6:511 (1976). Monoclonal antibodies, or the epitope- binding region of a monoclonal antibody, may alternatively be produced by recombinant methods. Thus, in some embodiments, chimeric or humanized forms of a monoclonal antibody are utilized, wherein the antibody of use includes the complementarity determining regions (CDRs) of an antibody that specifically binds PrP^D or PrP^Res.

In some emboindments monoclonal antibody can bind PrP^Res from subject with either scrapie or Nor98. Alternatively, the monoclonal antibody can bind PrP^D or PrP^Res from subjects with scrapie (but not Nor98), or the monoclonal antibody can bind PrP^D or PrP^Res from subjects with Nor98 (but not scrapie).

By way of example, where the protein of interest is a prion protein that is capable of changing conformation to form PrP^D or PrP^Res aggregates, the monoclonal antibody can be a murine monoclonal antibody that is generated by immunizing "knock out" mice with recombinant normal mouse cellular protein (PrP^c). Spleen cells (antibody producing lymphocytes of limited life span) from the immunized mice can then be fused with non- producing myeloma cells (tumor lymphocytes that are "immortal") to create hybridomas. The hybridomas can then be screened for the production of antibody specific to PrP^D, PrP^Res or PrP^Sc and the ability to be propagated in tissue culture. These hybridomas can then be cultured to provide a permanent and stable source for the specific monoclonal antibodies.

Particular monoclonal antibodies produced by this method are disclosed in U.S. Pat. No. 6,528,269. These monoclonal antibodies include 2F8, 5B2, 6H3, 8C6, 8H4 and 9H7 produced by cell lines PrP2F8, PrP5B2, PrP6H3, PrP8C6, PrP8H4 and PrP9H7, that can specifically bind human PrP^Res, and also bind PrP^Res from cows, sheep and other species, see also U.S. Published Patent Application No. 2005/0118720, which is incorporated herein by reference. The antibody can also be 6A12 or 8D5 (Masujin K et al. (2013), PLoS ONE 8(2): e58013).

The methods disclosed herein can also utilize monoclonal antibody 15B3, which is described in U.S. Published Patent Application No. 2008/0220447, published September 1,

2008, which is incorporated herein by reference. The antibody 15B3 is available from Prionics AG, Zurich, Switzerland and methods to generate this antibody are disclosed in PCT Publication No. WO 98/37210, which is incorporated herein by reference. This PCT Publication also describes antibodies that bind p_rP^BSE and PrP^Sc but not PrP^c. PCT Publication No. WO

98/37210 discloses that hybridomas that produce antibody 15B3 were deposited in accordance with the Budapest treaty at DSMZ— Deutsche Sammlung von Mikroorganismen und

Zellkulturen GmbH (Germany) (Zellkulturen GmbH, InhoffenstraBe 7 B38124 Braunschweig, Germany) under Accession Number: DSM ACC2298.

The IgM monoclonal antibody 15B3 binds PrP^Res from cows (see Korth et al., Nature 390: 74-77, 1997, incorporated by reference herein). 15B3 specifically recognizes the disease- associated form of the prion protein (i.e., PrP^BSE, PrP^Res or PrP^Sc) and is capable of detecting abnormal PrP in brain homogenates without the need of PK digestion (Korth et al., Nature 1997; 390:74-77, 1997, see also Nazor et al., EMBO J. 24(13):2472-80, 2005; Yakovleva et al., Transfusion 44:1700-5, 2004).

The capture-monoclonal antibody (such as 15B3, Ig 261, Ig W226 or 262) can be immobilized on a solid phase by insolubilizing the capture-monoclonal antibody before the assay procedure, as by adsorption to a water-insoluble matrix or surface (U.S. Pat. No.

3,720,760, herein incorporated by reference in its entirety) or non-covalent or covalent coupling, for example, using glutaraldehyde or carbodiimide cross-linking, with or without prior activation of the support with, e.g., nitric acid and a reducing agent (as described in U.S. Pat. No.

3,645,852 or in Rotmans et al., J. Immunol. Methods 57:87-98, 1983), or afterward, such as by immunoprecipitation.

The solid phase used for immobilization may be any inert support or carrier that is essentially water insoluble and useful in immunometric assays, including supports in the form of, for example, surfaces, particles, porous matrices, sepharose, etc. Examples of commonly used supports include small sheets, Sephadex, polyvinyl chloride, plastic beads, magnetic beads, and assay plates or test tubes manufactured from polyethylene, polypropylene, polystyrene, and the like including 96-well microtiter plates and 384-well microtiter well pates, as well as particulate materials, such as filter paper, agarose, cross-linked dextran, and other

polysaccharides. Alternatively, reactive water-insoluble matrices, such as cyanogen bromide- activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287;

3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are suitably employed for capture- monoclonal antibody immobilization. In one example, the immobilized capture-monoclonal antibodies are coated on a microtiter plate, and in particular the solid phase can be a multi-well microtiter plate. For example, the multi-well microtiter plate can be a microtest 96-well ELISA plate. The solid phase can be a magnetic bead, such as DYNABEADS® (Invitrogen) or other magnetic beads, such as those available from NEW ENGLAND BIOLABS® or DYNAL® magnetic beads.

Generally, the capture-monoclonal antibody (such as 15B3, Ig 261, Ig W226 or 262) is attached to the solid substrate. This attachment can be through a non-covalent or covalent interaction or physical linkage as desired. Techniques for attachment include those described in U.S. Pat. No. 4,376,110 and the references cited therein. If covalent binding is used, the plate, bead or other solid phase can be incubated with a cross-linking agent together with the capture reagent under conditions well known in the art.

The solid substrate can also have an antibody, such as a rabbit anti-mouse antibody or a rabbit anti-human antibody covalently linked to the solid substrate. The antibody attached to the solid substrate can then be incubated with a second antibody of interest (such as a mouse or human antibody) to achieve attachment of the second antibody to the solid substrate. In one specific non- limiting example, a rabbit anti-mouse antibody is coupled to the solid substrate, which is then incubated with a second antibody that specifically binds a prion protein, such as, but not limited to, such as 15B3, Ig 261, Ig W226, W262, 6A12 or 8D5.

Commonly used cross-linking agents for attaching a capture-monoclonal antibody to the solid phase substrate include, for example l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-l,8-octane. Derivatizing agents, such as methyl-3-[(p- azidophenyl)dithio]propioimidate yield photoactivatable intermediates capable of forming crosslinks in the presence of light. If micro-titer well plates (e.g., 96-well plates or 384-well plates) are utilized, they can be coated with affinity purified capture monoclonal antibodies (typically diluted in a buffer) at, for example, room temperature and for about 2 to about 3 hours. The plates can also be coated with the antibody that specifically binds PrP^D, PrP^Res or PrP^Sc directly. The plates may be stacked and coated long in advance of the assay itself, and then the assay can be carried out

simultaneously on several samples in a manual, semi-automatic, or automatic fashion, such as by using robotics.

Similarly, if DYNABEADS®, such as DYNABEADS® M-450 (rat anti-mouse IgM) are utilized, the beads can be coated with the antibody using any procedures known in the art. In one non-limiting examples, the DYNABEADS® are suspended in a vial using vortexing, and then an appropriate amount of the DYNABEADS®, is moved to a polypropylene or polystyrene tube. The tube is placed on a magnet for a short period of time, and then removed from the magnet. A coating buffer is added, and the beads are mixed, such as by using a vortex. In one non-limiting example, a coating buffer comprising about 0.01% to 1%, such as about 0.1% bovine serum albumin in phosphate buffered saline is utilized. Examples of additional blocking agents for the coating buffer might include, but are not limited to egg albumin, casein, and nonfat milk. The antibody of interest is added (such as, but not limited to, 15B3, IgG W226 or IgG 261), and the DYNABEADS® are incubated with the antibody of interest with gentle mixing for a sufficient time for the antibody to adhere to the beads. A magnet can then be used to separate the coated beads from the supernatant, and a coating buffer can be added. The DYNABEADS® coupled to the antibody can be washed repeatedly, and stored for future use.

In one example, the antibody (such as, but not limited to, 15B3, IgG W226 or IgG 261) can be coupled to the substrate as about 5 μg antibody per 100 μΐ DYNABEADS®. In another example, the antibody (such as, but not limited to, 15B3, IgG W226 or IgG 261) can be coupled to the substrate as per lxlO ⁶ DYNABEADS® per μg of antibody. In another example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20 or 30-fold more antibody can be utilized, such as 30-50 μg, such as 36 μg of antibody per 100 μΐ DYNABEADS® (for example, 4 x 10⁸ beads/ml). In other embodiments, lng to 10 μg of antibody can be used for 1 x 10⁸ beads. In yet another example, 100-300 μg of antibody per 1 x 10^"8 DYNABEADS® (for example, 4 xlO⁸ beads/ml) can be utilized. In some non-limiting examples, the concentration of the antibody on the magnetic beads is about 10-500 μg of antibody per 1 x 10⁸ beads. In one specific non-limiting example, the antibody is 15B3, 6A12, or 8D5.

Coated plates or beads optionally can be treated with a blocking agent that binds non- specifically to and saturates the binding sites to prevent unwanted binding of the free ligand to the excess sites on the wells of the plate. Examples of appropriate blocking agents for this purpose include gelatin, bovine serum albumin, egg albumin, casein, and non-fat milk.

After coating and blocking, a sample to be analyzed is added to the immobilized antibody. The sample can be a biological sample or an environmental sample. The sample can be homogenized (such as for a tissue sample, such as a brain sample), and appropriately diluted with, for example, a lysis buffer (e.g., phosphate-buffered saline (PBS) with 1% Nonidet P-40, 0.5% sodium deoxycholate, 5 mM EDTA, and pH 8.0). Other detergents can be used, such as anioinic, cationic or non-ionic detergents, including but not limited to sodium dodecyl sulfated (SDS) to homogenize a sample. Alternatively, mechanical means can be utilized, such as using pipetting or devices such as blenders and homogenizers. The biological sample can be a blood, serum, plasma, or a sample of another biological fluid, such as, but not limited to cerebral spinal fluid or a nasal fluid. In a non-limiting example, the biological sample is a nasal burshing. The sample can be a tissue sample, such as a brain sample or a lymphoid tissue sample (such as tonsils). The sample can be diluted, such as in buffer, for example a buffer including bovine serum albumin. In one embodiment, the sample is diluted in a buffer, such as TRIS® buffered saline (TBS) or phosphate buffered saline (PBS), optionally including a detergent. The detergent can be a cationic, anionic or non-ionic detergent. In one embodiment the detergent is Sarkosyl. For example, the beads can be contacted with the sample in the presence of about 0.1% to about 1%, such as about 0.4 % Sarkosyl in TBS. In another embodiment, the beads can be contacted with the sample in the presence of about 0.1% to about 1% Sarkosyl in TBS, such as 0.4% to about 1% Sarkosyl in a buffer, such as TBS or PBS. In some examples, about 0.1%, about 0.4%, about 1%, about 2%, about 3% or about 4% Sarkosyl in a buffer, such as TBS or PBS, is utilized.

For sufficient sensitivity, the amount of sample added to the immobilized capture monoclonal antibody can be such that the immobilized capture monoclonal antibodies are in molar excess of the maximum molar concentration of the conformational altered protein anticipated in the biological sample after appropriate dilution of the sample.

The conditions for incubation of the biological sample and immobilized monoclonal antibody are selected to maximize sensitivity of the assay and to minimize dissociation.

