EP0795133A1 - Spongiform encephalopathy detection methods - Google Patents

Spongiform encephalopathy detection methods

Info

Publication number
EP0795133A1
EP0795133A1 EP95937125A EP95937125A EP0795133A1 EP 0795133 A1 EP0795133 A1 EP 0795133A1 EP 95937125 A EP95937125 A EP 95937125A EP 95937125 A EP95937125 A EP 95937125A EP 0795133 A1 EP0795133 A1 EP 0795133A1
Authority
EP
European Patent Office
Prior art keywords
animal
agent
spongiform encephalopathy
body fluid
amount
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP95937125A
Other languages
German (de)
French (fr)
Inventor
Michael Central Veterinary Laboratory DAWSON
Trevor Conrad Central Veterinary Lab. MARTIN
Paula Central Veterinary Laboratory KEYES
Verity Central Veterinary Laboratory JONES
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Minister of Agriculture Fisheries and Food UK
Original Assignee
Minister of Agriculture Fisheries and Food UK
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
Priority claimed from GB9424068A external-priority patent/GB9424068D0/en
Application filed by Minister of Agriculture Fisheries and Food UK filed Critical Minister of Agriculture Fisheries and Food UK
Publication of EP0795133A1 publication Critical patent/EP0795133A1/en
Withdrawn legal-status Critical Current

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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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 methods for the detection of spongiform encephalopathies in animals, and in particular to the detection of bovine spongiform encephalopathy (BSE) in cattle.
  • BSE bovine spongiform encephalopathy
  • Spongiform encephalopathies are a class of diseases which include scrapie in sheep and Creutzfeldt-Jakob disease in humans.
  • BSE is a notifiable fatal neurodegenerative disease found in cattle. BSE is of major importance to the British farming industry.
  • Cases of BSE are identified by clinical manifestations in the animal. Cases are confirmed by post-mortem analysis of brain tissue, for instance by histopathology, by detection of scrapie associated fibrils or proteinase K resistant protein.
  • the present invention has now provided a method for detecting spongiform encephalopathy in an animal which addresses some, and in preferred forms all, of these problems.
  • a method for detecting the presence of a spongiform encephalopathy in an animal comprising determining the presence and/or amount of agent in a body fluid of the animal which has a molecular weight of between 35-39 Da. a pi of approximately 5-3 ⁇ and which does not cross-react with antibody raised against apoliprotein E and relating the result of this determination to the infection status of the animal.
  • the result of the determination is compared with a control value and the relationship between the two is correlated with the infection status of the animal.
  • the method is used to detect BSE.
  • the discovery that the spongiform encephalopathy infection in an animal may be correlated with the presence of, or an increase in the concentration of, an agent or agents in the body fluids of that animal forms the basis for the methods of the current invention.
  • Apolipoprotein E is cholesterol transporting protein produced in the peripheral and central nervous system. Its presence in either multiple- or single-forms has been categorised in cerebrospinal fluid (CSF) and serum. Its molecular weight (37 kDa) and pi (around $ A to 5.7) are similar to those of the agent described above. However the agent which is determined in the methods of the present invention does not cross-react with anti-apolipoprotein E antibody.
  • the control value may be derived from the agent concentration in a different animal (for which the infection status is known) and which is analysed in parallel with the test animal.
  • the control value may come from the same animal, or be a known standard.
  • control value may be determined using the same method used for the test animal or using a different analytical method.
  • the results from the 'control' animal may be used to derive a standard control value, or to calibrate the test animal result.
  • the body fluid analysed in the method is CSF since the proximity of the CSF to brain means that neurological disorders which produce alterations in the protein composition of the brain may be manifested in the CSF.
  • Methods for extracting samples of CSF are well known to those skilled in the art.
  • the invention embraces any method for determining the presence and/or amount of agent in a body fluid of an animal which is currently comprised in the art, and any methods which may later become available.
  • the presence and/or amount of agent in a body fluid of the animal is derived by the use of polyacrylamide gel electrophoresis (PAGE) to separate out the agents having both a molecular weight of between 35-39 kDa and a pi of approximately 5-3 from other materials in the body fluid, and then staining the gel and making densitometry measurements in the region of the gel containing the agent so as to determine the presence and/or amount of the agent present.
  • PAGE polyacrylamide gel electrophoresis
  • the PAGE is two dimensional polyacrylamide gel electrophoresis (2DPAGE) and the stain is silver stain.
  • the lack of cross reactivity between the agent and anti-apolipoprotein E antibody agent is confirmed by the use of antibody raised against apolipoprotein E.
  • Immunogen-based techniques for measuring cross-reactivity are well known to those skilled in the art eg. ELISA or Western Blotting.
  • these immunogenic techniques may be used both to identify the agent and to estimate its concentration. This can be achieved by raising antibodies against the agent, or a synthetic peptide derived from the sequence thereof.
  • the invention makes available methods for detecting the presence of spongiform encephalopathy in a test animal which address many, and in preferred forms all, of the problems of the prior art.
  • the balance between test certainty and ease of use will be dependent on the precise method of agent analysis chosen for use in the methods of the current invention.
  • the pre-mortem diagnosis of BSE in cattle opens up the possibility of mass-testing in herds, thereby reducing the likelihood of slaughtering uninfected animals or 'missing' infected ones.
  • Sample preparation CSF samples were collected from BSE-positive cattle and BSE-negative cattle. In each case the diagnosis was confirmed by post-mortem histopathology and electron microscopy. CSF samples were taken by cisternamagna puncture after death and concentrated 10-15 fold. Volumes of CSF containing 40 ⁇ g total protein were mixed in a 4:1 ratio with denaturing solution (lg sodium dodecyl sulphate (SDS) and 0.232g dithiothreitol in 10ml water) and heated at 95 * C for 5 minutes. Samples were then pulse centrifuged.
  • denaturing solution lg sodium dodecyl sulphate (SDS) and 0.232g dithiothreitol in 10ml water
  • Electrophoresis The prepared samples were 2D electrophoresed using a Millipore Investigator 2D electrophoresis system according to the method in the instruction manual. First dimensional iso-electric-focussing was carried out in 26 cm threaded glass tubes with 1 mm inner diameter in a pH gradient of 3"10 for 18000 volt hours after pre-focussing the gels for 1 hour to 1500 V. Second dimension SDS-PAGE was carried out using 1 mm thick large format gels (23 cm x 23 cm) with 12.5/- acrylamide and no stacking gel.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Steroid Compounds (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Nitrogen And Oxygen As The Only Ring Hetero Atoms (AREA)

