Nothing Special   »   [go: up one dir, main page]

WO2016149537A1 - Protéine de prion du campagnol roussâtre utilisée comme substrat à large spectre pour détection et discrimination de souches de prions basées sur une conversion induite par agitation en temps réel (rt-quic) - Google Patents

Protéine de prion du campagnol roussâtre utilisée comme substrat à large spectre pour détection et discrimination de souches de prions basées sur une conversion induite par agitation en temps réel (rt-quic) Download PDF

Info

Publication number
WO2016149537A1
WO2016149537A1 PCT/US2016/022945 US2016022945W WO2016149537A1 WO 2016149537 A1 WO2016149537 A1 WO 2016149537A1 US 2016022945 W US2016022945 W US 2016022945W WO 2016149537 A1 WO2016149537 A1 WO 2016149537A1
Authority
WO
WIPO (PCT)
Prior art keywords
rprp
prion protein
res
seq
reaction mixture
Prior art date
Application number
PCT/US2016/022945
Other languages
English (en)
Inventor
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 (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, Istituto Superiore Di Sanita filed Critical The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Publication of WO2016149537A1 publication Critical patent/WO2016149537A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease

Definitions

  • 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.
  • Prion diseases or transmissible spongiform encephalopathies
  • CJD Creutzfeldt- Jakob disease
  • GSS Gerstmann-Straussler-Scheincker syndrome
  • FFI fatal familial insomnia
  • sFI sporadic fatal insomnia
  • BSE bovine spongiform encephalopathy
  • CWD chronic wasting disease
  • the origin of prion diseases can be infectious, genetic or sporadic.
  • PrP D prion protein
  • PrP D 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 .
  • PrP D usually includes forms called PrP Res that, unlike the normal PrP Sen , are partially resistant to digestion by proteinase K (PK).
  • PK proteinase K
  • 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).
  • 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).
  • GSS P102L 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.
  • RT-QuIC Real Time Quaking Induced Conversion
  • 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:
  • rPrP Sen recombination protease K sensitive 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.
  • a method 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.
  • 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.
  • a method 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.
  • 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 r p D p resenl m 1 ⁇ 6 biological sample with the recombinant bank vole protease sensitive prion protein (BV rPrP Sen ).
  • BV rPrP Sen recombinant bank vole protease sensitive prion protein
  • 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.
  • 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
  • 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).
  • the methods can be used to determine if a cow, sheep or a goat has C-BSE, H-BSE or L-BSE.
  • FIG. 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 r PrP Sen as the substrate, 300mM NaCl, 0.002% SDS.
  • FIGS 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 SDS
  • FIGS 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.
  • AD Alzheimer's disease
  • FIG. 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.
  • FIGS 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).
  • 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.
  • FIGS 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 were seeded with 10 "4 dilutions of frontal cortex or brain stem from uninfected cattle.
  • Ha 90- 231) r PrP Sen (300mM NaCl and 0.002% SDS; (A)-(B), Ha (23-231) or Hu (23-231) r PrP Sen
  • 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 was diluted 10 "4 (A and D).
  • Equivalent dilutions of cerebellum or frontal cortex brain tissue dilutions were used as specificity controls (C and F).
  • Either Ha-S rPrP Sen (300mM NaCl and 0.002% SDS; A-C) or BV r PrP 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.
  • FIGS 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 r PrP Sen with 300mM NaCl and 0.002% SDS (A and B) or BV r PrP 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.
  • FIGS 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 r PrP 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.
  • 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).
  • FIG. 13 RT-QuIC detection of seeding activity from PK-digested purified PrP Res using bank vole r PrP 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 10 6 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, ⁇ 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.
  • FIGS 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.
  • FIGS 18A-18C Use of bank vole 90-231 for detection of human prion seeding activity from CSF. Prion seeding activity was detected from 20 ⁇ ⁇ 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.
  • FIG. 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.
  • Figure 21 RT-QuIC end-point dilution analyses of brain tissues from H-BSE-
  • FIGS 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.
  • FIGS 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 r PrP Sen in the same plate.
  • the graphs represent three examples of RT-QuIC kinetics observed using BV r PrP Sen 23-230, M109 and Sh ARR 25-234 rPrP sen 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.
  • FIGS. 25A and 25B Western blot analysis of BV r PrP Res products from RT-QuIC reactions seeded with C-, L- and H-type BSE strains. Reaction products were digested with 10 ⁇ g/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).
  • 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).
  • FIGS 26A-26C RT-QuIC analysis of CSF from C-, L- and H-type positive cattle using BV r PrP Sen 23-230, M109, BV r PrP Sen 90-230, M109 and Hamster r PrP 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 r PrP Sen 23-230, M109 (A), BV r PrP Sen 90-230; M109 (B) and Ha r p r ps e n 90-231 were used as substrates in the presence of 0.002% SDS and 300mM NaCl.
  • 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.
  • 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).
  • SEQ ID NO: 3 is an amino acid sequence of a recombinant human (129M) prion protein, starting at residue 23 (23-231).
  • SEQ ID NO: 4 is an amino acid sequence of a recombinant human (129V) prion protein, starting at reside 23 (23-231).
  • This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below.
  • 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).
  • This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below.
  • SEQ ID NO: 6 is amino acid sequence of amino acids 23-231 of Hamster prion protein.
  • This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below.
  • SEQ ID NO: 7 is an amino acid sequence of amino acids 90-231 of Hamster prion protein.
  • This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below.
  • SEQ ID NO: 8 is an amino acid sequence 23-230 of Bank Vole prion protein (residue
  • This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below.
  • 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.
  • This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below.
  • SEQ ID NO: 10 is an amino acid sequence of a recombinant bovine (6-octarepeat) prion protein.
  • This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below.
  • SEQ ID NO: 11 is the amino acid sequence of the mouse prion protein 23-231.
  • This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below.
  • SEQ ID NO: 12 is the amino acid sequence of bank vole 90-230 109M GQGGGTHNQWNKPSKPKTNMKHVAGAAAAGAVVGGLGGYMLGSAMSRPMIHFGND WEDRYYRENMNRYPNQVYYRPVDQYNNQNNFVHDCVNITIKQHTVTTTTKGENFTET DVKMMERVVEQMCVTQYQKESQAYYEGRS
  • This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below.
  • SEQ ID NO: 13 is the amino acid sequence of Bank vole 90-230 1091
  • This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below.
  • SEQ ID NO: 14 is the amino acid sequence of Human-BV Chimera (Human 23- 165[129M]/Bank vole 166-230)
  • This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below.
  • SEQ ID NO: 15 is the amino acid sequence of BV-Human Chimera 109M (Bank vole).
  • This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below.
  • SEQ ID NO: 16 is the amino acid sequence of BV-Human Chimera 1091 (Bank vole 23- 165/Human 166-231)
  • This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below.
  • SEQ ID NO: 17 is the amino acid sequence of bank vole 1-230 (109M)
  • SEQ ID NO: 18 is the amino acid sequence of Human (129M) 1-231:
  • SEQ ID NO: 19 is the amino acid sequence of Sheep (ARQ) 1-234:
  • SEQ ID NO: 20 is the amino acid sequence of mouse 1-231:
  • SEQ ID NO: 21 is the amino acid sequence 25-234 of a recombinant ovine (136A 154R 171R) prion protein.
  • SEQ ID NO: 22 is the amino acid sequence of Human-BV Chimera (Human 23-165 [129V]/Bank vole 166-230)
  • This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below.
  • SEQ ID NO: 23 is the amino acid sequence of Human (129V) 1-231:
  • SEQ ID NO: 24 is the amino acid sequence 1-234 of a recombinant ovine (136A 154R
  • SEQ ID NO: 25 is an amino acid sequence 1-230 of Bank Vole prion protein (residue
  • 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).
  • This protein can be generated with an N-terminal methionine and used in any of the methods disclosed below.
  • RT-QuIC Real Time Quaking Induced Conversion
  • rPrP Sen recombinant prion protein
  • This type of assay provides highly specific and ultra- sensitive for detection of multiple human and animal prion diseases.
  • 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.
  • rPrP Sen prion- associated seeds induce amyloid fibril formation of bacterially expressed recombinant PrP Sen
  • the resulting rPrP Res amyloid fibrils are then detected by the enhanced fluorescence of the amyloid-sensitive dye, thioflavin T (ThT).
  • ThT thioflavin T
  • 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.
  • 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.
  • 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.
  • GSS Gerstmann-Straussler-Scheincker syndrome
  • BV rPrP Sen bank vole PrP Sen
  • 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.
  • 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).
  • 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) ).
  • 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.
  • agitation includes the use of force sufficient to fragment rPrP-res (Sc) aggregates or amyloids, which disperses rPrP- res (Sc) a gg re g a tes and/or polymers to facilitate further amplification.
  • 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.
  • 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”).
  • 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.
  • a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds.