Preferably, the incubation is accomplished at fairly constant temperatures, ranging from about 0° C to about 40° C, such as at about 4 °C, room temperature (e.g., about 25° C), about 35 °C to about 39 °C, or at about 37 °C, or about 35 °C to 40 °C. In some embodiments the temperature is about 19 to about 40 °C, such as at room temperature. The time for incubation can be for example, 2 hours to 12 hours, such as overnight. In some examples, the incubation period is 2, 4, 6, 8, 10, 12, 20 or 24 hours, for example overnight at about 0° C to about 40° C, such as at about 4 °C, room temperature (e.g., about 25° C), or 37 °C. In specific non-limiting examples, the incubation is about 2 hours at room temperature or overnight at 4 °C, such as about 12 hours at 4 °C or for about 10 to 20 hours at room temperature, such as 20 hours at room temperature or 37 °C.

Following contact of the biological sample with the immobilized capture-monoclonal antibody (such as, but not limited to, 15B3, IgG W226 or IgG 261), the biological sample is washed. The washing medium is generally a buffer ("washing buffer") with a pH determined using the considerations and buffers typically used for the incubation step. The washing may be done, for example, one, two, three or more times. The washing can be performed at any temperature, such as from about 0°C to about 40° C, such as at room temperature (e.g., 25 °C) or at 37 °C. In additional embodiments, the method comprises using SDS in a buffer, such as 0.01% to 0.1% SDS, such as about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06% or 0.07% SDS, for example 0.04% to 0.06% SDS, such as about 0.05% SDS. Examples of washing buffers include, but are not limited to, phosphate buffered saline (PBS) and Tris buffered saline (TBS), optionally including Sarkosyl, such as about 0.05-0.5% Sarkosyl, such as 0.1%, 0.2%, 0.3% OR 0.4% Sarkosyl. One exemplary washing buffer is 0.2% Sarkosyl in TBS.

The solid substrate, such as magnetic beads that have been contacted with the sample, can then be processed to detect bound prion protein, such as using a Standard QuIC (SQ) reaction or a RT- QuIC (RTQ) reaction, as disclosed herein. In one embodiment, prion proteins (e.g. PrP^D or PrP^Sc) bound to the antibody are not released (eluted) prior to detecting the bound prion proteins, rather the reaction mix including both the solid substrate comprising the antibody and the prion proteins are directly used in an assay to detect PrP^D, PrP^Res or PrP^Sc, such as, but not limited to, QuIC or RT-QuIC. Thus, the immune complexes comprising the antibody that specifically binds PrP^Res are not separated from the reaction mixture, but used directly in a QuIC or RT-QuIC assay.

In additional embodiments, the method can include contacting a sample from the subject with an effective amount of a particulate or immobilized ligand (such as plasminogen) or a precipitant such as sodium phosphotungstic acid, that selectively binds prions or PrP^Res for sufficient time to form a precipitable complex and; isolating the complex from the remainder of the sample.

The disclosure is illustrated by the following non-limiting Examples. EXAMPLES

Example 1

Materials and Methods

Protein Expression and Purification: Recombinant prion protein (rPrP^Sen) substrates were purified as previously described (27). Briefly, PrP DNA sequences encoding for Syrian golden hamster (residues 23 to 231 ; accession no. K02234; or residues 90-231), Bank Vole (residues 23 to 230; accession no. AF367624) or hamster-sheep chimera (Syrian hamster residues 23 to 137 followed by sheep residues 141 to 234 of the R154Q171 polymorph [accession no. AY907689]) prion protein genes were ligated into the pET41 vector (EMD Biosciences). Vectors were transformed into Rosetta (DE3) Escherichia coli and were grown in Luria broth medium in the presence of kanamycin and chloramphenicol. Protein expression was induced using the autoinduction system (28, 29) and was purified from inclusion bodies under denaturing conditions using Ni-nitrilotriacetic acid (NT A) superflow resin (Qiagen) with an ΑΚΤΑ fast protein liquid chromatographer (GE Healthcare Life Sciences). The protein was refolded on the column using a guanidine HC1 reduction gradient and eluted using an imidazole gradient as described (27). The eluted protein was extensively dialyzed into 10 mM sodium phosphate buffer (pH 5.8), filtered (0.22-μιη syringe filter [Fisher]) and stored at -80°C. Protein concentration was determined by measuring absorbance at 280 nm.

Brain homogenate preparations: Brain homogenates (BH; 10% w/v, Table 1 and 2, see Figs. 14 and 15) were prepared as previously described (8) and stored at - 80°C. For RT-QuIC analysis BHs were serially diluted in 0.1 % SDS (sodium dodecyl sulfate, Sigma)/N2

(Gibco)/PBS as previously reported (25), or where indicated the last dilutions were performed to a final concentration of 0.05% SDS/N2/PBS.

RT-QuIC protocol: RT-QuIC reactions were performed as previously described (8). Reaction mix was composed of 10 mM phosphate buffer (pH 7.4), 300 or 130 mM NaCl, 0.1 mg/ml rPrP^Sen, 10 μΜ thioflavin T (ThT), 1 mM ethylenediaminetetraacetic acid tetrasodium salt (EDTA), and 0.002% or 0.001% SDS. NaCl and SDS concentrations were varied where indicated. Aliquots of the reaction mix (98 μί) were loaded into each well of a black 96-well plate with a clear bottom (Nunc) and seeded with 2 μΐ_^ of indicated BH dilutions. The plate was then sealed with a plate sealer film (Nalgene Nunc International) and incubated at 42°C in a

BMG FLUOstar Omega plate reader with cycles of 1 min shaking (700 rpm double orbital) and 1 min rest throughout the indicated incubation time. ThT fluorescence measurements (450 +/-10 nm excitation and 480 +/-10 nm emission; bottom read) were taken every 45 min.

To compensate for minor differences in baselines between fluorescent plate readers and multiple experiments, data sets were normalized to a percentage of the maximal fluorescence response of the plate readers, as described (24), and plotted versus reaction time. Reactions were classified as RT-QuIC positive base on criteria similar to those previously described for RT-QuIC analyses of brain specimens (8, 24).

Proteinase K (PK) digestion of RT-QuIC products and immunoblotting: RT-QuIC reaction products were collected from the plates by extensive scraping and pipetting and treated with 10 μg/ml Proteinase K (PK) for 1 hour at 37 °C with 400 rpm orbital shaking. Equal volumes of PK-treated reactions were run on 12% Bis-Tris NuPAGE gels (Invitrogen). Proteins were transferred to an Immobilon P membrane (Millipore) using the iBlot Gel Transfer System (Invitrogen). Membranes were probed with R20 primary antiserum (epitope: residues 218-231) (30) diluted 1: 15,000 and visualized with the Attophos AP fluorescent substrate system

(Promega) according to the manufacturer's recommendations.

Example 2

Lack of detection of GSS and atypical scrapie subtypes associated with 6-14 kDa PrP^Res fragments using previously described rPrP^Sen constructs.

Most mammalian PrP^Res types with predominant 21-32 kDa PrP^Res bands can seed Thioflavin T-positive (ThT) amyloid formation in RT-QuIC reactions (9, 16-20) using at least one of the following substrates: Syrian golden hamster rPrP^Sen residues 90-231 (8, 17, 18), hamster _rPrP^Sen 23-231 (11), human _rPrP^Sen 23-231 (21), murine _rPrP^Sen 23-231 (12) or hamster- sheep chimeric rPrP^Sen 23-231 (9, 22). For example, detection of human P102L GSS brain tissue using hamster rPrP^Sen 90-231 is shown in Figure 1. However, to date, no detection of RT-QuIC seeding activity has been reported using these rPrP^Sen substrates with cases of human GSS or sheep scrapie that give prominent low molecular weight PrP^Res fragments in immunoblots, despite extensive efforts. Specifically, these cases include human GSS with the F198S, A117V or H187R- and sheep Nor98 scrapie types giving 6-14 kDa PrP^Res fragments (3-6), and the human P102L-GSS with an ~8 kDa PrP^Res fragment (P102L*) (3). Our inability to detect these prion types is exemplified in Figure 1 using 10^~3 brain tissue dilutions of human GSS F198S and P102L* and sheep Nor98 scrapie with the hamster _rPrP^Sen 90-231. In contrast, P102L GSS (without the ~8-kDa fragment) gave positive reactions with 1,000,000-fold smaller amounts of brain tissue. Example 3

Detection of GSS F198S and A117V prion seeding activity using BV _rPrP^Sen

We then tested bank vole rPrP^Sen residues 23-230 (BV rPrP^Sen) as a substrate to detect seeding activity of two human prion subtypes that have not been detectable previously by RT- QuIC, namely F198S- and A117V-GSS. Concurrently, we varied two parameters that we have shown to be influential, namely the concentrations of NaCl (8) and SDS (17). Each reaction was seeded with 10^"4 dilutions of frontal cortex brain tissue from confirmed GSS cases carrying either the F198S or A117V mutation of the prion protein gene. We found that our standard concentrations of SDS (0.002%) in combination with either 130 or 300mM NaCl failed to allow a distinction in lag phase between prion positive and uninfected brain homogenate (BH) seeded reactions (Figure 2 A-B). Lowering the SDS concentration to 0.001% with either 130 or 300mM NaCl improved this distinction between prion positive and uninfected BH seeded reactions (Figure 2 C-D). Furthermore, using final concentrations of 300 mM NaCl and 0.001% SDS, we obtained much shorter lag phases in reactions seeded with the two GSS subtypes than with the cerebral ischemia negative control (Figure 2D). These results indicated that under these latter RT-QuIC conditions BV rPrP^Sen detected seeding activity associated with PrP^D conformers that had not otherwise been detectable by RT-QuIC or PMCA prion seed amplification techniques.

Next, we assessed the sensitivity of this new RT-QuIC assay for detecting GSS- associated prion seeding activity. Reactions were seeded with 10^~4 to 10^~9 dilutions of brain tissue from GSS patients carrying the P102L, P102L*, A117V, F198S and H187R mutation of the prion gene (Figure 3 A-E). A reaction time cutoff of 50h was chosen because in more than 20 independent RT-QuIC experiments seeded with negative control Alzheimer's disease (AD) or cerebral ischemia brain homogenates, no positive RT-QuIC reactions were observed until after 55h (in rare wells). We detected GSS P102L, A117V and F198S and H187R prion seeding activity in as little as 10^~9, 10^~4, 10^~7 and 10^~6 dilutions of brain (frontal cortex) tissue dilutions, respectively (Figure 3).

Example 4

Detection of 28 different prion types/strains of humans, sheep, cattle, deer, elk, mouse and hamster using BV rPrP^Sen

After finding that BV rPrP^Sen supported RT-QuIC detection of prion seeding activity from previously undetectable types of GSS, we tested whether BV rPrP^Sen could be used to detect other types of prion diseases. We tested 28 different types of prions in brain tissue from humans, sheep, mouse, hamster, cattle, elk, and deer (Tablel and 2) and found that all of them gave faster and stronger positive ThT fluorescence responses than a variety of uninfected negative control brain specimens (Figure 4). Among the 28 were the five prion types that have not been detectable by RT-QuIC under other conditions, namely human GSS F198S, Al 17V, H187R, and P102L* and sheep Nor98 scrapie (Figure 4, red traces). These results indicated that under these conditions, BV rPrP^Sen is the most broadly prion-seeded RT-QuIC substrate described to date.

