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WO2014071359A1 - Détection de maladies neurologiques par mesure de la neuromélanine dans des phagocytes en recirculation - Google Patents

Détection de maladies neurologiques par mesure de la neuromélanine dans des phagocytes en recirculation Download PDF

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Publication number
WO2014071359A1
WO2014071359A1 PCT/US2013/068465 US2013068465W WO2014071359A1 WO 2014071359 A1 WO2014071359 A1 WO 2014071359A1 US 2013068465 W US2013068465 W US 2013068465W WO 2014071359 A1 WO2014071359 A1 WO 2014071359A1
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WIPO (PCT)
Prior art keywords
sample
fluid
biomarker
level
neuromelanin
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English (en)
Inventor
Ramesh C. Nayak
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MSDX Inc
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MSDX Inc
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Priority to CA2929711A priority Critical patent/CA2929711A1/fr
Publication of WO2014071359A1 publication Critical patent/WO2014071359A1/fr
Priority to US14/704,791 priority patent/US20150233904A1/en
Anticipated expiration legal-status Critical
Priority to US17/228,416 priority patent/US20220236288A1/en
Priority to US17/719,177 priority patent/US20220236291A1/en
Priority to US18/379,431 priority patent/US20240036065A1/en
Ceased legal-status Critical Current

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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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • 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
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • phagocytes In general, when tissue damage occurs, it incites inflammation, which usually aids in wound healing. For example, one of the normal functions of inflammation is to recruit phagocytes to clear away the cellular debris and prepare the injured site for repair and rebuilding. These phagocytes may be resident in the brain (e.g., dendritic ceils, microglial cells) or recruited from the blood stream (e.g., monocytes). Ceils that engulf debris are thought to enter the brain by crossing the blood-brain barrier but are not believed to return to the blood stream. For example, when phagocytes engulf tissue debris and exit the tissue, it is thought to be via the lymph notes.
  • phagocytes engulf tissue debris and exit the tissue, it is thought to be via the lymph notes.
  • the present invention also features methods for detecting Parkinson's disease by the detection of melanin (e.g., neuromelanin, e.g., neuromelanin from neurons of substantia nigra) and other neuronal antigens in recirculating phagocytes.
  • melanin e.g., neuromelanin, e.g., neuromelanin from neurons of substantia nigra
  • other neuronal antigens in recirculating phagocytes e.g., Parkinson's disease by the detection of melanin (e.g., neuromelanin, e.g., neuromelanin from neurons of substantia nigra) and other neuronal antigens in recirculating phagocytes.
  • the present invention features a method of detecting Parkinson's disease in a mammal.
  • the method comprises detecting a level of a biomarker associated with Parkinson's disease in a first sample from outside a brain tissue of the mammal, the first sample comprising a first circulating phagocyte; and comparing the level of the biomarker in the first sample with a level of the biomarker in a second sample, the second sample being either (i) a control sample or (ii) a second sample from outside of a brain tissue, the second sample comprising a second circulating phagocyte, the second sample being collected prior to the first fluid sample, wherein if the level of the biomarker in the first sample is higher than that of the second sample then Parkinson's disease is detected.
  • the sample is derived from blood, peripheral blood mononuclear cells (PBMCs), cerebrospinal fluid (CSF), synovial fluid, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, ocular fluids, vitreai fluid, urine, the like, or a combination thereof.
  • the biomarker associated with Parkinson's disease comprises neuromelanin or a fragment thereof.
  • the circulating phagocyte includes a monocyte, a macrophage, a lymphocyte, or a combination thereof.
  • detecting the biomarker comprises subjecting the first sample and the second sample each to a peptide that binds to neuromelanin.
  • the peptide that binds to neuromelanin comprises 4B4 (SEQ ID NO:1 A).
  • the present invention also features a kit for detecting Parkinson's disease, said kit comprising a 4B4 peptide (SEQ ID NO:1 A), the 4B4 peptide is for detecting neuromelanin in a recirculating phagocyte.
  • the 4B4 peptide comprises a label.
  • the label comprises biotin.
