The present invention provides methods for diagnosing neurodegenerative disease, such as Alzheimer's Disease, Parkinson's Disease, and dementia with Lewy body disease by detecting a pattern of gene product expression in a cerebrospinal fluid sample and comparing the pattern of gene product expression from the sample to a library of gene product expression pattern known to be indicative of the presence or absence of a neurodegenerative disease. The methods also provide for monitoring neurodegenerative disease progression and assessing the effects of therapeutic treatment. Also provided are kits, systems and devices for practicing the subject methods.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention provides methods for diagnosing neurodegenerative disease, such as Alzheimer's Disease, Parkinson's Disease, and dementia with Lewy body disease by detecting a pattern of gene product (e.g., protein) expression in a cerebrospinal fluid sample and comparing the pattern of gene product expression from the sample to a library of gene product expression pattern known to be indicative of the presence or absence of a neurodegenerative disease. Also provided are kits, systems and devices for practicing the subject methods.

The present invention provides a method for detecting presence or absence of a neurodegenerative disease in a subject by detecting a pattern of gene product expression present in a cerebrospinal fluid sample obtained from a subject; and comparing the pattern of gene product expression from the cerebrospinal fluid sample to a library of gene product expression pattern known to be indicative of the presence or absence of a neurodegenerative disease, wherein the comparing indicates the presence or absence of a neurodegenerative disease.

In some embodiments, the gene product is a polypeptide. In some embodiments, the detecting is by mass spectrometry. In other embodiments, the detecting is by immunoassay. In certain embodiments, the immunoassay is enzyme linked immunosorbent assay (ELISA). In other embodiments, the detecting by a Luminex xMAP system. In certain embodiments, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or dementia with Lewy body disease.

The present invention also provides a method for monitoring progression of a neurodegenerative disease in a subject by detecting a first pattern of expression of gene products present in a cerebrospinal fluid sample obtained from a subject at a first time point, wherein said first pattern is indicative of a neurodegenerative disease; detecting a second pattern of expression of gene products present in a cerebrospinal fluid sample obtained from a subject at a second time point; and comparing the first and second patterns of expression of gene products from the cerebrospinal fluid samples, wherein the comparing provides for monitoring of the progression of the neurodegenerative disease from the first time point to the second time point.

In some embodiments, the gene product is a polypeptide. In some embodiments, the detecting is by mass spectrometry. In other embodiments, the detecting is by immunoassay. In certain embodiments, the immunoassay is enzyme linked immunosorbent assay (ELISA). In other embodiments, the detecting by a Luminex xMAP system. In certain embodiments, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or dementia with Lewy body disease.

The present invention also provides a method of providing a differential diagnosis between Alzheimer's disease (AD), Parkinson's disease (PD), and dementia with Lewy body disease (DLB) in a subject by detecting a pattern of gene product expression present in a cerebrospinal fluid sample obtained from a subject; and comparing the pattern of gene product expression from the cerebrospinal fluid sample to a library of gene product expression patterns known to be indicative of the presence or absence of AD, PD and DLB, wherein the comparing providing a differential diagnosis between AD, PD, and DLB.

In some embodiments, the gene product is a polypeptide. In some embodiments, the detecting is by mass spectrometry. In other embodiments, the detecting is by immunoassay. In certain embodiments, the immunoassay is enzyme linked immunosorbent assay (ELISA). In other embodiments, the detecting by a Luminex xMAP system.

The present invention also provides a system, including a computing environment; an input device, connected to the computing environment, to receive data from a user, wherein the data received includes a pattern of gene product expression from a cerebrospinal fluid sample obtained from a subject; an output device, connected to the computing environment, to provide information to the user; and a computer readable storage medium having stored thereon at least one algorithm to provide for comparing the pattern of gene product expression from the cerebrospinal fluid sample to a library of gene product expression pattern known to be indicative of the presence or absence of a neurodegenerative disease. In some embodiments, the computing environment includes a local computer local to the user and a remote computer at a site remote to the user, wherein the local computer and the remote computer are connected through a network, and wherein the computer readable storage medium is provided on the remote computer.

