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WO1991015773A1 - Procede de test diagnostique - Google Patents

Procede de test diagnostique Download PDF

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Publication number
WO1991015773A1
WO1991015773A1 PCT/GB1991/000545 GB9100545W WO9115773A1 WO 1991015773 A1 WO1991015773 A1 WO 1991015773A1 GB 9100545 W GB9100545 W GB 9100545W WO 9115773 A1 WO9115773 A1 WO 9115773A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
sample
region
disease
isoelectric point
Prior art date
Application number
PCT/GB1991/000545
Other languages
English (en)
Inventor
Iradj Hajimohammadreza
Michael Brammer
Original Assignee
Oxford Virology Plc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oxford Virology Plc filed Critical Oxford Virology Plc
Publication of WO1991015773A1 publication Critical patent/WO1991015773A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • 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
    • G01N2800/2821—Alzheimer

Definitions

  • This invention relates to a method of diagnosis for Alzheimer's Disease.
  • Alzheimer's Disease is a degenerative brain disorder producing a range of symptoms including dementia. Elevated aluminium levels, virus-like agents and pre ⁇ disposing genetic factors have all been put forward as casual agents. However, the absence of a specific diagnostic test for Alzheimer's Disease has greatly hampered further research and the development of therapeutic agents for the treatment of afflicted individuals.
  • This invention exploits the finding that certain polypeptides normally found in blood platelets are present in significantly lowered amounts or are absent from the platelets of Alzheimer's Disease sufferers.
  • the invention provides a method of diagnosing Alzheimer's Disease comprising detecting the presence of one or more polypeptides in a solubilised blood platelet polypeptide sample taken from a suspected Alzheimer's Disease sufferer, and if such a polypeptide or polypeptides are present, comparing the amount thereof with the amount present in the platelets of disease-free individuals.
  • the invention provides a method of diagnosing Alzheimer's Disease comprising separating the components of a solubilised blood platelet polypeptide sample taken from a suspected Alzheimer's Disease sufferer, detecting the presence of the separated polypeptides and comparing the amount thereof with the amount of the corresponding polypeptide observed in a corresponding sample taken from a disease-free individual.
  • separation techniques can be employed to separate particular polypeptides on the basis of such characteristics as size, solubility, charge and specific binding affinity,n using methods such as dialysis through a semi-permeable membrane; gel- filtration chromatography; ion-exchange chromatography and affinity chromatography (see Stryer L. Biochemistry 2nd Edition, [1981] Pg. 18 to 20, W.H. Freeman and Co. ) .
  • the separation of the components may, for example, be • carried out by chromatography or by gel electrophoresis such as polyacrylamide gel electrophoresis (PAGE) .
  • PAGE polyacrylamide gel electrophoresis
  • two dimensional PAGE is used to separate the polypeptide components in the samples.
  • Detection techniques such as immunoassay and colourimetry may be employed in the above methods to determine the presence and/or amount of particular polypeptides in a test sample.
  • the invention provides a diagnostic test method for Alzheimer's disease comprising detecting or determining the level of one or more particular polypeptides in a solubilised blood platelet polypeptide sample taken from a test subject, wherein the or each particular polypeptide is one which, if the components of the said sample are subjected to two-dimensional polyacrylamide gel electrophoresis are present in the 110 kDa Mwt region at an isoelectric point of 5.4 and/or the 50 kDa Mwt region at an isoelectric point of 5.5 and/or the 40 kDa Mwt region at an isoelectric point in the range of from 5.6 to 6.2.
  • the invention further provides a method of diagnosing Alzheimer's Disease comprising separating the components of a solubilised blood platelet polypeptide sample taken from a suspected Alzheimer's Disease sufferer by two dimensional PAGE, detecting the presence of the separated polypeptides by staining the gel, and comparing the degree of band staining, in one or more regions selected from the 100 Mwt region at an isoelectric point of approximately 5.4, the 50 kDa Mwt region at an isoelectric point of approximately 5.5, and the 40 kDa Mwt region at an isoelectric point in the range of from 5.6 to 6.2, with the degree of staining in the corresponding region or regions observed in a sample taken from a disease-free individual.
  • the gels are run in pairs, that is one sample from a suspected sufferer with one from a disease-free individual to account for any variability in running conditions.
  • the samples taken from suspected sufferers and disease-free individuals are matched as closely as possible for age and sex so that samples are more strictly comparable.
  • the presence of the separated polypeptide bands on the gel may be detected using a polypeptide stain such as "Coomassie blue", or by using immunoassay techniques.