  • the present invention also features the use of a system for detecting Parkinson's disease.
  • the system comprises a neuromeianin-binding peptide for binding to neuromelanin, the neuromelanin- binding peptide is incubated in a first sample comprising a first circulating phagocyte from outside of a brain tissue and a second sample comprising a control sample, wherein if the level of neuromelanin detected in the first sample via the neuromeianin-binding peptide is higher than the level of neuromelanin detected in the second sample via the neuromeianin-binding peptide then Parkinson's disease is detected.
  • the neuromeianin-binding peptide comprises 4B4 (SEQ ID NO:1A).
  • the first sample is derived from blood.
  • the first sample comprises PBMCs.
  • the present invention also features a system for detecting Parkinson's disease, wherein the system comprises a neuromeianin-binding peptide for binding to neuromelanin, the neuromeianin-binding peptide is incubated in a first sample comprising a first circulating phagocyte from outside of a brain tissue and a second sample comprising a control sample, wherein if the level of neuromelanin detected in the first sample via the neuromeianin-binding peptide is higher than the level of neuromelanin detected in the second sample via the neuromeianin-binding peptide then Parkinson's disease is detected.
  • the neuromeianin-binding peptide comprises 4B4 (SEQ ID NO:1 A).
  • the first sample is derived from blood.
  • the first sample comprises PBMCs.
  • the present invention also features a method of determining status of Parkinson's disease.
  • method comprises detecting a level of a biomarker associated with Parkinson's disease in a first fluid sample from outside a brain tissue of the mammal, the first fluid sample comprising a first circulating phagocyte; comparing the level of the biomarker in the first sample with a level of the biomarker in a second sample, the second sample being either (I) a control sample or (ii) a second fluid sample from outside of a brain tissue, the second fluid sample comprising a second circulating phagocyte, the second fluid sample being collected prior to the first fluid sample.
  • the biomarker level in the first sample is the same as the level of the biomarker in the second sample or in the control sample, then Parkinson's disease activity is the same. In some embodiments, if the biomarker level in the first sample is higher than the level of the biomarker in the second sample or in the control sample, then Parkinson's disease activity is increased in the first sample. In some embodiments, if the biomarker level in the first sample is lower than the level of the biomarker in the second sample or in the control sample, then Parkinson's disease activity is decreased in the first sample.
  • the sample is derived from blood, peripheral blood mononuclear cells (PBMCs), cerebrospinal fluid (CSF), synovial fluid, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, ocular fluids, vitreai fluid, urine, the like, or a combination thereof.
  • the circulating phagocyte includes a monocyte, a macrophage, a lymphocyte, or a combination thereof.
  • the biomarker comprises neuromeianin or a fragment thereof.
  • the present invention also features a method of detecting neuromeianin.
  • the method comprises introducing a neuromeianin binding protein comprising a labeled 4B4 peptide (SEQ ID NO:1 A) to a sample; and detecting the label on the 4B4 peptide.
  • the sample comprises a circulating phagocyte.
  • the sample comprises a circulating phagocyte derived from serum, plasma, peripheral blood mononuclear cells (PBMCs), cerebrospinal fluid (CSF), synovial fluid, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, ocular fluids, vitreai fluid, or a combination thereof.
  • the label comprises an enzyme.
  • the label comprises biotin.
  • the enzyme comprises horseradish peroxidase.
  • the present invention also features a method of detecting Parkinson's disease in a patient.
  • the method comprises obtaining from a patient a fluid sample from outside of a brain tissue of the patient, the fluid sample comprises peripheral blood mononuclear cells (PBMCs); and detecting neuromelanin in the fluid sample, wherein when neurome!anin is detected then Parkinson's disease is detected in the patient.
  • the fluid sample comprises a circulating phagocyte.
  • the circulating phagocyte includes a monocyte, a macrophage, or a lymphocyte.
  • FIG. 1 Groups of plasma samples from multiple sclerosis (MS) patients that had low, medium or high levels of MSDx compiex-1 were selected and then MMP-9 was measured.
