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WO1997018476A1 - Methode pour diagnostiquer la maladie d'alzheimer - Google Patents

Methode pour diagnostiquer la maladie d'alzheimer Download PDF

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Publication number
WO1997018476A1
WO1997018476A1 PCT/AU1996/000711 AU9600711W WO9718476A1 WO 1997018476 A1 WO1997018476 A1 WO 1997018476A1 AU 9600711 W AU9600711 W AU 9600711W WO 9718476 A1 WO9718476 A1 WO 9718476A1
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WO
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Prior art keywords
disease
app
alzheimer
cells
precursor protein
Prior art date
Application number
PCT/AU1996/000711
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English (en)
Inventor
Ralph Nigel Walter Martins
Original Assignee
The University Of Western Australia
Ramsay Health Care Pty. Ltd.
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 The University Of Western Australia, Ramsay Health Care Pty. Ltd. filed Critical The University Of Western Australia
Priority to AU74860/96A priority Critical patent/AU7486096A/en
Publication of WO1997018476A1 publication Critical patent/WO1997018476A1/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
    • 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

  • the present invention relates to a method for diagnosing Alzheimer's disease in a patient by identifying the presence of and or the relative abundance of a form of secreted amyloid precursor protein which has an average molecular weight of greater than about 130 kilodaltons
  • AD Alzheimer's disease
  • cerebral degeneration which leads to dementia It is characterised by the deposition of amyloid in intracellular and extracellular compartments of the cerebral cortex
  • the disease can only be conclusively diagnosed at death following postmortem examination of the brain for the presence of amyloid deposits
  • AD is characterised by the deposition of amyloid in amyloid plaque cores and as congophilic angiopathy which are localised in the extracellular compartments of the brain and as neurofibrillary tangles within neurons
  • a ⁇ beta- amyloid
  • APP amyloid precursor protein
  • APP is a transmembrane protein which consists of a family of different protein isoforms that are generated by alternate splicing of the 19 exons of the APP gene
  • Three major forms of APP are APP695, APP751 and APP770 APP695 differs from the other two major forms in that it lacks exon 7 which codes for a 57-am ⁇ no acid domain with homology to Kunitz-type protease inhibitors
  • L-APP leucocyte derived APP
  • the major forms of secreted APP from stimulated T-lymphocytes contain high molecular weight isoforms (125-130 kilodaltons (kD)) derived from L-APP
  • kD kilodaltons
  • a high molecular weight form of secreted APP has been reported to be produced in both early-onset and late-onset familial AD (FAD) from lymphoblastoid cell lines (Matsumoto, A. and Fujiwana, Y. (1991 ) Biochem, Biophys, Res Commun, 175, 367-365)
  • FAD early-onset and late-onset familial AD
  • this higher molecular form of 120 kD has been found to be a complex containing a protease and a 65 kD fragment of APP which lacks the beta amyloidogenic sequence
  • AD amyloidogenic proteolytic product beta amyloid
  • AD Alzheimer's disease
  • the present invention provides a method for assaying for Alzheimer's disease in a patient said method comprising the steps of
  • the present invention relates to the assaying of secreted amyloid precursor proteins in fluid samples
  • the relative concentration of secreted amyloid precursor proteins in fluid is often very low
  • the specificity of the method may, however, be increased by selecting and culturing in vitro, a cellular source of tissue that is known to produce secreted amyloid precursor proteins
  • the culture supernatant can then be assayed for the presence of secreted amyloid precursor proteins with an average molecular weight of greater than about 130 kilodaltons.
  • the invention consists in a method for detecting Alzheimer's disease in a human said method comprising the steps of: (i) selecting a biological sample from a patient suspected of having alzheimer's disease, wherein said sample is isolated from a cell source which in alzheimer's disease patients is known to produce secreted amyloid precursor protein.;
  • step (ii) growing the biological sample selected in step (i) under suitable cell culture conditions for sufficient time to allow amyloid precursor protein expression and secretion into the culture supernatant;
  • step (iii) reacting, the culture supernatant, with a binding substance specific for either the ⁇ -amyloid peptide or the amyloid precursor protein or both; and (iv) assaying the proteins selected in step (iii) for the presence of a higher than normal quantity of protein with an average molecular weight of greater than about 130 kilodaltons.
