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WO2005026740A1 - Procedes et kits pour detecter des maladies a prions - Google Patents

Procedes et kits pour detecter des maladies a prions Download PDF

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WO2005026740A1
WO2005026740A1 PCT/IL2004/000840 IL2004000840W WO2005026740A1 WO 2005026740 A1 WO2005026740 A1 WO 2005026740A1 IL 2004000840 W IL2004000840 W IL 2004000840W WO 2005026740 A1 WO2005026740 A1 WO 2005026740A1
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sample
gags
prion
urine
subject
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Ruth Gabizon
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Hadasit Medical Research Services and Development Co
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Hadasit Medical Research Services and Development Co
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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
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, Konjac gum, Locust bean gum or Guar gum
    • G01N2400/40—Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides
    • 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 methods for the diagnosis of a prion- associated neurodegenerative disorder in a mammalian subject. More particularly, the invention relates to methods and kits for the diagnosis of prion diseases by detection of GAGs (glycosaminoglycans) in urine samples.
  • the diagnostic methods and kits of the invention are based on the finding that aggregates containing GAGs are formed in urine samples of TSE infected subjects.
  • TSEs transmissible spongiform encephalopathies
  • BSE bovine spongiform encephalopathy
  • Mad Cow Disease Another animal prion disease is scrapie in sheep, which after transmission to rodents constitutes the main experimental prion animal model.
  • CJD Creutzfeldt Jakob Disease
  • Prion diseases are believed to be caused by the accumulation in the brain of PrP sc , an abnormally folded isoform of PrP c , a GPI anchored protein of unknown function. It has been postulated that prion diseases propagate by the conversion of PrP c molecules into protease-resistant and insoluble PrP sc by an as yet unknown mechanism.
  • the proteinase K (PK) resistant PrP in prion diseases was described by McKinley et al. [Cell 35(l):57-62 (1983)].
  • Diagnosis of prion diseases was based on the presence of this characteristic protease-resistant PrP in brain biopsies, as well as on clinical criteria.
  • Current methods for the conclusive identification of prion diseases include mostly a post-mortem analysis of the subject's brain homogenate. Clinical symptoms of the disease can many times be misleading.
  • sampling brain tissue from the living subject or patient involves a painful and risky surgical procedure and, moreover, does not give a definite answer since the distribution of PrP sc in the brain is not uniform.
  • vCJD Creutzfeldt Jakob disease
  • a major object of the present invention is the development of a reliable, non-invasive method for diagnosing prion diseases, which will allow the pre-clinical and clinical diagnosis of the disease in humans and in animals.
  • PrP sc Since most urine proteins originate from the blood, the present inventors speculated that during the incubation period some PrP sc , either from the brain or from a peripheral organ, is released into the blood serum in a non- aggregated form, although at low and undetectable concentrations. Due to its protease resistance, PrP sc is not digested by blood proteases. However, since the MW of PrP is below the cutoff size for filtering through kidney cells (about 40kDA) [Berne, R. M., and Levy, M. N., Physiology, 4th Ed (1998)], PrP may subsequently be secreted into the urine and thereby be concentrated, as other proteins, to about 120 folds of its concentration in blood [Kocisko, D. A., et al., Nature 370(6489):471-4 (1994)]. The concentration by the kidney makes it possible to detect PrP sc in urine more easily than in blood.
  • the present inventors have previously developed a method for the detectionof the protease resistance abnormal isoform of the prion protein, PrP sc , in a urine sample [WO 02/33420].
  • the procedure described in this publication is based on the enrichment of the protease-resistant isoform in the urine sample by dialyzing the sample through membrane having a pore range of about 6Kd-8Kd, followed by protease digestion and immunological assay.
  • the theoretical possibility for diagnosis of prion diseases in a variety of body fluids, such as urine, has been mentioned in several patent documents.
  • EP 0854364 discloses a diagnostic method for neurodegenerative disorders such as Alzheimer's disease and prion diseases.
  • This method is based on concentrating a protein associated with the specific neurodegenerative disease (such as PrP in prion diseases and APP in Alzheimer's disease) in a sample (urine, for example).
  • the concentration is carried out by contacting the sample with a solid, non-buoyant particulate material having free ionic valencies such as calcium phosphate.
  • WO 93/23432 discloses a diagnostic method for prion diseases in different body fluids such as CSF (cerebrospinal fluid) and theoretically, urine.