  • TiMP-1 levels were higher in subjects with low MSDx complex -1 levels (consistent with low proteolytic activity and potentially lower levels of leukocyte invasion and disease activity).
  • MS subjects with high MSDX compiex-1 level had lower levels of TIMP-1 (consistent with higher proteolytic activity and potentially higher levels of leukocyte invasion and disease activity; TIMP-1 in MSDx complex-1 high vs. Low p ⁇ 0.0033).
  • FIG. 2 Image (a) on the left shows neuromelanin-containing dopaminergic neurons in the human substantia nigra revealed by the Masson- Fontana stain. Image (b) on the right shows neuromelanin-containing dopaminergic neurons in the human substantia nigra revealed by the 4B4 peptide binding (4B4 peptide binds to neuromelanin in substantia nigra tissue sections).
  • FIG. 3 shows the binding of the 4B4 peptide to neuromelanin in extracts of retinal pigment epithelium immobilized on ELISA plates in two-fold dilution series.
  • the present invention features the detection and/or the monitoring of diseases, e.g., neurodegenerative diseases, Parkinson's disease, etc., by analysis of phagocytosed central nervous system (CNS) debris within phagocytes that have re-entered the blood circulation (e.g., recirculating phagocytes).
  • diseases e.g., neurodegenerative diseases, Parkinson's disease, etc.
  • CNS central nervous system
  • MSDx comp!ex-1 comprises Fibrinogen, Fibulin-1 and Fibronectin. Fibronectin and Fibulin-1 are basement membrane proteins, suggesting that the circulating complex may be generated as a consequence of leukocyte transmigration into target tissues. Transmigration of leukocytes is mediated by the enzyme activity of matrix metailoproteinases (MMPs).
  • MMPs matrix metailoproteinases
  • FIG. 1 shows that in plasma samples of Multiple Sclerosis patients, levels of MSDx complex-1 may be indirectly related to TIMP-1 levels (TSMPs are tissue inhibitors of metailoproteinases). For example, higher levels of TIMP-1 (a specific inhibitor of MMP-9) may be associated with lower activity of MMP-9 and lower level of MSDx complex-1 (see FIG. 1 ). Conversely, a lower level of TIMP-1 may be associated with a higher level of MMP-9 activity and level of MSDx complex-1. Intermediate levels of TIMP-1 correlate with intermediate levels of MSDx complex- 1 .
  • TIMP-1 tissue inhibitors of metailoproteinases
  • MSDx complex-1 may be generated by cell transmigration into tissues.
  • MSDx complex-1 may be generated by proteolytic activity of leukocytes (or other ceil types) crossing the blood vessel wail and tissue barriers in order to enter the target organ.
  • a disease that is characterized by movement of leukocytes (or other cell types) into tissues can be monitored by the measurement of MSDx complex-1.
  • Diseases that may be monitored or detected by measurement of MSDx compiex-1 include but are not limited to: autoimmune diseases, e.g., multiple sclerosis, rheumatoid arthritis, lupus, Sjogren's syndrome, thyroiditis, uveitis, Crohn's disease, ulcerative colitis, psoriasis, type 1 diabetes mel!itus, autoimmune Addison's disease, autoimmune hepatitis, celiac disease, pemphigous, chronic inflammatory demyelinating polyneuropathy, acute disseminated encephalomyeiopathy, sarcoidosis, dermatomyositis and behcet's disease; neurological diseases, e.g., stroke, concussion, chronic traumatic encephalopathy, neuromyelitis optica, transverse mye disease,
  • Various debris antigens may be found in recirculating phagocytes in the peripheral blood. Such debris antigens may be used to detect (or monitor) neurodegenerative or neuroinfiammatory diseases (e.g., diseases as described above).
  • Antigens include but are not limited to: a Tau protein (or fragment thereof), a Tau protein or fragment thereof comprising a phosphoryiated residue (e.g., a phosphoryiated serine reside, a phosphoryiated threonine reside; e.g., serine 214, serine 235, serine 282, serine 356, serine 398, serine 404, serine 413, serine 46, serine 515, serine 516, serine 519, serine 531 , serine 552, serine 810, serine 622, serine 641 , serine 713, serine 721 , serine 726, serine 730, serine 739, threonine 181 , threonine
  • Another debris antigen that may be found in recirculating phagocytes in the peripheral blood may include neuromelanin (or a fragment thereof).