The present invention also provides a computer readable medium including a program stored thereon, wherein the program provides for execution of one or more algorithms to provide for comparing a pattern of gene product expression from a cerebrospinal fluid sample obtained from a subject to a library of gene product expression pattern known to be indicative of the presence or absence of a neurodegenerative disease.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for diagnosing neurodegenerative disease, such as Alzheimer's Disease, Parkinson's Disease, and dementia with Lewy body disease by detecting a pattern of gene product (e.g., protein) expression in a cerebrospinal fluid sample and comparing the pattern of gene product expression from the sample to a library of gene product expression pattern known to be indicative of the presence or absence of a neurodegenerative disease. Also provided are kits and devices for practicing the subject methods.

Before the present invention is described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the marker" includes reference to one or more markers and equivalents thereof known to those skilled in the art, and so forth.

It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely", "only" and the like in connection with the recitation of claim elements, or the use of a "negative" limitation.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Overview

The present invention is based on the identification and quantification of cerebrospinal fluid (CSF) proteins using an unbiased quantitative proteomic approach called iTRAQ (isobaric Tagging for Relative and Absolute protein Quantification) to label pre-fractionated human CSF, and followed by MudPIT (Multidimensional Protein Identification Technology), prior to mass spectrometry (MS) analysis. This multiplex format allowed simultaneous comparison of the proteome of CSF in AD, PD, DLB patients and healthy controls. This analysis not only identified 1,540 CSF proteins (see FIGS. 6A-6T and FIGS. 7A-7V, see Original Patent), thereby greatly expanding the current knowledge about the human CSF proteome, but also detected 136, 73, and 100 proteins that displayed quantitative changes unique to AD, PD, and DLB, respectively. Finally, the sensitivity at 95% specificity of each of eight exemplary markers or composite markers was calculated, demonstrating that the combination of several markers could distinguish between AD, PD and DLB patients not only from controls, but also from each other with high sensitivity at 95% specificity.

In addition, several exemplary panels of unique makers are capable of distinguishing AD, PD and DLB patients from each other as well as from controls with high sensitivity at 95% specificity (see e.g., FIGS. 5A-5YY, see Original Patent).

Methods of the Invention

The invention features methods for diagnosing neurodegenerative disease, such as Alzheimer's Disease, Parkinson's Disease, and dementia with Lewy body disease by detecting a pattern of gene product (e.g., proteins/peptides) expression in a cerebrospinal fluid sample and comparing the pattern of gene product expression from the sample to a library of gene product expression pattern (e.g., FIGS. 5A-5YY, see Original Patent) known to be indicative of the presence or absence of a neurodegenerative disease. In general, the detection of a pattern of gene product expression in a CSF sample obtained form a subject as described herein can be accomplished using any acceptable methodology.

The term's "neurodegeneration" and "neurodegenerative condition or disease" as used in the present application stand for the same and are used interchangeable throughout the application. These terms include any condition of the brain that is associated with a neuronal malfunctioning. Various diseases associated with neurodegeneration include Alzheimer's disease, Parkinson disease, dementia with Lewy Body, Huntington' disease, Creutzfeld Jacob disease, frontal temporal lobe dementia, normal Pressure Hydrocephalus, and amyotrophic lateral sclerosis. However, this list is not complete. Other diseases known to be associated with neuronal malfilrictioning are included as well. In certain embodiments of the present invention, the neurodegenerative disease or condition to be specifically detected, monitored, quantified and/or differentially diagnosed is chosen from the group consisting of Alzheimer's disease, and dementia with Lewy Body.

In general, the method for detecting the presence or absence of a neurodegenerative disease in a subject includes detecting a pattern of gene product expression present in a cerebrospinal fluid sample obtained from a subject; and comparing the pattern of gene product expression from the cerebrospinal fluid sample to a library of gene product expression pattern known to be indicative of the presence or absence of a neurodegenerative disease, wherein the comparing indicates the presence or absence of a neurodegenerative disease.

Any possible combination of gene product(s), such as proteins and peptides, that have an altered level in a CSF sample obtained form a subject under a certain neurological condition can be used for the detection of the presence or absence of a neurological disease, the monitoring of a neurological disease, including assessing therapeutic effects of a treatment regimen (e.g., administration of a therapeutic drug), or the differential diagnosis of AD, PD, or DLB. An exemplary list of candidate gene products that are suitable for use in the detection, monitoring, and differential diagnosis methods of the present invention are summarized in FIGS. 5A-5YY.

Detection of an alerted marker expression pattern(s) in a CSF sample obtained from a subject as compared to that of a normal subject (e.g., a subject known to not have a neurodegenerative disease) is an indicator of neurodegenerative disease, such as AD, PD, or DLB. As with all controls mentioned herein, the control is preferably derived from CSF of subjects without any neurological diseases or taking any medicines for any conditions that might influence neurological functions.

