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WO1995005604A2 - Procedes de diagnostic de la maladie d'alzheimer - Google Patents

Procedes de diagnostic de la maladie d'alzheimer Download PDF

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Publication number
WO1995005604A2
WO1995005604A2 PCT/US1994/008903 US9408903W WO9505604A2 WO 1995005604 A2 WO1995005604 A2 WO 1995005604A2 US 9408903 W US9408903 W US 9408903W WO 9505604 A2 WO9505604 A2 WO 9505604A2
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Prior art keywords
disease
proteins
alzheimer
protein
concentration
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PCT/US1994/008903
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English (en)
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WO1995005604A3 (fr
Inventor
Ginger Johnson
Hossein A. Ghanbari
Benjamin Wolozin
Carl R. Merril
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Molecular Geriatrics Corporation
The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services
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Application filed by Molecular Geriatrics Corporation, The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services filed Critical Molecular Geriatrics Corporation
Priority to AU75569/94A priority Critical patent/AU7556994A/en
Publication of WO1995005604A2 publication Critical patent/WO1995005604A2/fr
Publication of WO1995005604A3 publication Critical patent/WO1995005604A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to methods for the diagnosis of Alzheimer's Disease and determination of effectiveness of treatment of Alzheimer's Disease.
  • the methods involve measuring certain proteins in a body fluid or tissue sample which are found to be increased or decreased in concentration in patients with Alzheimer's Disease.
  • the invention also relates to these certain proteins and antibodies to these proteins, and to diagnostic test kits.
  • AD Alzheimer's Disease
  • Disease is a progressive dementia, characterized by degeneration of nerve cells in the cerebral cortex, presence of neuritic plaques and neurofibrillary tangles.
  • Alzheimer's Disease Early symptoms of Alzheimer's Disease differ widely from patient to patient, but commonly include psychological symptoms such as depression, paranoia and anxiety. There is a slow disintegration of personality and intellect. Along with the cognitive dysfunction, specific disturbances of speech (aphasia) , motor activity (apraxia) , and recognition of perceptions (agnosia) are observed. As the disease progresses, memory impairment increases.
  • Alzheimer's Disease The diagnosis of Alzheimer's Disease is a difficult one. There are many disease states which exhibit the same clinical symptoms as Alzheimer's Disease, including dementia secondary to treatable conditions such as infectious diseases and thyroid dysfunction, as well as psychiatric disorders. Currently, a definitive diagnosis of Alzheimer's Disease requires histopathologic examination at autopsy. A Work Group on the Diagnosis of Alzheimer's Disease has established criteria for the clinical diagnosis of the disease. rMcKhann et al.. "Clinical Diagnosis of Alzheimer's Disease: Report of the NINCDS-ADRDA Work Group Under the Auspices of Department of Health and Human Services Task Force on Alzheimer's Disease", Neurology, Vol. 34, pp. 939-944 (July 1984)]. While this report of the Work Group attempted to refine the clinical diagnostic criteria for Alzheimer's Disease, there are to date no laboratory tests which would give a definite diagnosis of the disease.
  • the present invention relates to obtaining a definitive diagnosis of Alzheimer's Disease by observing changes in the concentration of certain proteins in body fluids or tissues from patients suspected of having Alzheimer's Disease as compared to normal controls.
  • the present invention is directed to a method of determining the effectiveness of a course of treatment for Alzheimer's Disease for its ability to reverse or eliminate the disease, by observing a change in protein concentration of certain proteins in a body fluid or tissue sample to normal levels.
  • the present invention relates to certain proteins which have been found to be increased or decreased in concentration in body fluid or tissue samples from patients with Alzheimer's Disease as compared to normal controls. Antibodies to these proteins, monoclonal or polyclonal, are contemplated in the present invention.
  • the present invention relates to a diagnostic test kit, which can be used in the clinical laboratory for the diagnosis of Alzheimer's Disease.
  • Figure 1 Silver-stained, two-dimensional gel of olfactory neuroblast total protein (40 ⁇ g) indicating five proteins that were found to be altered in relative amount when comparing 6 AD and 6 control cell lines (proteins 503, 612, 673, 822 and 1095). Relative molecular weight (y-axis) and isoelectric point (x-axis) values are indicated. Phosphoproteins were identified on autoradiogra s of 2D gels by incorporation of 3 P and are indicated on Figure 1 with a "p" prefix. None of the five AD-associated proteins were identified as phosphoproteins.