  • Neuromelanin may be used to detect Parkinson's disease.
  • neuromelanin may be detected in the debris of degenerated dopaminergic neurons (by recirculating phagocytes).
  • Neuromelanin can be measured in several ways, e.g., via the binding of labeled melanin selective peptides (e.g., 4B4 peptide (SEQ ID NO:1A), e.g., biotinyiated 4B4 peptide, a control peptide P601 G (DGASYSWMYGA (SEQ ID NO:2A)) may be used as a control); the binding of monoclonal or polyclonal antibodies to melanin; measurement of metal binding to melanin; measurement of the semiconductor properties of melanin; measurement of the fluorescence properties of melanin; and extraction of melanin from recirculating phagocytes and subsequent quantification of melanin, it's components or adducts (both natural or synthetic); physical methods such as gas chromatography, liquid chromatography or mass spectrometry; and combinations of these methods.
  • labeled melanin selective peptides e.g., 4B4 peptide (SEQ ID NO:1A
  • the 4B4 peptide of sequence YERKFWHGRH (SEQ SD NO:1 A) binds to neuromelanin granules in the dopaminergic neurons of the human substantia nigra.
  • FIG., 3 shows the binding of the 4B4 peptide. Extracts of retinal pigment epithelium were immobilized on ELISA plates in two-fold dilution series of the extract and incubated with biotinyiated 4B4 peptide. Unbound peptide was washed off and bound peptide was detected with streptavidin-HRP. In comparison, PB Cs from healthy human subjects show little binding of 4B4 peptide.
  • more than one biomarker is defected in the sampie(s).
  • the biomarker(s) is a neural-derived biomarker.
  • the biomarker(s) is not limited to neural-derived biomarkers.
  • one or more biomarkers are detected in the sample, wherein the biomarkers are neural-derived, non-neural-derived biomarkers, or a combination thereof.
  • peripheral phagocyte refers to anything outside of brain tissue.
  • a peripheral phagocyte may be obtained from cerebrospinal fluid (CSF).
  • Phagocytes may include monocytes, macrophages, and/or lymphocytes.
  • Such circulating phagocytes may be found in tissues, cells, and/or fluids in the body, for example in blood, peripheral blood mononuclear cells (PBMCs), synovial fluid, cerebrospinal fluid (CSF), central nervous system tissues, synovial fluid, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, ocular fluids, vitreal fluid, urine the like, or a combination thereof.
  • the biomarker is an intracellular component.
  • the biomarker may be obtained from within a macrophage.
  • the macrophage sample is permeabi!ized.
  • the biomarker is an intracellular component.
  • the biomarker may be obtained from within a macrophage.
  • the macrophage sample is perme
  • macrophage is lysed via various means, e.g., hypotonic solution treatment, detergent solution treatment, mechanical stress, etc.
  • the present invention features the detection of and/or the monitoring of various diseases via detection/measurement of various biomarkers in recirculating phagocytes.
  • the a disease detected or monitored includes (but is not limited to): autoimmune diseases, e.g., multiple sclerosis, rheumatoid arthritis, lupus, Sjogren's syndrome, thyroiditis, uveitis, Crohn's disease, ulcerative colitis, psoriasis, type 1 diabetes mellitus, autoimmune addison's disease, autoimmune hepatitis, celiac disease, pemphigous, chronic inflammatory demye!inating polyneuropathy, acute disseminated encephalomyelopathy, sarcoidosis, dermatomyositis and behcet's disease; neurological diseases, e.g., stroke, concussion, chronic traumatic encephalopathy, neuromyelitis optica, transverse myelitis, intractable epilepsy and CNS infections; Parkinson's disease; primary tumor growth, metastasis of tumors; etc.