  • the or each particular polypeptide is extracted from the sample or gel using conventional techniques and is used tc immunise a mammal such as a rabbit in order to raise antisera specific for said polypeptide.
  • antisera can be used as an immunoassay reagent in further methods of the invention.
  • the invention provides a method for separating from a solubilised blood platelet sample a polypeptide which, if the components of the sample are subjected to two-dimensional polyacrylamide gel electrophoresis, is present in the 110 kDa Mwt
  • SUBSTITUTE SHEET region at an isoelectric point of 5.4 and/or the 50 kDa Mwt region at an isoelectric point of 5.5 and/or the 40 kDa Mwt region at an isoelectric point in the range of from 5.6 to 6.2 which method comprises: (i) applying said sample to an affinity chromatography column including antibodies specific for said polypeptide, which antibodies are bound to a solid support; (ii) washing the support to remove unbound components of the sample to which the antibodies do not bind specifically; (iii) eluting said polypeptide from the support so that it is obtained in substantially purified form.
  • the solid support to which said antibodies bind comprises plastic beads.
  • the invention also provides said polypeptide in an isolated and substantially purified form, which polypeptide is present in a reduced concentration in a solubilised blood platelet sample from a sufferer from Alzheimer's disease and which, if the components of such a sample are subjected to two-dimensional polyacrylamide gel electrophoresis are present in the 110 kDa Mwt region at an isoelectric point of 5.4 and/or the 50 kDa Mwt region at an isoelectric point
  • the invention provides an immunoassay method for determining the amount of a particular polypeptide as described previously which method comprises (i) coating a solid support surface with antibodies specific for the particular polypeptide; (ii) washing the support to remove unbound antibodies; (iii) applying a solubilised blood platelet polypeptide sample from a test sample; (iv) washing the support to remove unbound components of the sample; (v) applying labelled antibodies specific for the particular polypeptide; (vi) washing the support to remove any unbound labelled antibodies; (vii) determining the amount of labelled antibody present, which amount is proportional to the amount of the particular polypeptide present in the sample.
  • the labelled antibodies are labelled with enzymes (such as peroxidase and phosphatase) which are labelled with enzymes (such as peroxidase and phosphatase) which are labelled with enzymes (such as peroxidase and phosphatase) which are labelled with enzymes (such as peroxidase and phosphatase) which are labelled with enzymes (such as peroxidase and phosphatase) which
  • SUBSTITUTESHEET leave a coloured deposit on reaction with substrate.
  • the coloured deposit being measurable using colourmetric techniques.
  • the invention provides an immunoassay kit for diagnosing Alzheimer's Disease in a solubilised blood platelet polypeptide sample from a test subject comprising
  • the above kit further comprises a multiwell plastic plate for using the kit according to the methods of the invention.
  • the sample from the test subject is assayed alongside the comparative sample from the disease-free individual so
  • examples of the label of the labelled antibodies include an enzyme as mentioned previously, a fluorescent dye which can be seen using ⁇ V microscopy, or a radioactive substance which can be detected by deposition of silver grains from a photographic emulsion or by using counting equipment.
  • Fig. 1 shows a diagnostic test according to the inven ion.
  • AD Alzheimer's Disease
  • CON 5 disease-free control
  • Coomassie blue staining was used to detect the presence of the separated polypeptide bands.
  • differential staining of certain blood platelet polypeptide bands between Alzheimer's Disease patients and controls provides a specific marker or markers for the diagnosis of the disease in suspected sufferers.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Neurology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Neurosurgery (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Procédé servant à diagnostiquer la maladie d'Alzheimer et consistant à détecter la quantité d'un ou plusieurs polypeptides se trouvant dans un échantillon dissous d'un polypeptide de plaquette sanguine prélevé sur un malade présumé souffrant de la maladie d'Alzheimer, et, si ledit polypeptide est présent, à en comparer la quantité avec la quantité de polypeptide(s) présente dans un échantillon correspondant prélevé sur des personnes saines. La présence d'une quantité sensiblement réduite de ce(s) polypeptide(s) dans l'échantillon, par rapport à la quantité se trouvant dans les échantillons prélevés sur les personnes saines, indique que la personne est atteinte de la maladie d'Alzheimer.
PCT/GB1991/000545 1990-04-07 1991-04-08 Procede de test diagnostique WO1991015773A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB909007922A GB9007922D0 (en) 1990-04-07 1990-04-07 A diagnostic test method
GB9007922.9 1990-04-07

Publications (1)

Publication Number Publication Date
WO1991015773A1 true WO1991015773A1 (fr) 1991-10-17

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PCT/GB1991/000545 WO1991015773A1 (fr) 1990-04-07 1991-04-08 Procede de test diagnostique