  • FIG. 2 Graphs showing comparison of relative concentration values for five AD-associated proteins in olfactory neuroblasts from 8 control and 6 AD donors. Protein concentration values are reported as the percent total integrated density (% TID) of the protein of interest in comparison to all other proteins on the gel (y-axis) . All samples were electrophoresed and analyzed in duplicate with similar results. "*” indicates that p ⁇ 0.05, "**” indicates p ⁇ 0.01, and "*** » indicates p ⁇ 0.005.
  • the present invention relates to the identification of certain proteins which have been found to be present in increased or decreased amounts in cell cultures of olfactory neuroblasts of patients that had been diagnosed as having Alzheimer's Disease. It is contemplated that these proteins will also found at increased or decreased levels in body fluids and tissues of patients suspected of having Alzheimer's Disease. It is contemplated that the increased or decreased levels of these proteins may be detected by any test which can measure separate proteins in a body fluid or tissue sample, providing an objective test to definitively diagnosis Alzheimer's Disease. Any test which can measure the amount or concentration of separate proteins in the sample can be used in the present invention. In particular, immunological tests and electrophoresis are contemplated as useful in the present invention, although the invention is not limited to these procedures.
  • the effectiveness of a course of treatment for Alzheimer's Disease can be determined by observing these proteins for proportional decreases or increases in levels to those of normal controls.
  • the present inventors obtained cultures of neuronal cells of adult human olfactory epithelium from Alzheimer's Disease donors and age-matched controls according to known methods (Wolozin et al. supra) .
  • AD Alzheimer's Disease
  • olfactory neurons from patients suffering from Alzheimer's Disease (AD) develop AD- associated neurofibrillary pathology [Wolozin et al. t Journal of Neuroscience Research. Vol. 33, pp. 163-169 (1992)].
  • AD-olfactory neuroblasts also exhibit elements of AD brain pathophysiology, including abnormal processing of amyloid precursor protein and an increase in potentially amyloidogenic C-terminal derivatives [Talamo et al.. supra. and Wolozin (In press) ] .
  • the combination of the ability of olfactory neuroblasts to proliferate in cell culture and to express pathophysiological elements of AD makes these cells an ideal tool for the study of Alzheimer's Disease.
  • the present inventors Using two-dimensional gel electrophoresis, the present inventors have identified five unique proteins that are altered in concentration when comparing olfactory neuroblast cell cultures from AD patients to controls.
  • the five proteins detected ranged in molecular weight from approximately 17kd to approximately 50kd, and all were acidic in varying degrees with pi's ranging from 4.8 to 6.7.
  • Four of the five proteins (proteins 503, 612, 673 and 822) were increased at least 4-fold in olfactory neuroblasts from AD-donors, with two of the proteins being not present in normal control cells (proteins 673 and 822) .
  • One of the proteins (protein 1095) was decreased 2.5-fold in the cells from patients with Alzheimer's disease as compared to normal controls.
  • the cell lines analyzed were matched for both age of donor, passage number and cell viability. Hence, the differences detected in these proteins appear to be related to the only clearly different variable, the effects of AD.
  • Alzheimer's Disease Altered protein phosphorylation has been implicated in the pathogenesis of Alzheimer's Disease [Saitoh et al.. Alzheimer's Disease and Related Disorders, pp. 769-780 (1989) ] .
  • olfactory neuroblasts were metabolically labeled with 32 P-orthophosphate.
  • 2D gel electrophoresis showed that none of the disease- related proteins were phosphorylated suggesting that increases in phosphorylation state did not directly account for the disease-related differences in the five proteins detected.
  • the lysosomal system has also been implicated in the pathogenesis of Alzheimer's Disease. Lysosomal catabolism appears to be necessary for constitutive production of ⁇ - amyloid, and blockade of lysosomal catabolism in Alzheimer olfactory neuroblasts elicits large disease-related differences in amyloid precursor protein [Wolozin. supra. (In press); Haas. Nature. Vol. 359, pp. 322-324 (1992)]. The effects of lysosomal blockage on the five proteins detected by the present inventors were investigated. No significant changes in the levels of these five proteins were detected however. This suggests that, unlike ⁇ - a yloid, these proteins were not products of lysosomal catabolism.
  • body fluid or tissue samples obtained from a patient will be used to test for altered concentrations of these proteins for the diagnosis of Alzheimer's Disease.