  • autoimmune diseases e.g., multiple sclerosis,
  • a biomarker detected or measured in recirculating phagocytes includes (but is not limited to): Neuromelanin (or a fragment thereof); a Tau protein (or fragment thereof), a Tau protein or fragment thereof comprising a phosphorylated residue (e.g., a phosphorylated serine reside, a phosphorylated threonine reside; e.g., serine 214, serine 235, serine 262, serine 358, serine 398, serine 404, serine 413, serine 46, serine 515, serine 518, serine 519, serine 531 , serine 552, serine 810, serine 622, serine 841 , serine 713, serine 721 , serine 728, serine 730, serine 739, threonine 181 , threonine 205, threonine 470, threonine 492, threonine 498, threonine
  • Table A shows non-limiting examples of biomarkers that may be associated with disease states (e.g., degenerative disease states) with various organs.
  • the present invention may be used to detect a diabetes condition by detecting somatostatin in a similar manner as described herein, e.g., similar to methods for detecting neuromelanin for Parkinson's disease.
  • hydrolase L1 also known as PARKS protein; neuronal-speclflc protein gene product 9,5; SwissProt P09936; proteoilpid protein; myelin oligodendrocyte glycoprotein.
  • Thyroid eg Graves disease, Hashi motos Thyroglobuli n
  • Retina eg macular degeneration, retinitis Rhodopsin
  • Pancreatic islets Insulin, Glucagon , somatostati n,
  • SBDPs severe traumatic brain injury patients ail-spectrin breakdown products
  • MS Multiple Sclerosis
  • CNS central nervous system
  • MS is a demyelinating disease, where myelin, the insulating layer on nerve fibers, is destroyed in the CNS, which consists of the brain, optic nerves, and spinal column. There is an accompanying inflammatory response and the blood brain barrier (BBB) is breached. Axon damage can occur and the optic nerve is commonly affected. Myelin damage makes it more difficult for nerves to transmit impulses, leading to symptoms of MS.
  • the diagnostic McDonald Criteria (1 ) were revised in 2005 to include magnetic resonance imaging (MR! criteria of different types of lesions of the brain and spinal cord in the diagnosis of MS. Prognosis is difficult to determine, and many brain lesions do not necessarily correlate with seventy of disease. There are medications available to alleviate some symptoms and a few others to modify and hopefully delay the onset or severity of relapses of MS.
  • RRMS reiapsing-remitting multiple sclerosis
  • a first episode is referred to as Clinically Isolated Syndrome (CIS) pending a more certain diagnosis of MS corresponding to clinical signs and/or brain lesions visualized by MRL or possibly a spinal tap to check for immunoglobulin oligoclonal bands (OCB) in the cerebral spinal fluid (CSF). None of these diagnostic methods is 100% specific. (2). Its drawbacks include the expense and the fact that a patient must wait one to three months between scans to determine if new lesions have formed during the intervening period. There is a clear need for identification of a biomarker or set of biomarkers that indicate presence and/or severity of disease for MS patients. A simple blood test would be ideal for diagnosing MS, however at this time, no commercial blood test exists.
  • the present invention features a method of detecting multiple sclerosis or a risk of multiple sclerosis.
  • the present invention also features methods of determining the status of a disease or condition or monitoring disease activity and drug efficacy.
  • the method comprises detecting a multiple sclerosis-associated biomarker, e.g., an antigen, wherein detecting an elevated level of such multiple sderosis-associated biomarker indicates the presence of multiple sclerosis or a risk of multiple sclerosis.
  • the antigens detected in accordance with the present invention includes, for example, Ubiquitin carboxy- terminal hydrolase L1 (UCHL1 ) also known as PARKS protein; neuronai-specific protein gene product 9.5; SwissProt P09936.
  • the present invention features a method of detecting an inflammatory condition.
  • the method comprises providing a first sample (e.g., a fluid sample) that contains a peripheral (e.g., circulating) phagocyte, and detecting one or more biomarkers, e.g., an antigen, inside a phagocyte of said fluid sample, wherein the biomarker is associated with an inflammatory condition.
