WO1998040748A1 - Diagnostic de troubles neurologiques - Google Patents

Diagnostic de troubles neurologiques Download PDF

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Publication number
WO1998040748A1
WO1998040748A1 PCT/US1998/005045 US9805045W WO9840748A1 WO 1998040748 A1 WO1998040748 A1 WO 1998040748A1 US 9805045 W US9805045 W US 9805045W WO 9840748 A1 WO9840748 A1 WO 9840748A1
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Prior art keywords
protein
disease
cit
proteins
protein profile
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PCT/US1998/005045
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English (en)
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Michael G. Harrington
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Neuromark
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Priority to AU64657/98A priority Critical patent/AU6465798A/en
Publication of WO1998040748A1 publication Critical patent/WO1998040748A1/fr

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    • 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
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/302—Schizophrenia

Definitions

  • TECHNICAL FIELD This invention relates to diagnosis of neurologic disorders. More specifically, the invention provides methods of obtaining a disease-specific protein profile by determining proteins from a biological sample which are altered relative to normal controls. The invention also relates to methods of diagnosing neurologic disorders by determining the levels of the proteins which make up the disease-specific protein profile. The invention provides methods of diagnosing schizophrenia, Alzheimer's disease, Parkinson's disease, multiple sclerosis, subacute sclerosing panencephalitis (SSPE) and other neurologic disorders using disease-specific protein profiles.
  • SSPE subacute sclerosing panencephalitis
  • AD Alzheimer's Disease
  • Symptoms of AD include difficulty with memory and loss of intellectual abilities severe enough to interfere with routine work or social activities.
  • Confusion, language problems (such as trouble finding words), poor or decreased judgment, disorientation in place and time and changes in behavior or personality also occur.
  • AD leaves its victims totally unable to care for themselves.
  • the only way to confirm a diagnosis of AD is by post-mortem autopsy.
  • Parkinson's Disease affects over 1 million people in the U.S., with 20 new cases of PD per 100,000 people per year.
  • PD is a chronic disease in which certain dopamine-producing neurons are destroyed.
  • CT or MRI brain scans
  • a diagnosis is based on a neurological examination, the severity of the symptoms and brain scans (CT or MRI) to help rule out other neurological diseases. If the symptoms are significant, a trial test of anitparkinsonian drugs may be used to diagnose the disease.
  • Primary symptoms include stiffness, tremor, slowness and poverty of movement, difficulty with balance and walking. Secondary symptoms are depression; sleep disturbances; weight loss; dementia; drooling; difficulty with speech, breathing or swallowing; constipation and stopped posture.
  • Schizophrenia usually develops between adolescence and age 30 and is characterized by positive symptoms (delusions or hallucinations), negative symptoms (blunted emotions and lack of interest), and disorganized symptoms (confused thinking and speech, or disorganized behavior and perception). Schizophrenia can generally be treated with antipsychotic drugs which control most of the clinical symptoms of the disease.
  • neurologic disorders are usually only tentatively diagnosed by pre- mortem clinical evaluation or, definitively, by post-mortem autopsy of the brain.
  • clinical evaluation of neurologic disorders is complex as the physician must rule out other problems or disorders which exhibit like symptoms.
  • PD can be confused with multiple sclerosis, ALS or Huntington's chorea.
  • schizophrenia drug abuse, seizures, major depressive or manic episodes, autism, and other personality disorders may present with psychotic symptoms similar to those seen in schizophrenia.
  • the search for protein markers which are specific for a neurological disorder has ⁇ proven elusive.
  • Using two-dimensional gel electrophoresis (2DE) Hsich et al. (1996) NEJM 335(13):924-929 report that transmissible spongiform encephalopathies can be detected by the presence or amount of the protein in cerebrospinal fluid.
  • U.S. Patent No. 5,006,462 to Gattaz et al. describes diagnosis of schizophrenia by measuring increased enzymatic activity of phospholipase A2 (PLA2) in plasma samples when compared to nonschizophrenic controls.
  • PKA2 phospholipase A2
  • WO 95/05604 discloses methods of diagnosing Alzheimer's disease by detecting the presence of proteins altered in the olfactory neuroblast ly sates of AD patients.
  • WO 95/05395 discloses a method of diagnosing AD by measuring elevated levels of alpha- 1 anti-trypsin or fibrinogen.
  • U.S. Patent No. 5,429,947 to Merril et al. describes a method for diagnosing schizophrenia and Alzheimer's Diseases (AD) by detecting elevated levels of specific haptoglobin proteins in the bodily fluids of patients. This method is not specific for either of the two disorders listed.
  • 5,234,814 describes a method for assisting the diagnosis of AD by detecting amyloid precursor related proteins (APP) using an antibody which is reactive with the C-terminal of human amyloid precursor protein.
  • Japanese Patent Nos. 3211461 and 2543606 disclose diagnosis of multiple sclerosis by measuring the amount of human tumor necrosis factor-alpha (h-TNF) in cerebrospinal fluid.
  • h-TNF human tumor necrosis factor-alpha
  • the cerebrospinal fluid CSF
  • High resolution, _ two dimensional (2DE) separation techniques have yielded a detailed protein map of more than 2,000 individual components from CSF samples. The first dimension of 2DE separates proteins on the basis of charge, while the second dimension separates based on mass.
  • the present invention provides a specific method of generating disease-specific molecular markers.
  • the disease-specific markers are obtained by screening a large number of proteins from a biological sample, identifying the particular proteins which are altered in subjects with neurological diseases by comparing their levels to levels found in normal subjects. Individually, these proteins do not distinguish the disorders from normal or other disease controls, but in combination, these protein profiles are very accurate at differentiating specific disorders.
  • the particular proteins can then be isolated, purified, and used to generate antibodies.
  • the antibodies are then used, for example, in Western blots or other immunoassays to diagnosis a specific disease condition in a sample.
  • the disease-specific protein profiles are also called "bar-codes" in reference to the unique marking system used for merchandise.