In general, at least enough gene products from FIGS. 5A-5YY are selected for the subject methods that provide for the specific detection of the presence or absence of a neurodegenerative disease. In most embodiments, at least two ore more gene products from FIGS. 5A-5YY are selected for determining the presence or absence of a neurodegenerative disease. In some embodiments, a least three or more genes are selected, including about four or more gene products, and about five or more gene products.

The present invention also provides a method for differential diagnosis between Alzheimer's disease (AD), Parkinson's disease (PD), and dementia with Lewy body disease (DLB) in a subject by detecting a pattern of gene product expression present in a cerebrospinal fluid sample obtained from a subject; and comparing the pattern of gene product expression from the cerebrospinal fluid sample to a library of gene product expression patterns known to be indicative of the presence or absence of AD, PD and DLB, wherein said comparing providing a differential diagnosis between AD, PD, and DLB.

It will be appreciated that the number of gene products selected for use in the present methods will be in part dictated by the specific gene products that are selected for the analysis and whether a general diagnosis of neurodegenerative disease is desired or a differential diagnosis of PD, AD, or DLB is desired. As will be readily apparent to one having skill in the art, the expression level of certain gene products will be modulated as compared to a control in certain conditions and will not be modulated (i.e., decrease or increased) in other conditions as compared to a control. For example, as shown in FIG. 5A, a decrease in expression of BDNF1 is witnessed in AD, while no change (NC) in expression as compared to a control is witnessed in PD or DLB. Likewise, as shown in FIG. 5B, a decrease in expression of Chromogranin B is witnessed in AD, while an increase in expression is witnessed in PD and no change in expression is witnessed in DLB.

As such, in some embodiments, the pattern of gene product expression will be detected and compared to the library of gene product expression patterns known to be indicative of the presence or absence of a neurodegenerative disease. In certain embodiments, the assessment of gene product expression of a single gene product will provide a preliminary result and will be followed up with the assessment of at least a second gene product expression.

The present invention also provides a method for monitoring progression of a neurodegenerative disease in a subject by detecting a first pattern of expression of gene products present in a cerebrospinal fluid sample obtained from a subject at a first time point, wherein said first pattern is indicative of a neurodegenerative disease; detecting a second pattern of expression of gene products present in a cerebrospinal fluid sample obtained from a subject at a second time point; and comparing the first and second patterns of expression of gene products from the cerebrospinal fluid samples, wherein the comparing provides for monitoring of the progression of the neurodegenerative disease from the first time point to the second time point.

In certain embodiments, the method of monitoring progression of a neurodegenerative disease in a subject will include detecting a pattern of expression of gene products present in a CSF sample obtained from a subject at more than two time points, such as three or more. In general, the time points for detecting a pattern of expression of gene products can be separated by any amount of time that is desired. For example, the first time point and second time point can be separated by about 3 months, about 6 months, or about 1 year or more, such as about 3 or more years.

In general, it will be appreciated by one of skill in the art that the duration of time between the first time point and the second time point must be sufficient to provide for a monitoring of the progression of the neurodegenerative disease.

In certain embodiments, the monitoring of the neurodegenerative disease in the subject will be conducted in parallel with a treatment regimen for the neurodegenerative disease. In such embodiments, the method of monitoring the neurodegenerative disease during treatment will provide information of whether the treatment is improving the condition, or having no effect or an adverse effect on the condition. In such embodiments, the first time point may be either just before, concurrent with, or just after the in initiation of a treatment regimen and the second time point may be a time point following a desired treatment period. For example, in such embodiments, the second time point may be about 6 month or more following initiation of treatment, including about 1 year, about 2 years, or more. For example, the detection of the pattern of expression of gene products present in a CSF sample obtained from the subject may be determined about once every 6 months to monitor progression of the disease and efficacy of the treatment regimen.

In general, methods of the invention involving detection of a gene product (e.g., proteins or polypeptides). In one embodiment, the methods involve contacting a sample with a probe specific for the gene product of interest (e.g., marker polypeptide). "Probe" as used herein in such methods is meant to refer to a molecule that specifically binds a gene product of interest (e.g., the probe binds to the target gene product with a specificity sufficient to distinguish binding to target over non-specific binding to non-target (background) molecules). "Probes" include, but are not necessarily limited to, antibodies (e.g., antibodies, antibody fragments that retain binding to a target epitope, single chain antibodies, and the like), or other polypeptide, peptide, or molecule (e.g., receptor ligand) that specifically binds a target gene product of interest.