Example 5

Prion strain/type-dependent RT-QuIC products from reactions using BV rPrP^Sen Prion strain-dependence has not been observed previously in the immunoblot banding profile of PK-treated recombinant PrP^Res (rPrP^Res) products of RT-QuIC reactions. However, using B V rPrP^Sen we observed consistently distinct products of RT-QuIC reactions seeded with different types of human prions (Figure 5 and Table 1, Figure 14). The observed banding patterns could be grouped based on the type of seed: GSS cases (F198S, A117V, H187R) with the -8-14 Kda protease-resistant bands and sFI gave 2 bands: a major -lOkDa band and a -6- 9kDa band; the GSS (P102L), gCJD (E200K, V210I, octapeptide repeat insertion), and the iatrogenic CJD (iCJD) cases with -21-32 kDa PrP^Res bands gave multiple bands with a major ~12Kda band and multiple minor bands between -6-10 kDa; variant CJD, GSS (P102L*) and FFI (D178N) cases gave a single predominant band at -lOkDa; and sporadic CJD in some cases gave two bands between -10-12 kDa, while in other cases gave a predominant band at - lOkDa. Repeated analyses (>4) of individual sCJD cases indicated that they consistently seeded the formation of only one or the other of the latter two rPrP^Res products. This observation provided evidence that the different sCJD-seeded rPrP^Res products were dictated by differential templating activity in the tissue samples rather than stochastic events during the RT-QuIC reaction.

Additionally, because these immunoblots used an antiserum to the C-terminus of PrP, the fragments likely differed primarily at their N-termini.

We also compared the BV rPrP^Res products of reactions seeded with different rodent, bovine, cervine and ovine prion strains (Table 2, Figure 15). As with the human prion seeds, we observed distinct strain-dependent BV rPrP^Res banding profiles from reactions seeded with different prion types. Mouse 22L scrapie-seeded BV rPrP^Res products consistently showed a -10 and -12 kDa PK-resistant band, whereas BV rPrP^Res products from reactions seeded with Chandler, ME7, 87V and anchorless 22L (22L GPI^") scrapie (Figure 6A) displayed a predominant -10 kDa band. The lack of the GPI anchor in the 22L GPI^" scrapie seed resulted in an RT-QuIC product that was distinct from the wild-type GPI-anchored 22L scrapie. Additionally, closely related hamster prion strains (Hyper and 263 K; Figure 6B) showed similar BV rPrP^Res banding profiles (-10 and -12 kDa PK-resistant bands) which were distinct from the Drowsy-seeded BV rPrP^Res products (primarily a -10 kDa band; Figure 6B). Deer and elk CWD-seeded reactions each gave -8, 9, 10, and 12 kDa bands, but differed in the relative intensities of the top two bands between the two (Figure 6C). Furthermore, distinct strain- dependent BV rPrP^Res banding profiles were observed between classical (C-BSE) and atypical (L-BSE) (-10 kDa vs. -9, 10, and 12 kDa bands, respectively; Figure 6C), as well as between classical and atypical Nor98 sheep scrapie (-10 kDa vs. -9, 10, and 12 kDa bands, respectively; Figure 6D). Collectively, these immunoblotting results suggested that certain human and animal prion diseases can be discriminated in part based on analysis of the rPrP^Res products of B V rPrP^Sen-based RT-QuIC reactions.

Example 6

Detection and discrimination of classical and atypical BSE using BV and hamster rPrP^Sen substrates

We previously reported that classical and atypical L-type BSE strains can be

discriminated on the basis of relative RT-QuIC reactivities with hamster rPrP^Sen 90-231 and hamster-sheep chimeric rPrP^Sen 23-231 substrates 9). Here we found that BV rPrP^Sen can similarly detect both classical and L-type BSE providing an alternative substrate for

discrimination between the two bovine strains. Specifically, detection of seeding activity with BV rPrP^Sen (Figure 7) but not with three other rPrP^Sen substrates that detected only L-type BSE, namely human 23-231, hamster 23-231 or hamster 90-231 9), can be used to identify these two bovine prion types.

Example 7

Discrimination of classical and Nor98 sheep scrapie using BV and hamster-sheep chimeric rp_rpSen _substrates

Having detected Nor98 sheep scrapie with BV rPrP^Sen, (Figure 4) we tested whether a strategy similar to the one described above for C- vs. L-type BSE using different rPrP^Sen substrates would allow discrimination of Nor98 and classical sheep scrapie. Brain tissue from eight sheep with classical scrapie [ARQ/ARQ (n=6), VRQ/VRQ (n=2) PrP genotypes, Table 2, see Fig. 15] were readily detected using the hamster-sheep chimeric rPrP^Sen 23-231 within ~40hs (Figure 7A). However, brain tissue from eight cases of Nor98 scrapie (ARR/AHQ (n=l), ARQ/ARQ (n=4), ARQ/AHQ (n=2) and ARR/ARR (n=l) genotypes, Table 2, Fig. 15) gave no positive responses using the same substrate (Figure 8B). In contrast, consistent with the results in Figure 4, seven of these cases gave positive responses when BV rPrP^Sen was used in reactions seeded with 10^"4 brain tissue dilutions (Figure 8-E, orange lines) and those that were weaker were positive when seeded with 10^"3 dilutions (Figure 8-E, red lines). To compare the sensitivities of the assay for detection of classical and atypical scrapie using these two substrates, we diluted representative brain homogenates from classical and Nor98 scrapie positive sheep (Figure 9) and tested them using both BV and Ha-S rPrP^Sen. We detected classical scrapie down to 10^"8 dilutions using Ha-S rPrP^Sen and down to 10^"6 using BV rPrP^Sen. Consistent with the data in Figure 7, no fluorescence increases were seen in reactions seeded with the same dilutions of a Nor98 atypical scrapie sample when using Ha-S rPrP^Sen. In contrast, parallel reactions with BV rPrP^Sen gave positive reactions when seeded with Nor98 brain dilutions down to 10^"6. These results suggested that if ovine brain samples give strong positive RT-QuIC responses with BV rPrP^Sen, but not with the hamster-sheep chimeric rPrP^Sen 23-231, the host is likely to have had an atypical, e.g. Nor98, rather than classical, strain of scrapie.

Example 8

Detection and discrimination of human sCJD and vCJD using BV 23-230 and hamster 23-

231 rPrP^Sen substrates

To investigate the discrimination of two non-genetic human prion strains, we tested 10^"4 brain tissue dilutions from two confirmed cases of Type 1 sCJD (Figure 9, a and b) and two cases of vCJD (Figure 10, c and d, orange lines). We used previously described SDS conditions (0.002% final concentration of SDS;) with hamster 23-231 rPrP^Sen, and 0.001% SDS with BV rPrP^Sen, both in the presence of 300mM NaCl. We observed rapid amplification of prion seeding activity in the two Type 1 sCJD samples when using either hamster 23-231 or BV rPrP^Sen (Figure 9, top and bottom panel). Our detection of the sCJD samples with the hamster 23-231 substrate was consistent with previous demonstrations that all sCJD subtypes are detectable with this substrate. No increase in ThT fluorescence was seen in vCJD-seeded hamster 23-231 rPrP^Sen RT-QuIC reactions (Figure 10, top panel). However, in accordance with the results shown in Figure 4, seeding activity was detected in both vCJD samples using BV rPrP^Sen (Figure 10, bottom panel). Thus, sporadic and variant CJD sample were discriminated by differential reactivities with the BV and hamster 23-231 rPrP^Sen substrates.

Next we compared the RT-QuIC sensitivities for detection of sCJD and vCJD brain homogenates using hamster and BV rPrP^Sen. We performed end-point dilution RT-QuIC analysis of brain tissue from sCJD (Case a) and vCJD (Case c) (Figure 11). We detected sCJD down to 10- ⁸-10-⁹ with hamster 23-231 rPrP^Sen, (Figure 11-A) and 10 ⁸ with BV rPrP^Sen (Figure 11-C). Although sCJD gave slightly slower amplification kinetics with hamster 23-231 rPrP^Sen (Figure

11- A) compared to BV rPrP^Sen (Figure 11-B), the overall sensitivities using the two substrates were comparable. In contrast, markedly different sensitivities were observed with the two substrates in the vCJD-seeded reactions. Specifically, only weak seeding activity was occasionally detected in 10^"4 or 10^"5 brain dilutions with hamster 23-231 rPrP^Sen (representative data in Figure 11-B), but fast and sensitive detection of vCJD seeding activity down to 10^"7 brain tissue dilution was observed using BV rPrP^Sen (Figure 11-D). These results suggest that BV _rPrPs^en _is 100-10,000-fold more sensitive than hamster 23-231 rPrP^Sen in detecting vCJD brain derived prion seeding activity. Collectively, these findings further support the potential broad applicability of a BV rPrP^Sen prion discrimination strategy to a variety of prion types.

The lack of practical and cost-effective tests that are sensitive enough to detect the lowest infectious levels of prions has long been a major impediment in coping with prion diseases. Rapid commercially available immunoassays have allowed post-mortem detection of prion infections in high-titered tissues such as brain or lymphoid tissues, but diagnostic specimens that are most readily accessible in living hosts, such as blood, CSF and nasal brushings, have much lower prion titers that are undetectable with these assays. In contrast, RT-QuIC assays have been highly effective in detecting prion seeding activity in such low-titered specimens, and are being widely implemented as state-of-the-art diagnostic tests for humans and animals. Moreover, recent improvements have increased the speed and sensitivity of RT-QuIC assays such that sCJD testing based on human CSF samples can now be performed in a matter of hours rather than days (17).

In our experience, the most demanding and costly requirement for RT-QuIC testing is the availability of suitable rPrP^Sen substrates. Prior to the present study, testing facilities would typically have to produce or procure multiple rPrP^Sen sequences to be able to test for multiple prion types. However, we have now shown that all of the prion diseases that we have tested so far from humans and other mammals can be detected sensitively by using BV rPrP^Sen (Figure 4). This provides a useful platform for broad-based prion detection and strain discrimination. Thus, we envision that most initial screening for the presence of a wide variety of prions could be performed using BV rPrP^Sen. Once a prion-infected sample from a given host species is identified, one could then often discriminate between strains by targeted use of another rPrP^Sen substrate that is known to be differentially sensitive to seeding by prion strains of that host species (Figures 7, 8 and 10) and/or by performing immunoblots of the PK-resistant RT-QuIC products of the reactions (Figures 5 and 6). Although we have demonstrated detection of a wide variety of prion types, the relative sensitivities of B V rPrP^Sen -based RT-QuIC for brain homogenates of hosts with different prion diseases is presumably dependent on the concentrations of PrP^D in the tissue samples. Clearly PrP^D concentrations may vary markedly between individuals and different regions of the brain as a function of strain. Furthermore, because PrP^D can vary markedly in its properties, e.g. amyloid vs. non-amyloid, protease-sensitive vs. resistant, small vs. large particles, infectious vs. non-infectious, it is probable that the RT-QuIC seeding activity will vary per unit PrP^D between different prion strains and tissue sources. Thus, although we have shown the potential for BV rPrP^Sen-based RT-QuIC to detect and help discriminate prion strains, much additional work with each type of prion and sample type will be required to better establish the quantitative relationships between RT-QuIC seeding activity and the levels of various types of PrP^D in different tissues of diagnostic or scientific interest.