  • the profiles expressed as bar codes provide a unique marking system for neurologic disorders.
  • the bar-coding strategy of this invention allow for easy, rapid and specific assay for neurologic disease including schizophrenia, manic depressive disorder, Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS) and Creutzfeldt- Jakob disease (CJD). It was the surprising discovery of the inventor that when the levels of the protein profile obtained from a patient is compared to normal controls, neurologic disorders can be diagnosed.
  • the present invention provides a method of diagnosing a neurological disorder in a biological sample, the method comprising: (a) obtaining a disease-specific protein profile by detecting proteins whose levels are altered in a subject having a neurological disorder when compared to levels of a healthy control; (b) obtaining the biological sample; (c) measuring the levels of the disease-specific protein profile obtained in step (a) in the biological sample; and (d) comparing the levels of the disease-specific protein profile in the biological sample to levels found in the healthy control; wherein altered levels of the protein profile of the biological sample as compared to the healthy control is diagnostic of the neurological disorder.
  • the biological sample is cerebrospinal fluid.
  • the neurologic disorder is selected from the group consisting of Alzheimer's Disease (AD), schizophrenia, Parkinson's disease (PD), multiple sclerosis, subacute sclerosing panencephalitis (SSPE) and transmissible spongiform encephalopathies.
  • AD Alzheimer's Disease
  • PD Parkinson's disease
  • SSPE subacute sclerosing panencephalitis
  • the neurologic disorder is AD and the protein profile comprises proteins designated AD-1 through AD- 18.
  • the neurologic disorder is PD and the protein profile comprises CIT-A, CIT-B, CIT-C (protein 129), CIT-1 (alpha 1 microglobulin subunit) ,
  • the neurologic disorder is schizophrenia and the protein profile comprises protein 91, protein 48, protein 46, orosomucoid, prostaglandin D synthetase (PGDS), antithrombin III and fibrinogen degradation products.
  • the neurological disorders are multiple sclerosis, subacute sclerosing panencephalitis (SSPE), or Creutzfeldt- Jakob disease (CJD).
  • the disease-specific protein profile is obtained using 2 dimensional gel electrophoresis (2DE), Western blotting or immunoassay.
  • the protein profile is measured by visualizing a 2DE gel,
  • Figure 1 is a color reproduction of a 2DE gel electrophoresis of CSF fluid obtained from normal controls. Five positional standards, or landmarks, are identified in green.
  • FIG. 1 is a color reproduction of a 2DE gel electrophoresis of CSF fluid obtained from Parkinson's disease patients. Each of the protein spots comprising a PD-specific protein profile are marked in red.
  • Figure 3 is a color reproduction of a 2DE gel electrophoresis of CSF fluid obtained from Alzheimer's Disease patients. Eighteen spots comprising a AD-specific protein profile are marked in red.
  • FIG. 4 panels A through C, are plots depicting the relative quantity of CIT-5 (Apo-Al lipoprotein) in various Parkinson's patients. Each bar is representative of one patient. Panel A shows CIT-5 (Apo-Al lipoprotein) plotted against the "H + Y" rating, a standardized index of disease severity. As disease severity increases, the amount of CIT-5 (Apo-Al lipoprotein) decreases.
  • a "3" denotes long duration PD with dyskinesia, and moderate to high dopaminergic therapy
  • a "2” denotes medium duration
  • a "1” denotes short duration disease, no dyskinesia and no or minimum dopaminergic therapy.
  • Panel B shows CIT-5 (Apo-Al lipoprotein) plotted against the age of patient/duration of clinical disease.
  • OOL refers to patients in whom the clinical disease onset occurred when they were older (above 50) with a long duration of disease
  • ZL refers to a young-onset patients (below 50) with a long duration of disease
  • OS refers to old-onset and short duration disease
  • FIG. 1 is a graphical representation of a multiple discriminant function analysis plotting canonical discriminant functions obtained from the protein profiles of six PD patients (c); six AD patients (a) and two controls (b).
  • Figure 6 is a graphical representation of a multiple discriminant function analysis plotting canonical functions obtained from the protein profiles of six PD patients (c); four AD patients (b) and six controls (a).
  • Figure 7 shows the effect of various schizophrenia drug therapies on the level of protein 91.
  • protein 91 has a normalized protein quantity (density units) of approximately 8.
  • Individuals effected with schizophrenia have only around 4 units of the same protein.
  • Patients on any of the three therapy regimes tested have less protein 91 than untreated or normal individuals.
  • Figure 8 shows the effect of various schizophrenia drug therapies on the level of protein 48.
  • protein 48 has a normalized protein quantity of approximately 7.
  • Individuals affected with schizophrenia have around 13 units of the same protein.
  • Patients on any of the three therapy regimes tested show a protein 48 level which is less than untreated individuals, but closer to the normal levels.
  • Figure 9 shows the effect of various schizophrenia drug therapies on the level of one isoform of prostaglandin D synthetase (PGDS).
  • PGDS prostaglandin D synthetase
  • this PGDS isoform has a normalized protein quantity of approximately 8. Patients affected with schizophrenia have less than 4 units of the same protein. Samples obtained from patients on any one of the three therapy regimes show that PGDS levels remain lower than normal levels.
  • Figure 10 shows the effect of various schizophrenia drug therapies on the level of protein 46. In normal controls, protein 46 has a normalized protein quantity of approximately 8. Individuals affected with schizophrenia have around 13 units of the same protein. Patients on any of the three therapy regimes tested show a protein 46 level which were intermediate between untreated and normal levels.
  • Figure 11 depicts a two dimensional graph in which samples have been positioned based on their protein profile. A diagnosis of SSPE was made in sample labeled "E". This graph shows complete resolution of samples diagnosed as characteristic of SSPE from other samples, including multiple sclerosis patients.
  • neurodethelial disorder is intended to refer the diverse group of disorders which affect the central or peripheral nervous system.