  • the body fluid examples include blood, serum, plasma, urine, cerebrospinal fluid and nasal mucosa. Most preferably, the body fluid sample will be cerebrospinal fluid, because it is generally accepted that proteins produced by nerve cells will be present in cerebrospinal fluid.
  • the cerebrospinal fluid it may be necessary to concentrate the cerebrospinal fluid by methods known in the art before subjecting it to an immunoassay or gel electrophoresis in order to obtain a similar protein concentration as that of the cell sample and thus detectable levels of the five proteins of this invention.
  • im unoaffinity chromatography can be used to concentrate the proteins of interest in this invention in the CSF. Briefly, small amounts (eg., ⁇ 5 ml) of CSF would be loaded onto an immunoaffinity column containing either monoclonal antibodies or affinity- purified polyclonal antibodies covalently attached to the solid-phase matrix.
  • the affinity column can consist of one or more antibodies made to the disease specific olfactory neuroblast proteins.
  • tissue sample useful in the diagnostic tests of the present invention is skin, which can be subjected to the test directly or the skin fibroblasts can be grown in tissue culture (by methods well known in the art) prior to being tested.
  • the spots on the 2D gels can be viewed by any number of methods, including staining with Coomassie blue and silver staining. They can be visualized for relative protein density manually, but it is preferred that they be scanned with an appropriate camera system with a normalization standard and analyzed with a computer densitometer to measure relative protein spot staining intensities or densities.
  • the type of body fluid or tissue used as the sample will affect the two-dimensional gel pattern, although the five proteins of abnormal concentration found by the present inventors in the olfactory .neuroblasts should appear at roughly the same relative molecular weights and pi's. Rather, it is the entire gel pattern which changes from one type of sample to another.
  • two-dimensional gel patterns appear different in a sample of plasma as compared to a lymphoblast cell culture sample. While the present inventors have located the abnormal concentration proteins in gels from neuroblasts according to landmarked proteins on those gels, it is also possible to locate the same spots on a gel from a different type of sample even though in certain instances it may be necessary to refer to different landmark proteins. Ultimately, however, it is considered that a more precise method of identifying the proteins is by performing a Western blot on the gels from different body fluid or tissue samples, using antibodies which have been raised to the proteins of interest. It is also understood that the present invention is not limited to the five proteins found so far, but to any proteins appearing in abnormal concentration in patients with Alzheimer's disease. Thus, different types of samples obtained from patients may show other proteins which are altered in concentration or, more importantly, may provide a more recognizable concentration difference on the two-dimensional gel pattern, perhaps by visualization with the naked eye, providing a simpler way to score the sample as positive or negative.
  • the first step is to obtain antibodies to the proteins of interest.
  • proteins of interest There are many methods of accomplishing this which are well known to those skilled in the art. [For comprehensive laboratory methods, see Harlow et al.. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988) , which is incorporated by reference herein.]
  • the antigen For antibodies with sufficient specificity for Western blots and immunoassays, the antigen must be purified to homogeneity or the antigen should be used to prepare monoclonal antibodies. Since the proteins of interest in this invention are seen as unique spots on the second dimension polyacrylamide gel, preferable the gel can be used as the final purification step of the individual antigens.
  • This gel piece can be injected into an animal to raise antibodies.
  • Still another technique for processing the protein for injection after separation on gels is to electrophoretically transfer the proteins to nitrocellulose, locate the proteins of interest by staining (e.g., with Ponceau S) excise the spots and cut them into pieces for injection.
  • the particular method used is only limited by the ability to illicit an immune response to the proteins of interest.
  • the AD-specific proteins can be purified using preparative two-dimensional electrophoresis techniques involving liquid-phase isoelectric focusing in combination with preparative polyacrylamide gel electrophoresis (Sanchez et al..
  • monoclonal antibodies may be the best choice for immunochemical techniques.
  • Methods of monoclonal production are well known in the art and were first described by Kohler and Milstein in 1975. Briefly, antibody-secreting cells are fused to, for example, myeloma cells to create hybridoma cells which are cloned and screened by appropriate methods for the desired antibodies.
  • any of the known immunological tests can be used for either detecting the presence or absence of a particular protein of interest, or to quantitate antigen concentration.
  • An example of one assay is the two antibody sandwich assay described in Ghandbari et al. r J. Clin. Lab. Anal., Vol 4, pp. 189-192 (1990). Briefly, in this assay two antibodies that bind to overlapping epitopes of the same antigen are combined in a sandwich enzyme-linked immunoassay.