  • H heavy chain
  • L light chain
  • ⁇ lambda
  • k kappa
  • IgM immunoglobulin heavy chain classes
  • Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”).
  • 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".
  • CDRs complementarity-determining regions
  • 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.
  • a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found
  • 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).
  • VH refers to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.
  • VL refers 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.
  • affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol, 16:101-106, 1979.
  • binding affinity is measured by an antigen/antibody dissociation rate.
  • a high binding affinity is measured by a competition radioimmunoassay.
  • a high binding affinity is at least about 1 x 10 "8 M.
  • 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.
  • Epitopes or “antigenic determinant” refers to a site on an antigen to which B and/or T-cells respond.
  • 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.
  • C-BSE classical BSE
  • ruminant feed ban Ducrot et al., 2008, Vet Res. 39:15
  • phenotypically atypical forms of BSE have been identified in several countries (Jacobs et al., 2007, J Clin Microbiol. 45: 1821-1829).
  • H-BSE H-type atypical BSE
  • L-BSE L-type 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).
  • PK Proteinase K
  • 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).
  • the presently disclosed methods provide detection of C-BSE, L- BSE and H-BSE.
  • 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.
  • 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.
  • 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).
  • CWD Chronic Wasting Disease
  • TSE transmissible spongiform encephalopathy
  • deer elk
  • moose elk
  • 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.
  • 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
  • CJD fCJD
  • sCJD appears subjects without germline PRNP mutations.
  • prion protein variants 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.
  • 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.
  • 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) ).
  • 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.
  • 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.
  • 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%.
  • 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 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.
  • 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 r pRes or r p r p_ 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.
  • the term is construed broadly, and can have a variety of applications.
  • 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.
  • a DNA molecule can encode an RNA molecule (for instance, by the process of transcription incorporating a DNA-dependent RNA polymerase enzyme).
  • an RNA molecule can encode a peptide, as in the process of translation.
  • the term “encode” also extends to the triplet codon that encodes an amino acid.
  • an RNA molecule can encode a DNA molecule, for instance, by the process of reverse transcription incorporating an RNA-dependent DNA polymerase.
  • 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.
  • FFI Fatal familial insomnia
  • FFI sporadic fatal insomnia
  • 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 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.
  • the gene encoding prion protein 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)
  • 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.
  • 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- trifluoromethyl
  • GSS Gerstmann-Straussler-Scheinker syndrome
  • 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.
  • IP Immunoprecipitation
  • 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.
  • 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.
  • 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
  • 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.
  • TSE transmissible spongiform encephalopathies
  • CJD human Creutzfeldt-Jakob disease
  • GSS Gerstmann- Straussler-Scheincker syndrome
  • FFI fatal familial insomnia
  • sFI sporadic fatal insomnia
  • BSE bovine spongiform encephalopathy
  • CWD cervid chronic wasting disease
  • TEE transmissible mink encephalopathy
  • 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.
  • PrP D abnormally structured forms
  • PrP BSE for bovine spongiform encephalopathy
  • PrP vCJD for variant CJD
  • 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.
  • PrP D or PrP Sc which is typically, but not always, partially protease-resistant (i.e., PrP Res )
  • PrP Res partially protease-resistant
  • PrP-sen normal host-encoded protease- sensitive precursor
  • PrP Sen normal host-encoded protease-sensitive precursor
  • PrP c which is sensitive to proteinase K digestion
  • PrP D (and PrP Sc ) aggregates such as PrP Res aggregates
  • PrP Res aggregates are high in ⁇ -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.
  • rPrP-res 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 r pRes to ijjjjate 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.
  • polypeptide fragment refers to a portion of a polypeptide which exhibits at least one useful epitope.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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;
  • tissue can be any tissue of interest.
  • the tissue can be skin tissue or brain tissue.
  • 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
  • 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.
  • rPrP-res 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 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
  • 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.
  • 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.
  • 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.
  • bank vole rPrP Sen as a substrate for an amplification reaction performed on a biological sample from the subject.
  • 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.
  • 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.
  • 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
  • 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).
  • the subject is a human, and the transmissible spongiform encephalopathy is Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-
  • GSS Scheincker syndrome
  • FFI fatal familial insomnia
  • sFI sporadic fatal insomnia
  • the subject is a cow
  • encephalopathy is bovine spongiform encephalopathy (BSE).
  • BSE bovine spongiform encephalopathy
  • the subject is a cervid, and wherein the transmissible spongiform encephalopathy is chronic wasting disease (CWD).
  • CWD chronic wasting disease
  • the subject is a sheep and the transmissible spongiform encephalopathy is classical or atypical scrapie, including Nor98 scrapie.
  • the subject is a human, and has, or is suspected to have Gerstmann-Straussler- Scheincker (GSS) P102L, F198S, Al 17V and/or H187R.
  • GSS Gerstmann-Straussler- Scheincker
  • the subject is suspected of having CJD, such as sCJD, vCJD, or gCJD.
  • 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.
  • the subject can be diagnosed with the transmissible spongiform encephalopathy, and the methods disclosed herein can be used to confirm the diagnosis.
  • 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.
  • BSE bovine spongiform encephalopathy
  • the subject is a human, and the transmissible spongiform
  • the encephalopathy is GSS P102L, P102L*, F198S, Al 17V or H187R.
  • the subject is a human, and the transmissible spongiform encephalopathy is genetic CJD (gCJD), such as E200K, V210I or six octarepeat insertion gCJD.
  • the subject is a human, and the transmissible spongiform encephalopathy is FFI, such as caused by the D178N PRNP mutation, or sFI.
  • the subject is a human, and the transmissible spongiform encephalopathy is sporadic, variant, iatrogenic or genetic CJD.
  • the subject is a cervid and the transmissible spongiform encephalopathy is chronic wasting disease.
  • the subject is a bovine, sheep or goat, and the transmissible spongiform encephalopathy is bovine spongiform encephalopathy.
  • 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.
  • clinical symptoms are assessed.
  • 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 ).
  • 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.
  • 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) .
  • the additional recombinant bank vole rPrP Sen is added to the reaction mixture without serial rounds of amplification.
  • rPrP-res (Sc) is then detected.
  • 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 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 r p D p resenl m 1 ⁇ 6 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.
  • each shaking cycle comprises a period of rest and a period of shaking.
  • the period of rest and the period of shaking are substantially equal.
  • 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 ).
  • rPrP Sen second recombinant protease sensitive prion protein
  • 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.
  • the second recombinant protease sensitive prion protein is the recombinant hamster sheep protease sensitive prion protein
  • 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.
  • the second recombinant sensitive prion protein is the human protease sensitive prion protein
  • 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.
  • the second recombinant protease sensitive prion protein is the mouse protease sensitive prion protein
  • 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.
  • 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.
  • 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 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.
  • each shaking cycle comprises a period of rest and a period of shaking.
  • the period of rest and the period of shaking are substantially equal.
  • 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.
  • rPrP Sen second recombinant protease sensitive prion protein
  • step (viii) 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.
  • 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.
  • the 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • the second recombinant protease sensitive prion protein is the human protease sensitive prion protein
  • 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.
  • the second recombinant protease sensitive prion protein is the hamster protease sensitive prion protein
  • 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.
  • methods are also disclosed for determining whether a subject that has bovine spongiform encephalopathy has atypical L-type bovine spongiform
  • 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 ).
  • 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 ).