  • the disorders may be acute or chronic and include, but are not limited to, schizophrenia, manic depression, Alzheimer's disease, Parkinson's disease, multiple sclerosis, transmissible spongiform encephalopathies
  • AD Alzheimer's Disease
  • AD Alzheimer's Disease
  • AD Alzheimer's Disease
  • amyloid plaques and neurofibrillary tangles in the brain, which can be seen upon autopsy.
  • Parkinson's Disease or "PD” are used interchangeably to include the neurologic disease characterized by tremor, rigidity, akinesia (freezing), postural disturbance or balance problems. Fatigue, depression, anxiety, restlessness, poor handwriting, quieting of voice, change in posture, muscle aches, cramping or paresthesias
  • PD Parkinson's disease
  • PD can be divided into different subtypes based, for example, on age of onset, duration or amount of dyskinesia.
  • the term "schizophrenia" is intended to include the group of mental disorders characterized by disruptions in thinking and perception. In a clinical evaluation, schizophrenia is commonly marked by auditory hallucinations (especially hearing voices), disorganized thought processes, delusions, and the absence of emotion or affect.
  • the disorder known as schizophrenia can be divided into several subtypes or categories.
  • the term "schizoaffective” is intended to mean a category of schizophrenia patients who also exhibit mood disorders, for example, bipolar disorder. Schizoaffective disorder is generally characterized by common schizophrenia symptoms in combination with major depressive or manic episodes.
  • Post partum schizophrenia refers to a the category of schizophrenia which exhibits common symptoms but onsets in women after giving birth.
  • the term “schizophrenic” refers to a person who suffers from schizophrenia.
  • TSEs transmissible spongiform encephalopathies
  • CJD Creutzfeldt- Jakob disease
  • Gerstmann-Staussler-Scheinker syndrome Fatal Familial Insomnia
  • Kuru Kuru.
  • the TSEs include sheep scrapie, bovine spongiform encephalopathy, transmissible mink encephalopathy, and chronic wasting disease of captive mule deer and elk (Gajdusek (1990) Subacute Spongiform Encephalopathies: Transmissible Cerebral Amyloidoses
  • TSEs are characterized by the same hallmarks: a spongiform degeneration, reactive gliosis in the cortical and subcortical gray matters of the brain, and transmission when experimentally inoculated into laboratory animals including primates, rodents and transgenic mice.
  • a biological "sample” as used herein includes a variety of sample types obtained from an individual and is typically used in a diagnostic procedure or assay.
  • the definition encompasses blood, cerebrospinal fluid (CSF) and other liquid samples of biological origin, solid tissue samples such as biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. Methods of collecting and storing tissue or fluid samples are known to those of skill in the art.
  • the definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization or enrichment for certain components, such as proteins or polynucleotides.
  • biological sample encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid and tissue samples.
  • level or “levels” are used to refer to the presence and/or amount of protein, and can be determined qualitatively or quantitatively.
  • a “qualitative” change in the protein level refers to the appearance or disappearance of a protein spot that is not detectable or is present in samples obtained from normal controls.
  • a “quantitative” change in the levels one or more proteins of the profile refers to a measurable increase or decrease in the protein levels when compared to a healthy control.
  • protein profile refers to a pattern of specific proteins whose levels are altered in samples obtained from subjects with neurologic disorders when compared to a healthy control.
  • the profile may be expressed in many forms, including, for example, as a
  • “molecular bar code” composed of the proteins of the profile.
  • a disease-specific protein profile is obtained by comparing the level of a variety of proteins in a sample taken from a healthy control to the levels found in samples taken from affected individuals. Individually, the proteins which are altered in the affected individuals may not be diagnostic of the disorder, but, when viewed together as a profile, are disease-specific.
  • the proteins which comprise the profile may all be increased, decreased, present or absent with respect to healthy levels. More often, some of the proteins will be increased and some will be decreased as compared to the control.
  • the proteins which make up the disease-specific protein profile may be identified by their common name, their structure, their sequence, by the antibodies which recognize them or by their migration pattern in gel electrophoreses. One or two dimensional gels may be used. Methods of 2DE are known in the art, and described herein in Example 1.
  • a protein profile for AD comprising 18 proteins has been identified. Two of the proteins have been identified by their common names, Haptoglobin 1 precursor subunits, while sixteen are identified by their migration pattern in gel electrophoresis.
  • a protein profile for PD comprises 9 proteins, including CIT-A, CIT-B, protein 129, alpha 1 microglobulin subunits, two PGDS isoforms, complement 4 gamma and Apo-Al lipoprotein.
  • the profile for schizophrenia described in the present application includes proteins 48, 46 and 91. To date, the amino acid sequence of these proteins is not known. Accordingly, these proteins are identified herein by randomly-assigned numbers corresponding to the location on a 2DE gel where the protein appears under the conditions described herein. Proteins 5, 48, 46 and 91 have been previously identified in Harrington et al. (1985) Clinical Chemistry 31(5):724. As shown in Figure 1 of this reference, these proteins migrate in response to electrophoresis in a characteristic manner.
  • proteins of the protein profile may also be identified, for example, by their isoeiectric focusing point (pi) and molecular weight (MW) in kilodaltons (kD).
  • pi isoeiectric focusing point
  • MW molecular weight
  • kD kilodaltons
  • pi and molecular weight values are intended to include a range which includes any variations which may occur due to experimental conditions as would be known by one of skill in the art.
  • pis will generally range up or down 0.5 from the value given. At higher pHs, the range may be + 1.
  • Molecular weight ranges will usually be within 5 kilodaltons of the value given, although glycoproteins may migrate anomalously in SDS PAGE gels and give show MW values which are higher than the actual MW.
  • the proteins which comprise the disease-specific profiles can be quantified, for example, by measuring the units of protein concentration or normalized density units.