  • the antibodies can be either two monoclonals recognizing discrete sites or affinity purified polyclonals. One antibody is bound to a solid phase, antigen is bound, non-specifically bound proteins are washed away, and a labeled second antibody is bound. The assay is quantitated by measuring the amount of labeled second antibody bound to the matrix.
  • Many other immunoassays are well-known in the art and are useful in the present invention.
  • Olfactory neuroblasts were generated as described in Wolozin et al.. Journal of Molecular Neurosciences. Vol. 3, pp. 137-146 (1992), incorporated herein by reference.
  • Biopsies of olfactory epithelium were obtained from five neuropsychologically evaluated Alzheimer's donors (mean age 67.7 +/- 6.5 years) and seven age-matched controls (mean age 73.5 +/- 6.3 years). See Table 1. All samples were from subjects who had been evaluated by medical and neuropsychological testing. Diagnosis of Alzheimer's Disease was based upon the NINCDS-ADRDA criteria for "probable Alzheimer's Disease" (McKhann et al.. supra) .
  • the 4506 medium is a Hams- F12 based medium (neuroblast formulation, Whittaker) , supplemented with 6% fetal calf serum (GIBCO) , 150 ⁇ g/m£ insulin (Sigma), 40 pg/m£ thyroxine, 2.5 ng/m-C selenious acid and 60 ⁇ g/m£ gentamicin (Whittaker) .
  • GEBCO fetal calf serum
  • DMEM Dulbecco's Modified Eagle Medium
  • the cultures were incubated overnight and then harvested with a twenty minute incubation at 4°C in a buffer containing 100 mM TRIS, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 ⁇ g/m£ leupeptin, 10 ⁇ g/m£ pepstatin, 10 ⁇ g/m£ aprotinin and 10 ⁇ g/m£ phenylmethylsulfonylfluoride.
  • Levels of protein-incorporated radioactivity were determined by counting trichloracetic acid-precipitable counts in a scintillation counter.
  • olfactory neuroblasts Treatment of olfactory neuroblasts with the lysosomal inhibitor chloroquine results in the altered processing of the AD-associated amyloid precursor protein. To determine whether the five disease-related proteins were products of lysosomal degradation, olfactory neuroblasts were treated with the lysosomal inhibitor chloroquine. Olfactory neuroblasts were grown to confluence in 60 mm dishes in 4506 medium and then treated with 60 mM chloroquine overnight. Previous studies have shown that the cells survived this treatment without toxicity rwolozin et al.. Journal of Neuroscience Research. Vol. 33, pp. 163-169 (1992)].
  • Olfactory neuroblast cells were harvested by scraping the dishes, adding protease inhibitors and sonicating. Samples containing equal amounts of total protein (40 ⁇ g) as determined by the method of Bradford rAnalytical Biochemistry. Vol. 72, pp. 248 (1976)] were separated by high resolution two-dimensional gel electrophoresis according to the method of Hochstrasser et al. with minor revisions [Hochstrasser et al.. Analytical Biochemistry. Vol. 173, pp. 424-435 (1989)].
  • total protein was prepared with the addition of a denaturing solution containing 2% SDS, 2% CHAPS, 0.15 molar dithiothreitol (DTT) , and 20% glycerol and heated at 95°C for four minutes. Proteins were separated in the first dimension by isoelectric focussing on polyacrylamide tube gels (3% T, 1% C) containing 9.2 molar urea, 4.35% pH 3-10 ampholines (Hoefer), 4.35% pH 4-8 ampholines (Hoefer), with diacrylpiperazine (PDA, Bio-Rad) as the crosslinking agent.
  • a denaturing solution containing 2% SDS, 2% CHAPS, 0.15 molar dithiothreitol (DTT) , and 20% glycerol and heated at 95°C for four minutes. Proteins were separated in the first dimension by isoelectric focussing on polyacrylamide tube gels (3% T, 1% C) containing 9.2 molar urea, 4.35%
  • Isoelectric focussing proceeded at 200 volts per hour, followed by 500 volts for five hours and 1200 volts for 10.5 hours. Separation of proteins in the second dimension was according to molecular weight on polyacrylamide slab gels containing 12% acryla ide, 0.2 molar TRIS-HC1 pH 8.8, 0.7% (w/v) sodium thiosulfate, 0.3%
  • Phosphorylation state values of elongation factor-2 represent the ratio of the absolute TID values of the phosphorylated isoforms to the TID values of the nonphosphorylated isoforms as previously described rJohnson et al.. Molecular Brain Research. Vol. 15, pp. 319-326 (1992)].