Since the inception of prion-seeded cell-free PrP conversion reactions (Kocisko et al., Nature. 1994 Aug l l;370(6489):471-4), striking sequence- and strain-specificities have been observed that appeared to correlate, at least largely, with transmission barriers and strain phenotypes of prion diseases in vivo (Kocisko et al., Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3923-7; Raymond et al., Nature. 1997 Jul 17;388(6639):285-8; et al., EMBO J. 2000 Sep l ;19(17):4425-30; Bessen et al., Nature. 1995 Jun 22;375(6533):698-700). Indeed, sequence differences of as little as a single residue between the PrP^D seed and PrP^Sen substrate can block PrP^Res formation in such cell-free reactions (Bossers et al., Proc Natl Acad Sci U S A. 1997 May 13;94(10):4931-6), as it can in scrapie-infected cells (25) and in vivo (Goldmann et al., J Gen Virol. 1991 Oct;72 ( Pt 10):2411-7). However, RT-QuIC assays have tended to be less constrained by such sequence differences (Wilham et al., PLoS Pathogens 2010). We reason that this is due in part to the fact that in RT-QuIC reactions, it is only the C-terminal residues -160- 231 of the substrate molecules that must refold into the PK-resistant amyloid core (26) to give a positive reaction, i.e., an increase in ThT fluorescence. In contrast, earlier cell-free conversion (Kocisko et al., Nature 1994, op. cit.; Atarashi et al., Nature Medicine, 2007 & 2008 and PMCA reactions (Saborio et al., Nature. 2001 Jun 14;411(6839):810-3) have used the immunoblot- based detection of much larger PK-resistant cores, typically comprised of residues -90-231, as a positive readout. Thus, much more extensive packing of more N-proximal residues is required in the latter reactions, as it is in vivo, giving more opportunities for sequence differences between seed and substrate to influence conversion. Nonetheless, despite the lower sequence specificity of RT-QuIC reactions, we and others have observed multiple examples of rPrP^Sen substrates that can be converted by some types of prion seeds and not others (9, 10). Therefore, we were surprised to find that BV rPrP^Sen can be induced to convert to ThT-positive amyloid by every type of prion- associated seed that we have tried so far (n=28), including several that had never before been detected by RT-QuIC or PMCA. We also did not anticipate that different PK- resistant BV rPrP^Res products of RT-QuIC reactions would be seeded with different prion strains from a single host species, because we had never seen such distinct templating with the many other rPrP^Sen substrates that we have tested. These findings suggest that BV rPrP^Sen -based RT- QuIC reactions may provide a new means of probing the strain-dependent heterogeneity of prion seeding activities and conformational templates. Moreover, the availability of BV rPrP^Sen as an apparently universal RT-QuIC substrate may markedly improve the practicality, efficiency and cost-effectiveness of detecting and discriminating prions.

Example 9

Detection of PrP in Formalin Fixed Brain Samples

Slices of formalin fixed, paraffin embedded brain tissue from humanized transgenic mice inoculated with either sCJD (top) or vCJD (bottom), or uninoculated controls were tested in the RT-QuIC. sCJD and Alzheimer's diseased brain homogenates were used as reaction controls. The substrate used for these experiments was Bank Vole 23-230 109M.

Under these conditions seeding activity from both sCJD and vCJD infected animals, was

-3 -4

detected. This seeding activity was detected in 10 and 10 fixed brain tissue dilutions, respectively, without any spontaneous conversion occuring in the negative control reactions. See Figures 16-18.

Example 10

Detection of atypical H-type bovine spongiform encephalopathy and the discrimination of bovine prion strains by RT-QuIC

The first prion disease to be recognized in cattle was "classical" bovine spongiform encephalopathy (C-BSE). C-BSE was likely caused primarily by widespread prion

contamination of cattle feed (Bradley and Wilesmith 1993. Br. Med. Bull. 49:932-959). After peaking in the early 1990s, the incidence of C-BSE has now been greatly reduced by regulatory measures that limit its horizontal spread. C-BSE is the only known zoonotic prion disease, having caused variant Creutzfeldt- Jakob disease (vCJD) in humans who, presumably, consumed contaminated beef. Although new clinical cases of vCJD are rare, a recent survey of appendices in the UK suggests a high incidence of subclinical vCJD infections of -1:2000 population born between 1941 and 1985 (Gill, Spencer, Richard-Loendt A, et al. 2013 BMJ 347:f5675). Since the C-BSE epidemic, two "atypical" strains of BSE, H-type (H-BSE) and L-type (L-BSE), have also been identified in cattle. PrPRes is usually comprised of a mixture of glycosylated and unglycosylated molecules, and the various BSE strains can be differentiated biochemically using Western blot analysis of post-mortem brain tissue by comparing the proteinase K (PK)-treated PrPRes banding patterns (Casalone C, Zanusso G, Acutis P, et al. 2004 Proc Natl Acad Sci U S A 101:3065-3070; Baron T, Vulin J, Biacabe AG, et al. 2011 PLoS One 6:el5839; Torres JM, Andreoletti O, Lacroux C, et al. 2011 Emerging Infect. Dis. 17:1636-1644; and Biacabe AG, Laplanche JL, Ryder S, Baron T. 2004 EMBO Rep. 5:110- 115). The H- and L-types of BSE are classified by their respective high and low apparent molecular masses of the unglycosylated PrPRes band. These atypical BSE strains, which are rare (<100 cases identified worldwide), tend to affect older animals (Windl O, Dawson M. Subcell 2012 Biochem 65:497-516) and appear to represent sporadic forms of bovine prion diseases (Brown P, McShane LM, Zanusso G, et al. 2006 Emerging Infect. Dis. 12:1816-1821). Despite the apparent rarity of the various types of BSE, the facts that H- and L-BSE appear to arise spontaneously and have distinct transmissibilities (Baron T, Vulin J, Biacabe AG, et al. 2011 PLoS One 6:el5839; Fukuda S, Iwamaru Y, Imamura M, et al. 2009 Microbiol. Immunol. 53:704-707; Lombardi G, Casalone C, A DA, et al. 2008 PLoS Path. 4:el000075; Buschmann A, Gretzschel A, Biacabe AG, et al. 2006 Vet. Microbiol. 117:103-116; Masujin K, Shu Y, Yamakawa Y, et al. 2008 Prion 2:123-128; Kong Q, Zheng M, Casalone C, et al. 2008 J. Virol. 82:3697-3701 ; Okada H, Masujin K, Iwamaru Y, et al. 2011 Vet. Pathol. 48:942-947; Bencsik A, Leboidre M, Debeer S, et al. 2013 Exp. Neurol. 72:211-218; and Wilson R, Dobie K, Hunter N, et al. 2013 J. Gen. Virol. 94:2819-2827) make it important to be able to detect and differentiate them to reduce the risk of transmission to cattle or other species such as humans. Commercially available "rapid" immunochemical tests for PrP^D, can, in the best cases, give positive responses from 10^~3-10^~4 dilutions of post-mortem brain tissues with high levels of PrP^D (Gray JG, Dudas S, Graham C, et al. 2012 J Vet Diagn Invest 24:976-980 and Meloni D, Davidse A, Langeveld JP, et al. 2012 PLoS One 7:e43133). Immunoblotting for PrP^Res can detect BSE- infected tissues with similar sensitivity and also discriminate between the bovine strains based on the relative electrophoretic migration and glycoform ratios of the PrP^Res bands (Casalone C, Zanusso G, Acutis P, et al. 2004 Proc Natl Acad Sci U S A 101 :3065-3070; Baron T, Vulin J, Biacabe AG, et al. 2011 PLoS One 6:el5839; Torres JM, Andreoletti O, Lacroux C, et al. 2011 Emerging Infect. Dis. 17:1636-1644; and Biacabe AG, Laplanche JL, Ryder S, et al. 2004 EMBO Rep. 5:110-115). A more sensitive conformation-dependent immunoassay (CDI) has also been described for C-BSE, which has a sensitivity similar to that of end-point dilution bioassays in a line of transgenic mice expressing bovine PrP [Tg(BoPrP^+/+)4092/Pn¾9^0/0] , that is, with 50% positive responses at C-BSE brain dilutions of ~10^~5 (Safar JG, Scott M, Monaghan J, et al. 2002 Nat. Biotechnol. 20: 1147-1150). Another line of "bovinized" transgenic mice has been reported to be 5-10-fold more sensitive in detecting dilutions of BSE brain tissue

(Buschmann A, Groschup MH. 2005. J. Infect. Dis. 192:934-942). Still more sensitive protein misfolding cyclic amplification (PMCA) assays have also been developed for C-BSE in cattle, which can detect brain tissue dilutions down to 10^"10 (Murayama Y, Yoshioka M, Masujin K, et al. 2010 PrP(Sc). PLoS One 5; Murayama Y, Masujin K, Imamura M, et al. 20104 J. Gen. Virol. 95:2576-2588; and Lacroux C, Comoy E, Moudjou M, et al. 2014 PLoS Path. 10:el004202). However, these PMCA assays require 4-8 days for this level of sensitivity and are more technically demanding than is optimal for routine diagnostic purposes.

An RT-QuIC assay was developed for H-BSE that can rapidly detect as little as 10^"9 dilutions of brain tissue and neat cerebrospinal fluid from clinically affected cattle. Moreover, comparisons of reactivities with different recombinant prion protein substrates and/or immunoblot band profiles of proteinase K- treated RT-QuIC reaction products indicated that H-, L- and C -BSE have distinctive prion seeding activities and can be discriminated by RT-QuIC on this basis.

The following methods were used in these studies: Western blot analysis of PrPRes from C-, L- or H-BSE affected cattle: Brain tissues

(cortex) from uninfected, C-, L- or H-BSE affected cattle were homogenized [20 %

concentration (w/v)] in PBS using a multi-bead shocker. The brain homogenates (125 μΐ) were mixed with an equal volume of buffer containing 4% (w/v) Zwittergent 3-14 (Calbiochem), 1% (w/v) Sarkosyl, 100 mM NaCl and 50mM Tris-HCl (pH 7.6), and incubated with 1 mg/mL collagenase at 37°C for 1 h. Next, samples were subjected to PK (Roche Diagnostics) digestion (40 μg/ml) at 37 °C for 1 h. PK digestion was terminated with Pefabloc (Roche Diagnostics). Samples were mixed with an equal volume of 2-butanol: methanol mixture (5: 1) and centrifuged at 20,000 x g for 10 min. The pellets were resuspended in gel-loading buffer containing 2% (w/v) SDS and boiled for 10 min prior to loading the gel. The samples were separated by SDS- PAGE (12% acrylamide) and transferred onto a PVDF membrane (Millipore). The membrane was then incubated with horseradish peroxidase (HRP) -conjugated monoclonal antibody (mAb) T2 at 1:5000 dilution (45). Signals were detected by incubating the membrane with a chemiluminescent reagent (SuperSignal; Pierce Biotechnology). Recombinant prion protein expression and purification: Recombinant PrP (rPrPSen) substrates were prepared as previously described (Groveman BR, Kraus A, Raymond LD, et al. 2015 J. Biol. Chem. 290: 1119-1128). Briefly, the PrP sequence for bank vole (BV) [residues 23-230; methionine at residue 109 (M109): GENBANK® accession no. AF367642, residue 23- 230; isoleucine at 109 (1109) and residues 90-230 (M109)], Syrian golden hamster (Ha)

[residues 23-231: accession no. K02234 and residues 90-231], mouse [residues 23-231:

accession no. M13685], sheep (Sh) [residue 25-234; ARQ: alanine at 136 (A136)/arginine at 154 (R154)/ glutamine at 171 (Q171): accession no. AY907689, VRQ: valine at 136

(V136)/R154/Q171: accession no. AJ567988.1, ARR: A136/R154/R171], Human (Hu) [residues 23-231 ; methionine at 129 (M129)], human-bank vole chimera (Hu-BV) [human residues 23- 165 followed by bank vole residues 166-230 (M10929)] and a hamster-sheep (Ha-S) chimera [Syrian hamster residues 23-137 followed by sheep residues 141-234 of the R154Q171 polymorph: accession no. AY907689] were amplified and ligated into the pET41 vector (EMD Biosciences), and sequences verified. Vectors were transformed into Rosetta (DE3) Escherichia coli and were grown in Luria broth medium in the presence of kanamycin and chloramphenicol. Protein expression, purification and refolding were performed as previously described

(Groveman BR, Kraus A, Raymond LD, et al. 2015 J. Biol. Chem. 290: 1119-1128 and Orru CD, Groveman BR, Raymond LD, et al. 2015 PLoS Path. I l :el004983). The eluted protein was extensively dialyzed into 10 mM sodium phosphate buffer (pH5.8), then filtered with 0.22-μιη syringe filter (Fisher) and stored at -80°C until use. Protein concentration was determined by measuring absorbance at 280 nm.