The probe and sample suspected of having the gene product of interest are contacted under conditions suitable for binding of the probe to the gene product. For example, contacting is generally for a time sufficient to allow binding of the probe to the gene product (e.g., from several minutes to a few hours), and at a temperature and conditions of osmolarity and the like that provide for binding of the probe to the gene product at a level that is sufficiently distinguishable from background binding of the probe (e.g., under conditions that minimize non-specific binding). Suitable conditions for probe-target gene product binding can be readily determined using controls and other techniques available and known to one of ordinary skill in the art.

The probe can be an antibody or other polypeptide, peptide, or molecule (e.g., receptor ligand) that specifically binds a target polypeptide of interest.

The detection methods can be provided as part of a kit. Thus, the invention further provides kits for detecting the presence/absence and/or a level of expression of a marker of the invention, and/or a polypeptide in a human CSF sample. The kits of the invention for detecting a marker polypeptide generally comprise a moiety that specifically binds the polypeptide, which may be a specific antibody. The kit may optionally provide additional components that are useful in the procedure, including, but not limited to, buffers, developing reagents, labels, reacting surfaces, means for detection, control samples, standards, instructions, and interpretive information.

Detecting a Marker Polypeptide in Diagnosing Neurodegenerative Disease

The gene products according to the methods of the present invention can be detected by any suitable method. Detection paradigms that can be employed to this end include enzymatic methods, including immunological-based methods, optical methods, electrochemical methods (voltametry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g., multipolar resonance spectroscopy. It is to be understood that the present invention is not limited to a particular detection method. However, in some embodiments detection is by, for example, fluorescent detection, spectrometric detection, chemiluminescent detection, matrix assisted laser desorption-time-of flight (MALDI-TOF) detection, high pressure liquid chromatographic detection, charge detection, mass detection, radio frequency detection, and light diffraction detection. Exemplary detection methods that are suitable for use with the subject methods are described herein.

Detection by Capture Agent

In some embodiments, detection of gene products is by use of capture reagents specific to the gene products (e.g., polypeptides). In general, the biospecific capture reagent is bound to a solid phase, such as a bead, a plate, a membrane or a chip. Methods of coupling biomolecules, such as antibodies, to a solid phase are well known in the art. They can employ, for example, bifunctional linking agents, or the solid phase can be derivatized with a reactive group, such as an epoxide or an imidizole, that will bind the molecule on contact. Biospecific capture reagents against different gene products can be mixed in the same place, or they can be attached to solid phases in different physical or addressable locations. For example, one can load multiple columns with derivatized beads, each column able to capture a single gene product. Alternatively, one can pack a single column with different beads derivatized with capture reagents against a variety of gene products, thereby capturing all the analytes in a single place. Accordingly, antibody-derivatized bead-based technologies, such as Multi-Analyte Profiling (xMAP.TM.) technology of Luminex (Austin, Tex.) can be used to detect the gene products.

Luminex xMAP.TM. is based on polystyrene particles (microspheres) that are internally labeled with two different fluorophores. When excited by a 635-nm laser, the fluorophores emit light at different wavelengths, e.g., 658 and 712 nm. By varying the 658-nm/712-nm emission ratios, the beads are individually classified by the unique Luminex 100 IS analyzer. A third fluorophore coupled to a reporter molecule allows for quantification of the interaction that has occurred on the microsphere surface. The Luminex xMAP.TM. technology is described, for example, in U.S. Pat. Nos. 5,736,330, 5,981,180, and 6,057,107, all of which are specifically incorporated by reference.

In yet another embodiment, the surfaces of biochips can be derivatized with the capture reagents directed against specific gene products (e.g., selected from FIGS. 5A-5YY). Biochips generally comprise solid substrates and have a generally planar surface, to which a capture reagent (also called an adsorbent or affinity reagent) is attached. Frequently, the surface of a biochip comprises a plurality of addressable locations, each of which has the capture reagent bound there.

Detection by Mass Spectrometry

In some embodiments, the gene products (e.g., polypeptides) are detected by mass spectrometry, a method that employs a mass spectrometer to detect gas phase ions. Examples of mass spectrometers are time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these. In such embodiments, the relative levels of gene products in each sample can be determined with mass spectrometry where a standard curve can be generated using corresponding synthetic peptides without isotope labeling. Alternatively, the gene products (e.g., polypeptides) in the sample can be identified and quantified when the identical synthetic peptides are isotope labeled and spiked in the sample.