  • CSF cerebrospinal fluid
  • EP 0854364 this method is based on concentrating the prion protein by ammonium sulfate precipitation and affinity chromatography. This publication exemplifies CSF as a sample.
  • a tested sample (diluted brain homogenate of a scrapie-affected hamster) was incubated with brain homogenate from healthy hamsters as a -source of PrP c . This mixture was subjected to five cycles of incubation- sonication. The prion protein was then detected using an immunoassay (blot incubated with a PrP sc specific antibody).
  • an immunoassay blot incubated with a PrP sc specific antibody.
  • the present inventors have recently developed a simple, non-invasive, rapid and protease free method for the detection of PrP sc aggregates in urine samples of prion infected animals and humans (UPrP sc ) [PCT Application IL2004/000699].
  • This diagnostic method enhances the aggregation of the abnormal isoform of PrP in a sample, and thereby enables the detection of these aggregates in urine samples obtained from cattle and sheep, without using prion-specific antibodies.
  • This specific enhancement procedure includes the addition of a protein having a beta-sheet structure to the tested sample, preferably IgG light chain (LC), prior to a staining procedure using Congo Red.
  • LC IgG light chain
  • Congo Red has been used for many years to identify amyloid aggregates in different tissues.
  • Amyloid aggregates are believed to be composed of proteins and glycosaminoglycans such as heparan sulfate.
  • the proteins comprised within a specific amyloid aggregate may be related to the pathology of a specific disease, such as beta-amyloid for AD and PrP for TSEs.
  • the inventors thus hypothesized that Congo red recognizes amyloid aggregates in urine TSE. Therefore, the possibility of detecting GAGs in urine samples of TSE patients was next examined.
  • Proteoglycans are widely distributed in animal tissues, occurring predominantly in extracellular matrices, but are also found on cell surfaces. They consist of one or several glycosaminoglycans linked covalently to a protein core. The glycosaminoglycans are composed of repeating disaccharide units, containing carboxylic and/or sulfate ester groups making them acidic by nature [Heinegard, D. and Pausson, M. in Extracellular Matrix -Biochemistry (Piez, K.A. and Reddi, A.H. Eds.), 277-328 Elsevier, Amsterdam/New York (1984)].
  • GAGs in urine are present in several clinical conditions involving renal dysfunction such as diabetes.
  • the only known neurological syndromes in which GAGS are secreted in urine are the group of recessive genetic diseases known as mucopolysaccharidoses, in which diverse enzymes that degrade GAGS are defected.
  • mucopolysaccharidoses in which diverse enzymes that degrade GAGS are defected.
  • Sanfillipo syndrome heparan sulfate is known to accumulate in the brain as part of aggregates resembling amyloid deposits present in neurodegenerative diseases.
  • the diagnostic method of the invention may be used for the in- ⁇ ivo early diagnosis of ill as well as seemingly healthy but prion- infected individuals.
  • the present invention relates to a method for the diagnosis of a prion-associated neurodegenerative disorder in a mammalian subject comprising: (a) providing a body fluid sample of said subject; (b) qualitatively and/or quantitatively detecting glycosaminoglycans (GAGs) in said sample by suitable means, whereby the presence of GAGs in said sample indicates that said subject carries said neurodegenerative disorder.
  • GAGs glycosaminoglycans
  • the method of the invention may further comprise the step of concentrating the sample by a suitable means, before performing the detection.
  • suitable means for detecting GAGs in the sample may be any one of optical density spectrophotometry and chromatography methods.
  • the detection of GAGs in the tested sample may be performed by the following steps: (i) adding to the optionally concentrated sample a binding material capable of binding GAGs and forming a non-soluble precipitate; (ii) applying the precipitate obtained in step (i) onto a solid support; and (iii) detecting a visual signal indicating the presence of GAGs in the tested sample.
  • suitable means for detecting GAGs in a sample may be any chromatography method such as staining of TLC plates or acrylamide or agarose gels.
  • solid support used by the method of the invention may be any one of TLC plate, acrylamide gel and agarose gel.
  • the visual signal indicating the presence of GAGs, where these methods are used may be color signal resulting from staining with a specific dye, preferably, Congo red, Azure blue, Alcyan blue, silver and any combination thereof.
  • the method of the invention is intended for diagnosis of a prion-associated neurodegenerative disorder such as spongiform encephalopathy.