  • the sample may be provided from a mammal (e.g., a patient, a mouse, a rat, etc.).
  • the fluid obtained does not necessarily directly come into contact with the inflamed tissue being detected.
  • the fluid obtained may have once directly come into contact with the inflamed tissue, but at the time that it is being extracted in accordance with the present invention, it is being separated from the inflamed tissue by a barrier.
  • the method may further comprise comparing the level of the biomarker in the first sample with a level of the biomarker in a second sample (e.g., a fluid sample).
  • the second sample may be a control sample.
  • the second sample is a fluid sample from outside of a brain tissue comprising a peripheral (e.g., circulating phagocyte),
  • the second sample may be provided from a mammal (e.g., a patient, a mouse, a rat, etc.).
  • the second sample may have been collected prior to the first fluid sample.
  • peripheral phagocyte refers to anything outside of brain tissue.
  • a peripheral phagocyte may be obtained from cerebrospinal fluid (CSF).
  • Phagocytes may include monocytes, macrophages, and/or lymphocytes.
  • Such circulating phagocytes may be found in tissues, ceils, and/or fluids in the body, for example in blood, peripheral blood mononuclear ceils (PBMCs), synovial fluid, cerebrospinal fluid (CSF), central nervous system tissues, synovial fluid, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, ocular fluids, vitreai fluid, urine the like, or a combination thereof.
  • PBMCs peripheral blood mononuclear ceils
  • CSF cerebrospinal fluid
  • central nervous system tissues synovial fluid
  • cystic fluid cystic fluid
  • lymph fluid ascites, pleural effusion, interstitial fluid, ocular fluids, vitreai fluid, urine the like, or
  • the biomarker is an intracellular component.
  • the biomarker may be obtained from within a macrophage.
  • the macrophage sample is permeabilized.
  • the macrophage is lysed via various means, e.g., hypotonic solution treatment, detergent solution treatment, mechanical stress, etc.
  • the sample is a plasma sample.
  • a fluid that does not directly come into contact with the inflamed tissue is a fluid that is separated from the inflamed tissue by at least one barrier, e.g., a tissue membrane, a layer of ceils, etc.
  • one or more biomarkers are detected in the collected fluid sample.
  • a pattern of biomarkers may be detected in the sample. Detecting the biomarker or biomarkers indicates the presence of the inflammatory condition or a risk of the inflammatory condition.
  • detecting an increased level of the biomarker or biomarkers as compared to the level of the biomarker or biomarkers of a control sample indicates the presence of the inflammatory condition or a risk thereof.
  • a control sample is discussed below.
  • detecting a decreased level of the biomarker or biomarkers as compared to the level of the biomarker or biomarkers of a control sample indicates the presence of the inflammatory condition or a risk thereof.
  • the inflammation condition that may be monitored or detected includes Rheumatoid Arthritis, Systemic Lupus Erythematosis, Shogren's Syndrome, and the like.
  • the biomarker(s) is a neural-derived biomarker.
  • the biomarker(s) is not limited to neural-derived biomarkers.
  • one or more biomarkers are detected in the sample, wherein the biomarkers are neural-derived, non-neural-derived biomarkers, or a combination thereof.
  • the biomarker(s) may be detected using a variety of methods. Methods may include an immunoassay, a histological assay, a flow cytometry assay, the like, or a combination thereof.
  • the step of detecting the biomarker(s) in the sample may comprise introducing an antibody to the sample, wherein the antibody binds to the biomarker or is specific for the biomarker.
  • this method of detecting an inflammatory condition is used in combination with one or more different methods for detecting the inflammatory disease. In some embodiments, this method is used to differentiate between one or more inflammatory conditions.
  • the present invention also features a method of detecting a neurological condition.
  • the method comprises providing a first sample (e.g., a fluid sample) that comprises a peripheral (e.g., circulating) phagocyte.
  • the first sample may be derived from outside of a brain tissue.
  • the method further comprises detecting one or more biomarkers, e.g., an antigen, inside a phagocyte of said sample, wherein the biomarker is associated with a neurological condition (e.g., a neurological condition-associated protein).