  • the protein profile can be visualized, for example, from electrophoresis, in particular, 2DE. Electrophoretic methods are well-known to those skilled in the art and are described in detail herein. Visualizing proteins may be performed by any method known in the art. Coomassie blue and silver stain, for instance, are well known methods of visualizing proteins on gels. Western blotting and autoradiography of gels can also used to identify the disease-specific protein profiles.
  • the protein profiles used in the methods described herein be composed of at least 2 different proteins, or alternatively, at least two isoforms or glycoforms of the same protein.
  • the protein profiles are comprised of between 3 and 20 proteins or glycoforms, more preferably, between 4 and 15, and even more preferably 5 to 10.
  • Glycoforms of the same protein can be distinguished by methods known in the art. As described above, glycoforms of proteins are known to migrate differently in 2DE, as determined by molecular weight and isoeiectric focusing points. Therefore, a protein profile could be prepared by measuring or visualizing glycoforms of the same protein. Although the only limit on the number of proteins which can make up a profile is the number of proteins in the sample, for ease of analysis, less than 20 is preferred.
  • the protein profiles can be used to differentiate between diagnostic categories of individual diseases.
  • schizophrenia for example, post-partum schizophrenia or schizoaffective disorder may produce protein profiles which are distinct from conventional schizophrenia.
  • early-onset Alzheimer's disease may present with a different protein profile from the more common age-associated AD.
  • an individual subject may have a unique protein profile, for instance, because they are suffering from more than one neurologic disorder.
  • the protein profile can be used to distinguish specific cell damage or brain region damage based on the appearance of proteins unique to their origin.
  • a "healthy control” or "normal control” is a biological sample taken from an individual who does not suffer from a neurologic disorder.
  • a "negative control” is a sample that lacks any of the specific analyte the assay is designed to detect and thus provides a reference baseline for the assay.
  • cerebrospinal fluid or "CSF” is intended to include whole cerebrospinal fluid or derivatives or fractions thereof well known to those of skill in the art.
  • a cerebrospinal fluid sample can include various fractionated forms of cerebrospinal fluid or can include various diluents added to facilitate storage or processing in a particular assay.
  • Such diluents are well known to those of skill in the art and include various buffers, preservatives and the like.
  • 2-dimensional gel electrophoresis or “2-D gel electrophoresis” or “2DE” is intended to refer to the two dimensional migration of proteins in solution or suspension in the presence of an electrical field.
  • Methods of 2DE are well known to those of skill in the art. See generally, R. Scopes (1982) Protein Purification, Springer- Verlag, N.Y. and Deutscher (1990) Methods in Enzymology Vol. 182: Guide to
  • 2DE relies on isoeiectric focusing (IEF) in either carrier ampholyte gradient gels or immobilized pH gradients for one dimension and SDS-polyacrylamide gels for the second dimension.
  • IEF isoeiectric focusing
  • SDS-polyacrylamide gels for the second dimension.
  • the amount of polyacrylamide to be used in making the gels can be readily determined by a skilled artisan.
  • an "immunoassay” is an assay that utilizes an antibody to specifically bind to the antigen.
  • the immunoassay is thus characterized by detection of specific binding of the proteins of the protein profile to antibodies as opposed to the use of other physical or chemical properties to isolate, target, and quantify the analyte. Direct immunoassay and competitive immunoassays are described in detail below.
  • an “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • the basic immunoglobulin (antibody) structural unit is known to comprise a tetramer or dimer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V,) and variable heavy chain (V H ) refer to these variable regions of the light and heavy chains respectively.
  • Antibodies may exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2 , a dimer of Fab which itself is a light chain joined to V H -C H 1 by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the F(ab)' 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially an Fab with part of the hinge region (See, Fundamental Immunology, W.E.
  • antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology.
  • antibody as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.
  • the specified antibodies preferentially bind to a particular protein and relative binding to other proteins does not occur in significant amounts.
  • Specific binding to a protein under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • antibodies can be raised to the protein markers specific for schizophrenia.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies. A Laboratory Manual, Cold Spring Harbor Publications, New ⁇ York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • the term "subject” refers to a mammal and includes, but is not limited to, humans, bovine, mink, sheep, elk, cats and deer.
  • the present invention includes a method of obtaining a disease-specific protein profile which may be expressed as a molecular bar code.
  • a disease-specific protein profile which may be expressed as a molecular bar code.
  • the familiar bar code system of grocery and other retail stores used to price goods reduces errors, saves time and helps keep inventory.
  • unique bar codes will provide accurate diagnosis, patient management and therapeutic monitoring.
  • the protein profiles expressed as molecular bar codes are obtained from a biological sample taken from a subject known to have a particular disorder, and preferably a neurologic disorder. Sample collection is discussed in detail below.
  • the proteins of the sample are then detected.
  • the sample is first subjected to two-dimensional gel electrophoresis (2DE) to distinguish individual proteins by their migration patterns.
  • the 2DE gel is then visualized, preferably by silver staining and quantified, for example by image analysis.
  • Altered levels of particular proteins in the sample are then obtained by comparing the protein levels in the sample to levels of a healthy control or a control known to have a disorder, including a neurologic disorder.
  • a computer algorithm and a database of protein levels from CSF is used to determine which particular proteins are increased or decreased in the sample.
  • a computer algorithm and a database of protein levels from CSF is used to determine which particular proteins are increased or decreased in the sample.
  • those which are altered in particular diseases can be identified. All or some of the specific proteins which are altered are called the protein profile.
  • Protein spots which are present in every gel are designated as positional standards or "landmarks.” These landmark spots are aligned as between at least two gels, and the rest of the spots digitized according to their relation to the landmarks.
  • a computer generated algorithm or the like can be programmed to compare the test CSF with healthy controls and determine which spots are different in one gel from another, and which are similar.
  • the algorithm used to determine the protein profile is a multi-step process.
  • Computer programs can used to compare and contrast intensity of spots on 2DE gels, for instance commercially available programs such as "Melony" (BioRad).