  • spongiform encephalopathy may be any one of Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker Syndrome (GSS), Kuru, scrapie and bovine spongiform encephalopathy (BSE).
  • CJD Creutzfeldt-Jakob disease
  • GSS Gerstmann-Straussler-Scheinker Syndrome
  • Kuru scrapie
  • BSE bovine spongiform encephalopathy
  • the method of the invention is particularly applicable for mammalian subjects such as humans, sheep, goats, bovines, minks, hamsters and felines such as cats.
  • the body fluid sample used by the method of the invention may be a sample of blood, lymph, milk, urine, faeces, semen, brain extracts, spinal cord fluid (SCF), appendix, spleen and tonsillar tissue extracts.
  • Preferred sample may be a urine sample.
  • the tested sample may be concentrated before detection by using centrifugation and precipitation, preferably by using Amicon tubes.
  • the present invention thus provides a method for the diagnosis of a prion disease in a mammalian subject.
  • this method comprises: (a) providing a urine sample of said subject; (b) optionally concentrating the sample; (c) detecting glycosaminoglycans (GAGs) in said sample by any one of optical density spectrophotometry and chromatography methods as defined by the invention, whereby the presence of GAGs in said sample indicates that said subject carries said prion disease.
  • GAGs glycosaminoglycans
  • the present invention relates to a method for detecting the presence of a neurodegenerative disorder-associated aggregates containing GAGs, in a sample of a subject, which method comprises the steps of: (a) concentrating said sample by a suitable means; and (b) qualitatively and/or quantitatively detecting glycosaminoglycans (GAGs) in the sample by suitable means as described by the invention.
  • a neurodegenerative disorder-associated aggregates containing GAGs in a sample of a subject, which method comprises the steps of: (a) concentrating said sample by a suitable means; and (b) qualitatively and/or quantitatively detecting glycosaminoglycans (GAGs) in the sample by suitable means as described by the invention.
  • GAGs glycosaminoglycans
  • the present invention relates to kit for the diagnosis of a prion-associated neurodegenerative disorder in a mammalian subject
  • kit may comprise: (a) means for obtaining a sample from a tested mammalian subject; (b) means for concentrating the tested sample; (c) means for measuring GAGs in the sample; (d) optionally, suitable buffers; and (e) instructions for carrying out the detection of the presence of aggregates comprising GAGs in the tested sample.
  • the kit of the invention may be designed for the diagnosis of a prion-associated neurodegenerative disorder in a mammalian subject according to the methods of the invention.
  • Fig. 1 Congo red staining of samples obtained from prion infected (BSE) or normal bovine (N), with (+) or without (-) Human IgG Light chain (Lc).
  • Fig. 2 Congo red staining of samples obtained from healthy individual control (N), AD and from CJD patients. The samples were treated (+) or not- treated (-) with PK prior to separation on 12% SDS PAGE.
  • Fig. 3 Congo red staining of normal (C), AD and CJD samples which were deproteinized by treatment with Guanidium prior to PK treatment (blot in the left, PK aG). The blot in the right is a control, without Guanidium treatment.
  • Fig. 4 Urine samples obtained from healthy controls (C), CJD, AD and Sanfillipo (SF) patients were precipitated with CPC and run on a TLC plate. The plate was subsequently stained with Azure Blue.
  • Urine samples of three CDJ patients (P), healthy control (C), and heparan sulphate standard (HS) were separated on 1% Agarose gel. The gel was subsequently stained with Alcyan blue.
  • Urine samples of three CDJ patients (P), three healthy controls (C), and heparan sulphate standard (HS) were separated on 12% acrylamide gel. The gel was subsequently stained with Alcyan and silver.
  • Fig. 7A-7B Urine samples of three CDJ patients (P), one suspected patient (S), three healthy controls (C), one AD patient (AD) and Heparan Sulphate standard (HS) were subjected to PK treatment (+/- PK) and separated on two identical 5-20% acrylamide gradient gels.
  • Fig. 7A One gel was transferred into nitrocellulose membrane that was subsequently incubated with mAb 3F4 (Western blot analysis).
  • Fig. 7B The second gel was subsequently stained with Alcyan and silver (As).
