WO2004046728A1 - Methode de detection de prpsc - Google Patents
Methode de detection de prpsc Download PDFInfo
- Publication number
- WO2004046728A1 WO2004046728A1 PCT/EP2002/012900 EP0212900W WO2004046728A1 WO 2004046728 A1 WO2004046728 A1 WO 2004046728A1 EP 0212900 W EP0212900 W EP 0212900W WO 2004046728 A1 WO2004046728 A1 WO 2004046728A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- prp
- protease
- digestion
- chaotropic agent
- sample
- Prior art date
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Classifications
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- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2828—Prion diseases
Definitions
- the present invention relates to diagnosis of transmissible spongiform encepha- lopathies using PrP Sc (abnormal Prion Protein) as a surrogate marker and methods to distinguish the abnormal from the normal form of the prion protein (PrP c ).
- PrP Sc abnormal Prion Protein
- Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that affect mammals including cattle (BSE), human (Creutzfeldt- Jakob- Disease) and sheep (scrapie).
- the disease is characterized by the accumulation of an abnormal partially protease-resistant form (PrP-res or PrP Sc ) of the host- encoded cellular prion protein (PrP c ). It has been postulated that the infectious agent or prion is partly or entirely composed of PrP Sc and that it propagates itself by inducing conversion of PrP to PrP .
- PrP Sc represents an abnormal conformational form with significantly increased ⁇ -sheet content.
- the ⁇ -sheet rich conformation is prone to aggregation and forms amyloid fibrils accumulating in distinct amyloid plaques in brain tissue (for review see Collinge J. Annu.Rev.Neurosci. 24:519-550,2001).
- a priority of research in the TSE field is the development of methods to detect TSE-infected animals and materials to prevent their spread and entry into the food chain.
- the most common diagnostic methods for TSEs used are histopa- thological detection of the typical spongiform changes found exclusively in the central nervous system and the immunological detection of the TSE-specific iso- form (PrP Sc or PrP-res) of the prion protein that can be distinguished from the normal isoform (PrP ) by its partial resistance to protease digestion (Oesch et al. Cell 40:735-746, 1985).
- PrP Sc the detection of PrP Sc is not confined to the central nervous system but can be found in other tissues and also in body fluids.
- PrP Sc occurs only in TSEs and therefore its presence is a specific marker for these disorders. After experimental inoculation of rodents with TSE agents both PrP Sc and infectivity levels rise until the animals die (Bueler et al. Molecular Medicine 1 : 19-30, 1994). Therefore PrP Sc is often used as a surrogate marker for measuring the amount of infectivity in biological samples.
- Prote- olytic treatment e.g. proteinase K digestion
- PrP Sc removes 60 - 70 amino acid residues from the N-terminal region of the polypeptide chain yielding a pro- tease-resistant core designated PrP27-30.
- PrP Sc The protease resistance of PrP Sc is, however, not an inherent property of the monomeric conformational state of PrP but rather a direct consequence of the aggregation due to its ⁇ -sheet rich secondary structure.
- Treatment of PrP Sc with high concentrations (e.g. >1.5 M guanid- inium thiocyanate) of chaotropic agents alters the physical properties of PrP Sc preparations, leads to denaturation and disaggregation of PrP Sc and renders PrP Sc protease sensitive (Oesch et al. Biochemistry 33:5926-5931,1994).
- prion infectivity is completely and irreversibly lost upon incubation of purified prion preparations with >3 M guanidinium thiocyanate for 24 hours (Prusiner et al. PNAS 90:27932797,1993).
- low concentrations e.g. 0.2 M guanidinium hydrochloride
- chaotropic agents have been used for in vitro conversion reactions to partly denature PrP Sc and make it capable of converting monomeric PrP c molecules into new PrP c molecules following in vitro incubation for extended time periods, e.g. > 24 hours.
- PrP Sc had to be partially denatured with 2 M guanidinium hydrochloride and the reaction mixtures were then diluted to 0.2 M for the conversion reaction and the subsequent protease digestion. There was no indication in that study as to which extent guanidinium would have an effect on the prote- olytic degradation of PrP.