  • the protein spots on the 2DE gel are first identified using any spot-finding method known in the art. Preferably, an edge detector or peak identifier is used. Various parameters are recorded and stored for each spot, including, for example, x and y co-ordinates corresponding to pH and molecular weight along with total integrated density. Next, all spots are compared from one sample to one another, using a master gel retaining all data. The protein spots are either matched or unmatched and noted by their location on the gel.
  • the differences can be qualitative (e.g. unmatched spots) or quantitative (statistically significant variations of normalized protein quantity).
  • the compared gel data is then split into groups of each disease and normal controls. Similarities and differences are noted as described above. All of the different proteins are then further analyzed in subsets of patients within each disease compared to controls.
  • steps each use conventional statistics for judging differences in data.
  • means, standard deviations, a student T-test, and analysis with appropriate degrees of freedom are employed.
  • Other methods known to those in the art may also be suitable. Any changes are verified from one population by testing in a second population and only those changes that are replicable are confirmed as valid.
  • the novel method described here we can define a diagnostically powerful profile for each disease and even for disease sub-types. This is achieved simply when an individual protein is so discriminative, but this is rare and likely to be unusual.
  • One known example is the 14-3-3 marker for CJD. More commonly, a number of protein spots are altered in a statistically significant manner between disease and controls, but with overlap that is not useful for any one individual protein, but when analyzed together possess powerful discrimination. This can be achieved in a number of different ways, but most appropriately using multi-dimensional discriminant function analysis.
  • the algorithm is standard statistical software from Unistat. Other statistical methods will be known to those skilled in the art.
  • Either linear or nonlinear discriminant function analysis can be performed, and alternative approaches can include principal component analysis, factor analysis, or cluster analysis. All variable proteins were examined for combinations that would provide the best discrimination and then the best discrimination is investigated for the least number of proteins. Once this is achieved (usually 10 or less proteins with most discrimination coming from the first 5 proteins) then these proteins were used to test a second, new population of samples. Only when these same proteins gave discriminating power in the second population was this group of proteins considered for the profile. Once so identified, the profile can be used to define a multidimensional space of discriminant function that allows subsequent samples to be placed with confidence in one group or another.
  • the present invention provides a method for identifying protein profiles which are specific for a disease or an individual.
  • the diseases which can be diagnosed and monitored using the novel method described herein include, but are not limited to, schizophrenia, Alzheimer's disease, Parkinson's disease, multiple sclerosis, manic depression, Huntington's disease, viral encephalitis, stroke, dementia, headaches, bacterial meningitis, insomnia and transmissible spongiform encephalopathies.
  • AD Alzheimer's Disease
  • a protein profile specific for AD is identified. At least about 18 protein spots have been found to make up a protein profile. These spots, AD-1 through AD-18 are identified by their molecular weight and isoeiectric point in Table 1.
  • the AD profile identified herein is comprised of 18 qualitative markers.
  • Another profile specific for AD has been identified using a combination of proteins that include 2 of those qualitative markers with 7 other proteins that are altered in various diseases.
  • the present invention also includes a protein profile for PD.
  • the present inventor has discovered at least 3 qualitative and 5 quantitative proteins making up a disease- specific protein profile.
  • the 3 qualitative markers of the profile i.e. spots that are not present in normal controls
  • the quantitative markers, or spots which increase or decrease compared to normal are: Apo-Al lipoprotein; alpha 1 microglobulin subunit; two PGDS isoforms and complement 4 gamma. All spots decrease in PD patients except one PGDS isoform (CIT-2) which is increased.
  • CIT-2 Protein Profile for Schizophrenia
  • a protein profile specific for schizophrenia is identified. As described in the Examples, using the methods of the present invention a schizophrenia- specific profile made up of seven proteins was identified. These seven proteins, protein
  • 91, protein 48, protein 46, orosomucoid, prostaglandin D synthetase (PGDS), antithrombin III and fibrinogen degradation products are altered in schizophrenic subjects when compared to normal controls.
  • protein 91, orosomucoid (also known as ⁇ ,- acid glycoprotein), PGDS, and antithrombin III have a relative concentration which is decreased relative to normal.
  • Proteins 48 and 46 are increased relative to normal.
  • Fibrinogen degradation products are absent in normal controls and appear in schizophrenic samples.
  • FIG. 5 shows that PD patients (c) and AD patients (a) can be distinguished from each other and from healthy controls (b) where the multidimensional discriminating space is plotted in 2-dimensions.
  • Figure 6 PD patients (c) separate from AD (b) and from controls (a).
  • Figure 11 illustrates the diagnosis of subacute sclerosing panencephalitis (SSPE) patients in Group 5, individually labeled E with complete resolution from the other patient groups.
  • SSPE subacute sclerosing panencephalitis
  • protein profiles specific for multiple sclerosis, Parkinson's disease, subacute sclerosing panencephalitis (SSPE) or Creutzfeldt- Jakob disease are disclosed.
  • the ability to distinguish between SSPE and multiple sclerosis is an especially interesting and novel finding as conventional biochemical techniques cannot distinguish between these two disorders.
  • the disease-specific protein profile obtained as described above can then be used for diagnosis of the specific disorder.
  • the individual proteins of the protein profiles can be detected or quantified by any of a number of means well known to those of skill in the art.
  • analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, partial amino acid sequence determination and the like, or various immunological methods such as fluid or gel precipitation reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, western blotting, and the like.
  • analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, partial amino acid sequence determination and the like
  • immunological methods such as fluid or gel precipitation reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent
  • a qualitative change in one or more proteins of the protein profile is determined.
  • Qualitative changes include the appearance of a protein spot that is not detectable in samples obtained from normal controls or the disappearance of a protein spot which is detectable in normal controls but not in the sample taken from an affected subject.
  • a quantitative change in one or more proteins of the profile is measured.
  • the protein profile is quantified using 2DE, immunoassay or Western blotting.
  • concentration of protein levels may be expressed in absolute terms, for example, optical density as read by image analysis. Alternatively, the concentrations can be expressed as a fraction, relative to normal levels of the same protein.