  • the conformationally modified protein may be implicated in the disease by direct toxic activity, by the lack of biological function of normally-folded protein, or by improper trafficking [Thomas et al., (1995), supra]. In cases where the protein is toxic, it usually self-associates and becomes deposited as amyloid fibrils in diverse organs, inducing tissue damage [Thomas et al., (1995), supra; Kelly, Curr. Opin. Struct. Biol. 6:11-17, (1996); Soto, (1999), supra].
  • Amyloid is a generic term that describes fibrillar aggregates that have a common structural motif, i.e., the ⁇ -pleated sheet conformation [Serpell et al., Cell Mol. Life Sci. 53:887 (1997); Sipe et al., Ann. Rev. Biochem. 61:947-975, (1992)]. These aggregates exhibit specific properties, including the ability to emit a green glow after staining with Congo red, and the capacity to bind the fluorochrome thioflavin S [Sipe, (1992), supra; Ghiso et al., Mol. Neurobiol. 8:49-64, (1994)].
  • amyloid is basically a problem of protein folding, whereby a mainly random coil soluble peptide becomes aggregated, adopting a ⁇ -pleated sheet conformation [Kelly, (1996), supra; Soto, (1999), supra].
  • Amyloid formation proceeds by hydrophobic interactions among conformationally altered amyloidic intermediates, which become structurally organized into a ⁇ -sheet conformation upon peptide interaction.
  • the hydro hobicity appears to be important to induce interaction of the monomers leading to aggregation, while the ⁇ -sheet conformation might determine the ordering of the aggregates in amyloid fibrils.
  • PrP sc a conformational isomer of host-derived prion protein
  • PrP host-derived prion protein
  • PrP c is soluble in non-denaturing detergents, PrP sc is insoluble; PrP c is readily digested by proteases, while PrP sc is partially resistant, resulting in the formation of an N-terminally truncated fragment.
  • Prion diseases are characterized by an extremely long incubation period, followed by a brief and invariably fatal clinical disease. To date, no therapy is available.
  • the present inventors have previously established a method for the detection of the abnormal prion protein in urine samples, which was based on specific enrichment procedure including dialysis of the urine sample through membrane having pore range of about 6KD to about 8Kd, followed by protease digestion and immunoassay [WO 02/33420].
  • PrP sc the aberrant isoform and the only known marker for prion diseases
  • PrP sc the aberrant isoform and the only known marker for prion diseases
  • the inventors have now surprisingly found, and this is an object of the invention, that GAGs can be detected in urine samples of TSE patients, using well known GAG detecting methods.
  • the present invention thus provides a rapid, sensitive and specific method for the diagnosis of prion diseases avoiding dialysis, ultracentrifugation, protease digestion and immunological detection steps.
  • the present invention relates to a specific and sensitive method for the diagnosis of a prion-associated neurodegenerative disorder in a mammalian subject comprising: (a) providing a body fluid sample of said subject; (b) qualitatively and/or quantitatively detecting glycosaminoglycans (GAGs) in said sample by suitable means, whereby the presence of GAGs in said sample indicates that said subject carries said neurodegenerative disorder.
  • GAGs glycosaminoglycans
  • the GAGs-containing aggregates which are detected in accordance with the invention may be non-covalent or non-cross linked aggregates.
  • Covalent aggregates may be formed by disulphide bridges between two cysteine residues present in different proteins and GAGs.
  • Non-covalent aggregates are not necessarily formed through disulphide bonds, but e.g. through adoption of a high ⁇ -structure content.
  • Aggregation typically occurs by nucleation. Nucleation occurs when intermolecular bonds form between polypeptides in a partially or fully denatured state. Important parameters for nucleation, typically include variations in solvents, polypeptide concentration, salt, ligands, temperature and pH. A skilled person will be able to determine suitable conditions for any given GAG.
  • GAGs can be measured by any technique known in the art. Techniques -typically used include optical density spectrophotometry and chromatography methods.
  • detection of GAGs may comprise the following steps: (i) adding to a sample, preferably to a concentrated sample, a binding material capable of binding GAGs and forming a non-soluble precipitate; (ii) applying the precipitate obtained in step (i) onto a solid support; and (iii) detecting a visual signal indicating the presence of GAGs in the tested sample.
  • a "binding material capable of binding GAGs” may include any material such as a protein, peptide, sequence of either, antibody, Congo red, Thioflavin-T, or any species capable of the binding so described. In the case of antibodies, this binding is site-specific. In other cases, it can be nonspecific. In a specifically preferred embodiment, the binding material is CPC (cetylpyridinium chloride), which specifically binds and precipitates GAGs.