  • the subject invention may be used to detect long forms of secreted APP (i.e. forms of secreted APP of greater than 130 kilodaltons in molecular weight) from a range of biological sources.
  • the secreted APPs Preferably, have an average molecular weight of approximately 130 to 140 kD. Increased production of secreted APP's of a molecular weight of 130 kD to 140 kD have not previously been identified in high abundance in AD patients. Detection and quantitation of such proteins provides a means for identifying and quantifying the onset of AD.
  • the method of the present invention should be carried out with parallel control experiments using fluid or tissue samples from known AD negative patients.
  • the identification of a difference between the control and sample sources is taken as being indicative of the onset of AD. If visual differences can not be readily identified between the control and sample sources the results of the method should be quantified.
  • Methods for quantifying the amounts of protein in an electrophoretic band are known in the art. Preferably, the results are quantified using densitometry.
  • Examples of fluid sources from in which secreted APPs are found in humans include blood plasma and cerebral spinal fluid.
  • Examples of cellular sources which are known to secrete APPs include, lymphoblastoid cells and skin fibroblasts.
  • the cellular source material is lymphoblastoid cells.
  • Binding substances suitable for use in the invention may be of any form provided that they are capable of selecting either the ⁇ -amyloid peptide or the amyloid precursor protein or both proteins.
  • the binding substances individually or in combination recognise part or all of the first 24 amino acids of the A ⁇ peptide or the C-terminus of APP.
  • the binding substance may be an antibody which recognises part or all of one or more of the following sequences: amino acids 17 through 24 of the A ⁇ peptide; amino acids 1 through 16 of the A ⁇ peptide; or the C-terminus of APP.
  • binding substances When the binding substances interact with the secreted APPs they form a complex and can be readily selected.
  • the selected secreted APPs can then be assayed for and examined by molecular weight analysis.
  • Methods of molecular weight analysis include, for example, gel exclusion chromatography, gel filtrations, ultra centrifugation, SDS PAGE electrophoresis analysis, western blots etc.
  • proteins obtained from either the fluid sample or the culture supernatant may be concentrated and/or precipitated before being incubated with the binding substance specific.
  • Methods of concentrating and precipitating proteins are well known in the art and include, for example, centrifugation, altering the conditions in the sample to precipitate the proteins and like methods.
  • lymphoblastoid cells are selected as the biological sample and are grown under suitable cell culture conditions for sufficient time to allow amyloid precursor protein expression and release into the culture supernatant.
  • the method of the invention may also be modified to accommodate a qualitative comparison of the difference between secreted APPs in fluid and APPs in the cellular tissue
  • the cellular tissue grown in cell culture is separated from the culture supernatant
  • the cells are grown in the presence of a biosynthetic label which labels produced proteins.
  • the cells are then disrupted using methods known in the art
  • the lysate and culture supernatant is then reacted separately with binding substances capable of selecting either part or all of the first 24 ammo acids of the A ⁇ peptide or the C-terminus of APP
  • the selected proteins samples are then analysed for APPs of 130 to 140 kD molecular weight
  • the method of the present invention may have general application for identifying various forms of AD where abnormalities in the cleavage of A ⁇ result in the production of a secreted APPs of 130 to 140 kD molecular weight
  • the present method provides a means for assaying for familial AD (FAD) caused by abnormal processing of APP resulting in a secreted form of APP with a molecular weight of 130 to 140 kD
  • the molecular weight of processed secreted APP in AD negative patients is typically in the order of about 100 to 110 kD molecular weight
  • the 130 to 140 kD high molecular weight form of secreted APP is typically observed in affected individuals with a missense mutation at codon 717 in the APP gene (i e. it results from a point mutation in exon 17 of APP which results in a substitution of isoleucine for a valine at residue 717 (APP770))
  • the same high molecular weight form of secreted APP is also observed in early onset FAD lymphoblastoid cell lines containing a defect in chromosome 14 Both of these defects have been linked to early onset FAD. While little is presently known about this high molecular weight form of APP it is expected that the protein may consist of either the entire or substantially all of the amylogenic precursor protein before it is proteolytically cleaved.