Brain homogenate preparation for RT-QuIC: Brain homogenates (BH: 10% w/v) were prepared as previously described (Wilham JM, Orru CD, Bessen RA, et al. 2010 PLoS Path. 6:el001217) and stored at -80°C. For RT-QuIC analysis, brain homogenates were serially diluted in 0.1% SDS/N2 (Gibco)/PBS as previously reported (Sano K, Satoh K, Atarashi R, et al. 2013 PLoS One 8:e54915), where indicated the last dilution were performed in 0.05% SDS/N2/PBS.

RT-QuIC analysis: RT-QuIC reactions were performed as previously described (Wilham JM, Orru CD, Bessen RA, et al. 2010 PLoS Path. 6:el001217). The RT-QuIC reaction mixture was composed of 10 mM phosphate buffer (pH 7.4), 300 mM or 130 mM NaCl, 10 μΜ thioflavin T (ThT), ImM EDTA, 0.1 mg/ml of rPrPSen and 0.002% or 0.001% SDS. Aliquots of this mixture (98 μΐ for BH or 80 μΐ for CSF) were loaded into each well of a black 96- well plate with a clear bottom (Nunc) and seeded with 2 μΐ of BH dilution or 20 μΐ of CSF.

Uninfected bovine BH dilutions or CSF were used as negative controls. The plate was sealed with a plate- sealer film (Nalgen Nunc International) and incubation at ether 42 °C or 55 °C for 40 to 90 h in a BMG FLUOster Omega plate reader with cycles of 1 min shaking (700 rpm double orbital) and 1 min of rest throughout the incubation. ThT fluorescence measurements

(excitation: 450 + 10 nm, emission: 480 + 10 nm, bottom read) were taken every 45 min. The RT-QuIC data was analyzed as previously described (Orru CD, Bongianni M, Tonoli G, et al. 2014 New Engl. J. Med. 371:519-529). Briefly, to compensate for differences between the fluorescence plate readers, data sets from replicate wells were averaged and normalized to a percentage of the maximal fluorescence response of the instrument, and the obtained values were plotted against the reaction times. Samples were judged to be RT-QuIC positive using criteria as previously described (Orru CD, Groveman BR, Raymond LD, et al. 2015 PLoS Path. I l :el004983). Data are displayed as the average of four technical replicates except where indicated.

SD50 calculations. The 50% seeding dose (SD50) was determined by end point dilution RT-QuIC as previously described (Wilham JM, Orru CD, Bessen RA, et al. 2010 PLoS Path. 6:el001217). Briefly, a dilution series with at least one dilution giving 100% ThT positive replicates and at least one dilution giving 0% ThT positive replicates was chosen for Spearman- Karber analysis. SD50 is defined as the amount giving positive ThT fluorescence in 50% of the replicate wells.

PK digestion of RT-QuIC products and Western blot analysis: PK treatment of RT-QuIC BV rPrPSen conversion products and immunoblotting was performed as previously described (Orru CD, Groveman BR, Raymond LD, et al. 2015 PLoS Path. I l:el004983). RT-QuIC reaction products were collected from the bottom of plates by extensive scraping and pipetting using the same tip for all replicate wells and treated with 10 μg/ml PK for 1 h at 37 °C with 400 rpm orbital shaking to leave PK-resistant rPrP products. The samples were then mixed with an equal volume of gel-loading buffer containing SDS and 8M Urea and were boiled for 10 min for Western blot analysis. The samples were separated using a 12% SDS-PAGE gel and transferred onto a PVDF membrane (Millipore). The blotted membrane was then incubated with R20 primary antiserum [hamster epitope: residues 218-231] (50), followed by an anti-rabbit Alkaline Phosphatase (AP) conjugated secondary antibody. Signals were visualized using Attophos AP fluorescent substrate system (Promega). The rPrPRes band ratio was calculated by using the Image analysis software (ImageQuant TL, GE Healthcare).

The following results were obtained: Immunoblot profile of PrPRes in brains from BSE-infected cattle.

The brain samples (cortex) were collected from cattle affected by C-, L-, or H-BSE. To confirm the presence of PrP^Res in the brain specimens and the presence of the typical banding pattern profiles of C-, L- and H-BSE, the samples were PK treated and analyzed by Western blot. As expected (Fig. 19) and previously described (Casalone C, Zanusso G, Acutis P, et al. 2004 Proc Natl Acad Sci U S A 101:3065-3070), the H-BSE samples all showed a

predominance of the highest molecular mass glycoform and unglycosylated (lower) and mono- glycosylated (middle) bands that migrated slightly above the corresponding bands in the C- and L-BSE profiles.

H-BSE detection using bank vole (BV) rPrPSen

Given that full-length wild- type BV rPrP^Sen with residues 23-230 and methionine at residue 109 (BV rPrP^Sen 23-230, M109) is a universal substrate for RT-QuIC detection of prions, BV rPrP^Sen was tested as a substrate for detecting H-BSE. Reactions seeded with 10^"5 dilutions of brain tissue from three cattle clinically affected with H-BSE gave rapid increases in ThT fluorescence within 5 h (Fig. 20). In contrast, reactions seeded with the same dilution of brain samples from C-BSE-infected cattle gave much longer lag phases of between 24-40 h, while L- BSE reactions consistently displayed intermediate lag phases of 8-13 h. For one C-BSE sample, when reactions were seeded with 10^~5 dilutions of brain tissue, RT-QuIC kinetics were only -20% faster than an uninfected negative control brain homogenate, which started to give spontaneous, prion-independent positive fluorescence after 50 h. The latter time therefore marked the point at which it became difficult to clearly discriminate prion-seeded from spontaneously positive reactions; this point is known to vary with substrate, reaction conditions and sample matrix effects (Wilham JM, Orru CD, Bessen RA, et al. 2010 PLoS Path.

6:el001217; Orru CD, Groveman BR, Raymond LD, et al. 2015 PLoS Path. Il:el004983;

Vascellari S, Orru CD, Hughson AG, et al. 2012 PLoS One 7:e48969; and Orru CD, Groveman BR, Hughson AG, et al. 2015 MBio 6). These results indicated that, with RT-QuIC using the BV rPrP^Sen substrate, H-BSE was consistently more rapidly detectable than either C-BSE or L- BSE at the same concentration of brain homogenate from clinically affected animals.

To assess the analytical sensitivity of detection of H-BSE seeding activity in brain tissue from clinical animals, end point dilutions analysis was performed on samples from the three infected cattle. For each case, all brain tissue dilutions down to 10^"8 gave positive reactions in 3/4 (H-BSE #1) and 4/4 (#2 and #3) replicate wells within 40 h (Fig. 21). At 10^"9, 1 out of 4 replicate wells were positive for two of the H-BSE brains, while the third brain was negative for all 4 replicates. Using Spearman-Karber analysis, 50% seeding dose (SD50) titers of 108.20- 108.45 SD50/mg were estimated, which were comparable to the highest that we have seen with BV rPrP^Sen 23-230, M109 and most other substrates, for prion strains from other host species (Orru CD, Groveman BR, Raymond LD, et al. 2015 PLoS Path. I l:el004983).

H-BSE, L-BSE and C-BSE prion seeding activity detection using other rPrPSen substrates

Eleven other rPrP^Sen substrates were tested for their relative utilities in detecting these bovine prion strains (see above Materials and Methods and Fig. 22). All of these substrates detected seeding activity in a 10^"4 dilution of H-BSE brain homogenate in 4/4 replicate reactions with average lag phases of 5-20 h, while uninfected brain homogenate gave no positive reactions within 40 h (Fig. 22A-L). The shortest lag phases were observed with those substrates containing BV sequence. Overall, these results showed that H-BSE brain homogenate was able to seed amyloid formation by 12 different rPrP^Sen substrate molecules in RT-QuIC reactions.

Each of these same substrates also detected 10^"4 dilutions of L-BSE brain homogenates in all replicate reactions (Fig. 22A-L). With a majority of these substrates (BV rPrP^Sen 90-230, M109; BV _rPrP^Sen 23-230, 1109; Hu-BV _rPrP^Sen 23-230, M109; Mo _rPrP^Sen 23-231; Hu _rPrP^Sen 23-231, M129; Ha _rPrP^Sen 23-231 ; Ha _rPrP^Sen 90-231), the L-BSE seeds gave longer lag phases than those seen with H-BSE. With BV _rPrP^Sen 23-230, M109 and Hu-BV _rPrP^Sen 23-230, M109 in particular, the L-BSE-seeded reactions always had a longer lag phase than was observed in simultaneous reactions seeded with comparable dilutions of H-BSE brain homogenate (Figure 22A). However, comparable, or shorter lag phases were seen for L-BSE with the substrates containing sheep sequence (Fig. 22I-L). The shortest lag phase for L-BSE was seen with the sheep (Sh) ARR sequence (Fig. 22K).

Only half of these various rPrP^Sen substrates detected seeding activity in 10^"4 dilutions of C-BSE within 40 h, and in all cases the lag phases were much longer, and the mean ThT fluorescence levels weaker, than was observed with comparable dilutions of H-BSE or L-BSE brain homogenates (Fig. 22). Thus, altogether, these results provided evidence that the H-, L- and C-BSE in these brains differed in their relative abilities to seed amyloid formation by these 12 _rPrP^Sen substrates.

The strain-dependent differences in RT-QuIC kinetics that was observed with BV rPrP^Sen 23-230 and Sh _rPrP^Sen ARR 25-234 substrates suggested the following potential strategy for discriminating these strains by RT-QuIC: If an unknown bovine sample were positive at a given dilution using the BV substrate, it could contain prion seeds of any one of the three strains. However, if the same dilution were tested concurrently with the Sh rPrP^Sen ARR 25-234 substrate, the lag phase should be markedly longer if the unknown were H-BSE, shorter if it were L-BSE, and undetectable if it were C-BSE (Fig. 23). This diagnostic algorithm was tested on brain samples from cattle clinically affected with each of the BSE strains (3 cattle per strain) (Fig. 24). Three dilutions (10^~3, 10^"4 and 10^"5) of each brain were tested using the BV rPrP^Sen 23- 230 and Sh rPrP^Sen ARR 25-234 substrates. Consistent with the above results, all of the brains gave positive reactions with the BV rPrP^Sen 23-230 substrate (darker colors) while only the H- BSE and L-BSE brains gave positive reactions with the Sh rPrP^Sen ARR 25-234 substrate (lighter colors). Uninfected brains were negative for all replicate reactions for at least 50 h. Also, at each dilution of each H-BSE brain, the lag phase with the Sh _rPrP^Sen ARR 25-234 substrate was approximately double that obtained with the BV rPrP^Sen 23-230 substrate. In contrast, for each dilution of the L-BSE brains, the lag phase with the Sh _rPrP^Sen ARR 25-234 substrate was shorter than with the BV substrate. Thus, these results provide evidence that C-, L- and H-BSE can be discriminated based on relative reactivities and lag phases obtained using the BV rPrPSen 23-230 and Sh rPrPSen ARR 25-234 substrates.