  • the proteins which make up the protein profile can be individually isolated and, optionally purified. Methods of isolating and purifying proteins from 2DE gel are known in the art. The isolated proteins are then identified, for example, by partial amino acid sequence, mass spec or carbohydrate analysis. Monoclonal or polyclonal antibodies can then be generated to the isolated proteins by methods known in the art. See Harlow and Lane (1988) Antibodies. A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of methods of generating antibodies. Used in combination, these specific antibodies are used to diagnosis the specific disease in a subject by analyzing a biological sample from the subject.
  • the disease-specific protein profile is preferably quantified in a biological sample derived from a mammal, more preferably from a human patient.
  • a biological sample is a sample of biological tissue or fluid that contains a protein profile concentration that may be correlated with protein profile levels of a healthy control.
  • Particularly preferred biological samples include but are not limited to, plasma, urine, serum, neurological tissue and cerebrospinal fluid.
  • the biological sample may be pretreated as necessary by dilution in an appropriate buffer solution or concentrated, if desired.
  • an appropriate buffer solution or concentrated, if desired.
  • Any of a number of standard aqueous buffer solutions, employing one of a variety of buffers, such as phosphate, Tris, or the like, at physiological pH can be used.
  • assays are performed using cerebrospinal fluid (CSF).
  • CSF cerebrospinal fluid
  • Obtaining and storing CSF are well known to those of skill in the art.
  • CSF is obtained by lumbar puncture.
  • the CSF may be diluted by the addition of buffers or other reagents well known to those of skill in the art and may be stored for up to 24 hours at 2-
  • the CSF is stored at -70°C without preservative indefinitely.
  • a protein profile specific for schizophrenia may be obtained form cerebrospinal fluid using electrophoretic methods.
  • Means of detecting proteins using electrophoretic techniques are well known to those of skill in the art (see generally, R. Scopes (1982) Protein Purification, Springer-Verlag, N.Y.; Deutscher, (1990) Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc., N.Y.).
  • the protein profile is obtained using two-dimensional electrophoresis.
  • a particularly preferred separation relies on isoeiectric focusing (IEF) in carrier ampholyte gradient gels or immobilized pH gradients for one dimension and 14% polyacrylamide gels for the second dimension.
  • IEF isoeiectric focusing
  • Proteins separated on 2DE gels can be visualized by any method known in the art. For example, Coomassie blue staining, gold staining, silver staining and the like may be used to visualize proteins directly on the gels, (see, generally, Current Protocols in Molecular Biology, Ausubel et al. eds. (1996) ⁇ 10.6 to 10.8 and references therein). Alternatively, the proteins on the 2DE gel can be transferred or blotted onto a membrane and probed with an antibody. Methods of blotting and immunostaining are known to those of skill in the art. (see, e.g., Ausubel (1996), supra, ⁇ 10.8). In a preferred embodiment, the present invention obtains a protein profile by silver staining and, subsequently, by immunostaining.
  • the protein profile is obtained or measured using any of a number of well recognized immunological binding assays (see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
  • immunological binding assays see also Methods in Cell Biology Volume 37. Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, eds. (1991).
  • Immunological binding assays typically utilize a "capture agent" to specifically bind to and often immobilize the analyte (in this case a protein of the specific protein profile).
  • the capture agent is a moiety that specifically binds to the analyte.
  • the capture agent is an antibody that specifically binds a protein of the protein profile.
  • Antibodies may be produced by any of a number of means well known to those of skill in the art (see, e.g. Methods in Cell Biology Volume 37 Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York (1993); and Basic and Clinical Immunology 7th Edition, Stites & Terr, eds. (1991)).
  • the antibody may be a whole antibody or an antibody fragment. It may be polyclonal or monoclonal, and it may be produced by challenging an organism (e.g. mouse, rat, rabbit, etc.) with a protein of the profile or an epitope derived therefrom. Alternatively, the antibody may be produced de novo using recombinant DNA methodology.
  • the antibody can also be selected from a phage display library screened against the protein or interest (see, e.g. Vaughan et al. (1996) Nature Biotechnology, 14:309-314 and references therein) .
  • Immunoassays also often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte
  • the labeling agent may itself be one of the moieties comprising the antibody/analyte complex.
  • the labeling agent may be a labeled profile protein or a labeled anti-profile protein antibody, Alternatively, the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/protein complex.
  • Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the label agent. These proteins are normal constituents of the cell walls of streptococcal bacteria.
  • incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C.
  • Immunoassays for detecting proteins may be either competitive or noncompetitive.
  • Noncompetitive immunoassays are assays in which the amount of captured analyte (in this case the protein profile) is directly measured.
  • the capture agent can be bound directly to a solid substrate where they are immobilized. These immobilized antibodies then capture the protein of interest present in the test sample.
  • the protein thus immobilized is then bound by a labeling agent, such as a second antibody bearing a label.
  • the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • the second can be modified with a detectable moiety, such as biotin, to which a third labeled, molecule can specifically bind, such as enzyme-labeled streptavidin.
  • the amount of analyte (proteins of the protein profile) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte displaced (or competed away) from a capture agent by the analyte present in the sample.
  • a known amount of, in this case, the protein profile is added to the sample and the sample is then contacted with a capture agent.
  • the amount of protein bound to the antibody is inversely proportional to the concentration of the protein present in the sample before the exogenous protein is added to the reaction mix.
  • the antibody is immobilized on a solid substrate.
  • the amount of protein bound to the antibody may be determined either by measuring the amount of protein present in an protein/antibody complex, or alternatively by measuring the amount of remaining uncompeted protein.
  • the amount of protein may be detected by providing a labeled protein molecule.
  • a hapten inhibition assay is another preferred competitive assay. In this assay a known analyte, in this case the protein profile is immobilized on a solid substrate. A known amount of antibody is added to the sample, and the sample is then contacted with the immobilized protein. In this case, the amount of antibody bound to the immobilized protein is inversely proportional to the amount of protein present in the sample. Again the amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above.