  • Mutations on the APP gene located on chromosome 21 account for only a small percentage of FAD families. It is now known that most forms of FAD result from genetic defects in other chromosomes in addition to chromosome 21. Recent research suggests that mutations on other chromosomes may have an impact on the processing of the APP gene which in turn may be responsible for the generation of abnormal APP products.
  • One such defect is on chromosome 14 where a mutation in the presenilin 1 (PS1 ) gene (formerly known as the S182 gene) may influence abnormal APP processing.
  • PS1 presenilin 1
  • Evidence has been accumulated to show that mutations in the PS1 gene are associated with increased levels of A ⁇ in blood, skin fibroblasts and the brains of FAD patients.
  • Binding substances suitable for use in the invention include antibodies, antibody fragments or antibody complexes which recognise at least an epitope within the A ⁇ peptide such as an epitope within part or all of the first 24 amino acids of the A ⁇ peptide and/or the C-terminus of the APP.
  • Antibodies suitable for use in the invention may be either monoclonal or polyclonal antibodies. In a particularly preferred form the invention the monoclonal antibodies are used in the method which are capable of detecting part or all of the first 16 amino acids of the A ⁇ peptide or part or all of amino acids 17 to 24 in the A ⁇ peptide.
  • the binding substances used in the method of the invention may be employed in a variety of ways to identify the presence of secreted APPs of a molecular weight 130 to 140 kD.
  • the binding substances may be used to selectively extract secreted APPs for a fluid sample.
  • they may be used to selectively extract APP from the total protein content of, for example, disrupted cells.
  • the selected proteins are preferably dissociated from the binding substance and then analysed on a gel. Detection of the protein may then be achieved by detection means known in the art such as, by using radioactive or fluorescent labelled probes (eg labelled antibodies) or by silver staining techniques
  • Methodologies that may be used to identify the long forms of secreted APP may include, for example, enzyme linked immunoabsorbant assays (ELISA), radioimmunoassays (RIA), or immunoblot assays
  • ELISA enzyme linked immunoabsorbant assays
  • RIA radioimmunoassays
  • immunoblot assays the proteins in the biological sample are first separated according to their molecular weight differences
  • the ba ⁇ d(s) corresponding to greater than 130 kD is then identified
  • the presence of secreted APPs with a molecular weight greater than 130 kD may be identified using such substances as labelled binding substances (e.g using labelled antibodies that are specific for the binding substances), or by other method known in the art which facilitates such detection
  • Labels which may be coupled to the binding substances are those known in the art and include but are not limited to enzymes, radionucleotides, fluourogenic and chromogenic substrates, cofactors and biotin-avidin, coll
  • Techniques especially useful for large scale clinical screening of suspected AD patients include ELISA and radioimmunoassays Such techniques are preferred for their speed, and their ability to test numerous samples simultaneously and ease of automation.
  • Protocols upon which ELISA assays may be based include, for example, competition assays, direct reaction assays and sandwich type assays
  • binding substances or antibodies directed against APP's generally are preferable coated into wells in, for example, a microtitre tray where an immunological complex forms if abnormal APP's or APP's generally are present in the sample
  • a signal generating means may be added to detect complex formation
  • a detectable signal is produced if abnormal APP's are present in the sample
  • lymphoblastoid cells are selected as the biological sample.