Abstract

A method for detecting the presence of a spongiform encephalopathy (e.g. Bovine Spongiform Encephalopathy) in an animal comprising determining the presence and/or amount of agent (e.g. by 2D-polyacrylamide gel electrophoresis and staining) in a body fluid (e.g. cerebrospinal fluid) of the animal which has a molecular weight of between 35-39 kDa, and a pI of 5.3 and which does not cross-react with antibody raised against apoliprotein E and relating the result of this determination to a control value (e.g. from a known uninfected animal) so as to be able to detect the likely presence of spongiform encephalopathy in the animal.

Description

SPONGIFORM ENCEPHALOPATHY DETECTION METHODS
The present invention relates to methods for the detection of spongiform encephalopathies in animals, and in particular to the detection of bovine spongiform encephalopathy (BSE) in cattle.
Spongiform encephalopathies are a class of diseases which include scrapie in sheep and Creutzfeldt-Jakob disease in humans. BSE is a notifiable fatal neurodegenerative disease found in cattle. BSE is of major importance to the British farming industry.
Currently cases of BSE are identified by clinical manifestations in the animal. Cases are confirmed by post-mortem analysis of brain tissue, for instance by histopathology, by detection of scrapie associated fibrils or proteinase K resistant protein.
These methods have the disadvantage that they necessitate the slaughter of potentially-infected animals which may turn out to be disease-free. Alternatively, clinical signs may be absent or go undetected, thus leaving infected animals in the herd.
Thus there exists a need for a pre-mortem test for BSE which can be used when diagnosing potentially-infected animals.
The present invention has now provided a method for detecting spongiform encephalopathy in an animal which addresses some, and in preferred forms all, of these problems.
According to one aspect of the present invention there is provided a method for detecting the presence of a spongiform encephalopathy in an animal comprising determining the presence and/or amount of agent in a body fluid of the animal which has a molecular weight of between 35-39 Da. a pi of approximately 5-3ι and which does not cross-react with antibody raised against apoliprotein E and relating the result of this determination to the infection status of the animal.
Preferably the result of the determination is compared with a control value and the relationship between the two is correlated with the infection status of the animal.
Preferably the method is used to detect BSE.
Thus the discovery that the spongiform encephalopathy infection in an animal may be correlated with the presence of, or an increase in the concentration of, an agent or agents in the body fluids of that animal forms the basis for the methods of the current invention.
Apolipoprotein E is cholesterol transporting protein produced in the peripheral and central nervous system. Its presence in either multiple- or single-forms has been categorised in cerebrospinal fluid (CSF) and serum. Its molecular weight (37 kDa) and pi (around $ A to 5.7) are similar to those of the agent described above. However the agent which is determined in the methods of the present invention does not cross-react with anti-apolipoprotein E antibody.
It should be noted that there is no requirement to accurately determine the agent concentration because the spongiform encephalopathy infection status of the animal may be detected by comparison with a control.
The control value may be derived from the agent concentration in a different animal (for which the infection status is known) and which is analysed in parallel with the test animal. Alternatively, the control value may come from the same animal, or be a known standard.
In either case the control value may be determined using the same method used for the test animal or using a different analytical method.
The results from the 'control' animal may be used to derive a standard control value, or to calibrate the test animal result.
Preferably the body fluid analysed in the method is CSF since the proximity of the CSF to brain means that neurological disorders which produce alterations in the protein composition of the brain may be manifested in the CSF. Methods for extracting samples of CSF are well known to those skilled in the art.
The invention embraces any method for determining the presence and/or amount of agent in a body fluid of an animal which is currently comprised in the art, and any methods which may later become available.