  • CPC cetylpyridinium chloride
  • the GAGs-containing aggregates further comprising proteins associated with neurodegenerative disease.
  • proteins mean proteins associated with a prion-associated neurodegenerative disease having sufficient binding capacity to bind to other molecules associated with the neurodegenerative disorder (including like molecules or GAGs), to form fibrils or aggregates characteristic of neurodegenerative disease.
  • aggregate-forming proteins typically are characterized by a change in molecule conformation, relative to sequence- homologous, healthy counterparts, allowing them to bind more readily to like or similar molecules. In some cases, such aggregate -forming proteins have the capability to convert proteins from non-aggregate-forming conformation into aggregate-forming conformation.
  • suitable means for detecting GAGs in a sample may be any chromatography method such as staining of TLC platesor acrylamide or agarose gels.
  • solid support used by the method of the invention may be any one of TLC plate, acrylamide gel and agarose gel.
  • the visual signal indicating the presence of GAGs, where these methods are used may be a color signal resulting from staining with a specific dye, preferably, Congo red, Azure blue, Alcyan blue, silver and any combination thereof.
  • the method of the invention is intended for diagnosis of a neurodegenerative disorder, preferably disorder related to amyloidosis or a conformational disease.
  • conformational diseases refers to that group of disorders arising from propagation of an aberrant conformational transition of an underlying protein, leading to protein aggregation and tissue deposition. Such diseases can also be transmitted by an induced conformational change, propagated from a pathogenic conformer to its normal or non-pathogenic conformer and in this case they are called herein "transmissible conformational disease”. Examples of such diseases are spongiform encephalopathies.
  • spongiform encephalopathy may be any one of Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker Syndrome (GSS), Kuru and FFI (Fatal familial insomnia) in humans, scrapie in sheep and goats and bovine spongiform encephalopathy (BSE) in cattle, spongiform encephalopathy of exotic ruminants (nyala, gemsbok, Arabian Oryx, eland, kudu, scimitar- homed Oryx, ankole, and bison); feline spongiform encephalopathy (domestic cat, puma, cheetah, ocelot, tiger), CWD (Chronic Wasting Disease) of mule, deer and elk and TME (Transmissible Mink Encephalopathy).
  • CJD Creutzfeldt-Jakob disease
  • GSS Gerstmann-Straussler-Scheinker Syndrome
  • GSS Garnier-Strassler-Scheinker Disease
  • the method of the invention is particularly applicable for -mammalian subjects such as humans, sheep, goats, bovines, minks, hamsters and felines such as cats.
  • the body fluid sample used by the method of the invention may be a sample of blood, lymph, milk, urine, faeces, ocular fluids, saliva, semen, brain extracts, spinal cord fluid (SCF), appendix, spleen and tonsillar tissue extracts, and where necessary can be obtained by biopsy.
  • Preferred sample may be a urine sample.
  • a preferred sample may be a body fluid sample
  • the method of the invention may be applicable for any sample.
  • sample refers to any cell, tissue, or fluid from a biological source, or any other medium that can advantageously be evaluated in accordance with the invention including, but not limited to, a biological sample drawn from a human patient, a sample drawn from an animal, a sample drawn from food designed for human consumption, a sample including food designed for animal consumption such as livestock feed, an organ donation sample, or the like.
  • the tested sample is concentrated by centrifugation or precipitation.
  • the sample is collected and concentrated using Amicon tubes. It is to be appreciated that any other suitable concentration methods such as dialysis, centrifugation and ethanol precipitation, may be used by the method of the invention.
  • the present invention thus provides for a method for the diagnosis of a prion disease in a mammalian subject.
  • this method comprises: (a) providing a urine sample of said subject; (b) concentrating proteins comprised within the sample by using Amicon tube; (c) detecting glycosaminoglycans (GAGs) in the sample by any one of optical -density spectrophotometry and chromatography methods as described in the Examples, whereby the presence of GAGs in the sample indicates that said subject carries said prion disease.
  • GAGs glycosaminoglycans
  • diagnosis of prion disease may be used for diagnosing a prion disease in a human or animal subject, by obtaining a urine sample of the subject and detecting the presence of GAGs in the urine sample by the detection method the invention, the presence of the GAGs in the urine of the subject indicating that said subject carries a prion disease.