  • the selection of lymphoblastoid cells from human patients may be achieved by suitable methods known in the art
  • the cell are then preferably immortalised and grown in the presence of a biosynthetic label which is capable of labelling proteins
  • One method that is particularly preferred for identifying longer forms of secreted APP's involves growing familial AD, sample and control lymphoblastoid cells in the presence of labelled ammo ac ⁇ d(s) under suitable conditions to allow the label to be incorporated into proteins that are synthesised by the cells At a suitable time after incubation the culture media is then separated from the cells and the cells are washed to remove excess label The cells are then solubilised to release all of their cytoplasmic and membrane proteins APP's of a molecular weight of 130 to 140 kD may then be precipitated from the cell lysate and the culture medium using the binding substances described above. Detection of any of the APP's may be achieved using electrophoretic gel or by employing standard detection means known in the art
  • Immortalisation may be achieved by fusing the lymphoblastoid cells to a variety of stabilised cell lines to maintain stability
  • the lymphoblastoid cells may be infected with transforming viruses (e.g. Sendai virus or Epstein Barr virus), or transfected with transforming genes to create permanent or semi ⁇ permanent cell lines
  • the lymphoblastoid cells may be fused to established cell lines
  • Methods of cell fusion are known in the art and may include, for example, the use of fusion agents such as polyethylene glycol
  • the cells are immortalised using Epstein Barr virus.
  • Conditions suitable for culturing, for example, lymphoblastoid cells are generally known in the art
  • the procedure involves incubating cells in, for example, RPMI 1640 medium containing 5 to 30% foetal calf serum at about 30 to 40°C in the presence of 1 to 10% C0 2 .
  • the cells are incubated in RPMI 1640 medium containing 20% foetal calf serum at 37°C in the presence of 5% C0 2
  • the time period for incubation of the biological sample will vary depending on the environmental conditions in which the cell sample is grown.
  • the cells should, however, be grown for a suitable time period to allow APP production and secretion into the cell culture supernatant
  • the cells are incubated under such conditions in the presence of a radioactive ammo acid for at least 10 hours, preferably for 16 to 72 hours and most preferably 24 to 48 hours to permit optimal APP detection.
  • Labels that may be used include: fluorescent, chemiluminescent, radioactive or dye molecules.
  • the lymphoblastoid cells are incubated in the presence of at least a radioactively labelled ammo acid(s) for at least 1 hr More preferably they are incubated in the presence of the labelled am o ac ⁇ d(s) for 1 to 48 hrs. Between 4 to 24 hrs and more specifically 5 to 16 hrs are the most desirable time periods for incubating with the label.
  • the method of the present invention may also be made available in the form of a kit.
  • the kit comprises a binding substance specific for either the ⁇ -amyloid peptide or the amyloid precursor protein or both and instructions consistent with the method of the present invention.
  • the kit may also comprise one or more of suitable media for growing a biological sample, solutions for disrupting a cellular sample, binding substances specific for part or all of the first the first 24 am o acid residues of ⁇ -amyloid peptide and or a binding substance specific for the C-terminus of APP, one or more solutions or media suitable for detecting high molecular weight (ie 130 to 140 kD) APP.
  • Figure 1 depicts a family tree of an Australian family with a mutation in the APP gene at codon 717 where the am o acid valine is substituted with isoleucine. Those family members whose lymphocytes were immortalised by infection with Epstein-Barr virus in accordance with the following methods and used for the study of APP metabolism are indicated by an associated asterisk.
  • Figure 2 is a photographic representation of an electrophoretic gel visualisation by phosphonmaging of APPs after biosynthetic labelling of the lymphoblastoid cell lines with 35 S methionine followed by a 2 hour chase and immunoprecipitation of the cell lysates with an antibody specific for the C-terminus of APP.
  • Lane 1 represents APP analysis of the affected individual Lanes 3 and 4 represent control cell lines.
  • Lane 5 represents APP analysis of the affected individual's "at risk” son
  • Lane 6 represents APP analysis of the affected individual's "at risk” brother.