Preferably the presence and/or amount of agent in a body fluid of the animal is derived by the use of polyacrylamide gel electrophoresis (PAGE) to separate out the agents having both a molecular weight of between 35-39 kDa and a pi of approximately 5-3 from other materials in the body fluid, and then staining the gel and making densitometry measurements in the region of the gel containing the agent so as to determine the presence and/or amount of the agent present.
Preferably the PAGE is two dimensional polyacrylamide gel electrophoresis (2DPAGE) and the stain is silver stain.
Preferably the lack of cross reactivity between the agent and anti-apolipoprotein E antibody agent is confirmed by the use of antibody raised against apolipoprotein E. Immunogen-based techniques for measuring cross-reactivity are well known to those skilled in the art eg. ELISA or Western Blotting.
In alternative embodiments of the invention, these immunogenic techniques may be used both to identify the agent and to estimate its concentration. This can be achieved by raising antibodies against the agent, or a synthetic peptide derived from the sequence thereof.
Thus the invention makes available methods for detecting the presence of spongiform encephalopathy in a test animal which address many, and in preferred forms all, of the problems of the prior art. The balance between test certainty and ease of use will be dependent on the precise method of agent analysis chosen for use in the methods of the current invention. However, the pre-mortem diagnosis of BSE in cattle opens up the possibility of mass-testing in herds, thereby reducing the likelihood of slaughtering uninfected animals or 'missing' infected ones.
The methods of the present invention will now be described, by way of illustration only, through reference to the following example and figures. Other embodiments falling within the scope of the invention will occur to those skilled in the art in the light of this.
EXAMPLE - IDENTIFICATION OF BSE IN CATTLE
Sample preparation: CSF samples were collected from BSE-positive cattle and BSE-negative cattle. In each case the diagnosis was confirmed by post-mortem histopathology and electron microscopy. CSF samples were taken by cisternamagna puncture after death and concentrated 10-15 fold. Volumes of CSF containing 40 μg total protein were mixed in a 4:1 ratio with denaturing solution (lg sodium dodecyl sulphate (SDS) and 0.232g dithiothreitol in 10ml water) and heated at 95*C for 5 minutes. Samples were then pulse centrifuged.
Electrophoresis: The prepared samples were 2D electrophoresed using a Millipore Investigator 2D electrophoresis system according to the method in the instruction manual. First dimensional iso-electric-focussing was carried out in 26 cm threaded glass tubes with 1 mm inner diameter in a pH gradient of 3"10 for 18000 volt hours after pre-focussing the gels for 1 hour to 1500 V. Second dimension SDS-PAGE was carried out using 1 mm thick large format gels (23 cm x 23 cm) with 12.5/- acrylamide and no stacking gel.
Staining and Image analysis: The 2D gels were silver stained according to the Millipore manual. Gels were scanned with an Omnimedia scanner XRS and analysed using Bioimage software and Investigator Database programme (Millipore) using a sunSPARC station computer.
Confirmation of the identity of the agent: The 2D gels were electroblotted onto Immobilon-P membranes overnight at 30V using a Bio-Rad Trans-Blot cell. The blots were blocked using Tween 80 for 1 hour and then incubated for 90 minutes with sheep antiserum containing polyclonal antibody raised against apolipoprotein E. Bound sheep antibodies were detected using rabbit anti-sheep IgG and a horseradish peroxidase detection system.
Comparison of BSE-negative and BSE-positive cattle: A comparison of the stained gels from typical BSE-positive and -negative samples is shown in Fig 1(a) and Fig 1(b). As can be seen the number and intensity of the silver stained spots in the region corresponding to agents having an approximate molecular weight of 3^"3δ kDa, and a pi of around 5-4 - 5-7 (labelled 'Apo E' ) is higher in the BSE-positive sample. Those spots in the region labelled 'Apo E' were found to cross react with anti-apolipoprotein E antibody. However, the 2-3 spots, molecular weight 35~39 kDa and pi 5-3 (arrowed in each gel), corresponding to one or more agents which did not cross react with this antibody, are undetectable in the BSE-negative sample but are present in clearly detectable amounts in the BSE-positive sample
Using a number of samples of body fluid from different cattle samples it was found that the agent was consistently present in detectable amounts in BSE-positive animals but was undetectable in BSE-negative animals, thus indicating that the presence and/or amount of this agent may be used to detect the likely presence of BSE in potentially infected animals.