- a method for reducing the risk of false positives in a PrP test is disclosed in WO 00/48003.
- the sample is firstly digested with a protease in order to distinguish PrP Sc from PrP c . Then the digested sample is exposed to high concentrations of guanidine thiocyanate (gdnSCN) or a functional equivalent in order to increase the signal size of the immunological test carried out subsequently.
- gdnSCN guanidine thiocyanate
- the method takes advantage of the fact that PrP Sc is brought into an unfolded state by chaotropic agents like 3-4 M gdnSCN, thereby exposing epitopes which were not accessible to certain antibodies before. This leads to a relative enhancement of the PrP Sc signal in the respective test but does not meet the problem of incomplete digestion of PrP .
- the method makes use of the effect that, when providing suitable conditions (concentration of the chaotropic agent, digestion time and temperature) also a protease resistant PrP c fraction possibly present in the sample is rendered sensitive to protease, whereas PrP Sc remains insensitive.
- the present invention provides an assay method for the detection of PrP Sc in the diagnosis of TSEs that takes advantage of the kinetics of PrP denaturation by chaotropic agents.
- Submitting a prion containing preparation to controlled protease digestion in the presence of low concentrations of chaotropic agents leads to a rapid and thorough digestion of PrP but only partially denatures the multimeric PrP aggregates with full retention of its protease resistance. From the diagnostic perspective, this method reduces background signals due to incomplete digestion of PrP c and thus, increases signal-to noise ratio of such a diagnostic method.
- the method for detection of PrP Sc in the diagnosis of TSEs comprises
- the tissue sample is preferably brain or another tissue known to express the prion protein like lymphoid tissues such as tonsils. Also preferred samples are body fluids like blood, saliva, urine or cerebrospinal fluid.
- the tissue sample is homogenized in an appropriate volume of buffer to solubi- lise said proteins.
- Any homogenization buffer that contains detergent for membrane solubilisation can be used.
- Preferred detergents are, sodium laurylsarcosine, sulfobetaine, Triton X-100, NP-40, ZWITTERGENT, Tween-20, Tween-80.
- Homogenization can be carried out in a device that is suitable for tissue homogenization. In a preferred embodiement of the invention homogenization is carried out in a Prypcons® homogenization device.
- the homogenate is then treated with the digestion buffer containing said chaotropic agent.
- denaturation is perfo ⁇ ned with urea or guanidine but other chemicals that are capable of denaturing proteins are expected to work in the same way.
- Conditions are used that completely digest PrP but will retain PrP Sc in its protease-resistant form.
- Preferred is a concentration of 0.25 M guanidinium hydrochloride, but since denaturation is an equilibrium process it is conceivable that any concentration lying in the range of 0.2 - 1 M will lead to similar results.
- Other denaturing agents preferred are guanidinium thiocyanate and urea.
- any person skilled in the art can easily determine the concentration at which these chemicals lead to a complete digestion of PrP m said method but keep PrP Sc protease-resistant.
- the treated homogenate containing the prion pro- teins is then subjected to proteolysis using a protease that completely digests PrP but removes only the N-terminal 60-70 amino acids of PrP So giving rise to the protease-resistant core PrP27-30.
- a preferred protease is proteinase K but other proteases known to the person skilled in the art are expected to work in a similar fashion.
- the products from the proteolytic digest are detected by an analytical method to identify the protease-resistant core PrP27-30 of the abnormal form of the prion protein.
- Preferred methods use immunological detection reagents such as antibodies that specifically recognize the prion protein.
- a preferred antibody is the monoclonal anti-PrP antibody 6H4 (Korth et al. Nature 390:74-77) which is able to recognize PrP27-30 from a vast variety of species and in various immunological techniques.
- Such methods comprise Western blot or ELISA methods, both technologies that are well known to any person skilled in the art.
- a TSE is diagnosed if products that are specific for the abnormal form of the prion protein are detected.