  • 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.
  • detecting the presence of rPrP-res (Sc) in the first sample and the second biological sample includes the use of thioflavin T (ThT).
  • the first reaction mixture has a first lag phase to the conversion of the bank vole rPrP Sen to the first rPrP-res (Sc)
  • the second reaction mixture has a second lag phase to the conversion of the second rPrP Sen to the second rPrP-res (Sc) .
  • 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.
  • 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.
  • detecting the presence of rPrP-res (Sc) in a sample comprises the use of thioflavin T (ThT).
  • 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.
  • the tissue can be skin tissue or brain tissue.
  • rPrP-res (Sc) such as the rPrP-res (Sc) in the first reaction mixture and in the second reaction mixture, is quantitated.
  • the shaking cycles include a period of rest that precedes the period of shaking.
  • 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.
  • 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.
  • 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.
  • the shaking cycle is about 120 seconds in length, and includes 100 seconds of shaking and 20 seconds for rest.
  • 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.
  • PrP D e.g., PrP CJD , PrP BSE or PrP Res
  • 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.
  • the tissue can be skin tissue or brain tissue.
  • 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.
  • a sample from the subject such as the sheep, human, cow, goat, or cervid
  • 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.
  • rPrP Sen substrates 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.
  • 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.
  • 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).
  • the subject is a cow, sheep or a goat, and the transmissible spongiform encephalopathy is bovine spongiform encephalopathy (BSE).
  • BSE bovine spongiform encephalopathy
  • the subject is a cervid, and the transmissible spongiform encephalopathy is chronic wasting disease (CWD).
  • CWD chronic wasting disease
  • 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.
  • the N-terminal methionine can also be not present in a rPrP Sen of use in the disclosed methods.
  • 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.
  • the N-terminal methionine is "optional," indicating that a methionine can be added at the N-terminus of these amino acid sequences.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • At most 1, 2, 3, 4, or 5 amino acids is deleted from the N- or C-terminus of the polypeptide.
  • an N-terminal methionine is present.
  • a portion of the protein is from one species, and a portion of the protein is from another species.
  • 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 .
  • the chimeric protein includes hamster rPrP Sen and sheep r p r pSen Additional chimeric proteins include bank vole rPrP Sen and rPrP Sen from another species, such as human.
  • 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.
  • 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.
  • 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.
  • 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.
  • sheep rPrP Sen is used in QuIC and/or RT-QuIC reactions, such as to distinguish between C-BSE, L-BSE and H-BSE.
  • 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 r pSen 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 .
  • rPrP Sen polypeptide can be used in the assays disclosed herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • prion proteins can also be utilized, such as those that have 1, 2, 3, 4, or 5 amino acids deleted from the carboxy terminus.
  • 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.
  • 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.
  • 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.
  • rPrP Sen 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 .
  • the cell is a bacterial cell, such as an E. coli cell. Purified r p r pSen f rom r p r pSen expressing ce rj s ⁇ m some cases, raw cell lysates, can be used as the source of the non-pathogenic protein.
  • the recombinant protein is fused with an additional amino acid sequence.
  • 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
  • GST Glutathione S-transferase
  • MBP Maltose binding protein
  • GFP green fluorescent protein
  • 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.
  • 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.
  • 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.
  • a protease cleavage sequence is added to allow inactivation of protein after it is converted into prion form.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • these tags do not interfere with conversion but allow much more rapid, fluorescence-based detection of the reaction product.
  • 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.
  • rPrP-res (Sc) Free pyrenes released from, or on, unconverted rPrP Sen molecules are unlikely to form excimer pairs.
  • 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 can be produced by any methods known to those of skill in the art.
  • in vitro nucleic acid amplification such as polymerase chain reaction (PCR)
  • PCR polymerase chain reaction
  • 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.
  • a target nucleic acid molecule that includes the prion protein-encoding sequence.
  • DNA or RNA can serve as a suitable target nucleic acid molecule for PCR reactions.
  • 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. ,
  • 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.
  • RT-PCR reverse transcriptase
  • 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).
  • primers typically, at least 10 consecutive nucleotides of prion-encoding nucleic acid 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
  • nucleic acid sequences 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • prion protein variants can have no more than 3, 5, 10, 15, 20, 25, 30, 40, or 50 conservative amino acid changes.
  • Table A 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.
  • 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.
  • 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.
  • SQ Standard QuIC
  • 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.
  • 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.
  • the tissue can be skin tissue or brain tissue.
  • PMCA protein misfolding cyclic amplification
  • PrP Sen protease sensitive prion protein
  • 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.
  • 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.
  • 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.
  • rPrP Sen can be easily mutated or strategically labeled with probes to simplify and accelerate the detection of relevant products.
  • 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.
  • rPrP-PMCA sonication
  • QuIC shaking
  • 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) .
  • RT-QuIC allows detection of the amyloid RT-QuIC product using thioflavin T (ThT).
  • 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.
  • 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
  • rPrP Sen purified rPrP Sen
  • This primary reaction includes incubating the reaction mix to permit the PrP D to initiate the conversion of r p r pSen 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.
  • serial amplification of rPrP-res is carried out by removing a portion of the reaction mix and incubating it with additional r p r pSen 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.
  • 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.
  • 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)
  • rPrP-res 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
  • 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.
  • the rPrP Sen can be replenished by adding additional rPrP Sen substrate to the reaction mix.
  • 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.
  • the reaction alternates 60 seconds of shaking and 60 seconds of no shaking (rest).
  • the reaction alternates 30 seconds of shaking and 30 seconds of no shaking (rest).
  • 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.
  • the period of rest and the period of shaking are equal.
  • the period of rest and the period of shaking are unequal.
  • the period of rest and the period of shaking are about 120 seconds in length for the total cycle.
  • 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.
  • the period of rest and the period of shaking are unequal.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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) .
  • NaCl sodium chloride
  • a RT-QuIC assay the reaction product is detected in real time (RT).
  • RT real time
  • 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.
  • a solid substrate such as a bead, such as magnetic beads
  • 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.
  • 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.
  • IP-RTQ is highly sensitive.
  • the overall sensitivity of the RTQ was increased by at least 1000-fold and overall reaction time is greatly reduced.
  • the concentration of substrate can be 0.1 mg/ml, such as 0.05 to 0.2 mg/ml.
  • 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 r p r pSen ajnyioid fibrils.
  • ThT thioflavin T
  • 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 r p r pSen ajnyioid fibrils.
  • ThT thioflavin T
  • 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
  • the prion-initiated rPrP-res (Sc) in the reaction mix is detected.
  • ThT is included in the reaction (RT-QuIC)
  • 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.
  • the reaction mix does not include chaotropes or detergents.
  • ThT is included, the reaction mix does not include chaotropic agents or detergents that can alter the rPrP-res (Sc) - sensitivity of ThT.
  • 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).
  • RT real time
  • 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 can be used to discrimate between different forms of a disease.
  • the length of the lag phase can be used to discriminate L-BSE, H-BSE, and C-BSE.
  • the first substrate is bank vole rPrP Sen
  • 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.
  • 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.
  • a first lag phase for the first reaction wherein bank vole rPrP Sen is convereted rPrP- res (Sc)
  • 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.
  • the detergent is sodium dodecyl sulfate (SDS).
  • PrP Res can be detected by means other than ThT
  • 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) .
  • proteinase K which digests the remaining rPrP Sen in the reaction mix
  • Sc rPrP-res
  • 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.