Claims

1. A method for detecting the presence of a spongiform encephalopathy in an animal comprising determining the presence and/or amount of agent in a body fluid of the animal which has a molecular weight of between 35 and 39 kDa, a pi of approximately 5*3. and which does not cross-react with antibody raised against apoliprotein E and relating the result of the determination to the infection status of the animal.
2. A method as claimed in claim 1 wherein the result of the determination is compared with a control value and the relationship between the two is correlated with the infection state of the animal.
3. A method as claimed in claim 1 or claim 2 wherein the spongiform encephalopathy is bovine spongiform encephalopathy.
4. A method as claimed in any one of the preceding claims wherein the body fluid analysed in the method is cerebrospinal fluid.
5. A method as claimed in any one of the preceding claims wherein the presence and/or amount of agent in a body fluid of the animal is derived by the use polyacrylamide gel electrophoresis (PAGE) to separate out the agents having both a molecular weight of between 35 and 39 kDa and a pi of 5-3 from other materials in the body fluid, and then staining the gel and making densitometry measurements in the region of the gel containing the agent so as to determine the presence and/or amount of the agent present.
6. A method as claimed in claim 5 wherein the PAGE is two dimensional polyacrylamide gel electrophoresis.
7. A method as claimed in claim 5 or claim 6 wherein the stain is silver stain.
8. A method as claimed in any preceding claim wherein the lack of cross reactivety between the agent and anti-apolipoprotein E antibody is confirmed by the use of antibody raised against apolipoprotein E.
9. A method as claimed in any of claims 1 to 4 wherein antibodies are raised against the agent, or a synthetic peptide derived generated from the sequence thereof, and the presence and/or amount of agent in a body fluid of the animal is determined by use of these antibodies.
10. A method as claimed in any one of claims 2 to 9 wherein the control value is derived by the same method as that used with the animal but using a further animal for which the infection status is known.
11. A method for detecting the presence of spongiform encephalopathy in an animal substantially as described hereinbefore.
EP95937125A 1994-11-29 1995-11-28 Spongiform encephalopathy detection methods Withdrawn EP0795133A1 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GB9424068 1994-11-29
GB9424068A GB9424068D0 (en) 1994-11-29 1994-11-29 Spongiform encephalopathy detection methods
GB9424768 1994-12-07
GBGB9424768.1A GB9424768D0 (en) 1994-11-29 1994-12-07 Spongiform encephalopathy detection methods
PCT/GB1995/002767 WO1996017250A1 (en) 1994-11-29 1995-11-28 Spongiform encephalopathy detection methods

Publications (1)