- Samples were then processed for Western blot analysis by adding 100 ⁇ l of SDS sample buffer (0.25 M Tris-HCl, pH 6.8; 20% glycerol; 0.36 M SDS; 0.6 mM bromphenolblue; 2-mercaptoethanol) and heating the samples for 5 min at 96°C.
- SDS sample buffer (0.25 M Tris-HCl, pH 6.8; 20% glycerol; 0.36 M SDS; 0.6 mM bromphenolblue; 2-mercaptoethanol
- An aliquot of 10 ⁇ l of the sample was applied to a 12% SDS polyacrylamide gel and electrophoresis was performed at 200 V for 1 hour (Laemmli, 1951).
- the proteins were then transferred to to to polyvinylidene difluoride (PVDF) membranes (Millipore) at 150V for 1 hour at 4°C in a transfer tank equipped with plate electrodes (Bio-Rad).
- PVDF polyvinylidene difluoride
- PrP Sc is detected independent of the concentration of guanidinium hydrochloride used during digestion. Remaining PrP c in the negative sample is still detected at concentrations of 0 - 0.1 M guanidinium hydrochloride. Total digestion of PrP is achieved at higher concentrations (0.2 - 1 M). It can be assumed that, at these concentrations, a positive signal will be caused by PrP Sc , whereas false positives can be excluded.
- Example 2 Detection of BSE by identification of protease-resistant PrP in a luminescence immunoassay ( Figure 2).
- Brain tissue from cattle diagnosed with clinical BSE was used to obtain a defined piece of the medulla oblongata from the brain stem.
- the tissue was homogenized for 45 seconds in 10 volumes (w/v) of homogenization buffer to give a 10% ho- mogenate containing 20 mM Hepes, pH 7; 15 mM EDTA; 0.25 M sucrose; 2% sodium laurylsarcosine and 3 mM dithiotreitol using an automated FASTH (Consul) homogenizer.
- One hundred microliters of brain homogenate were mixed with 30 ⁇ l homogenisation buffer, 10 ⁇ l digestion buffer containing guanidinium hydrochloride and 10 ⁇ l of proteinase K (20 Units/ml).
- guanidinium hydrochloride Final concentrations of guanidinium hydrochloride in the digest were 0, 0.12, 0.16 and 0.2 M.
- the prote- olytic reaction was performed for 60 minutes at 47°C in a microplate incubator.
- the protease digestion was teraiinated by the addition of 10 ⁇ l of Pefabloc (40 mM) (Pentapharm Basel, Switzerland). Fifteen microliters of the digested homogenate were denatured with 15 ⁇ l assay buffer (8 M guanidinium hydrochloride) for 2 minutes at room temperature followed by addition of 10 ⁇ l preincuba- tion buffer (40 mM hydrogen peroxide) for 2 minutes at room temperature.
- Results are shown in Figure 2. Signals of PrP remain at a stable level of around 1000 relative light units (RLU) during digestion between 15 and 60 min at 0 M guanidinium hydrochloride. PrP signals reach a background level of 245 RLU when digestion is performed in the presence of 0.2 M guanidinium hydrochloride for 60 minutes.