  • discrimination between the former and the latter can be done on the basis of differing protein fragment sizes generated upon exposure to proteinase K.
  • RT-QuIC which includes thioflavin T
  • 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.
  • 0.001% of SDS is included in the reaction.
  • lower final detergent concentration such as 0-0.001% SDS
  • higher concentrations can interfere with the thioflavin T fluorescence detection of rPrP- res (Sc)_
  • 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.
  • 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 .
  • rPrP-res 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.
  • reaction mixtures are incubated with, for example, Proteinase K (PK).
  • PK Proteinase K
  • An exemplary protease treatment includes digestion of the protein, for instance, rPrP Sen , in the reaction mixture with 1-20 ⁇ g/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 ⁇ g/ml of PK generally is sufficient to remove rPrP Sen .
  • rPrP-res 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.
  • reaction mixtures fractioned or treated with protease to remove r p r pSen are ⁇ 6 ⁇ 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.
  • a membrane such as nitrocellulose or PVDF
  • antibodies for detection of prion protein include the 3F4 monoclonal antibody, monoclonal antibody D13 (directed against residues 96-106 (Peretz et al.
  • An exemplary washing procedure includes washing with a solution such as
  • the immunoreactive bands are visualized by a variety of assays known to those in the art.
  • the enhanced chemoluminesence assay (Amersham, Piscataway, N.J.) can be used.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the amount of prion in the sample can be quantified.
  • 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.
  • a plate with either antigen or antibody 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.
  • a secondary or tertiary detection means rather than a direct procedure with ELISAs, though this is not always the case.
  • 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
  • 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.
  • 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.
  • the second or third antibody generally will have an associated label to allow detection.
  • this is an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate.
  • 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).
  • 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.
  • 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.
  • the recombinant rPrP Sen substrate protein can be labeled to enable high sensitivity of detection of protein that is converted into rPrP-res (Sc) .
  • r p r pSen 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 125 1, 32 P, 33 P, and 35 S.
  • 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.
  • the protein can be labeled in such a way that a signal can be detected upon the conformational changes induced during conversion.
  • a signal can be detected upon the conformational changes induced during conversion.
  • 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).
  • 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.
  • these tags do not interfere with conversion but allow much more rapid, fluorescence-based detection of the reaction product.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • a capture-monoclonal antibody or epitope -binding fragment thereof
  • 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 .
  • CDRs complementarity
  • monoclonal antibody can bind PrP Res from subject with either scrapie or Nor98.
  • 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).
  • 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.
  • PrP c normal mouse cellular protein
  • 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 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.
  • 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.
  • supports in the form of, for example, surfaces, particles, porous matrices, sepharose, etc.
  • 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
  • the immobilized capture-monoclonal antibodies are coated on a microtiter plate, and in particular the solid phase can be a multi-well microtiter plate.
  • 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.
  • 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.
  • 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.
  • 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.
  • micro-titer well plates e.g., 96-well plates or 384-well plates
  • 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
  • the beads can be coated with the antibody using any procedures known in the art.
  • 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.
  • a coating buffer comprising about 0.01% to 1%, such as about 0.1% bovine serum albumin in phosphate buffered saline is utilized.
  • 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.
  • the antibody (such as, but not limited to, 15B3, IgG W226 or IgG 261) can be coupled to the substrate as about 5 ⁇ g antibody per 100 ⁇ DYNABEADS®.
  • 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 ⁇ g of antibody.
  • 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 ⁇ g, such as 36 ⁇ g of antibody per 100 ⁇ DYNABEADS® (for example, 4 x 10 8 beads/ml).
  • lng to 10 ⁇ g of antibody can be used for 1 x 10 8 beads.
  • 100-300 ⁇ g of antibody per 1 x 10 "8 DYNABEADS® (for example, 4 xlO 8 beads/ml) can be utilized.
  • the concentration of the antibody on the magnetic beads is about 10-500 ⁇ g of antibody per 1 x 10 8 beads.
  • 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.
  • 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.
  • appropriate blocking agents for this purpose include gelatin, bovine serum albumin, egg albumin, casein, and non-fat milk.
  • 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).
  • PBS phosphate-buffered saline
  • 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.
  • SDS sodium dodecyl sulfated
  • 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.
  • 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.
  • 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.
  • the detergent is Sarkosyl.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PBS phosphate buffered saline
  • TBS Tris buffered saline
  • 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.
  • prion proteins e.g. PrP D or PrP Sc
  • 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.
  • 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.
  • 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.
  • a particulate or immobilized ligand such as plasminogen
  • a precipitant such as sodium phosphotungstic acid
  • 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).
  • the eluted protein was extensively dialyzed into 10 mM sodium phosphate buffer (pH 5.8), filtered (0.22- ⁇ 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
  • 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 ⁇ 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 ⁇ ) were loaded into each well of a black 96-well plate with a clear bottom (Nunc) and seeded with 2 ⁇ ⁇ 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.
  • RT-QuIC reaction products were collected from the plates by extensive scraping and pipetting and treated with 10 ⁇ g/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
  • 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 r PrP Sen 23-231 (11), human r PrP Sen 23-231 (21), murine r PrP Sen 23-231 (12) or hamster- sheep chimeric rPrP Sen 23-231 (9, 22).
  • Thioflavin T-positive (ThT) amyloid formation in RT-QuIC reactions 9, 16-20
  • the following substrates Hu golden hamster rPrP Sen residues 90-231 (8, 17, 18)
  • hamster r PrP Sen 23-231 11
  • human r PrP Sen 23-231 21
  • murine r PrP Sen 23-231 (12
  • BV rPrP Sen is the most broadly prion-seeded RT-QuIC substrate described to date.
  • 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.
  • BV rPrP Sen can similarly detect both classical and L-type BSE providing an alternative substrate for
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • sCJD top
  • vCJD bottom
  • 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.
  • C-BSE bovine spongiform encephalopathy
  • C-BSE is the only known zoonotic prion disease, having caused variant Creutzfeldt- Jakob disease (vCJD) in humans who, presumably, consumed contaminated beef.
  • vCJD Creutzfeldt- Jakob disease
  • 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).
  • 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.
  • PK proteinase K
  • C-BSE conformation-dependent immunoassay
  • PMCA protein misfolding cyclic amplification
  • 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.
  • 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)
  • 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
  • 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.
  • 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 ⁇ thioflavin T (ThT), ImM EDTA, 0.1 mg/ml of rPrPSen and 0.002% or 0.001% SDS.
  • SD50 50% seeding dose
  • 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 ⁇ g/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 brain samples were collected from cattle affected by C-, L-, or H-BSE.
  • the samples were PK treated and analyzed by Western blot.
  • the H-BSE samples all showed a
  • 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.
  • 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.
  • SD50 50% seeding dose 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).
  • Each of these same substrates also detected 10 "4 dilutions of L-BSE brain homogenates in all replicate reactions (Fig. 22A-L).
  • the L-BSE seeds gave longer lag phases than those seen with H-BSE.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Dans certains modes de réalisation, l'invention concerne un procédé de détection d'une encéphalopathie spongiforme transmissible chez un sujet. Dans certains modes de réalisation, le procédé comprend la réalisation d'un dosage d'ensemencement d'amyloïde de protéine de prion sur un échantillon biologique provenant du sujet à détecter PrPD. Dans d'autres modes de réalisation, l'invention concerne un procédé pour déterminer si un bovin est atteint par l'encéphalopathie spongiforme bovine classique ou atypique de type L ou de type H, si un ovin est affecté par la tremblante Nor98 du mouton classique ou atypique, ou si un être humain est affecté par la maladie de Creutzfeldt-Jakob sporadique ou variante.
PCT/US2016/022945 2015-03-17 2016-03-17 Protéine de prion du campagnol roussâtre utilisée comme substrat à large spectre pour détection et discrimination de souches de prions basées sur une conversion induite par agitation en temps réel (rt-quic) WO2016149537A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201562134476P 2015-03-17 2015-03-17
US62/134,476 2015-03-17
US201562148679P 2015-04-16 2015-04-16
US62/148,679 2015-04-16