In certain embodiments the mass spectrometer is a laser desorption/ionization mass spectrometer. In laser desorption/ionization mass spectrometry, the analytes are placed on the surface of a mass spectrometry probe, a device adapted to engage a probe interface of the mass spectrometer and to present an analyte to ionizing energy for ionization and introduction into a mass spectrometer. A laser desorption mass spectrometer employs laser energy, typically from an ultraviolet laser, but also from an infrared laser, to desorb analytes from a surface, to volatilize and ionize them and make them available to the ion optics of the mass spectrometer.

In general, a probe with an adsorbent surface is contacted with the CSF sample obtained from a subject for a period of time sufficient to allow gene products (e.g., peptides) that may be present in the sample to bind to the adsorbent surface. After an incubation period, the substrate is washed to remove unbound material. Any suitable washing solutions can be used; such as an aqueous solution. The extent to which molecules remain bound can be manipulated by adjusting the stringency of the wash. The elution characteristics of a wash solution can depend, for example, on pH, ionic strength, hydrophobicity, degree of chaotropism, detergent strength, and temperature. An energy absorbing molecule is then applied to the substrate with the bound gene products.

The gene products bound to the substrate are then detected in a gas phase ion spectrometer such as a time-of-flight mass spectrometer or an ion trap mass spectrometer. The gene products are ionized by an ionization source such as a laser, the generated ions are collected by an ion optic assembly, and then a mass analyzer disperses and analyzes the passing ions. The detector then translates information of the detected ions into mass-to-charge ratios. Detection of a gene product typically will involve detection of signal intensity. Thus, both the quantity and mass of the gene product can be determined.

In another mass spectrometry method, the gene product(s) (e.g., polypeptides) can be first captured on a chromatographic resin that binds the target molecules. For example, the resin can be derivatized with anti-gene product proteins antibodies. Alternatively, this method could be preceded by chromatographic fractionation before application to the bio-affinity resin. After elution from the resin, the sample can be analyzed by MALDI, electrospray, or another ionization method for mass spectrometry. In another alternative, one could fractionate on an anion exchange resin and detect by MALDI or electrospray mass spectrometry directly. In yet another method, one could capture the gene product(s) on an immuno-chromatographic resin that comprises antibodies that bind the target molecules, wash the resin to remove unbound material, elute the bound molecules from the resin and detect the eluted proteins by MALDI, electrospray mass spectrometry or another ionization mass spectrometry method.

Detection by Immunoassay

Any of a variety of known immunoassay methods can be used for detection, including, but not limited to, immunoassay, using an antibody specific for the encoded polypeptide, e.g., by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and the like; and functional assays for the encoded polypeptide, e.g., binding activity or enzymatic activity.

For example, an immunofluorescence assay can be easily performed on fractionated or non-fractioned human CSF. It is also possible to perform such assays in plasma if sufficient markers are diffused from human CSF to plasma.

To increase the sensitivity of the assay, the immunocomplex may be further exposed to a second antibody, which is labeled and binds to the first antibody, which is specific for the encoded polypeptide. Typically, the secondary antibody is detectably labeled, e.g., with a fluorescent marker. The cells which express the encoded polypeptide will be fluorescently labeled and easily visualized under the microscope. See, for example, Hashido et al. (1992) Biochem. Biophys. Res. Comm. 187:1241-1248.

As will be readily apparent to the ordinarily skilled artisan upon reading the present specification, the detection methods and other methods described herein can be varied. Such variations are within the intended scope of the invention. For example, in the above detection scheme, the probe for use in detection can be immobilized on a solid support, and the test sample (e.g., human CSF or plasma) contacted with the immobilized probe. Binding of the test sample to the probe can then be detected in a variety of ways, e.g., by detecting a detectable label bound to the test sample.

Thus generally the methods comprise: a) contacting the sample with an antibody specific for a gene product (e.g., a marker selected from FIGS. 5A-5YY); and b) detecting binding between the antibody and molecules of the sample. The level of antibody binding (either qualitative or quantitative) indicates the susceptibility of the patient to a neurodegenerative disease. For example, where the marker polypeptide is present at a level greater than that associated with a negative control level, then the patient is susceptive to neurodegenerative disease.