  • Protein 673 appears in this study to be of particular interest for a diagnostic marker because of the lack of overlap between the AD and control samples.
  • a model where in order to have a diagnosis of AD, a subject must have a positive % TID value for more than one protein.
  • the power of the test is equal to the product of the perspective p-values.
  • An example of the increased significance using this criteria is shown when it is stipulated that AD is diagnosed by having positive % TID values for both proteins 216 and 673. In this case, the p value is increased from ⁇ 0.005 for each of the individual proteins to ⁇ 0.0001 for the two proteins in combination.
  • a body fluid or tissue sample preferably cerebrospinal fluid
  • cerebrospinal fluid would be obtained from an Alzheimer's Disease patient prior to the treatment.
  • a two-dimensional gel would be run, stained and analyzed for levels of one or more of the proteins listed in Table 2 to obtain a baseline.
  • body fluid or tissue samples are taken from the patient and analyzed for levels of the same protein or proteins which were initially analyzed.
  • a proportional increase or decrease to normal levels signifies that the treatment is successful.
  • the gel After two-dimensional gel electrophoresis is performed on a patient with confirmed Alzheimer's Disease, the gel would be stained with Coomassie blue in order to locate a protein of interest (for example, one of the five proteins in Table 2) .
  • the gel would be rinsed with deionized water for a few minutes, changing the water several times.
  • the spot containing a protein would be cut out of the gel with a scalpel, and placed on a piece of parafilm or plastic wrap. The edge of a paper towel is used to remove any standing water by capillary action.
  • the plungers from the barrels of two 5 cc syringes are removed and the gel piece is placed into one of the barrels.
  • the plunger is then replaced and the syringe outlet is positioned in the barrel of the second syringe.
  • the gel is pushed into the barrel of the second syringe. This process is repeated several times back and forth between the two syringes. Then, 21-gauge needles are placed onto the outlet of the syringes, and the process is repeated.
  • a small amount of buffer (PBS) may be necessary to keep the small fragments passing back and forth between the syringes. The samples are now ready for injection.
  • Antibodies are raised in rabbits immunized by injecting the antigen preparation (above) .
  • An initial subcutaneous injection of approximately 150 ⁇ g of one of the protein preparations would be followed by two monthly injections of approximately 100 ⁇ g of the antigen. This will lead to a sufficient antibody titer for use in an immunoassay.
  • Monoclonal antibodies may be prepared according to the method of Kohler and Milstein. This method involves immunizing mice with an antigen bearing one or more epitopes (i.e., one of the Alzheimer's Disease proteins of Table 2) . The mice develop spleen cells making anti-epitope (SN) which appear as an antibody (or antibodies) in the serum. The spleen is removed and the individual cells fused in polyethyleneglycol with constantly dividing (i.e., immortal) B-tumor cells selected for a purine enzyme deficiency and often for their inability to secrete Ig.
  • an antigen bearing one or more epitopes i.e., one of the Alzheimer's Disease proteins of Table 2
  • the mice develop spleen cells making anti-epitope (SN) which appear as an antibody (or antibodies) in the serum.
  • the spleen is removed and the individual cells fused in polyethyleneglycol with constantly dividing (i.e., immortal) B-tumor cells selected for a
  • the resulting cells are distributed into microwell plates in HAT (hypoxanthine, aminopterin, thymidine) medium which kills off the profusion partners, at such a high dilution that, on average, each well will contain less than one hybridoma cell.
  • HAT hyperxanthine, aminopterin, thymidine
  • Each hybridoma being the fusion product of a single antibody-forming cell and a tumor cell will have the ability of the former to secrete a single species of antibody and the immortality of the latter enabling it to proliferate continuously, clonal progeny providing an unending supply of antibody.
  • the hybridomas which secrete the monoclonal antibody of interest are screened by using a solid phase assay with an enzyme reaction product which provides a positive signal, i.e., an ELISA.