Publications (1)

Publication Number Publication Date
WO2016149537A1 true WO2016149537A1 (fr) 2016-09-22

Family

ID=55642909

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2016/022945 WO2016149537A1 (fr) 2015-03-17 2016-03-17 Protéine de prion du campagnol roussâtre utilisée comme substrat à large spectre pour détection et discrimination de souches de prions basées sur une conversion induite par agitation en temps réel (rt-quic)

Country Status (1)

Country Link
WO (1) WO2016149537A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108037279A (zh) * 2017-12-08 2018-05-15 中国人民解放军军事科学院军事医学研究院 一种提高elisa试剂盒信噪比的通用型酶标板洗涤液及其使用方法
KR20190050505A (ko) * 2017-11-03 2019-05-13 성신여자대학교 연구 산학협력단 109번째 아미노산이 아이소류신인 제방 들쥐의 프리온 단백질 유전자로 상동재조합된 녹인 생쥐 및 이의 응용
KR20190050507A (ko) * 2017-11-03 2019-05-13 성신여자대학교 연구 산학협력단 109번째 아미노산이 메티오닌이고 23번째에서 89번째 아미노산을 코딩하는 서열을 제거한 제방 들쥐의 프리온 단백질 유전자로 상동재조합된 녹인 생쥐 및 이의 응용
KR20190050508A (ko) * 2017-11-03 2019-05-13 성신여자대학교 연구 산학협력단 109번째 아미노산이 메티오닌인 제방 들쥐의 프리온 단백질 유전자로 상동재조합된 녹인 생쥐 및 이의 응용

Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3645852A (en) 1967-05-23 1972-02-29 Pharmacia Ab Method of binding water-soluble proteins and water-soluble peptides to water-insoluble polymers using cyanogen halide
US3691016A (en) 1970-04-17 1972-09-12 Monsanto Co Process for the preparation of insoluble enzymes
US3720760A (en) 1968-09-06 1973-03-13 Pharmacia Ab Method for determining the presence of reagin-immunoglobulins(reagin-ig)directed against certain allergens,in aqueous samples
US3969287A (en) 1972-12-08 1976-07-13 Boehringer Mannheim Gmbh Carrier-bound protein prepared by reacting the protein with an acylating or alkylating compound having a carrier-bonding group and reacting the product with a carrier
US4195128A (en) 1976-05-03 1980-03-25 Bayer Aktiengesellschaft Polymeric carrier bound ligands
US4229537A (en) 1978-02-09 1980-10-21 New York University Preparation of trichloro-s-triazine activated supports for coupling ligands
US4247642A (en) 1977-02-17 1981-01-27 Sumitomo Chemical Company, Limited Enzyme immobilization with pullulan gel
US4330440A (en) 1977-02-08 1982-05-18 Development Finance Corporation Of New Zealand Activated matrix and method of activation
US4376110A (en) 1980-08-04 1983-03-08 Hybritech, Incorporated Immunometric assays using monoclonal antibodies
WO1998037210A1 (fr) 1997-02-21 1998-08-27 KANTON ZÜRICH vertreten durch DIE ERZIEHUNGSDIREKTION Detection immunologique de prions
US5866366A (en) 1997-07-01 1999-02-02 Smithkline Beecham Corporation gidB
WO2002004954A2 (fr) 2000-07-07 2002-01-17 Applied Research Systems Ars Holding N.V. Diagnostic precoce de maladies conformationelles
US6528269B1 (en) 1998-06-22 2003-03-04 Case Western Reserve University Immunological agents specific for prion protein (PRP)
US6855503B2 (en) 2002-12-20 2005-02-15 Amersham Biosciences Corp Heterocyclic FRETdye cassettes for labeling biological molecules and their use in DNA sequencing
US20050118720A1 (en) 2002-03-04 2005-06-02 Carlos Bloch Jr Method for the detection of proteins of animal origin in complex mixtures
US20080220447A1 (en) 2004-05-14 2008-09-11 Prionics Ag Method for the Detection of Disease-Related Prion
WO2009015091A1 (fr) * 2007-07-20 2009-01-29 The Government of the United States of America as represented by the Secretary of the Détection de protéine prion infectieuse par conversion à germes de protéine prion recombinante

Patent Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3645852A (en) 1967-05-23 1972-02-29 Pharmacia Ab Method of binding water-soluble proteins and water-soluble peptides to water-insoluble polymers using cyanogen halide
US3720760B1 (fr) 1968-09-06 1984-02-07 Pharmacia Ab
US3720760A (en) 1968-09-06 1973-03-13 Pharmacia Ab Method for determining the presence of reagin-immunoglobulins(reagin-ig)directed against certain allergens,in aqueous samples
US3691016A (en) 1970-04-17 1972-09-12 Monsanto Co Process for the preparation of insoluble enzymes
US3969287A (en) 1972-12-08 1976-07-13 Boehringer Mannheim Gmbh Carrier-bound protein prepared by reacting the protein with an acylating or alkylating compound having a carrier-bonding group and reacting the product with a carrier
US4195128A (en) 1976-05-03 1980-03-25 Bayer Aktiengesellschaft Polymeric carrier bound ligands
US4330440A (en) 1977-02-08 1982-05-18 Development Finance Corporation Of New Zealand Activated matrix and method of activation
US4247642A (en) 1977-02-17 1981-01-27 Sumitomo Chemical Company, Limited Enzyme immobilization with pullulan gel
US4229537A (en) 1978-02-09 1980-10-21 New York University Preparation of trichloro-s-triazine activated supports for coupling ligands
US4376110A (en) 1980-08-04 1983-03-08 Hybritech, Incorporated Immunometric assays using monoclonal antibodies
WO1998037210A1 (fr) 1997-02-21 1998-08-27 KANTON ZÜRICH vertreten durch DIE ERZIEHUNGSDIREKTION Detection immunologique de prions
US5866366A (en) 1997-07-01 1999-02-02 Smithkline Beecham Corporation gidB
US6528269B1 (en) 1998-06-22 2003-03-04 Case Western Reserve University Immunological agents specific for prion protein (PRP)
WO2002004954A2 (fr) 2000-07-07 2002-01-17 Applied Research Systems Ars Holding N.V. Diagnostic precoce de maladies conformationelles
US20050118720A1 (en) 2002-03-04 2005-06-02 Carlos Bloch Jr Method for the detection of proteins of animal origin in complex mixtures
US6855503B2 (en) 2002-12-20 2005-02-15 Amersham Biosciences Corp Heterocyclic FRETdye cassettes for labeling biological molecules and their use in DNA sequencing
US20080220447A1 (en) 2004-05-14 2008-09-11 Prionics Ag Method for the Detection of Disease-Related Prion
WO2009015091A1 (fr) * 2007-07-20 2009-01-29 The Government of the United States of America as represented by the Secretary of the Détection de protéine prion infectieuse par conversion à germes de protéine prion recombinante