Suitable controls include a sample known not to contain the marker polypeptide; a sample contacted with an antibody not specific for the marker polypeptide; a sample having a level of polypeptide associated with neurodegenerative disease. A variety of methods to detect specific antibody-antigen interactions are known in the art and can be used in the method, including, but not limited to, standard immunohistological methods, immunoprecipitation, an enzyme immunoassay, and a radioimmunoassay.

In general, the specific antibody will be detectably labeled, either directly or indirectly. Direct labels include radioisotopes; enzymes having detectable products (e.g., luciferase, .beta.-galactosidase, and the like); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, and the like); fluorescence emitting metals, e.g., .sup.152Eu, or others of the lanthanide series, attached to the antibody through metal chelating groups such as EDTA; chemiluminescent compounds, e.g., luminol, isoluminol, acridinium salts, and the like; bioluminescent compounds, e.g., luciferin, aequorin (green fluorescent protein), and the like.

The antibody may be attached (coupled) to an insoluble support, such as a polystyrene plate or a bead. Indirect labels include second antibodies specific for antibodies specific for the encoded polypeptide ("first specific antibody"), wherein the second antibody is labeled as described above; and members of specific binding pairs, e.g., biotin-avidin, and the like. The biological sample may be brought into contact with and immobilized on a solid support or carrier, such as nitrocellulose, that is capable of immobilizing cells, cell particles, or soluble proteins. The support may then be washed with suitable buffers, followed by contacting with a detectably-labeled first specific antibody. Detection methods are known in the art and will be chosen as appropriate to the signal emitted by the detectable label. Detection is generally accomplished in comparison to suitable controls, and to appropriate standards.

Polypeptide Arrays

Polypeptide arrays provide a high throughput technique that can assay a large number of polypeptides in a sample. This technology can be used as a tool to test for expression of a marker polypeptide and assessment of neurodegenerative disease. Of particular interest are arrays which comprise a probe for detection of one or more of the gene products selected from FIGS. 5A-5YY.

A variety of methods of producing arrays, as well as variations of these methods, are known in the art and contemplated for use in the invention. For example, arrays can be created by spotting polypeptide probes onto a substrate (e.g., glass, nitrocellulose, etc.) in a two-dimensional matrix or array having bound probes. The probes can be bound to the substrate by either covalent bonds or by non-specific interactions, such as hydrophobic interactions.

Samples of polypeptides can be detectably labeled (e.g., using radioactive or fluorescent labels) and then hybridized to the probes. Alternatively, the polypeptides of the test sample can be immobilized on the array, and the probes detectably labeled and then applied to the immobilized polypeptides. In most embodiments, the "probe" is detectably labeled. In other embodiments, the probe is immobilized on the array and not detectably labeled. In such embodiments, the sample is applied to the polypeptide array and bound gene products (e.g., peptides) are detected using secondary labeled probes

Examples of such protein arrays are described in the following patents or published patent applications: U.S. Pat. No. 6,225,047; PCT International Publication No. WO 99/51773; U.S. Pat. No. 6,329,209, PCT International Publication No. WO 00/56934 and U.S. Pat. No. 5,242,828.

Computer-Based Systems and Methods

The invention also provides a variety of computer-related embodiments. Specifically, the automated means for performing the methods described above may be controlled using computer-readable instructions, i.e., programming. Accordingly, in some embodiments the invention provides computer programming for analyzing and comparing a pattern of gene product expression present in a CSF sample obtained from a subject to a library of gene product expression patterns known to be indicative of the presence or absence of a neurodegenerative disease, wherein the comparing indicates the presence or absence of a neurodegenerative disease.

In another embodiment, the invention provides computer programming for analyzing and comparing a first and a second pattern of expression of gene products from CSF samples takes from a subject in at least two different time points, wherein the first pattern is indicative of a neurodegenerative disease. In such embodiments, the comparing provides for monitoring of the progression of the neurodegenerative disease from the first time point to the second time point.

In yet another embodiment, the invention provides computer programming for analyzing and comparing a pattern of gene product expression from CSF sample to a library of gene product expression patterns known to be indicative of the presence or absence of AD, PD and DLB, wherein the comparing providing a differential diagnosis between AD, PD, and DLB.