  • Lane 7 represents a third control cell line.
  • Figure 3 is a photographic representation of an electrophoretic gel showing a low molecular weight form of 100 kD in all the control cell lines after 5 hours of biosynthetic labelling (Fig. 3A, lanes 5-10) and the I SO- O kD secreted APP in the affected patient with the APP717 mutation (Fig. 3A, lanes 3,4).
  • the antibody used in this study was specific for the first 16 residues of A ⁇
  • the "at risk' individual (Fig 3A, lanes 1 ,2) and an FAD individual with a defect on chromosome 14 (Fig 3B, lanes 1 , 2) also exhibited the longer molecular form of secreted APP
  • Figure 4 is a photographic representation of an electrophoretic gel showing that labelling with 35 S methionine for a total of 14 hours results in an even more marked increase in the 130-140 kD secreted APP for the affected patient with the APP717 mutation (F ⁇ g.4A & Fig 4B, lane 1 ) as well as the "at risk” individuals carrying the APP717 mutation ( Fig. 4A & Fig. 4B, lanes 4,5).
  • Reagents - Monoclonal antibodies 6E10 and 4G8 were purchased from Drs. Kim and Wisniewski (Institute for Basic Research, Staten Island). Their preparation is described in Kim, K.S., Wen, G.Y., Bancher, C, Chen, C.J.M., Sapienza, V.J., Hong, H. and Wisniewski, H.M. (1990) NeuroSci. Res. Commun. 2, 121 -130.
  • Affinity-purified rabbit-C-terminal APP antibody termed 369 was produced according to the method in Buxbaum, J.D., Gandy, S.E., Cicchetti, P., Ehrlick, M.E., Czemik, A.J., Fracasso, R.P., Ramabhadran, T.V., Unterbeck, A.J. & Greengard, P. (1990) Proc. Natl. Acad.Sci. USA 87, 6003-6006.
  • Gamma Bind Plus Sepharose and Protein A-Sepharose CL-4B was obtained from Pharmacia LKB. 10-20% Tris/ tricine gradient gels were purchased from Novex (San Diego, CA). [ 35 S] methionine was purchased from Australian Biosearch (Perth, WA).
  • Lymphoblastoid cell Iines were established, from a recently identified early onset Australian FAD pedigree with a mutation on codon 717 (Brooks, W.S., Martins, R.N., De Voecht, J., Nicholson, G.A., Schofield, P.R., Kwok, J.B., Fisher, C, Yeung, LU. and Van Broeckhoven C. (1995) Neurosci. Lett. 199, 1 - 4.).
  • Cell Iines were immortalised by infecting with Epstein Barr virus employing standard procedures (Ventura, M., Gibaud, A., Le Pendu, J., Hillaire, D., Gerard, G., Vitrac, D. and Orbiol, R.
  • Biosynthetic Labelling - Cells were pelleted by centrifugation for 5 minutes at 200 x g. The media was aspirated and the cells resuspended in Hanks Balanced Salts (HBS) solution and centrifuged again as described above. The cells were resuspended again in HBS and centrifuged once more for 5 minutes at 200 x g. HBS was replaced with methionine free medium and the cells incubated for 45 minutes at 37°C. The cells were then incubated with 250 ⁇ Ci (9.75 MBq) [ 35 S] methionine in 1 ml of methionine-free DMEM. Metabolic labelling was carried out for either 40 minutes, 5 hours or 16 hours at 37°C.
  • HBS Hanks Balanced Salts
  • the 40 minute pulse was followed by a chase period of 2 hours in media containing 200 mM unlabelled methionine. After the 2 hour chase, the cells were centrifuged as described above and washed twice with HBS. After the second wash, the cell pellet was solubilised in lysis buffer (50 mM TrisCl pH7.4 containing 150 mM NaCl, 0.5% NP40, 0.5% Sodium deoxycholate, 0.25% SDS, 5 mM EDTA, 50 mg/ml leupeptin, 0.25 mM PMSF and 10 mg/ml aprotinin). The solubilised lysate was centrifuged at 10,000 x g for 5 minutes. The supernatant's were removed and normalised for total protein by diluting the high protein samples in lysis buffer.