Non-Patent Citations (137)

* Cited by examiner, † Cited by third party
Title
"Methods in Molecular Biology", vol. 22, 2003, HUMANA PRESS, article "PCR Protocols"
"Pierce Catalog and Handbook", 1994, PIERCE CHEMICAL CO.
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ATARASHI ET AL., NAT METHODS, vol. 4, 2007, pages 645 - 650
ATARASHI ET AL., NAT METHODS, vol. 5, 2008, pages 211 - 212
ATARASHI ET AL., NATURE MEDICINE, 2007
ATARASHI R ET AL., NATURE MEDICINE, vol. 17, 2011, pages 175 - 178
ATARASHI R; SATOH K; SANO K ET AL., NAT. MED., vol. 17, 2011, pages 175 - 178
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1992, GREENE PUBL. ASSOC. AND WILEY-INTERSCIENCES
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1992, GREENE PUBLISHING ASSOCIATES
AUSUBEL ET AL.: "Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed.,", 1999, WILEY & SONS
BARON T; VULIN J; BIACABE AG ET AL., PLOS ONE, vol. 6, 2011, pages EL5839
BEN BASSAT ET AL., J. BACTERIOL., vol. 169, 1987, pages 751 - 757
BENCSIK A; LEBOIDRE M; DEBEER S ET AL., EXP. NEUROL., vol. 72, 2013, pages 211 - 218
BENJAMIN LEWIN: "Genes V", 1994, OXFORD UNIVERSITY PRESS, ISBN: 0-19-854287-9
BESSEN ET AL., NATURE, vol. 375, no. 6533, 22 June 1995 (1995-06-22), pages 698 - 700
BESSEN RA ET AL., NATURE, vol. 375, 1995, pages 698 - 700
BESSEN RA; WILHAM JM; LOWE D ET AL., J.VIROL., vol. 86, 2011, pages 1777 - 1788
BIACABE AG; LAPLANCHE JL; RYDER S ET AL., EMBO REP., vol. 5, 2004, pages 110 - 115
BIACABE AG; LAPLANCHE JL; RYDER S; BARON T, EMBO REP., vol. 5, 2004, pages 110 - 115
BOSSERS ET AL., PROC NATL ACAD SCI USA., vol. 94, no. 10, 13 May 1997 (1997-05-13), pages 4931 - 6
BRADLEY; WILESMITH, BR. MED. BULL., vol. 49, 1993, pages 932 - 959
BROWN ET AL., EMERG INFECT DIS., vol. 12, 2006, pages 1816 - 1821
BROWN P; MCSHANE LM; ZANUSSO G ET AL., EMERGING INFECT. DIS., vol. 12, 2006, pages 1816 - 1821
BUSCHMANN A; GRETZSCHEL A; BIACABE AG ET AL., VET. MICROBIOL., vol. 117, 2006, pages 103 - 116
BUSCHMANN A; GROSCHUP MH, J. INFECT. DIS., vol. 192, 2005, pages 934 - 942
BUSCHMANN A; GROSCHUP MH, J. INFECT. DIS., vol. 192, pages 934 - 942
CARTONI C ET AL: "Identification of the pathological prion protein allotypes in scrapie-infected heterozygous bank voles (Clethrionomys glareolus) by high-performance liquid chromatography-mass spectrometry", JOURNAL OF CHROMATOGRAPHY, ELSEVIER SCIENCE PUBLISHERS B.V, NL, vol. 1081, no. 1, 15 July 2005 (2005-07-15), pages 122 - 126, XP027722950, ISSN: 0021-9673, [retrieved on 20050715] *
CASALONE C; ZANUSSO G; ACUTIS P ET AL., PROC NATL ACAD SCI U S A, vol. 101, 2004, pages 3065 - 3070
CASALONE ET AL., PROC NATL ACAD SCI USA., vol. 101, 2004, pages 3065 - 3070
CAUGHEY ET AL., J. VIROL., vol. 65, 1991, pages 6597 - 6603
CAUGHEY; BARON, NATURE, vol. 443, 2006, pages 803 - 810
CHESEBRO B; STRIEBEL J; RANGEL A ET AL., MBIO, vol. 6, 2015, pages 57 - 60
CHRISTINA D. ORRÚ ET AL: "Rapid and Sensitive RT-QuIC Detection of Human Creutzfeldt-Jakob Disease Using Cerebrospinal Fluid", MBIO, vol. 6, no. 1, 27 February 2015 (2015-02-27), pages e02451 - 14, XP055274242, DOI: 10.1128/mBio.02451-14 *
COLBY DW ET AL., PROC NATL ACAD SCI USA, vol. 104, 2007, pages 20914 - 20919
CORPET ET AL., NUCLEIC ACIDS RESEARCH, vol. 16, 1988, pages 10881 - 10890
CRAMM M ET AL., MOL NEUROBIOL, vol. 51, 2015, pages 396 - 405
CRAMM M ET AL., MOL NEUROBIOL., 2015
DELEAULT ET AL., J. BIOL. CHEM., vol. 280, 2005, pages 26873 - 26879
DUCROT ET AL., VET RES., vol. 39, 2008, pages 15
DUDAS ET AL., PLOS ONE, vol. 5, 2010, pages E10638
EFSA JOURNAL, vol. 12, 2014, pages 3798
EMBO J., vol. 19, no. 17, 1 September 2000 (2000-09-01), pages 4425 - 30
ERIC MINIKEL: "A proposal: how to tell if a cell-free assay is predictive of therapeutic potency in vivo", 1 January 2014 (2014-01-01), XP055274434, Retrieved from the Internet <URL:http://www.cureffi.org/2014/06/04/a-proposal-how-to-tell-if-a-cell-free-assay-is-predictive-of-therapeutic-potency-in-vivo/> [retrieved on 20160523] *
FRANKEL ET AL., MOL. IMMUNOL., vol. 16, 1979, pages 101 - 106
FUKUDA S; IWAMARU Y; IMAMURA M ET AL., MICROBIOL. IMMUNOL., vol. 53, 2009, pages 704 - 707
GILL ON; SPENCER Y; RICHARD-LOENDT A ET AL., BMJ, vol. 347, pages F5675
GILL, SPENCER, RICHARD-LOENDT A ET AL., BMJ, vol. 347, 2013, pages F5675
GOLDMANN ET AL., J GEN VIROL., vol. 72, October 1991 (1991-10-01), pages 2411 - 7
GOTTE DR ET AL., PLOS ONE, vol. 6, 2011, pages E27510
GRAY JG; DUDAS S; GRAHAM C ET AL., J VET DIAGN INVEST, vol. 24, 2012, pages 976 - 980
GRAY JG; DUDAS S; GRAHAM C ET AL., J VET DIAGN INVEST, vol. 24, pages 976 - 980
GROVEMAN BR; KRAUS A; RAYMOND LD ET AL., J. BIOL. CHEM., vol. 290, 2015, pages 1119 - 1128
HALEY NJ; VAN DE MOTTER A; CARVER S ET AL., PLOS ONE, vol. 8, 2013, pages E81488
HARLOW; LANE: "Antibodies, A Laboratory Manual", 1988, COLD SPRING HARBOR PUBLICATIONS
HENDERSON DM; DAVENPORT KA; HALEY NJ ET AL., J. GEN. VIROL., vol. 96, 2015, pages 210 - 219
HIGGINS; SHARP, CABIOS, vol. 5, 1989, pages 151 - 153
HIGGINS; SHARP, GENE, vol. 73, 1988, pages 237 - 244
HOCHULI ET AL., BIOLTECHNOLOGY, vol. 6, 1988, pages 1321 - 1325
HORNEMANN ET AL., EMBO REP., vol. 5, 2004, pages 1159 - 1164
HUANG ET AL., COMPUTER APPLICATIONS IN THE BIOSCIENCES, vol. 8, 1992, pages 155 - 165
INNIS ET AL.: "PCR Protocols, A Guide to Methods and Applications", 1990, ACADEMIC PRESS
INNIS ET AL.: "PCR Protocols: A Guide to Methods and Applications", 1990, ACADEMIC PRESS
J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
JACOBS ET AL., J CLIN MICROBIOL., vol. 45, 2007, pages 1821 - 1829
JOEL C. WATTS ET AL: "Evidence That Bank Vole PrP Is a Universal Acceptor for Prions", PLOS PATHOGENS, vol. 10, no. 4, 3 April 2014 (2014-04-03), pages e1003990, XP055274405, DOI: 10.1371/journal.ppat.1003990 *
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
KAWASAKI ET AL.: "PCR Protocols, A Guide to Methods and Applications", 1990, ACADEMIC PRESS, INC., pages: 21 - 27
KENDREW ET AL.: "The Encyclopedia of Molecular Biology", 1994, BLACKWELL SCIENCE LTD., ISBN: 0-632-02182-9