The methods and systems described herein can be implemented in numerous ways. In one embodiment of particular interest, the methods involve use of a communications infrastructure, for example the internet. Several embodiments of the invention are discussed below. It is also to be understood that the present invention may be implemented in various forms of hardware, software, firmware, processors, or a combination thereof. The methods and systems described herein can be implemented as a combination of hardware and software. The software can be implemented as an application program tangibly embodied on a program storage device, or different portions of the software implemented in the user's computing environment (e.g., as an applet) and on the reviewer's computing environment, where the reviewer may be located at a remote site (e.g., at a service provider's facility).

For example, during or after data input by the user, portions of the data processing can be performed in the user-side computing environment. For example, the user-side computing environment can be programmed to provide for defined test codes to denote platform, carrier/diagnostic test, or both; processing of data using defined flags, and/or generation of flag configurations, where the responses are transmitted as processed or partially processed responses to the reviewer's computing environment in the form of test code and flag configurations for subsequent execution of one or more algorithms to provide a results and/or generate a report in the reviewer's computing environment.

The application program for executing the algorithms described herein may be uploaded to, and executed by, a machine comprising any suitable architecture. In general, the machine involves a computer platform having hardware such as one or more central processing units (CPU), a random access memory (RAM), and input/output (I/O) interface(s). The computer platform also includes an operating system and microinstruction code. The various processes and functions described herein may either be part of the microinstruction code or part of the application program (or a combination thereof) which is executed via the operating system. In addition, various other peripheral devices may be connected to the computer platform such as an additional data storage device and a printing device.

As a computer system, the system generally includes a processor unit. The processor unit operates to receive information, which generally includes test data (e.g., specific gene products assayed), and test result data (e.g., the pattern of gene product expression for a sample). This information received can be stored at least temporarily in a database, and data analyzed in comparison to a library of gene product expression patterns known to be indicative of the presence or absence of a neurodegenerative disease, including PD, AD, and DLB, as described above.

Part or all of the input and output data can also be sent electronically; certain output data (e.g., reports) can be sent electronically or telephonically (e.g., by facsimile, e.g., using devices such as fax back). Exemplary output receiving devices can include a display element, a printer, a facsimile device and the like. Electronic forms of transmission and/or display can include email, interactive television, and the like. In an embodiment of particular interest, all or a portion of the input data and/or all or a portion of the output data (e.g., usually at least the library of gene product expression patterns known to be indicative of the presence or absence of a neurodegenerative disease) are maintained on a server for access, preferably confidential access. The results may be accessed or sent to professionals as desired.

A system for use in the methods described herein generally includes at least one computer processor (e.g., where the method is carried out in its entirety at a single site) or at least two networked computer processors (e.g., where gene product expression data for a CSF sample obtained from a subject is to be input by a user (e.g., a technician or someone performing the activity assays)) and transmitted to a remote site to a second computer processor for analysis (e.g., where the pattern of gene expression is compared to a library of gene product expression patterns known to be indicative of the presence or absence of a neurodegenerative disease), where the first and second computer processors are connected by a network, e.g., via an intranet or internet). The system can also include a user component(s) for input; and a reviewer component(s) for review of data, and generation of reports, including detection of neurodegenerative disease, differential diagnosis of PD, AD, and DLB, or monitoring the progression of a neurodegenerative disease. Additional components of the system can include a server component(s); and a database(s) for storing data (e.g., as in a database of report elements, e.g., a library of gene product expression patterns known to be indicative of the presence or absence of a neurodegenerative disease, or a relational database (RDB) which can include data input by the user and data output. The computer processors can be processors that are typically found in personal desktop computers (e.g., IBM, Dell, Macintosh), portable computers, mainframes, minicomputers, or other computing devices.

The networked client/server architecture can be selected as desired, and can be, for example, a classic two or three tier client server model. A relational database management system (RDMS), either as part of an application server component or as a separate component (RDB machine) provides the interface to the database.

In one embodiment, the architecture is provided as a database-centric user/server architecture, in which the user application generally requests services from the application server which makes requests to the database (or the database server) to populate the activity assay report with the various report elements as required, especially the assay results for each activity assay. The server(s) (e.g., either as part of the application server machine or a separate RDB/relational database machine) responds to the user's requests.

The input components can be complete, stand-alone personal computers offering a full range of power and features to run applications. The user component usually operates under any desired operating system and includes a communication element (e.g., a modem or other hardware for connecting to a network), one or more input devices (e.g., a keyboard, mouse, keypad, or other device used to transfer information or commands), a storage element (e.g., a hard drive or other computer-readable, computer-writable storage medium), and a display element (e.g., a monitor, television, LCD, LED, or other display device that conveys information to the user). The user enters input commands into the computer processor through an input device. Generally, the user interface is a graphical user interface (GUI) written for web browser applications.