To further test the ability of this assay to discriminate the BSE strains by RT-QuIC using the above algorithm, all of the available H- (n=3), L-(n=3) and C- (n=3) BSE samples described above were re-tested after the samples were blinded by a colleague not otherwise involved in this study. In all cases, the correct strain identification was made when brain homogenate dilutions of 10^~3-10^~5 were used. With more extreme dilutions, the greater variability in lag phase, which is typically seen in RT-QuIC reactions seeded with low levels of prion seeding activity (Wilham JM, Orru CD, Bessen RA, et al. 2010 PLoS Path. 6:el001217), could confound strain identification. Furthermore, it was important to use a single plate to compare the relative kinetics with BV rPrPSen 23-230 and Sh _rPrP^Sen ARR 25-234 substrates for a given test sample, along with positive controls of each strain. This precaution was needed because inconsistent reaction conditions between two reactions can influence lag phases.

Discrimination of H-BSE from L- and C-BSE by immunoblotting of RT-QuIC products Another RT-QuIC -based approach to discriminating prion strains is the comparison of the profile of PK-resistant products of RT-QuIC reactions using the BV _rPrP^Sen 23-230, M109 by Western blotting (Orru CD, Groveman BR, Raymond LD, et al. 2015 PLoS Path.

l l :el004983). This approach was tested by comparing the products of reactions seeded with brain samples from H-, L- and C-BSE cattle (Fig. 25 A). H- and C-BSE-seeded reaction products were difficult to distinguish from one another with each having a predominant 10 kDa band and a weaker band or a diffuse smear at -12 kDa (Fig. 25B). In contrast, the L-BSE-seeded products had stronger relative ratios of intensities of the 12 kDa band to 10 kDa band (t-test: p<0.001) compared to C- and H-BSE (Fig. 25B). Thus, these relative banding patterns might provide an additional means of discriminating L- and H-BSE and be helpful in future analyses to confirm BSE strain identifications. Detection of prion seeding activity in the cerebrospinal fluid ( CSF) of H- and L-BSE-infected cattle

Because prion seeding activity has been detected in the CSF of prion-infected humans, sheep, rodents and deer (Atarashi R, Satoh K, Sano K, et al. 2011 Nat. Med. 17: 175-178; Orru CD, Groveman BR, Hughson AG, et al. 2015 MBio 6; Orru CD, Wilham JM, Hughson AG, et al. 2009 Protein Eng. Des. Sel. 22:515-521; McGuire LI, Peden AH, Orru CD, et al 2012 Ann. Neurol. 72:278-285; Orru CD, Hughson AG, Race B, et al. 2012 J.Clin.Microbiol. 50: 1464- 1466; and Haley NJ, Van de Motter A, Carver S, et al. 2013 PLoS One 8:e81488), it was tested whether the same is true in BSE-infected cattle. For these experiments, 20 μΐ of neat CSF from two C-BSE, four L-BSE, two H-BSE-affected cattle and two uninfected cattle was tested using either the BV _rPrP^Sen 23-230, M109, BV _rPrP^Sen 90-230, Ml 09 or Ha _rPrP^Sen 90-231 substrates. Both H-BSE cattle (Fig. 26) gave positive reactions in 2/2 replicate reactions performed with BV rPrPSen 23-230, M109, and 2/3 or 3/3 reactions with Ha _rPrP^Sen 90-231 (Fig. 26A and 26C, respectively). However, only one of the H-BSE cattle was clearly positive, relative to uninfected controls, using the BV _rPrP^Sen 90-230, M109 substrate (Fig. 26B). CSFs from three of four L- BSE cattle (red) gave 3/3 positive replicate reactions with the Ha rPrPSen 90-231 substrate, while the fourth was negative (Fig. 26C). The other two substrates appeared to be less sensitive at detecting L-BSE in CSF (Fig. 26A and 26B). Finally, no positive reactions were detected in CSF samples from two C-BSE cattle with any of the three substrates (Fig. 26A and 26C).

Collectively, these results indicate preliminarily that the Ha rPrP^Sen 90-231 substrate was the most effective in detecting both H- and L-BSE in bovine CSF samples and that prion seeding activity was detectable in at least some of the H- and L-BSE cattle, but not in C-BSE or uninfected cattle. Thus, thus rPrP^Sen substrate for these particular discrimination assays must be selected based on the bodily fluids or tissues being tested.

RT-QuIC assays are capable of sensitive detection and discrimination of C-BSE and L- BSE using various rPrPSen substrates (Orru CD, Favole A, Corona C, et al. 2015 J. Clin.

Microbiol. 53: 1115-1120 and Orru CD, Groveman BR, Raymond LD, et al. 2015 PLoS Path. I l:el004983, 47). An ultrasensitive RT-QuIC assay is disclosed herein that also detects H-BSE prion seeding activity in 10^"5 H-BSE brain homogenate dilutions within 5-10 h and as little as lO ⁸ dilutions within 24 h. With sensitivities that are orders of magnitude greater than the commercially available rapid immunochemical tests, these RT-QuIC tests should be able to detect BSE in a larger proportion of cattle and in tissue specimens with much lower, but still potentially infectious, levels of contamination. This improves the likelihood that, as is the case for sCJD in humans (Atarashi R, Satoh K, Sano K, et al. 2011 Nat. Med. 17: 175-178; Orru CD, Bongianni M, Tonoli G, et al. 2014 New Engl. J. Med. 371:519-529; Orru CD, Groveman BR, Hughson AG, et al. 2015 MBio 6; and McGuire LI, Peden AH, Orru CD, et al. 2012 Ann.

Neurol. 72:278-285), antemortem diagnostic tests can be developed that are based on analyses of tissue or fluids that can be obtained from live cattle. The experiments disclosed herein demonstrate detection of prion seeding activity in the CSF of cattle with H- and L-, but not C- BSE (Fig. 25); however, CSF is not likely to be practical as antemortem diagnostic specimen for cattle, so testing of other more accessible tissue specimens is warranted. As noted above, RT- QuIC assays can be quantitative (Wilham JM, Orru CD, Bessen RA, et al. 2010 PLoS Path. 6:el001217; Bessen RA, Wilham JM, Lowe D, et al. 2011 J.Virol. 86: 1777-1788; Shi S, Mitteregger-Kretzschmar G, Giese A, et al. 2013 Acta Neuropathol Commun 1:44; Henderson DM, Davenport KA, Haley NJ, et al. 2015 J. Gen. Virol. 96:210-219; and Chesebro B, Striebel J, Rangel A, et al. 2015 MBio 6, 57-60), facilitating assessments of the relative amounts of prion seeding activity in various diagnostic specimens and tissues that might end up in the food supply (EFSA SRo. 2014.. EFSA Journal 12:3798).

Through comparisons of H-, L- and C-BSE reactivities with 12 different recombinant PrP substrates, provided herein are means of clearly discriminating each of three major bovine prion strains. In practice, test samples are run simultaneously with the BV rPrP^Sen 23-230 and sheep rPrP^Sen ARR 25-234 substrates in the same plate allowing the detection and strain discrimination in one assay. In such an assay, positive control standards of each strain are used to allow direct internal comparisons. An algorithm is disclosed above (Fig. 23) that is effective for 10 ³-10^"5 dilutions of brain tissue from clinically affected cattle, but not more extreme dilutions. Thus, test samples that have reduced concentrations of prion seeding activity are more difficult to detect. Such samples might include brain tissue from preclinical BSE-infected cattle or non-CNS tissues with lower levels of seeding activity.

RT-QuIC assays are less labor-intensive, time consuming and technically demanding than comparably sensitive PMC A tests for BSE (Murayama Y, Yoshioka M, Masujin K, et al. 2010 PLoS One 5 and Murayama Y, Masujin K, Imamura M, et al. 20104 J. Gen. Virol.

95:2576-2588), which require sonication rather than shaking and western blotting rather than fluorescence as a readout. Thus, an RT-QuIC assay for BSE is the most practical means of detecting all infectious levels of the three major BSE strains. Furthermore, when brain samples contain sufficient seed concentrations, RT-QuIC can discriminate these strains from one another.

In view of the many possible embodiments to which the principles of our invention may be applied, it should be recognized that illustrated embodiments are only examples of the invention and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method of determining whether a subject has a prion disease or transmissible spongiform encephalopathy, comprising:

a) performing a prion protein amyloid seeding assay a biological sample from the subject comprising:

(i) contacting the first biological sample with a recombinant bank vole protease sensitive prion protein (rPrP^Sen) to form a first reaction mixture;

(ii) incubating the reaction mixture to permit coaggregation of disease-associated prion protein (PrP^D) present in the biological sample with the recombinant bank vole protease sensitive prion protein (rPrP^Sen);

(iii) maintaining incubation conditions that promote coaggregation of the

recombinant bank vole protease sensitive prion protein (rPrP^Sen) with the PrP^D to result in a conversion of the recombinant bank vole sensitive prion protein to recombinant protease resistant and/or amyloid prion protein (rPrP-res^(Sc)) while inhibiting development of

spontaneously forming recombinant protease resistant and/or amyloid prion protein (rPrP- res^(sP°ⁿ⁾);

(iv) agitating aggregates formed during step (iii), wherein the reaction conditions comprise shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking; and detecting rPrP-res^(Sc) in the reaction mixture,

wherein detection of rPrP-res^(Sc) in the reaction mixture indicates that the subject has the prion disease or transmissible spongiform encephalopathy.

2. The method of claim 2, wherein the subject is a human, and wherein the prion disease or transmissible spongiform encephalopathy is Creutzfeldt- Jakob disease (CJD), Gerstmann-

Straussler-Scheincker syndrome (GSS), fatal familial insomnia (FFI) and sporadic fatal insomnia (sFI).

3. The method of claim 2, wherein the subject is a human, and wherein the prion disease is GSS P102L*, F198S, A117V or H187R.

4. The method of claim 1, wherein the subject is a cow, and wherein the transmissible spongiform encephalopathy is bovine spongiform encephalopathy (BSE).

5. The method of claim 1, wherein the subject is a cervid, and wherein the transmissible spongiform encephalopathy is chronic wasting disease (CWD).

6. The method of claim 2, wherein the subject is a sheep, and wherein the transmissible spongiform encephalopathy is scrapie.

7. The method of any one of claims 1-6, wherein detecting the presence of rPrP-res^(Sc) amyloid in the first sample and/or the second sample comprises the use of thioflavin T (ThT).

8. The method of any one of claims 1-7, wherein the biological sample is a nasal brushing, saliva, blood, serum, plasma, cerebral spinal fluid, feces, urine or tissue sample.

9. The method of any one of claims 1-8, wherein agitating aggregates in step (iv) comprises agitating aggregates in the absence of sonication.