  • Western blot (immunoblot) analysis is used to detect and quantify the presence of protein in the sample.
  • the technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind protein.
  • the anti-protein antibodies specifically bind to protein on the solid support.
  • These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the anti-protein.
  • labeled antibodies e.g., labeled sheep anti-mouse antibodies
  • LOA liposome immunoassays
  • Labels The particular label or detectable group used in the assay is not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the antibody used in the assay.
  • the detectable group can be any material having a detectable physical or chemical property.
  • detectable labels have been well developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g.
  • DynabeadsTM DynabeadsTM
  • fluorescent dyes e.g., fluorescein isothiocyanate, texas red, rhodamine, and the like
  • radiolabels e.g., ⁇ H, 125 ⁇ ? 35 ⁇ 5 14 or 32p
  • enzymes e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA
  • colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • the ligand then binds to an anti-ligand (e.g. , streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • an anti-ligand e.g. , streptavidin
  • a number of ligands and anti-ligands can be used.
  • a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be ⁇ used in conjunction with the labeled, naturally occurring anti-ligands.
  • any haptenic or antigenic compound can be used in combination with an antibody.
  • the molecules can also be conjugated directly to signal generating compounds, e.g. , by conjugation with an enzyme or fluorophore.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • Chemiluminescent compounds include luciferin, and 2,3-dihydrophtlialazinediones, e.g., luminol. (For a review of various labeling or signal producing systems which may be used, see, U.S. Patent No. 4,391,904).
  • Means of detecting labels are well known to those of skill in the art.
  • means for detection include a scintillation counter or photographic film as in autoradiography.
  • the label is a fluorescent label, it may be detected by exciting the fluorophore with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • CCDs charge coupled devices
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
  • agglutination assays can be used to detect the presence of the target antibodies.
  • antigen-coated particles are agglutinated by samples comprising the target antibodies.
  • none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
  • an indicator of disease severity can be the absolute or relative amount by which one or more proteins of the profile have increased or decreased as compared to healthy control levels.
  • an indicator of disease severity can be the absolute or relative amount by which one or more proteins of the profile have increased or decreased as compared to healthy control levels.
  • FIG. 4 panels A through C, severity of PD is correlated to decrease in CIT-5 (Apo-Al lipoprotein).
  • Panel A shows that the relative quantity of Apo-Al lipoprotein decrease as the "H+Y" rating of disease severity increases.
  • the least severely affected patients shown as "1" have more Apo-Al lipoprotein than the more severely affected patients (shown as
  • Panel B shows that a decrease in Apo-Al lipoprotein also correlates to age and duration of PD. Young patients with short disease duration (“YS”) have relatively more Apo-Al lipoprotein than young patients with long duration (“YL”) or old patients with either short (“OS”) or long (“OL”) duration disease.
  • Panel C expresses Apo-Al lipoprotein amounts plotted against clinical parameters. A rating of " 1 " indicates short duration PD with no dyskinesia, and no or minimum dopamine therapy. A “2” indicates medium duration disease, dyskinesia and low dopaminergic therapy. A “3” indicates long duration, dyskinesia and moderate to high dopaminergic therapy. As the rating increases, the relative amount of Apo-Al lipoprotein decreases.
  • the disease specific protein profiles which are identified as described herein are also useful in therapeutic monitoring and patient management. For example, using the schizophrenia protein profile described herein, changes in the levels of the individual proteins in response to drug treatment can be evaluated. As shown in Figures 7 to 10, when the protein profile of untreated schizophrenics is compared to those on some conventional treatment regimes, differences in the levels of the individual proteins is observed. In some cases, the treatments drive the protein level closer to normal ( Figures 8 and 10). In this way, it is contemplated that new treatment regimes could be developed by examining the levels of the protein profile. Similarly, the protein profiles of individual subjects could be monitored for example, to determine progression of the disease. The present invention, therefore, provide a rapid and efficient way to monitor treatment regimes and to determine if an individual is deteriorating or improving.
  • the protein profiles are also useful in developing drugs using animal models of disease.
  • Human disease models can be created in laboratory animals, for instance, by inserting a human gene(s) into the animal.
  • the protein profiles obtained by the methods described herein are applicable to these animal models and can be used to monitor treatment protocols and regimes in these animal models.
  • Cerebrospinal fluid samples were kindly provided by various neurologists and veterinarians. The diagnoses were made by the referring physicians according to standard clinical criteria in addition to pathological studies as appropriate. The CSF was collected and the samples were immediately frozen at -70°C before shipment to our laboratory. Upon receipt, samples were thawed and aliquoted for use. Samples for 2-DE were prepared by the addition of 9M urea, 2% 2-mercaptoethanol, 2% NP-40, 0.8% BioLyte pH 3-10 carrier ampholytes, and 0.002% Bromophenol blue. For SDS-PAGE, 50 ⁇ L CSF was added to 20 ⁇ L Laemmli sample buffer, heated for 5 minutes at 95°C, and loaded.
  • Enzymology 3:135-141 Two dimensional electrophoresis consisted of isoeiectric focusing followed by electrophoresis using a polyacrylamide gel. Isoeiectric focusing was done in immobilized pH gradients. The 180 mM linear pH 3-10 gradient (Pharmacia, LKB, Piscataway, New Jersey, USA, catalog #80-1128-30) were used. These gradients were rehydrated, electrophoresed, and equilibrated as described in Bjellqvist et al. (1993)
  • the second dimension was performed in 14% T polyacrylamide gels and silver stained as described in Harrington et al. (1991) Methods: A Companion to Methods in Enzymology 3:98-108 or electroblotted as described in Towbin et al. (1979) Proc. Nat'l. Acad. Sci. 76:4350-4354 onto nitrocellulose (Schleicher & Schuell, catalog # 0830N) or PVDF (Millipore, Massachusetts, USA). The blots were stained with Coomassie Brilliant Blue R-250 or silver stained.