The server component(s) can be a personal computer, a minicomputer, or a mainframe and offers data management, information sharing between clients, network administration and security. The application and any databases used can be on the same or different servers.

Other computing arrangements for the user and server(s), including processing on a single machine such as a mainframe, a collection of machines, or other suitable configuration are contemplated. In general, the user and server machines work together to accomplish the processing of the present invention.

Where used, the database(s) is usually connected to the database server component and can be any device which will hold data. For example, the database can be any magnetic or optical storing device for a computer (e.g., CDROM, internal hard drive, tape drive). The database can be located remote to the server component (with access via a network, modem, etc.) or locally to the server component.

Where used in the system and methods, the database can be a relational database that is organized and accessed according to relationships between data items. The relational database is generally composed of a plurality of tables (entities). The rows of a table represent records (collections of information about separate items) and the columns represent fields (particular attributes of a record). In its simplest conception, the relational database is a collection of data entries that "relate" to each other through at least one common field.

Additional workstations equipped with computers and printers may be used at point of service to enter data and, in some embodiments, generate appropriate reports, if desired. The computer(s) can have a shortcut (e.g., on the desktop) to launch the application to facilitate initiation of data entry, transmission, analysis, report receipt, etc. as desired.

Computer-Readable Storage Media

The invention also contemplates a computer-readable storage medium (e.g. CD-ROM, memory key, flash memory card, diskette, etc.) having stored thereon a program which, when executed in a computing environment, provides for implementation of algorithms to carry out all or a portion of the methods described herein, including detection of neurodegenerative disease, differential diagnosis of PD, AD, and DLB, or monitoring the progression of a neurodegenerative disease. Where the computer-readable medium contains a complete program for carrying out the methods described herein, the program includes program instructions for collecting, analyzing and comparing a pattern of gene product expression patterns from a CSF sample obtained from a subject to a library of gene product expression patterns known to be indicative of the presence or absence of a neurodegenerative disease, and generally includes computer readable code devices for interacting with a user as described herein, processing that data in conjunction with analytical information, and generating unique printed or electronic media for that user.

Where the storage medium provides a program which provides for implementation of a portion of the methods described herein (e.g., the user-side aspect of the methods (e.g., data input, report receipt capabilities, etc.)), the program provides for transmission of data input by the user (e.g., via the internet, via an intranet, etc.) to a computing environment at a remote site. Processing or completion of processing of the data may be carried out at the remote site to provide for detection of neurodegenerative disease, differential diagnosis of PD, AD, and DLB, or monitoring the progression of a neurodegenerative disease. The computer-readable storage medium can also be provided in combination with one or more reagents for carrying out one or more of the activity assays (e.g., control compounds, cells, probes, arrays, or other activity assay test kit components).

Kits

Also provided by the subject invention are kits for practicing the subject methods, as described above, including detection of neurodegenerative disease, differential diagnosis of PD, AD, and DLB, or monitoring the progression of a neurodegenerative disease. The subject kits include at least one or more of: a probe or primer for detection of a marker polynucleotide, a marker polypeptide, or an anti-marker polypeptide antibody. Other optional components of the kit include: restriction enzymes, control primers and plasmids; nucleic acid or polypeptide standards; buffers; reaction mixtures (e.g., for carrying out the assay); enzymes (e.g., DNA polymerase, reverse transcriptase, and the like); cells; and the like. The various components of the kit may be present in separate containers or certain compatible components may be precombined into a single container, as desired.

In addition to above-mentioned components, the subject kits typically further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
 

Claim 1 of 6 Claims

1. A method of aiding in a differential diagnosis between Alzheimer's disease (AD), Parkinson's disease (PD), and dementia with Lewy body disease (DLB) in a subject having clinical presentations of neurodegenerative diseases, said method comprising: detecting a pattern of gene product expression for at least five gene products listed in FIGS. 5A-5YY in a cerebrospinal fluid sample from the subject; and comparing the detected pattern of gene product expression from the cerebrospinal fluid sample to a library of gene product expression patterns known to provide a differential diagnosis between AD, PD and DLB as represented in FIGS. 5A-5YY, wherein when the detected pattern corresponds to a pattern as represented in FIGS. 5A-5YY a differential diagnosis between AD, PD, and DLB is made.

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