  • the sample may be provided from a mammal (e.g., a patient, a mouse, a rat, etc.). Detecting the neurological condition-associated protein indicates the presence of the neurological condition or a risk of the neurological condition.
  • the sample (e.g., fluid) obtained does not necessarily directly come into contact with the inflamed tissue being detected.
  • the fluid obtained may have once directly come into contact with the inflamed tissue, but at the time that it is being extracted in accordance with the present invention, it is being separated from the inflamed tissue by a barrier.
  • the method may further comprise comparing the level of the biomarker in the first sample with a level of the biomarker in a second sample (e.g., a fluid sample).
  • the second sample may be a control sample.
  • the second sample is a fluid sample from outside of a brain tissue comprising a peripheral (e.g., circulating phagocyte),
  • the second sample may be provided from a mammal (e.g., a patient, a mouse, a rat, etc.).
  • the second sample may have been collected prior to the first fluid sample.
  • detecting an increased level of the neuroiogical condition-associated protein as compared to the level of the neurological condition-associated protein of a control sample indicates the presence of the neurological condition or a risk thereof.
  • detecting a decreased level of the neurological condition-associated protein as compared to the level of the neuroiogical condition-associated protein of a control sample indicates the presence of the neurological condition or a risk thereof.
  • the neurological condition that may be monitored or detected includes Alzheimer's Disease, Parkinson's Disease, Neuromyelitis Optica, transverse myelitis, Acute and chronic Stroke, Traumatic Brain Injury, and the like.
  • the neurological condition-associated protein is derived from a brain source. In some embodiments, the neuroiogical condition- associated protein is derived from a non-brain source. In some embodiments, one or more neurological condition-associated proteins is derived from a brain source, a non-brain source, or a combination thereof.
  • the neuroiogical condition-associated protein may be present in a circulating phagocyte.
  • Phagocytes may include monocytes, macrophages, and/or lymphocytes.
  • Such circulating phagocytes may be found in tissues, cells, and/or fluids in the body, for example in blood, peripheral blood mononuclear ceils (PBMCs), cerebrospinal fluid (CSF), central nervous system tissues, synovial fluid, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, ocular fluids, vitreai fluid, urine, the like, or a combination thereof.
  • PBMCs peripheral blood mononuclear ceils
  • CSF cerebrospinal fluid
  • central nervous system tissues for example in blood, peripheral blood mononuclear ceils (PBMCs), cerebrospinal fluid (CSF), central nervous system tissues, synovial fluid, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, ocular fluids
  • the neurological condition-associated protein may be obtained from within a macrophage.
  • the macrophage sample is permeabilized.
  • the macrophage is iysed via various means, e.g., hypotonic solution treatment, detergent solution treatment, mechanical stress, etc.
  • the neurological condition-associated protein may be detected using a variety of methods. Methods may include an immunoassay, a histological assay, a flow cytometry assay, the like, or a combination thereof. In some embodiments, the step of detecting the neurological condition-associated protein in the sample may comprise introducing an antibody to the sample, wherein the antibody binds to the protein or is specific for the protein.
  • this method is used in combination with one or more different methods for detecting the neurological condition. In some embodiments, this method is used to differentiate between one or more neurological conditions.
  • the present invention also features methods of detecting multiple sclerosis or a risk of multiple sclerosis.
  • the methods of the present invention may allow for monitoring, detecting and/or predicting a relapse or a remission of multiple sclerosis.
  • the method of detecting multiple sclerosis is used in combination with one or more methods of detecting multiple sclerosis.
  • the present methods may be used in conjunction with other modalities to monitor, detect or predicting a relapse or a remission of multiple sclerosis.
  • the method of detecting multiple sclerosis comprises (1 ) providing a first sample (e.g., a fluid sample) that comprises a peripheral (e.g., circulating) phagocyte.
  • the first sample may be derived from outside of a brain tissue, and (2) detecting a multiple sclerosis-associated biomarker in the phagocyte.
  • the sample may be provided from a mamma! (e.g., a patient, a mouse, a rat, etc.).