Publication Number Publication Date
EP0795133A1 true EP0795133A1 (en) 1997-09-17

Family

ID=26306060

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95937125A Withdrawn EP0795133A1 (en) 1994-11-29 1995-11-28 Spongiform encephalopathy detection methods

Country Status (11)

Country Link
EP (1) EP0795133A1 (en)
AU (1) AU3932995A (en)
CA (1) CA2205228A1 (en)
CZ (1) CZ164197A3 (en)
FI (1) FI972254A (en)
GB (1) GB2308658B (en)
HU (1) HUT77339A (en)
NO (1) NO972340L (en)
NZ (1) NZ295722A (en)
SK (1) SK67797A3 (en)
WO (1) WO1996017250A1 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2319607A (en) * 1996-11-23 1998-05-27 Electrophoretics International Detection of prion proteins in mononuclear cells
GB9701045D0 (en) * 1997-01-18 1997-03-05 Narang Harash K Diagnosis of neuro-degenerative disorders
CA2286279C (en) * 1998-02-06 2009-05-12 Harash Kumar Narang Diagnosis of neuro-degenerative disorders

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4892814A (en) * 1987-06-22 1990-01-09 The United States Of America As Represented By The Department Of Health And Human Services Method for distinguishing Creutzfeldt-Jakob disease from other dementias

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9617250A1 *

Also Published As

Publication number Publication date
NO972340D0 (en) 1997-05-22
FI972254A0 (en) 1997-05-28
CZ164197A3 (en) 1997-11-12
CA2205228A1 (en) 1996-06-06
GB2308658A (en) 1997-07-02
NO972340L (en) 1997-05-22
WO1996017250A1 (en) 1996-06-06
HUT77339A (en) 1998-03-30
AU3932995A (en) 1996-06-19
FI972254A (en) 1997-07-28
NZ295722A (en) 1999-03-29
SK67797A3 (en) 2000-02-14
GB2308658B (en) 1998-11-18
GB9708654D0 (en) 1997-06-18

Similar Documents

Publication Publication Date Title
CA2480466C (en) Detection and/or monitoring of synuclein-related diseases
US20230139188A1 (en) Markers for renal disease
EA004953B1 (en) Method for detecting and separating abnormal prion protein and kit therefor
Brechlin et al. Cerebrospinal fluid‐optimized two‐dimensional difference gel electrophoresis (2‐D DIGE) facilitates the differential diagnosis of Creutzfeldt–Jakob disease
Nonno et al. Molecular analysis of cases of Italian sheep scrapie and comparison with cases of bovine spongiform encephalopathy (BSE) and experimental BSE in sheep
BG65933B1 (en) Method for diagnosing a transmissible spongiform subacute encephalopathy caused by an unconventional transmissible agent strain in a biological sample
US7368247B2 (en) Diagnostic assay for transmissible spongiform encephalopathies
US4892814A (en) Method for distinguishing Creutzfeldt-Jakob disease from other dementias
Choe et al. Apolipoprotein E and other cerebrospinal fluid proteins differentiate ante mortem variant Creutzfeldt‐Jakob disease from ante mortem sporadic Creutzfeldt‐Jakob disease
WO1996017249A1 (en) Spongiform encephalopathy detection methods
EP0795133A1 (en) Spongiform encephalopathy detection methods
EP2313782B1 (en) Proteomic fingerprint for the diagnosis of non-alcoholic steatohepatitis (nash) and/or steatosis
US9927429B2 (en) Tropomyosin isoforms related to alzheimers disease and mild cognitive impairment
Narang et al. Sensitive detection of prion protein in human urine
WO1998026293A1 (en) Methods of detecting transmissible spongiform encephalopathies by detecting 14-3-3 protein isoforms
WO1998026293A9 (en) Methods of detecting transmissible spongiform encephalopathies by detecting 14-3-3 protein isoforms
US20040175775A1 (en) Method of detecting PrPsc in eye fluid
US20030044868A1 (en) Method for detecting prion proteins in tissue samples
Russell et al. Fibrinogen heterogeneity in horses
BG64291B1 (en) Method and kit for extracting prion protein
WO2010020037A1 (en) The identification of disease-induced biomarkers in the urine of bse infected cattle
JP2010523978A (en) Prion ELISA
ZA200105304B (en) Method and kit for extracting prion protein.

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19970506

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU NL PT SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20000531