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 for the detection of bovine spongiform encephalopathy (BSE) in cattle.
• BSE bovine spongiform encephalopathy
• Spongiform encephalopathies are a group of diseases which include scrapie in sheep and Creutzfeldt-Jakob disease (CJD) 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 encephalopathies in animals which addresses some, and in preferred forms all, of these problems.
• a method for detecting the presence of spongiform encephalopathy in an animal comprising determining the presence and/or amount of agent in a body fluid of the animal which cross-reacts 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.
• Apolipoprotein E is a 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.
• CSF cerebrospinal fluid
• agent or agents are cross reactive with anti-apolipoprotein E, have a molecular weight of around 34-38 kDa, and have a pi of around 5-4 - 5-7- This is consistent with their identification as apolipoprotein E, and the term 'Apo E agent' as used hereinafter is intended to embrace any agent which has these properties (including apolipoprotein E itself and isoforms or multiple-forms thereof).
• the control value may be derived from the Apo E 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.
• the control value may be determined using the same method used for the test animal, or may be derived by a different analytical method.
• the results from the 'control' animal may be used to derive a standard positive- or negative-control value, or to calibrate the test animal result.
• the body fluid analysed in the method is CSF since authentic apolipoprotein E is the major apolipoprotein found in this fluid.
• 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 analysing the concentration of Apo E 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 Apo E agent in a body fluid of the animal is derived by the use of PAGE or 2DPAGE to separate out the Apo E agent from other agents in the body fluid, and then staining the gel and making densitometry measurements in the region of the gel of interest in order to determine the presence and/or amount of the Apo E agent.
• the identity of the Apo E agent is confirmed by use of immunogenic material, for instance antibody raised against Apo E agent or a synthetic peptide based on a sequence thereof.
• immunogenic material for instance antibody raised against Apo E agent or a synthetic peptide based on a sequence thereof.
• Suitable immunogen-based techniques for identifying the presence of cross-reactive agents are well known to those skilled in the art eg. ELISA or Western Blotting.
• these immunogenic techniques may be used both to identify the Apo E agent, and also to estimate its concentration.
• 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 Apo E 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. Staining and Image analysis: The 2D gels were silver stained according to the Millipore manual. Gels were scanned with an O ⁇ -nimedia scanner XRS and analysed using Bioimage software and Investigator Database programme (Millipore) using a sunSPARC station computer.
• 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 34-38 kDa, and a pi of around 5.4 - 5-7 (labelled 'Apo E' ) is higher in the BSE-positive sample.

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Immunology (AREA)
• Biomedical Technology (AREA)
• Urology & Nephrology (AREA)
• Hematology (AREA)
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• Molecular Biology (AREA)
• Physics & Mathematics (AREA)
• Biochemistry (AREA)
• Cell Biology (AREA)
• Virology (AREA)
• Biotechnology (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Pathology (AREA)
• Analytical Chemistry (AREA)
• Microbiology (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Tropical Medicine & Parasitology (AREA)
• Neurology (AREA)
• Neurosurgery (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Peptides Or Proteins (AREA)

Priority Applications (8)

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Publications (1)

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Cited By (5)

Citations (1)

• 1995
  • 1995-11-28 GB GB9708663A patent/GB2308659B/en not_active Expired - Fee Related
  • 1995-11-28 EP EP95937124A patent/EP0795132A1/en not_active Withdrawn
  • 1995-11-28 AU AU39328/95A patent/AU3932895A/en not_active Abandoned
  • 1995-11-28 SK SK678-97A patent/SK67897A3/sk unknown
  • 1995-11-28 WO PCT/GB1995/002766 patent/WO1996017249A1/en not_active Application Discontinuation
  • 1995-11-28 CA CA002205179A patent/CA2205179A1/en not_active Abandoned
  • 1995-11-28 HU HU9702327A patent/HUT77340A/hu unknown
  • 1995-11-28 NZ NZ295721A patent/NZ295721A/xx unknown
  • 1995-11-28 CZ CZ971642A patent/CZ164297A3/cs unknown
• 1997
  • 1997-05-22 NO NO972339A patent/NO972339L/no unknown
  • 1997-05-28 FI FI972253A patent/FI972253A/fi unknown

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Non-Patent Citations (4)

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