10. The method of any one of claims 1-9, further comprising adding additional recombinant bank vole sensitive prion protein to the first reaction mixture of step (iii) without removing rPrP-res^(Sc) prior to detecting the presence of rPrP-res^(Sc).

11. The method of claim 10, wherein the additional recombinant bank vole sensitive prion protein is added to the reaction mixture without serial rounds of amplification.

12. The method of any one of claims 1-11, further comprising quantitating the rPrP- res^(Sc) in the reaction mixture.

13. The method of any one of claims 1-12, wherein the shaking cycle in step (iv) comprises a period of rest that precedes the period of shaking, and wherein the period of rest and the period of shaking are substantially equal.

14. The method of claim 13, wherein the shaking cycle in step (iv) is 20 to 180 seconds in length.

15. The method of claim 14, wherein the period of rest and the period of shaking are about 60 seconds in length.

16. The method of any one of claims 1-15, wherein step (iv) and step (ix) are repeated from about 1 to about 200 times.

17. The method of any one of claims 1-16, comprising contacting a sample from the subject with an effective amount of an antibody that specifically binds prions, PrP^D or PrP^Res for sufficient time to form an immune complex and;

separating the immune complex from the sample, thereby producing the biological sample.

18. The method of any one of claims 1-17, wherein the recombinant bank vole sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 22, optionally with an N-terminal methionine, or the amino acid sequence set forth as SEQ ID NO: 17 or SEQ ID NO: 25, optionally without a N-terminal methionine.

19. A method of discriminating whether a sheep subject has atypical (Nor98) scrapie or classical scrapie, comprising:

a) performing a prion protein amyloid seeding assay on a first biological sample from the subject comprising:

(ii) incubating the first reaction mixture to permit coaggregation of disease- associated prion protein (PrP^D) present in the biological sample with the recombinant bank vole protease sensitive prion protein (rPrP^Sen);

(iii) maintaining incubation conditions that promote coaggregation of the recombinant bank vole protease sensitive prion protein (rPrP^Sen) with the PrP^D or PrP^Res to result in a conversion of the recombinant bank vole protease sensitive prion protein to recombinant protease resistant and/or amyloid prion protein (rPrP-res^(Sc)) while inhibiting development of spontaneously forming recombinant protease resistant and/or amyloid prion protein (rPrP-res^(sP^on));

(iv) agitating aggregates formed during step (iii), wherein the reaction conditions comprise shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking; and (v) detecting rPrP-res^(Sc) in the first reaction mixture; and

b) performing a second prion protein amyloid seeding assay reaction on a second biological sample from the subject comprising:

(vi) contacting the second biological sample with a second recombinant protease sensitive prion protein (rPrP^Sen), wherein the second protease sensitive prion protein is a chimeric hamster-sheep, human, mouse, bovine or sheep protease sensitive prion protein to form a second reaction mixture;

(vii) incubating the second reaction mixture to permit coaggregation of PrP^D present in the second sample with the second recombinant protease sensitive prion protein (rPrP^Sen);

(viii) maintaining incubation conditions that promote coaggregation of the second recombinant protease sensitive prion protein (rPrP^Sen) with the PrP^D to result in a conversion of the second recombinant protease sensitive prion protein to a second recombinant protease resistant and/or amyloid prion protein (rPrP-res^(Sc)) while inhibiting development of

(ix) agitating aggregates formed during step (viii), wherein the reaction conditions comprise shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking; and

(x) detecting rPrP-res^(Sc) in the second reaction mixture,

wherein detection of rPrP-res^(Sc) in the first reaction mixture but not the second reaction mixture indicates that the subject has atypical (Nor98) scrapie, and wherein detection of rPrP-res^(Sc) in the first reaction mixture and the second reaction mixture indicates that the subject has classical scrapie.

20. The method of claim 19, wherein detecting the presence of rPrP-res^(Sc) in the first sample and/or the second biological sample comprises the use of thioflavin T (ThT).

21. The method of claim 19 or claim 20, wherein the first biological sample and/or the second biological sample is a nasal brushing, saliva, blood, serum, plasma, cerebral spinal fluid, feces, urine or tissue sample.

22. The method of any one of claims 19-21, wherein agitating aggregates in step (iv) and (ix) comprises agitating aggregates in the absence of sonication.

23. The method of any one of claims 19-22, further comprising adding additional recombinant bank vole protease sensitive prion protein to the first reaction mixture of step (iii) without removing rPrP-res^(Sc) prior to detecting the presence of rPrP-res^(Sc).

24. The method of claim 23, wherein the additional recombinant bank vole protease sensitive prion protein is added to the reaction mixture without serial rounds of amplification.

25. The method of any one of claims 19-24, further comprising adding additional second recombinant protease sensitive prion protein to the second reaction mixture of step (viii) without removing rPrP-res^(Sc) prior to detecting the presence of rPrP-res^(Sc).

26. The method of claim 25, wherein the additional second recombinant protease sensitive prion protein is added to the reaction mixture without serial rounds of amplification.

27. The method of any one of claims 19-26, further comprising quantitating the rPrP- res^(Sc) in the first reaction mixture.

28. The method of any one of claims 19-27, further comprising quantitating the rPrP- res^(Sc) in the second reaction mixture.

29. The method of any one of claims 19-28, wherein the shaking cycle in step (iv) comprises a period of rest that precedes the period of shaking, and wherein the period of rest and the period of shaking are substantially equal.

30. The method of claim 29, wherein the shaking cycle in step (iv) is 20 to 180 seconds in length.

31. The method of any one of claims 19-30, wherein the shaking cycle in step (ix) comprises a period of rest that precedes the period of shaking, and wherein the period of rest and the period of shaking are substantially equal.

32. The method of claim 31, wherein the shaking cycle in step (ix) is 20 to 180 seconds in length.

33. The method of claim 30, wherein the period of rest and the period of shaking are about 60 seconds in length.

34. The method of claim 32, wherein the period of rest and the period of shaking are about 30 seconds in length.

35. The method of any one of claims 19-34, wherein step (iv) and step (ix) are repeated from about 1 to about 200 times.

36. The method of any one of claims 19-35, comprising contacting a sample from the subject with an effective amount of an antibody that specifically binds sheep prions or sheep PrP^D for sufficient time to form an immune complex and;

separating the immune complex from the sample, thereby producing the first biological sample.

37. The method of any one of claims 19-36, comprising contacting an additional sample from the subject with an effective amount of an antibody that specifically binds sheep prions or sheep PrP^D for sufficient time to form an immune complex and;

separating the immune complex from the sample, thereby producing the second biological sample.

38. The method of any one of claims 19-37, wherein

a) the second recombinant protease sensitive prion protein is the recombinant hamster sheep protease sensitive prion protein, and wherein the hamster sheep protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 5 or SEQ ID NO: 26, optionally with an N-terminal methionine; or

b) the second recombinant sensitive prion protein is the human protease sensitive prion protein, and wherein the human protease sensitive prion protein comprises i) the amino acid sequence set forth as SEQ ID NO: 3 or SEQ ID NO: 4, optionally with an N-terminal methionine, or ii) SEQ ID NO: 18 or SEQ ID NO: 23, optionally without an N-terminal methionine.

39. The method of any one of claims 19-39, wherein

a) the second recombinant protease sensitive prion protein is the mouse protease sensitive prion protein, and wherein the mouse protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 11, optionally with an N-terminal methionine, or SEQ ID NO: 20, optionally without an N-terminal methionine;

b) the second recombinant protease sensitive prion protein is the sheep protease sensitive prion protein, and wherein the sheep protease sensitive prion protein comprises 1) the amino acid sequence set forth as SEQ ID NO: 9 or 21, optionally with an N-termainl methionine, or ii) the amino acid sequence set forth as SEQ ID NO: 19 or SEQ ID NO: 24, optionally without an N-terminal methionine, or

c) the second recombinant protease sensitive prion protein is the bovine protease sensitive prion protein and wherein the bovine protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 10, optionally with an N-terminal methionine.

40. The method of any one of claim 19-38, wherein the recombinant bank vole protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 22, optionally with an N-terminal methionine, or the amino acid sequence set forth as SEQ ID NO: 17 or SEQ ID NO: 25, optionally without a N-terminal methionine.

41. A method of discriminating whether a human subject is affected with variant Creutzfeldt-Jakob disease or sporadic Creutzfeldt-Jakob disease, comprising:

(iii) maintaining incubation conditions that promote coaggregation of the

recombinant bank vole protease sensitive prion protein (rPrP^Sen) with the PrP^D to result in a conversion of the recombinant bank vole protease sensitive prion protein to recombinant protease resistant and/or amyloid prion protein (rPrP-res^(Sc)) while inhibiting development of spontaneously forming recombinant protease resistant and/or amyloid prion protein (rPrP- res^(sP°ⁿ⁾);

(iv) agitating aggregates formed during step (iii), wherein the reaction conditions comprise shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking; and

(v) detecting rPrP-res^(Sc) in the first reaction mixture; and

(vi) contacting the second biological sample with a second recombinant protease sensitive prion protein (rPrP^Sen), wherein the second protease sensitive prion protein comprises amino acids 23-213 of a hamster protease sensitive prion protein, to form a second reaction mixture;

(x) detecting rPrP-res^(Sc) in the second reaction mixture,

wherein detection of rPrP-res^(Sc) in the first reaction mixture but not the second reaction mixture indicates that the subject has variant Creutzfeldt-Jakob disease, and wherein detection of rPrP- res^(Sc) in the first reaction mixture and the second reaction mixture indicates that the subject has sporadic Creutzfeldt-Jakob disease.

42. The method of claim 41, wherein detecting the presence of rPrP-res^(Sc) amyloid in the first sample and/or the second biological sample comprises the use of thioflavin T (ThT).

43. The method of claim 41 or claim 42, wherein the first biological sample and/or the second biological sample is a nasal brushing, saliva, blood, serum, plasma, cerebral spinal fluid, feces, urine or tissue sample.

44. The method of any one of claims 41-43, wherein agitating aggregates in step (iv) and (ix) comprises agitating aggregates in the absence of sonication.

45. The method of any one of claims 41-44, further comprising adding additional recombinant bank vole protease sensitive prion protein to the first reaction mixture of step (iii) without removing rPrP-res^(Sc) prior to detecting the presence of rPrP-res^(Sc).

46. The method of claim 45, wherein the additional recombinant bank vole protease sensitive prion protein is added to the reaction mixture without serial rounds of amplification.

47. The method of any one of claims 41-45, further comprising adding additional second recombinant protease sensitive prion protein to the second reaction mixture of step (viii) without removing rPrP-res^(Sc) prior to detecting the presence of rPrP-res^(Sc).

48. The method of claim 47, wherein the additional second recombinant protease sensitive prion protein is added to the reaction mixture without serial rounds of amplification.

49. The method of any one of claims 41-48, further comprising quantitating the rPrP- res^(Sc) in the first reaction mixture.

50. The method of any one of claims 41-49, further comprising quantitating the rPrP- res^(Sc) in the second reaction mixture.

51. The method of any one of claims 41-50, wherein the shaking cycle in step (iv) comprises a period of rest that precedes the period of shaking, and wherein the period of rest and the period of shaking are substantially equal.

52. The method of claim 51, wherein the shaking cycle in step (iv) is 20 to 180 seconds in length.

53. The method of any one of calms 41-52, wherein the shaking cycle in step (ix) comprises a period of rest that precedes the period of shaking, and wherein the period of rest and the period of shaking are substantially equal.