- RLU relative light units
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- Neurology (AREA)
- Food Science & Technology (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
L'invention concerne une méthode de détection de PrPSc dans un échantillon contenant PrPC et probablement PrPSc. Selon cette méthode, ledit échantillon est soumis à un traitement à la protéase en vue de digérer PrPC, et des agents chaotropiques peuvent être ajoutés pour diminuer le risque de résultats positifs faux. La digestion protéasique est réalisée en présence de l'agent chaotropique et les conditions, dans lesquelles l'agent chaotropique est appliqué, sont adaptées pour améliorer la sensibilité de PrPC à la digestion protéasique, tandis que PrPSc reste insensible.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/EP2002/012900 WO2004046728A1 (fr) | 2002-11-18 | 2002-11-18 | Methode de detection de prpsc |
AU2002352042A AU2002352042A1 (en) | 2002-11-18 | 2002-11-18 | Method for the detection of prpsc |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/EP2002/012900 WO2004046728A1 (fr) | 2002-11-18 | 2002-11-18 | Methode de detection de prpsc |
Publications (1)
Publication Number | Publication Date |
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WO2004046728A1 true WO2004046728A1 (fr) | 2004-06-03 |
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ID=32319526
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/EP2002/012900 WO2004046728A1 (fr) | 2002-11-18 | 2002-11-18 | Methode de detection de prpsc |
Country Status (2)
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AU (1) | AU2002352042A1 (fr) |
WO (1) | WO2004046728A1 (fr) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006063437A1 (fr) * | 2004-12-15 | 2006-06-22 | University Of Guelph | Detecteurs de prions pour le diagnostic de l'encephalopathie spongiforme transmissible ou pour la detection de prions, et leur utilisation |
EP1674870A2 (fr) * | 2004-11-15 | 2006-06-28 | Roche Diagnostics GmbH | Essais de haut débit pour les prions |
US7439041B2 (en) | 2003-08-13 | 2008-10-21 | Novartis Vaccines And Diagnostics, Inc. | Prion-specific peptide reagents |
US7834144B2 (en) | 2005-09-09 | 2010-11-16 | Novartis Ag | Prion-specific peptoid reagents |
US20140220593A1 (en) * | 2006-06-09 | 2014-08-07 | Manfred Watzele | Inhibition of peroxidase enzymatic activity |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0861900A1 (fr) * | 1997-02-21 | 1998-09-02 | Erziehungsdirektion Of The Canton Zurich | Détection immunologique de prions |
WO2000048003A1 (fr) * | 1999-02-11 | 2000-08-17 | Id-Lelystad, Instituut Voor Dierhouderij En Diergezondheid B.V. | Test du prion |
-
2002
- 2002-11-18 AU AU2002352042A patent/AU2002352042A1/en not_active Abandoned
- 2002-11-18 WO PCT/EP2002/012900 patent/WO2004046728A1/fr not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0861900A1 (fr) * | 1997-02-21 | 1998-09-02 | Erziehungsdirektion Of The Canton Zurich | Détection immunologique de prions |
WO2000048003A1 (fr) * | 1999-02-11 | 2000-08-17 | Id-Lelystad, Instituut Voor Dierhouderij En Diergezondheid B.V. | Test du prion |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7439041B2 (en) | 2003-08-13 | 2008-10-21 | Novartis Vaccines And Diagnostics, Inc. | Prion-specific peptide reagents |
EP1674870A2 (fr) * | 2004-11-15 | 2006-06-28 | Roche Diagnostics GmbH | Essais de haut débit pour les prions |
EP1677115A2 (fr) * | 2004-11-15 | 2006-07-05 | Roche Diagnostics GmbH | Essais de haut débit pour les prions |
EP1677115A3 (fr) * | 2004-11-15 | 2006-09-20 | Roche Diagnostics GmbH | Essais de haut débit pour les prions |
EP1674870A3 (fr) * | 2004-11-15 | 2006-09-20 | Roche Diagnostics GmbH | Essais de haut débit pour les prions |
WO2006063437A1 (fr) * | 2004-12-15 | 2006-06-22 | University Of Guelph | Detecteurs de prions pour le diagnostic de l'encephalopathie spongiforme transmissible ou pour la detection de prions, et leur utilisation |
GB2435789A (en) * | 2004-12-15 | 2007-09-05 | Univ Guelph | Prion sensors for diagnosis of transmissible spongiform encephalopathy or for detection of prions,and use thereof |
US7834144B2 (en) | 2005-09-09 | 2010-11-16 | Novartis Ag | Prion-specific peptoid reagents |
US20140220593A1 (en) * | 2006-06-09 | 2014-08-07 | Manfred Watzele | Inhibition of peroxidase enzymatic activity |
US9453842B2 (en) | 2006-06-09 | 2016-09-27 | Roche Molecular Systems, Inc. | Inhibition of peroxidase enzymatic activity |
Also Published As
Publication number | Publication date |
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AU2002352042A1 (en) | 2004-06-15 |
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