KHURANA ET AL., J. STRUCTURAL BIOL., vol. 151, 2005, pages 229 - 238
KOCISKO ET AL., NATURE, 1994
KOCISKO ET AL., NATURE, vol. 370, no. 6489, 11 August 1994 (1994-08-11), pages 471 - 4
KOCISKO ET AL., PROC NATL ACAD SCI USA., vol. 92, no. 9, 25 April 1995 (1995-04-25), pages 3923 - 7
KOHLER; MILSTEIN, EUR. J. IMMUNOL., vol. 6, 1976, pages 511
KONG Q; ZHENG M; CASALONE C ET AL., J. VIROL., vol. 82, 2008, pages 3697 - 3701
KORTH ET AL., NATURE, vol. 390, 1997, pages 74 - 77
KUBY, J.: "Immunology, 3rd Ed.,", 1997, W.H. FREEMAN & CO.
LACROUX C; COMOY E; MOUDJOU M ET AL., PLOS PATH., vol. 10, 2014, pages EIO04202
LOMBARDI G; CASALONE C; A DA ET AL., PLOS PATH., vol. 4, 2008, pages E1000075
LYNNE I. MCGUIRE ET AL: "Real time quaking-induced conversion analysis of cerebrospinal fluid in sporadic Creutzfeldt-Jakob disease", ANNALS OF NEUROLOGY., vol. 72, no. 2, 1 August 2012 (2012-08-01), BOSTON, US, pages 278 - 285, XP055275068, ISSN: 0364-5134, DOI: 10.1002/ana.23589 *
M. A. DI BARI ET AL: "The bank vole (Myodes glareolus) as a sensitive bioassay for sheep scrapie", JOURNAL OF GENERAL VIROLOGY., vol. 89, no. 12, 1 December 2008 (2008-12-01), GB, pages 2975 - 2985, XP055274335, ISSN: 0022-1317, DOI: 10.1099/vir.0.2008/005520-0 *
MASUJIN K ET AL., PLOS ONE, vol. 8, no. 2, 2013, pages E58013
MASUJIN K; SHU Y; YAMAKAWA Y ET AL., PRION, vol. 2, 2008, pages 123 - 128
MCGUIRE LI ET AL., ANNALS OF NEUROLOGY, vol. 72, 2012, pages 278 - 285
MCGUIRE LI; PEDEN AH; ORRU CD ET AL., ANN. NEUROL., vol. 72, 2012, pages 278 - 285
MELONI D; DAVIDSE A; LANGEVELD JP ET AL., PLOS ONE, vol. 7, 2012, pages E43133
MELONI D; DAVIDSE A; LANGEVELD JP ET AL., PLOS ONE, vol. 7, pages E43133
MONACO S ET AL., PLOS ONE, vol. 7, 2012, pages E32382
MURAYAMA Y; MASUJIN K; IMAMURA M ET AL., J. GEN. VIROL., vol. 95, pages 2576 - 2588
MURAYAMA Y; YOSHIOKA M; MASUJIN K ET AL., PLOS ONE, 2010, pages 5
MURAYAMA Y; YOSHIOKA M; MASUJIN K ET AL., PRP(SC). PLOS ONE, vol. 5, 2010
N. PIENING ET AL: "Conversion Efficiency of Bank Vole Prion Protein in Vitro Is Determined by Residues 155 and 170, but Does Not Correlate with the High Susceptibility of Bank Voles to Sheep Scrapie in Vivo", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 281, no. 14, 7 April 2006 (2006-04-07), US, pages 9373 - 9384, XP055274482, ISSN: 0021-9258, DOI: 10.1074/jbc.M512239200 *
NAZOR ET AL., EMBO J., vol. 24, no. 13, 2005, pages 2472 - 80
NEEDLEMAN; WUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443
OKADA H; MASUJIN K; IWAMARU Y ET AL., VET. PATHOL., vol. 48, 2011, pages 942 - 947
O'REGAN ET AL., GENE, vol. 77, 1989, pages 237 - 251
ORRU CD; BONGIANNI M; TONOLI G ET AL., NEW ENGL. J. MED., vol. 371, 2014, pages 519 - 529
ORRU CD; FAVOLE A; CORONA C ET AL., J. CLIN. MICROBIOL., vol. 53, 2015, pages 1115 - 1120
ORRU CD; GROVEMAN BR; HUGHSON AG ET AL., MBIO, 2015, pages 6
ORRU CD; GROVEMAN BR; RAYMOND LD ET AL., PLOS PATH., vol. 11, 2015, pages E1004983
ORRU CD; GROVEMAN BR; RAYMOND LD ET AL., PLOS PATH., vol. I I, 2015, pages EIO04983
ORRU CD; GROVEMAN BR; RAYMOND LD ET AL., PLOS PATH., vol. I I, no. EI0049, 2015, pages 47
ORRU CD; HUGHSON AG; RACE B ET AL., J.CLIN.MICROBIOL., vol. 50, 2012, pages 1464 - 1466
ORRU CD; WILHAM JM; HUGHSON AG ET AL., PROTEIN ENG. DES. SEL., vol. 22, 2009, pages 515 - 521
ORRU ET AL., J CLIN MICROBIOL., vol. 53, no. 4, April 2015 (2015-04-01), pages 1115 - 20
ORRU ET AL., MBIO, vol. 2, no. 3, 2011, pages E00078 - 11
PAN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 10962 - 10966
PEARSON ET AL., METHODS IN MOLECULAR BIOLOGY, vol. 24, 1994, pages 307 - 331
PEARSON; LIPMAN, PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 2444
PERETZ ET AL., NATURE, vol. 412, 2001, pages 739 - 743
PICCARDO P ET AL., AM. J. PATHOL., vol. 158, 2001, pages 2201 - 2207
PIRISINU L ET AL., PLOS ONE, vol. 8, 2013, pages E66405
RAYMOND ET AL., NATURE, vol. 388, no. 6639, 17 July 1997 (1997-07-17), pages 285 - 8
ROBERT A. MEYERS: "Molecular Biology and Biotechnology: a Comprehensive Desk Reference", 1995, VCH PUBLISHERS, INC., ISBN: 1-56081-569-8
ROTMANS ET AL., J. IMMUNOL. METHODS, vol. 57, 1983, pages 87 - 98
RYUICHIRO ATARASHI ET AL: "Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion", NATURE MEDICINE., vol. 17, no. 2, 30 January 2011 (2011-01-30), US, pages 175 - 178, XP055274246, ISSN: 1078-8956, DOI: 10.1038/nm.2294 *
SABORIO ET AL., NATURE, vol. 411, no. 6839, 14 June 2001 (2001-06-14), pages 810 - 3
SAFAR JG; SCOTT M; MONAGHAN J ET AL., NAT. BIOTECHNOL., vol. 20, 2002, pages 1147 - 1150
SAFAR JG; SCOTT M; MONAGHAN J ET AL., NAT. BIOTECHNOL., vol. 20, pages 1147 - 1150
SAHIN-TOTH ET AL., PROTEIN SCI., vol. 3, 1994, pages 240 - 247
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual, 2d ed.,", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual, 2nd ed.,", vol. 1-3, 1989, COLD SPRING HARBOR LABORATORY PRESS, article "chapters 9 and 11"
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual, 3d ed.,", 2001, COLD SPRING HARBOR PRESS
SHI S; MITTEREGGER-KRETZSCHMAR G; GIESE A ET AL., ACTA NEUROPATHOL COMMUN, vol. 1, 2013, pages 44
SMITH; WATERMAN, ADV. APPL. MATH., vol. 2, 1981, pages 482
TATIANA ET AL., FEMS MICROBIOL. LETT., vol. 174, 1999, pages 247 - 250
TELLING GC ET AL., SCIENCE, vol. 274, pages 2079 - 2082
TORRES JM; ANDREOLETTI O; LACROUX C ET AL., EMERGING INFECT. DIS., vol. 17, 2011, pages 1636 - 1644
VASCELLARI S; ORRU CD; HUGHSON AG ET AL., PLOS ONE, vol. 7, 2012, pages E48969
WILHAM ET AL., PLOS PATHOG., vol. 6, no. 12, pages 1 - 15
WILHAM ET AL., PLOS PATHOGENS, 2010
WILHAM JM ET AL., PLOS PATHOGENS, vol. 6, 2010, pages E1001217
WILHAM JM; ORRU CD; BESSEN RA ET AL., PLOS PATH., vol. 6, 2010, pages E1001217
WILSON R; DOBIE K; HUNTER N ET AL., J. GEN. VIROL., vol. 94, 2013, pages 2819 - 2827
WINDL O; DAWSON M, SUBCELL 2012 BIOCHEM, vol. 65, 2012, pages 497 - 516
WINDL; DAWSON, SUBCELL BIOCHEM., vol. 65, 2012, pages 497 - 516
YAKOVLEVA ET AL., TRANSFUSION, vol. 44, 2004, pages 1700 - 5