  • lysis buffer 50 mM TrisCl pH7.4 containing 150 mM NaCl, 0.5% NP40, 0.5% Sodium deoxycholate, 0.25% SDS, 5
  • Immunoprecipitation The supernatant's were incubated at 4°C with monoclonal antibodies 6E10/4G8 or other antibodies directed at similar epitopes of the A ⁇ molecule overnight for media or with the polyclonal antibody 369 or similar antibodies directed against the C-terminus of APP for lysates. Immune complexes were immunoprecipitated with Gamma Bind Plus Sepharose or with Protein A Sepharose for the monoclonal and polyclonal antibodies respectively. The immune complexes were washed with 1 ml of 0.5M NaCl in Tris-buffered saline (100 mM Tris-HCl, 150 mM NaCl, pH 7.4) for 1 hour at 4°C.
  • pellets were resuspended in 0.1 ml of wash buffer and the suspension was applied to a 1 ml sucrose cushion and centrifuged at 10,000 x g for ten minutes. Samples were boiled in 20 ml of Tris/tricine sample buffer and separated on 10-20% Tris/Tricine gradient polyacrylamide gels. Gels were treated for fluorography with enhancer solution (Entensify; New England Nuclear-Dupont, Boston MA), dried, and quantitated with a Phosphorimager (Fuji).
  • enhancer solution Entensify; New England Nuclear-Dupont, Boston MA
  • Protein Quantitation The concentration of total protein in each lysate sample was estimated using a protein assay, as per the manufacturers' instructions (BSA Protein Assay Kit, Pierce (Rockford, IL, USA).
  • BSA Protein Assay Kit Pierce (Rockford, IL, USA).
  • Figure 1 A family tree depicting members of an Australian family with a mutation in the APP gene at codon 717 where the amino acid valine is substituted with isoleucine is shown in Figure 1. Those family members whose lymphocytes were immortalised by infection with Epstein-Barr virus and used for the study of APP metabolism are indicated by an associated asterisk (Fig. 1).
  • Biosynthetic labelling of the lymphoblastoid cell Iines with 35 S methionine followed by a 2 hour chase and immunoprecipitation of the cell lysates with antibody #369 was carried out followed by visualisation by phosphorimaging of APPs (Fig 2).
  • the three control cell Iines in lanes 3,4 and 7 exhibited the classical profile, as seen for other cell types, of an immature 100 kD form and a mature 110 kD form of APP.
  • the affected individual in lane 1 (Fig.2) exhibited a number of APP isoforms in addition to the immature and mature forms observed in the controls.
  • a prominent 130 to 140 kD protein was observed in the affected patient's lymphoblastoid cell line (lanel ) and was also observed in the affected's "at risk” son (lane 5; Fig.2) and "at risk” brother (lane 6; Fig.2).
  • the "at risk” son had abundant levels of a protein with a molecular weight between 100 kD and 110 kD (lane 6; Fig. 2).
  • the APP lysate profiles for the controls differ significantly from those carrying the APP mutation at codon 717 confirming that APP processing is impaired in the latter group.
  • the present method employs a specific APP antibody, that recognises the first 16 amino acid residues of A ⁇ , to establish that early onset FAD lymphoblastoid cells secrete abundant levels of a 130 -140 kD form of APP.