  • the invention provides a method for the detection of different prion diseases before or after onset of clinical symptoms.
  • the diagnostic method of the invention is particularly important for detecting carriers of CJD, for monitoring treatment of CJD patients and for estimating the patient's clinical stage as well as the severity of the disease. It is to be noted that when referring to CJD, all other TSE's are also included. Suspected carriers of pathogenic prion mutations are tested by molecular method for the presence of the mutation, which defines their carrier status. However, and since the age of disease onset can be between 35-85 years or more, there is yet no test to establish at early stages whether the disease is manifesting. Such test could be crucial for early or prophylactic treatment. The detection of carriers of the mutation leading to CJD disease may be used, for example, in genetic counseling.
  • the diagnostic method of the invention is useful in identifying infection of BSE, particularly in individuals that have been exposed to the disease. Identifying human carriers of BSE has importance, inter alia, in screening blood samples of human donors for the presence of a prion disease in the donors. Screening can be carried out, for example, by obtaining a urine sample from the donor, detecting the presence of GAGs in the urine sample by the detection method of the invention and ascribing the results of the -detection to said blood sample. Such screening would prevent the use of prion-infected blood, thus diminishing risks of blood transfusions.
  • the diagnostic method of the invention when applied to bovine animals, and also to other domestic animals like sheep and goats or any other animal of interest susceptible to BSE or any other prion disease, may assist in screening food products originating from the tested animals, like meat and dairy products, and reduce the risk of infection of human consumers.
  • the present invention relates to a method for detecting the presence of a prion-associated neurodegenerative disorder-associated aggregate containing GAGs in a sample of a subject, such method comprises the steps of: (a) concentrating the sample, preferably by using Amicon tubes; (b) qualitatively and/or quantitatively detecting glycosaminoglycans (GAGs) in said sample by suitable means.
  • a concentrating the sample preferably by using Amicon tubes
  • GAGs glycosaminoglycans
  • the present invention relates to kit for the diagnosis of a prion-associated neurodegenerative disorder in a mammalian subject, such kit comprising: (a) means for obtaining a sample from a tested mammalian subject; (b) means for concentrating the tested sample; (c) means for measuring GAGs in the sample; (d) optionally, suitable buffers; and (e) instructions for carrying out the detection of the presence of aggregates comprising GAGs in the tested sample.
  • a specific dye such as Congo red, Azure blue, Alcyan blue, silver and any combination thereof may be included in the kit.
  • kit according to the invention is particularly applicable for the detection of a prion disease in a mammalian subject, using a urine sample.
  • Such specific kit may preferably comprise means for obtaining a urine sample from the tested subject, means for concentrating the sample, -such as Amicon tubes, CPC for precipitating GAGs, solid support for attachment of GAGs in said sample (for example nitrocellulose membrane, TLC plates, acrylamide or agarose gels), further optional buffers and instructions for carrying out the detection of the presence of aggregates comprising GAGs in the tested urine sample.
  • the kit of the invention is intended for the diagnosis of a prion- associated neurodegenerative disorder such as a spongiform encephalopathy, and is useful in carrying out all of the diagnostic methods of the invention.
  • prion disease and “prion-associated neurodegenerative disorder” are used herein interchangingly and synonymously.
  • the total urine GAG content was determined by a CPC (cetylpyridinium chloride) GAG method [Peonock, C.A., J. Clin. Pathol. 29:111 (1976); Di Ferrante, N. Anal. Biochem. 21:98 (1967)]. Under controlled pH and electrolyte concentration conditions, CPC reacts with GAGs to form an insoluble precipitate. In the presence of a citrate buffer at pH 4.8 this precipitate is sufficiently stabilized and dispersed to measure the absorbanceof 680 mm in a spectrophotometer (Mod Du 650, Beckman Industries, Fullerton, CA, USA). Ch-4-S was used as a reference standard.
  • CPC cetylpyridinium chloride
  • GAG excretion was expressed as a GAGs/creatinine ratio (mg GAG excreted per gram of creatinine) [Peonock, (1976) ibid. ; Di Ferrante, (1967) ibid.].
  • Isolation of GAGs One milliliter of CPC reagent was added to an aliquot of urine. After overnight CPC precipitation at 4°C, the harvested precipitate was mixed with 2-3 mL of potassium acetate in ethanol, and the precipitates were washed with an aliquot of alcohol reagent, harvested by centrifugation, washed once with a few milliliters of diethyl ether, air dried, and taken up in deionized water [Peonock, (1976) ibid.].