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AU (1) AU7677991A (fr)
GB (1) GB9007922D0 (fr)
WO (1) WO1991015773A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993025911A1 (fr) * 1992-06-17 1993-12-23 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Diagnostic de la maladie d'alzheimer et de la schizophrenie
WO1997013152A1 (fr) * 1995-09-29 1997-04-10 Yeda Research And Development Co., Ltd. Dosage permettant de diagnostiquer la demence
US5714471A (en) * 1995-01-06 1998-02-03 Sibia Neurosciences, Inc. Peptide and peptide analog protease inhibitors
US5863902A (en) * 1995-01-06 1999-01-26 Sibia Neurosciences, Inc. Methods of treating neurodegenerative disorders using protease inhibitors
US7166202B2 (en) * 2001-07-16 2007-01-23 Protein Forest, Inc. Matrixes, arrays, systems and methods

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5272055A (en) * 1991-12-24 1993-12-21 The University Of Kentucky Research Foundation Detection of Alzheimer's disease and other diseases using a photoaffinity labeling method

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4666829A (en) * 1985-05-15 1987-05-19 University Of California Polypeptide marker for Alzheimer's disease and its use for diagnosis
WO1989007657A1 (fr) * 1988-02-10 1989-08-24 The Children's Medical Center Corporation Precurseurs de proteines amyloides, sondes genetiques, anticorps, et modes d'utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4666829A (en) * 1985-05-15 1987-05-19 University Of California Polypeptide marker for Alzheimer's disease and its use for diagnosis
WO1989007657A1 (fr) * 1988-02-10 1989-08-24 The Children's Medical Center Corporation Precurseurs de proteines amyloides, sondes genetiques, anticorps, et modes d'utilisation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Biotechnology, volume 7, February 1989, C.R. Abraham et al.: "Alzheimer's disease: Recent advances in understanding the brain amyloid deposits", pages 147-152 *
Science, volume 248, no. 4956, May 1990, W.E. Van Nostrand et al.: "Protease nexin-II (amyloidb-protein precursor): A platelet x-granule protein", pages 745-748 *
Science, volume 248, no. 4959, 1 June 1990, R.P. Smith et al.: "Platelet coagulation factor XIa-inhibitor, a form of Alzheimer myloid precursor protein" *
The Journal of Biological Chemistry, volume 265, no. 26, 15 September 1990, The American Society for Biochemistry and Molecular Biologoy, Inc., (US), A.I. Bush et al.: "The amyloid precursor protein of Alzheimer's disease is released by human platelets", pages 15977-15983 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993025911A1 (fr) * 1992-06-17 1993-12-23 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Diagnostic de la maladie d'alzheimer et de la schizophrenie
US5962419A (en) * 1995-01-06 1999-10-05 Sibia Neurosciences, Inc. Peptide and peptide analog protease inhibitors
US5714471A (en) * 1995-01-06 1998-02-03 Sibia Neurosciences, Inc. Peptide and peptide analog protease inhibitors
US5804560A (en) * 1995-01-06 1998-09-08 Sibia Neurosciences, Inc. Peptide and peptide analog protease inhibitors
US5863902A (en) * 1995-01-06 1999-01-26 Sibia Neurosciences, Inc. Methods of treating neurodegenerative disorders using protease inhibitors
US5872101A (en) * 1995-01-06 1999-02-16 Sibia Neurosciences, Inc. Methods of treating neurodegenerative disorders using protease inhibitors
US5969100A (en) * 1995-01-06 1999-10-19 Sibia Neurosciences, Inc. Peptide, peptide analog and amino acid analog protease inhibitors
US6015879A (en) * 1995-01-06 2000-01-18 Sibia Neurosciences, Inc. Peptide and peptide analog protease inhibitors
US6017887A (en) * 1995-01-06 2000-01-25 Sibia Neurosciences, Inc. Peptide, peptide analog and amino acid analog protease inhibitors
US6051684A (en) * 1995-01-06 2000-04-18 Sibia Neurosciences Inc. Methods of treating neurodegenerative disorders using protease inhibitors
US6153171A (en) * 1995-01-06 2000-11-28 Sibia Neurosciences, Inc. Methods for identifying compounds effective for treating neurodegenerative disorders and for monitoring the therapeutic intervention therefor
WO1997013152A1 (fr) * 1995-09-29 1997-04-10 Yeda Research And Development Co., Ltd. Dosage permettant de diagnostiquer la demence
US7166202B2 (en) * 2001-07-16 2007-01-23 Protein Forest, Inc. Matrixes, arrays, systems and methods
US7914656B2 (en) 2001-07-16 2011-03-29 Protein Forest, Inc. Matrixes, arrays, systems and methods

Also Published As

Publication number Publication date
GB9007922D0 (en) 1990-06-06
AU7677991A (en) 1991-10-30

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