  • APP antibody that recognises the first 16 amino acid residues of A ⁇

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Abstract

L'invention concerne une méthode permettant de détecter la maladie d'Alzheimer chez un patient, comprenant les étapes suivantes: (i) on prélève chez un patient, chez qui l'on soupçonne une maladie d'Alzheimer, un échantillon de fluide contenant une protéine précurseur amyloïde; (ii) on met à réagir les protéines de l'échantillon de fluide avec une substance de liaison spécifique soit du peptide β-amyloïde, soit de la protéine précurseur amyloïde, soit des deux; (iii) on recherche la présence de la protéine précurseur amyloïde ayant un poids moléculaire moyen supérieur à 130 kilodaltons environ, et on détermine si elle est en quantité supérieure à la normale par rapport à des patients qui ne sont pas atteints de maladie d'Alzheimer.
PCT/AU1996/000711 1995-11-10 1996-11-08 Methode pour diagnostiquer la maladie d'alzheimer WO1997018476A1 (fr)

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AU74860/96A AU7486096A (en) 1995-11-10 1996-11-08 A method for diagnosing alzheimer's disease

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AUPN6493A AUPN649395A0 (en) 1995-11-10 1995-11-10 A method for diagnosing alzheimer's disease
AUPN6493 1995-11-10

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WO2000051632A3 (fr) * 1999-03-03 2001-10-04 Konrad Beyreuther Agoniste du cuivre qui se lie au site de fixation de cuivre de l'app et/ou exerce une action d'inhibition sur la liberation du peptide beta-amyloide
WO2004048979A1 (fr) * 2002-11-27 2004-06-10 Evotec Neurosciences Gmbh Utilisation diagnostique et therapeutique de la proteine h-rev107 pour la maladie d'alzheimer
US8877190B2 (en) 2006-11-30 2014-11-04 Abbvie Inc. Aβ conformer selective anti-Aβ globulomer monoclonal antibodies
US8895004B2 (en) 2007-02-27 2014-11-25 AbbVie Deutschland GmbH & Co. KG Method for the treatment of amyloidoses
US8987419B2 (en) 2010-04-15 2015-03-24 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins
US9062101B2 (en) 2010-08-14 2015-06-23 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins
US9146244B2 (en) 2007-06-12 2015-09-29 Ac Immune S.A. Polynucleotides encoding an anti-beta-amyloid monoclonal antibody
US9175094B2 (en) 2007-06-12 2015-11-03 Ac Immune S.A. Monoclonal antibody
US9176150B2 (en) 2003-01-31 2015-11-03 AbbVie Deutschland GmbH & Co. KG Amyloid beta(1-42) oligomers, derivatives thereof and antibodies thereto, methods of preparation thereof and use thereof
US9221900B2 (en) 2010-07-30 2015-12-29 Ac Immune S.A. Methods for identifying safe and functional humanized antibodies
US9403902B2 (en) 2007-10-05 2016-08-02 Ac Immune S.A. Methods of treating ocular disease associated with amyloid-beta-related pathology using an anti-amyloid-beta antibody
US9540432B2 (en) 2005-11-30 2017-01-10 AbbVie Deutschland GmbH & Co. KG Anti-Aβ globulomer 7C6 antibodies
US10208109B2 (en) 2005-11-30 2019-02-19 Abbvie Inc. Monoclonal antibodies against amyloid beta protein and uses thereof

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WO1990015331A1 (fr) * 1989-06-01 1990-12-13 E.I. Du Pont De Nemours And Company Dosage diagnostique pour depister la maladie d'alzheimer
WO1992013069A1 (fr) * 1991-01-21 1992-08-06 Imperial College Of Science, Technology & Medicine Test et modele pour la maladie d'alzheimer
WO1993007296A1 (fr) * 1991-10-03 1993-04-15 Indiana University Foundation Procede pour depister la maladie d'alzheimer
WO1993010459A1 (fr) * 1991-11-12 1993-05-27 The University Of Melbourne Procede de diagnostic et de traitement de la maladie d'alzheimer
WO1994005811A1 (fr) * 1992-08-31 1994-03-17 Bristol-Myers Squibb Company CLONAGE ET EXPRESSION DU RECEPTEUR β-APP-C100

Patent Citations (5)

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