  • the antigen of interest is bound to a solid support, such as a 96 well plate. Then a sample of the culture medium from each of the hybridomas is placed in the well containing the bound antigen. The medium containing the antibody to the antigen of interest will react with that antigen which has been bound to the plate. Unbound antibody is washed away and a second antibody which reacts with the mouse antibody, and which binds to the hybridoma antibody already bound to the antigen, is added. This second antibody contains an enzyme tag, such as peroxidase or alkaline phosphatase. By then adding the appropriate substrate, the enzyme produces a colored reaction product which may then be observed visually or through the use of a machine that can measure color intensity. Once positive hybridomas are found through the screening procedure, the hybridomas are cloned so that eventually a pure culture of a single hybridoma line which produces the antibody of interest is obtained, providing an unlimited supply of the monoclonal antibody.
  • a solid support such as a 96 well plate.
  • test kits for use with a radioimmunoassay or ELISA test will contain: (A)
  • a plate with absorbed rabbit FAB fragment IgG (to any of the proteins set forth in Table 2) , or nitrocellulose sheets with the absorbed rabbit IgG.
  • kits may also contain appropriate buffers such as PBS, blocking solution and appropriate enzyme substrates (for ELISAs) . These materials may be provided with the kit or may be separately provided or prepared.
  • test kit A (above) would be employed as follows:
  • test kit B would be employed as follows: 1. Incubate a substrate (plate, nitrocellulose paper, etc.) with an unknown sample (such as cerebrospinal fluid) for an appropriate time and temperature. 2. Wash with BSA/PBS.

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Abstract

L'invention concerne un procédé permettant d'établir un diagnostic de la maladie d'Alzheimer. Le procédé fait appel à un ensemble unique de protéines que l'on trouve modifiées en concentration chez les malades souffrant de la maladie d'Alzheimer. L'invention se rapporte également à ces protéines et à leurs anticorps, ainsi qu'à des nécessaires pouvant être utilisés pour les tests de diagnostic.
PCT/US1994/008903 1993-08-13 1994-08-15 Procedes de diagnostic de la maladie d'alzheimer WO1995005604A2 (fr)

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WO1997007403A1 (fr) * 1995-08-16 1997-02-27 Hoechst Marion Roussel, Inc. PROCEDE PERMETTANT DE DETERMINER LE DEGRE D'AGREGATION DU PEPTIDE βA4
WO1998003869A1 (fr) * 1996-07-17 1998-01-29 Valbiofrance Methodes de diagnostic in vitro de maladies neurodegeneratives et trousses pour la mise en oeuvre de ces methodes
WO1998040748A1 (fr) * 1997-03-14 1998-09-17 Neuromark Diagnostic de troubles neurologiques
US5817626A (en) * 1995-03-14 1998-10-06 Praecis Pharmaceuticals Incorporated Modulators of beta-amyloid peptide aggregation
US5854204A (en) * 1995-03-14 1998-12-29 Praecis Pharmaceuticals, Inc. Aβ peptides that modulate β-amyloid aggregation
US5854215A (en) * 1995-03-14 1998-12-29 Praecis Pharmaceuticals Incorporated Modulators of β-amyloid peptide aggregation
US5919631A (en) * 1996-07-17 1999-07-06 Hoechst Marion Roussel, Inc. Method of determining the degree of aggregation of the β-A4 peptide
US5958684A (en) * 1995-10-02 1999-09-28 Van Leeuwen; Frederik Willem Diagnosis of neurodegenerative disease
US5985581A (en) * 1996-07-25 1999-11-16 The Mclean Hospital Corporation Use of presenilin-1 for diagnosis of alzheimers disease
US5985242A (en) * 1995-10-27 1999-11-16 Praecis Pharmaceuticals, Inc. Modulators of β-amyloid peptide aggregation comprising D-amino acids
WO2000039580A1 (fr) * 1998-12-23 2000-07-06 University Of Sydney Essai pour la detection d'un partenaire de liaison
US6277826B1 (en) 1996-08-27 2001-08-21 Praecis Pharmaceuticals, Inc. Modulators of β-amyloid peptide aggregation comprising D-amino acids
US6303567B1 (en) 1995-03-14 2001-10-16 Praecis Pharmaceuticals, Inc . Modulators of β-amyloid peptide aggregation comprising D-amino acids
US6610658B1 (en) 1999-03-04 2003-08-26 Praecis Pharmaceuticals Inc. Modulators of μ-amyloid peptide aggregation
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US6610658B1 (en) 1999-03-04 2003-08-26 Praecis Pharmaceuticals Inc. Modulators of μ-amyloid peptide aggregation
US7803774B2 (en) 1999-03-04 2010-09-28 Praecis Pharmaceuticals, Inc. Modulators of β-amyloid peptide aggregation
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