  • SDS-PAGE Eletrophoresis of glycosaminoglycans was performed on a PhastSystem (pharmacia/LKB) using 12% or 5-20% gels. SDS-PAGE was carried out under non-reducing conditions following the protocol described by the manufacturer. Samples were diluted in sample buffer (final composition of 7.8 mM Tris//HCI, 6% (W/V) urea, 0.875% SDS (W V), 2.5% glycerol (V/V), 0.625 mM EDTA, and 0.00025% bromophenol blue, pH 8.9) and heated at 100°C for 4 min.
  • Alcyan blue/neutral silver stain Immediately after electrophoresis, the gels were placed in the development chamber of the PhastSystem and stained according to Table 1.
  • the method is a combination of Alcyan blue staining (steps 1-7) and a neutral silver staining protocol (steps 8-20) from PhastGel Silver Kit, which is a modification of a method by Heukeshoven and Dernick [Electrophoresis 6:108-112 (1985)].
  • Example 1 CR staining detects amyloid aggregates in CJD urine samples.
  • the inventors developed a protocol for TSE urine testing, -based on Congo Red (CR) staining of urine prion aggregates.
  • urine from CJD patients and scrapie-infected hamsters could be stained with CR following concentration of the urine sample, and identified as positive by a dot blot protocol.
  • the inventors further found that addition of protein having a beta-sheet conformation to a sample containing the prion protein may enhance aggregation and thus increase the signal.
  • the inventors have developed a protocol for a sensitive and specific detection of PrP protein in prion-infected samples.
  • This protocol is based on the addition of external protein having beta- sheet structure, preferably, IgG light chain (LC), to the concentrated urine samples. Following incubation for 2 to 20h, Congo Red is added and the samples are subjected to a dot blot assay as indicated in the experimental procedures.
  • IgG light chain LC
  • CR Congo red
  • urine samples obtained from a CJD patient, a patient suffering from Alzheimer Disease (AD) and a healthy individual control were treated with PK (Proteinase K) and were separated on 12% SDS PAGE prior to staining with CR.
  • PK Proteinase K
  • a variety of fragments were recognized by CR in AD and CJD untreated samples.
  • only samples obtained from CJD patients were recognized by CR after PK treatment.
  • the staining pattern of the PK-treated CJD sample further indicates that PrP is not the sole protease resistant protein in prion disease urine.
  • the inventors therefore hypothesized that prion urine comprises amyloid "seed" which forms a center for aggregation of variety of protein which leads to formation of amyloid accumulation.
  • CR binds any components other than protease resistant proteins in urine samples of CJD patients. Therefore, urine samples obtained from two CJD patients, one AD patient and two healthy individuals (control), were subjected to Guanidium treatment, which leads to deproteinization by denaturation of proteins, prior to treatment with PK. The samples were then subjected to dot-blot and stained with CR. Fig. 3 shows that only CJD samples were recognized by CD, after deproteination followed by PK treatment. It should be noted that Guanidium treatment significantly reduced the CR signal, indicating that CR binds to amyloid aggregates composed of proteins and some other components.
  • Example 2 Diagnosis of CJD by the detection of GAGs Since amyloid aggregates are known as being composed of proteins and glycosaminoglycans (GAGs), the inventors next examined the presence of GAGs in urine samples obtained from CJD patients.
  • GAGs glycosaminoglycans
  • Several methods have been previously developed for this purpose since GAGs in urine are present in several clinical conditions involving renal dysfunction such as diabetes and in a group of recessive genetic diseases called mucopolysaccharidoses.
  • urine samples obtained from two CJD patients, one AD, three healthy individuals (control) and from a patient who suffers from Sanfillipo syndrome (positive control) were subjected to CPC precipitation followed by separation on TLC plates and staining with Azure Blue as indicated in the Experimental procedures.
  • Sanfillipo syndrome is a mucopolysaccharidose disorder characterized by accumulation of heparan sulfate in the brain as part of amyloid-like aggregates.
  • Fig. 4 the presence of GAGs in urine samples of CJD patients, as well as in the Sanfillipo sample, was clearly detected. No signal was detected in control and AD samples.