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20190050505A (ko) * 2017-11-03 2019-05-13 성신여자대학교 연구 산학협력단 109번째 아미노산이 아이소류신인 제방 들쥐의 프리온 단백질 유전자로 상동재조합된 녹인 생쥐 및 이의 응용
KR20190050507A (ko) * 2017-11-03 2019-05-13 성신여자대학교 연구 산학협력단 109번째 아미노산이 메티오닌이고 23번째에서 89번째 아미노산을 코딩하는 서열을 제거한 제방 들쥐의 프리온 단백질 유전자로 상동재조합된 녹인 생쥐 및 이의 응용
KR20190050508A (ko) * 2017-11-03 2019-05-13 성신여자대학교 연구 산학협력단 109번째 아미노산이 메티오닌인 제방 들쥐의 프리온 단백질 유전자로 상동재조합된 녹인 생쥐 및 이의 응용
KR102070257B1 (ko) 2017-11-03 2020-01-28 성신여자대학교 연구 산학협력단 109번째 아미노산이 메티오닌이고 23번째에서 89번째 아미노산을 코딩하는 서열을 제거한 제방 들쥐의 프리온 단백질 유전자로 상동재조합된 녹인 생쥐 및 이의 응용
KR102100510B1 (ko) 2017-11-03 2020-04-13 성신여자대학교 연구 산학협력단 109번째 아미노산이 메티오닌인 제방 들쥐의 프리온 단백질 유전자로 상동재조합된 녹인 생쥐 및 이의 응용
KR102100509B1 (ko) 2017-11-03 2020-04-13 성신여자대학교 연구 산학협력단 109번째 아미노산이 아이소류신인 제방 들쥐의 프리온 단백질 유전자로 상동재조합된 녹인 생쥐 및 이의 응용
CN108037279A (zh) * 2017-12-08 2018-05-15 中国人民解放军军事科学院军事医学研究院 一种提高elisa试剂盒信噪比的通用型酶标板洗涤液及其使用方法

Similar Documents

Publication Publication Date Title
AU2012207454B2 (en) Methods for amplification and detection of prions
AU2012207454A1 (en) Methods for amplification and detection of prions
EP1119773A1 (fr) Essai de detection de la conformation d&#39;une proteine liee a une maladie
WO2005047860A2 (fr) Anticorps a l&#39;alpha-synucleine
WO2007021255A1 (fr) Anticorps de l’alpha-synucléine
RU2769568C2 (ru) REP белок в качестве белкового антигена для диагностических анализов
JP2008537155A (ja) 自動化タンパク質異常折り畳みの周期的増幅によるプリオンの超高感度検出
WO2016149537A1 (fr) Protéine de prion du campagnol roussâtre utilisée comme substrat à large spectre pour détection et discrimination de souches de prions basées sur une conversion induite par agitation en temps réel (rt-quic)
Taguchi et al. Humanized knock-in mice expressing chimeric prion protein showed varied susceptibility to different human prions
CZ295412B6 (cs) Monoklonální protilátka a koktejl protilátek, které se specificky vážou ke konzervovanému epitopu prionových proteinů, buněčné linie produkující tyto protilátky a způsob detekce PrP-Sc proteinu
MXPA01008969A (es) Muestra de suero sanguineo para aislamiento de priones y prpsc.
JP6938372B2 (ja) ヒトｐ５３の直線状エピトープに結合する抗体及びその診断的応用
JP2018091844A (ja) α−シヌクレイン検出方法
JP4422396B2 (ja) ヒト起源の病原性プリオンを特異的に検出する抗体およびその体を用いて実施される検出方法
EP1368644A2 (fr) Anticorps specifiques de proteine prion d&#39;ongule
AU2001275977A1 (en) Antibodies specific for ungulate PrP
CA2452946C (fr) Methode de detection des symptomes et de diagnostic d&#39;encephalopathies spongiformes transmissibles
Priola et al. Molecular aspects of disease pathogenesis in the transmissible spongiform encephalopathies
WO2018007979A1 (fr) Essai pour la détection d&#39;alpha-synucléine totale et phosphorylée sur s129
WO2009040508A2 (fr) Procédés
鈴木章夫 Studies on methods for specific detection of abnormal isoform of prion protein and its application
JP2003215131A (ja) マススペクトル分析による病原性プリオンタンパク質の検出方法
AU2003276778A1 (en) Method for the detection of a pathogenic form of a prion protein
US20080113387A1 (en) Immunoassay for Prion Disease
PrP Molecular Profiling of Ovine Prion Diseases

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16713245

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16713245

Country of ref document: EP

Kind code of ref document: A1