  • the raw data file for each sample is then processed by computer-assisted algorithms.
  • the algorithm was performed as follows. First, the protein spots were identified using an edge detector or peak identifier. The x and y co-ordinates corresponding to pH and molecular weight along with total integrated density were recorded. Next, all spots were compared from one sample to another. A master gel was used to retain all the data.
  • the compared gel data was then split into groups of each disease and normal controls. Similarities and differences were noted. All of the different proteins were then further analyzed in subsets of patients within each disease compared to controls.
  • Proteins altered in neurologic disorders that may not uniquely discriminate the disease by themselves are studied to identify whether in combination a number of proteins can be form a profile which is specific for neurologic disorders as described in Example 2.
  • the proteins can be identified by their migration in the gel.
  • each protein in the profile can be identified by obtaining a molecular footprinting, for example, by one or more of the following: antibody assay, protein sequence determination, mass spectrometry and database comparison.
  • AD-1 through AD-18 are identified by name or by approximate molecular weight and isoeiectric point in Table 1.
  • AD Another profile specific for AD has been identified using a combination of proteins that include 2 of those qualitative markers with 7 other proteins that are altered in various diseases.
  • Parkinson's Disease Six samples from PD patients were used to determine a protein profile as described above. Eight proteins were found to be qualitatively or quantitatively altered in PD samples when compared to normal controls. The protein spots are identified in Table 1.
  • CIT-A, CIT-B and CIT-C were present in PD samples and were not present in healthy controls.
  • CIT-1 alpha 1 microglobulin
  • CIT-3 PGDS isoform
  • CIT-4 complement 4 gamma
  • CIT-5 Apo-Al lipoprotein
  • Example 1 Twenty samples from schizophrenic patients were used in the computation described in Example 1. The profile was composed of seven proteins, or spots, which were altered in presence or amount from 100 healthy controls matched for age and sex and 70 disease controls having disorders including manic depression, CJD, viral encephalitis, subacute sclerosing panencephalitis, Korsakoff s psychosis, benign essential tremor, Huntington's disease, cerebro vascular disease, Alzheimer's disease, motor neuron disease, bacterial meningitis, systemic lupus erythematosis and headache. Results are shown in
  • a protein profile specific for schizophrenia is characterized by decreased amounts of PGDS, orosomucoid, antithrombin III and protein 91 as well as increased amounts of protein 48 and protein 46 when compared to normal CSF levels. Fibrinogen degradation products are absent in normal controls and appear in samples obtained from schizophrenics.
  • Example 2 Twenty samples from multiple sclerosis patients were used in the computation described in Example 1. One hundred age and sex matched controls were used along with 70 other disorders including manic depression, CJD, viral encephalitis, subacute sclerosing panencephalitis, Korsakoff s psychosis, benign essential tremor, Huntington's disease, cerebro vascular disease, Alzheimer's disease, motor neuron disease, bacterial meningitis, systemic lupus erythematosis and headache. Using the methods described above, a protein profile for multiple sclerosis was obtained and is shown in Table 4 and 5.
  • the protein profile for multiple sclerosis includes ApoD (protein 108), ApoD (protein 110), protein 91, protein 92, , ⁇ -glycoprotein, ApoJ/K, transthyretin, C4 ⁇ , and Ig K and L chains. In affected individuals levels of these proteins are elevated as compared to normal controls. Fibrinogen degradation products also appear in these subjects.
  • CJD Creutzfeldt-Jakob Disease
  • CJD Twenty samples of CJD were used to compute a protein profile. The samples were matched by age and sex to 100 normal controls and to 70 disease controls having disorders including manic depression, multiple sclerosis, viral encephalitis, subacute sclerosing panencephalitis, Korsakoff s psychosis, benign essential tremor, Huntington's disease, cerebrovascular disease, Alzheimer's disease, motor neuron disease, bacterial meningitis, systemic lupus erythematosis and headache. As previously discussed, 99% of CJD patients show the appearance of 14-3-3 protein when compared to normal controls. In addition, 67% show the appearance of fibrinogen degradation products.
  • the seven protein markers which make up the protein profile identified in Example 2 were analyzed to determine if their levels were affected by drug treatment. Protein quantity of each marker from normal controls (normal), untreated schizophrenics (none), and schizophrenics taking fluphenazine and chloropromazine (Flu + CPZ) or fluphenazine and lithium (Flu + Li) or fluphenazine alone (Flu). As shown in Figures 2 and 4, levels of protein 48 and protein 46 in schizophrenics taking drugs is closer to normal levels than in untreated patients. Interestingly, as shown in Figure 1, protein 91, which is decreased in the CSF of schizophrenia patients, is further decreased in schizophrenic patients who are following drug treatment.
  • PGDS levels are decreased in both treated and untreated schizophrenic patients, however, in patients taking fluphenazine alone or fluphenazine and lithium, the levels of PGDS are closer to normal controls.
  • Figure 3 The inventor has previously shown that altered levels of PGDS glycoforms are correlated with symptoms of sleep disorders.
  • the present invention also indicates how PGDS levels correlate with sleep disorders.
  • the schizophrenic patients who were on treatment regimes in which PGDS levels were closer to normal also had fewer sleep related symptoms.
  • the present invention provides a method of monitoring treatment regimes and correlating protein levels to symptoms.

Abstract

L'invention concerne des procédés servant à identifier des profils de protéines qui sont spécifiques de troubles neurologiques. Des procédés permettant de diagnostiquer des troubles par une détection du taux de protéines individuelles dans le profil de protéines spécifiques à une maladie sont également décrits. Des profils de protéines spécifiques de la maladie d'Alzheimer, de la maladie de Parkinson, de la sclérose en plaques et de la schizophrénie sont décrits.
PCT/US1998/005045 1997-03-14 1998-03-13 Diagnostic de troubles neurologiques WO1998040748A1 (fr)

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