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Abstract

L'invention concerne une méthode et un kit pour diagnostiquer un trouble neurodégénératif associé à un prion chez un mammifère, cette méthode consistant à disposer d'un échantillon de fluide corporel du sujet, et à détecter en termes de qualité et/ou de quantité des glycosaminoglycanes (GAG) dans l'échantillon en se servant d'un moyen adapté. La présence de GAG dans l'échantillon indique que le sujet est porteur d'un trouble neurodégénératif associé à un prion, qui peut être une encéphalopathie spongiforme telle que la maladie de Creutzfeldt-Jakob (CJD), le syndrome de Gerstmann-Straussler-Scheinker (GSS), le Kuru, la scrapie et l'encéphalopathie spongiforme bovine (BSE).
PCT/IL2004/000840 2003-09-18 2004-09-13 Procedes et kits pour detecter des maladies a prions Ceased WO2005026740A1 (fr)

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IL15801003A IL158010A0 (en) 2003-09-18 2003-09-18 Methods and kits for the detection of prion diseases

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005080590A1 (fr) * 2004-01-20 2005-09-01 Biomerieux PROCEDE DE DETECTION DE LA PrP UTILISANT UNE MOLECULE AYANT AU MOINS UNE CHARGE POSITIVE ET/OU AU MOINS UNE LIAISON OSIDIQUE ET UN LIGAND AUTRE QU’UN LIGAND PROTEIQUE
US8158441B2 (en) 2005-07-21 2012-04-17 Biomerieux S.A. Method for detecting aggregate-forming circulating protein forms using an agent for aggregating said forms and an agent for capturing formed aggregates
EP2764114A4 (fr) * 2011-10-03 2015-05-06 Univ Los Andes Méthode pour la surveillance, le diagnostic et/ou le pronostic de troubles associés à l'hypoxie à l'aide de nfat5

Citations (3)

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Publication number Priority date Publication date Assignee Title
US5310646A (en) * 1988-05-13 1994-05-10 Regents Of The University Of Minnesota Method for the detection of mucopolysaccharide storage diseases
US6287789B1 (en) * 1996-09-13 2001-09-11 Biomarin Pharmaceuticals Chondroitin sulfate as a marker of bone resorption
US20020150948A1 (en) * 2000-11-03 2002-10-17 Gerardo Castillo Antibody PTI-HS7 for treatment of alzheimer's disease and other amyloidoses and parkinson's disease

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5310646A (en) * 1988-05-13 1994-05-10 Regents Of The University Of Minnesota Method for the detection of mucopolysaccharide storage diseases
US6287789B1 (en) * 1996-09-13 2001-09-11 Biomarin Pharmaceuticals Chondroitin sulfate as a marker of bone resorption
US20020150948A1 (en) * 2000-11-03 2002-10-17 Gerardo Castillo Antibody PTI-HS7 for treatment of alzheimer's disease and other amyloidoses and parkinson's disease

Non-Patent Citations (1)

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Title
THUY L P ET AL: "A new quantitative assay for glycosaminoglycans", CLINICA CHIMICA ACTA, AMSTERDAM, NL, vol. 212, 1992, pages 17 - 26, XP002978288, ISSN: 0009-8981 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005080590A1 (fr) * 2004-01-20 2005-09-01 Biomerieux PROCEDE DE DETECTION DE LA PrP UTILISANT UNE MOLECULE AYANT AU MOINS UNE CHARGE POSITIVE ET/OU AU MOINS UNE LIAISON OSIDIQUE ET UN LIGAND AUTRE QU’UN LIGAND PROTEIQUE
US7566530B2 (en) 2004-01-20 2009-07-28 Biomerieux Method for detecting PrP using at least one positive charge and/or at least one glycosidic bond and a ligand other than a protein ligand
US8158441B2 (en) 2005-07-21 2012-04-17 Biomerieux S.A. Method for detecting aggregate-forming circulating protein forms using an agent for aggregating said forms and an agent for capturing formed aggregates
EP2764114A4 (fr) * 2011-10-03 2015-05-06 Univ Los Andes Méthode pour la surveillance, le diagnostic et/ou le pronostic de troubles associés à l'hypoxie à l'aide de nfat5
US9631237B2 (en) 2011-10-03 2017-04-25 Universidad De Los Andes Method for monitoring, diagnosis and/or prognosis of hypoxia related disorders using NFAT5

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