54. The method of claim 53, wherein the shaking cycle in step (ix) is 20 to 180 seconds in length.

55. The method of claim 53, wherein the period of rest and the period of shaking are about 60 seconds in length.

56. The method of claim 55, wherein the period of rest and the period of shaking are about 30 seconds in length.

57. The method of any one of claims 41-56, wherein step (iv) and step (ix) are repeated from about 1 to about 200 times.

58. The method of any one of claims 41-57, comprising contacting a sample from the subject with an effective amount of an antibody that specifically binds sheep prions or sheep PrP^D for sufficient time to form an immune complex and;

59. The method of any one of claims 41-58, comprising contacting an additional sample from the subject with an effective amount of an antibody that specifically binds sheep prions or sheep PrP^D for sufficient time to form an immune complex and;

60. The method of any one of claims 41-59, wherein the second recombinant protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 2, optionally without an N-terminal methioinine, or the amino acid sequence set foth as SEQ ID NO: 6 or SEQ ID NO: 7, optionally with an N-terminal methionine.

61. The method of any one of claim 41-61, wherein the recombinant bank vole protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 22, optionally with an N-terminal methionine, or the amino acid sequence set forth as SEQ ID NO: 17 or SEQ ID NO: 25, optionally without a N-terminal methionine.

62. A method of discriminating whether a subject that has bovine spongiform encephalopathy has atypical L-type bovine spongiform encephalopathy (L-BSE) or classical bovine spongiform encephalopathy (BSE) comprising, comprising:

(iii) maintaining incubation conditions that promote coaggregation of the

(v) detecting rPrP-res^(Sc) in the first reaction mixture; and

b) performing a second prion protein amyloid seeding assay on a second biological sample from the subject comprising:

(vi) contacting the second biological sample with a second recombinant protease sensitive prion protein (rPrP^Sen), wherein the second protease sensitive prion protein is a hamster or a human protease sensitive prion protein to form a second reaction mixture;

spontaneously forming recombinant protease resistant and/or amyloid prion protein (rPrP- _res(^sP^on));

(x) detecting rPrP-res^(Sc) in the second reaction mixture,

wherein detection of rPrP-res^(Sc) in the first reaction mixture but not the second reaction mixture indicates that the subject has classical BSE, and wherein detection of rPrP-res^(Sc) in the first reaction mixture and the second reaction mixture indicates that the subject has L-type BSE, and wherein the subject is a cow, sheep or goat.

63. The method of claim 62, wherein detecting the presence of rPrP-res^(Sc) in the first sample and/or the second biological sample comprises the use of thioflavin T (ThT).

64. The method of claim 62 or claim 63, wherein the first biological sample and/or the second biological sample is a nasal brushing, saliva, blood, serum, plasma, cerebral spinal fluid, feces, urine or tissue sample.

65. The method of any one of claims 62-64, wherein agitating aggregates in step (iv) and (ix) comprises agitating aggregates in the absence of sonication.

66. The method of any one of claims 62-65, further comprising adding additional recombinant bank vole protease sensitive prion protein to the first reaction mixture of step (iii) without removing rPrP-res^(Sc) prior to detecting the presence of rPrP-res^(Sc).

67. The method of claim 66, wherein the additional recombinant bank vole protease sensitive prion protein is added to the reaction mixture without serial rounds of amplification.

68. The method of any one of claims 62-67, further comprising adding additional second recombinant protease sensitive prion protein to the second reaction mixture of step (viii) without removing rPrP-res^(Sc) prior to detecting the presence of rPrP-res^(Sc).

69. The method of claim 68, wherein the additional second recombinant protease sensitive prion protein is added to the reaction mixture without serial rounds of amplification.

70. The method of any one of claims 62-69, further comprising quantitating the rPrP- res^(Sc) in the first reaction mixture.

71. The method of any one of claims 62-70, further comprising quantitating the rPrP- res^(Sc) in the second reaction mixture.

72. The method of any one of claims 62-71, wherein the shaking cycle in step (iv) comprises a period of rest that precedes the period of shaking, and wherein the period of rest and the period of shaking are substantially equal.

73. The method of claim 72, wherein the shaking cycle in step (iv) is 20 to 180 seconds in length.

74. The method of any one of calms 62-73 wherein the shaking cycle in step (ix) comprises a period of rest that precedes the period of shaking, and wherein the period of rest and the period of shaking are substantially equal.

75. The method of claim 74, wherein the shaking cycle in step (ix) is 20 to 180 seconds in length.

76. The method of claim 74, wherein the period of rest and the period of shaking are about 60 seconds in length.

77. The method of claim 74, wherein the period of rest and the period of shaking are about 30 seconds in length.

78. The method of any one of claims 62-77, wherein step (iv) and step (ix) are repeated from about 1 to about 200 times.

79. The method of any one of claims 62-78, comprising contacting a sample from the subject with an effective amount of an antibody that specifically binds sheep prions or sheep PrP^D for sufficient time to form an immune complex and;

80. The method of any one of claims 62-79, comprising contacting an additional sample from the subject with an effective amount of an antibody that specifically binds sheep prions or sheep PrP^D for sufficient time to form an immune complex and;

81. The method of any one of claims 62-80, wherein the second recombinant protease sensitive prion protein is the human protease sensitive prion protein, wherein the human protease sensitive prion protein comprises i) the amino acid sequence set forth as SEQ ID NO: 3 or SEQ ID NO: 4, optionally with an N-terminal methionine, or ii) SEQ ID NO: 18 or SEQ ID NO: 23, optionally without an N-terminal methionine.

82. The method of any one of claims 62-81, wherein the second recombinant protease sensitive prion protein is the hamster protease sensitive prion protein, and wherein the hamster protease sensitive prion proteins comprises the amino acid sequence set forth as SEQ ID NO: 2, optionally without an N-terminal methioinine, or the amino acid sequence set foth as SEQ ID NO: 6 or SEQ ID NO: 7, optionally with an N-terminal methionine.

83. The method of any one of claim 62-82, wherein the recombinant bank vole protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 22, optionally with an N-terminal methionine, or the amino acid sequence set forth as SEQ ID NO: 17 or SEQ ID NO: 25, optionally without a N-terminal methionine.

84. A method of determining whether a subject that has bovine spongiform

encephalopathy has atypical L-type bovine spongiform encephalopathy (L-BSE), atypical H- type bovine spongiform encephalogpathy, or classical bovine spongiform encephalopathy (C- BSE) comprising, comprising:

(iii) maintaining incubation conditions that promote coaggregation of the recombinant bank vole protease sensitive prion protein (rPrP^Sen) with the PrP^D to result in a conversion of the recombinant bank vole protease sensitive prion protein to recombinant protease resistant and/or amyloid prion protein (rPrP-res^(Sc)) while inhibiting development of spontaneously forming recombinant protease resistant and/or amyloid prion protein (rPrP- res^(sP°ⁿ⁾);

(v) detecting rPrP-res^(Sc) in the first reaction mixture; and

(vi) contacting the second biological sample with a second recombinant protease sensitive prion protein (rPrP^Sen), to form a second reaction mixture;

(ix) agitating aggregates formed during step (viii), wherein the reaction conditions comprise shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking; and (x) detecting rPrP-res^(Sc) in the second reaction mixture,

wherein detection of rPrP-res^(Sc) in the first reaction mixture but not the second reaction mixture indicates that the subject has classical BSE, wherein detection of rPrP-res^(Sc) in the first reaction mixture and the second reaction mixture indicates that the subject has either L-type BSE or H- type BSE, and wherein the subject is a cow, sheep or goat.

85. The method of claim 84, further comprising quantitating the rPrP-res^(Sc) in the first reaction mixture and quantitating the rPrP-res^(Sc) in the second reaction mixture.

86. The method of claim 84 or claim 85, wherein detecting the presence of rPrP-res^(Sc) in the first sample and the second biological sample comprises the use of thioflavin T (ThT).

87. The method of any one of claims 84-86, wherein the first reaction mixture has a first lag phase to the conversion of the bank vole rPrP^Sen to the first rPrP-res^(Sc), and wherein the second reaction mixture has a second lag phae to the conversion of the second rPrP^Sen to the second rPrP-res^(Sc), and wherein a shorter length of the second lag phase as compared to the first lag phase indicates that the subject has L-BSE, and wherein a longer length of the second lag phase as compared to the first lag phase indicates that the subject has H-BSE.

88. The method of any one of claims 84-87, wherein the first biological sample and/or the second biological sample is a nasal brushing, saliva, blood, serum, plasma, cerebral spinal fluid, feces, urine or tissue sample.

89. The method of any one of claims 84-88, wherein agitating aggregates in step (iv) and (ix) comprises agitating aggregates in the absence of sonication.

90. The method of any one of claims 84-89, further comprising adding additional recombinant bank vole protease sensitive prion protein to the first reaction mixture of step (iii) without removing rPrP-res^(Sc) prior to detecting the presence of rPrP-res^(Sc).

91. The method of claim 90, wherein the additional recombinant bank vole protease sensitive prion protein is added to the reaction mixture without serial rounds of amplification.

92. The method of any one of claims 84-91, further comprising adding additional second recombinant protease sensitive prion protein to the second reaction mixture of step (viii) without removing rPrP-res^(Sc) prior to detecting the presence of rPrP-res^(Sc).

93. The method of claim 92, wherein the additional second recombinant protease sensitive prion protein is added to the reaction mixture without serial rounds of amplification.

94. The method of any one of claims 84-93, wherein the shaking cycle in step (iv) comprises a period of rest that precedes the period of shaking, and wherein the period of rest and the period of shaking are substantially equal, and/or wherein the shaking cycle in step (ix) comprises a period of rest that precedes the period of shaking, and wherein the period of rest and the period of shaking are substantially equal.

95. The method of claim 94, wherein the shaking cycle in step (iv) and/or the shaking cycle in step (ix) is 20 to 180 seconds in length.

96. The method of claim 94, wherein the period of rest and the period of shaking in the shaking cycle in step (iv) and/or step (ix) are each about 60 seconds in length.

97. The method of claim 94, wherein the period of rest and the period of shaking in the shaking cycle in step (iv) and/or step (ix) are each about 30 seconds in length

98. The method of any one of claims 84-97, wherein step (iv) and step (ix) are repeated from about 1 to about 300 times.

99. The method of any one of claims 84-98, wherein the second protease sensitive prion protein is a sheep protease sensitive prion protei.

100. The method of claim 99, wherein the sheep rPrP^Sen comprises the amino acid sequence set forth as SEQ ID NO: 9 or 21, optionally with an N-terminal methionine, or ii) the amino acid sequence set forth as SEQ ID NO: 19 or SEQ ID NO: 24, optionally without an N- terminal methionine.

101. The method of claim 100, wherein the recombinant sheep protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 21, optionally with an N- terminal methionine.

102. The method of any one of claims 84-101, wherein the recombinant bank vole protease sensitive prion protein comprises the amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 22, optionally with an N-terminal methionine, or the amino acid sequence set forth as SEQ ID NO: 17 or SEQ ID NO: 25, optionally without a N-terminal methionine.

103. The method of any one of claims 84-102, comprising contacting a sample from the subject with an effective amount of an antibody that specifically binds bank vole prions or bank vole PrP^D for sufficient time to form an immune complex and;

104. The method of any one of claims 99-103, comprising contacting an additional sample from the subject with an effective amount of an antibody that specifically binds sheep prions or sheep PrP^D for sufficient time to form an immune complex and;