  • one or more multiple sclerosis-associated biomarkers is detected in the sample.
  • the multiple sclerosis-associated biomarkers are associated with multiple sclerosis.
  • the method may further comprise comparing the level of the biomarker in the first sample with a level of the biomarker in a second sample (e.g., a fluid sample).
  • the second sample may be a control sample.
  • the second sample is a fluid sample from outside of a brain tissue comprising a peripheral (e.g., circulating phagocyte),
  • the second sample may be provided from a mammal (e.g., a patient, a mouse, a rat, etc.).
  • the second sample may have been collected prior to the first fluid sample. Detecting the multiple sclerosis- associated biomarker may indicate the presence of multiple sclerosis or a risk of multiple sclerosis.
  • detecting an increased level of the multiple scierosis-associated biomarker as compared to the level of the multiple sclerosis- associated biomarker of a control sample indicates the presence of multiple sclerosis or a risk thereof. In some embodiments, detecting a decreased level of the multiple sclerosis-associated biomarker as compared to the level of the multiple sclerosis-associated biomarker of a control sample indicates the presence of multiple sclerosis or a risk thereof.
  • detecting an increased level of one class of multiple sclerosis-associated biomarker and a decreased of another class of multiple sclerosis-associated biomarker as compared to the respective level of the multiple sclerosis-associated biomarker of a control sample indicates the presence of multiple sclerosis or a risk thereof.
  • the sample is obtained from the mammal immediately following a relapse (e.g., exacerbation of symptoms) before a drug (e.g., a steroid) treatment has begun.
  • a drug e.g., a steroid
  • the sample is obtained from the mammal before a relapse.
  • the sample is obtained during the course of the drug (e.g., steroid) treatment.
  • the multiple sclerosis-associated biomarker may be present in a circulating phagocyte.
  • Phagocytes may include monocytes, macrophages, and/or lymphocytes.
  • macrophages are a type of monocyte and are phagocytic cells important in both specific cell-mediated immunity and nonspecific innate immunity.
  • Circulating phagocytes may be found in tissues, ceils, and/or fluids in the body, for example in blood, peripheral blood mononuclear cells (PBMCs), cerebrospinal fluid (CSF), central nervous system tissues, synovial fluid, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, ocular fluids, vitreai fluid, urine, the like, or a combination thereof.
  • PBMCs peripheral blood mononuclear cells
  • CSF cerebrospinal fluid
  • central nervous system tissues synovial fluid
  • cystic fluid cystic fluid
  • lymph fluid ascites
  • interstitial fluid interstitial fluid
  • ocular fluids vitreai fluid
  • urine the like
  • the neurological condition-associated protein is an intracellular component.
  • the neurological condition-associated protein may be obtained from within a macrophage.
  • the macrophage sample is permeabilized.
  • a mammal includes a human, a mouse, a rat, a llama, a rabbit, a dog, a primate, a guinea pig, a cat, a hamster, a pig, a chicken, a goat, a horse, or a cow.
  • the multiple sclerosis-associated biomarker is a Tau protein (or a fragment thereof) or a Tau protein (or fragment thereof) comprising a phosphorylated residue (e.g., a phosphorylated serine reside, a phosphory!ated threonine reside).
  • a phosphorylated residue e.g., a phosphorylated serine reside, a phosphory!ated threonine reside.
  • the phosphorylated residue is serine 214, serine 235, serine 282, serine 356, serine 396, serine 404, serine 413, serine 48, serine 515, serine 516, serine 519, serine 531 , serine 552, serine 810, serine 622, serine 841 , serine 713, serine 721 , serine 726, serine 730, serine 739, threonine 181 , threonine 205, threonine 470, threonine 492, threonine 498, threonine 522, threonine 529, threonine 534, threonine 548, the like, or a combination thereof.
  • phosphorylation of Tau can decrease its solubility.
  • the method of detecting multiple sclerosis comprises detecting a level of insoluble Tau protein in the sample.
  • an increased level of insoluble Tau protein as compared to a control level of insoluble Tau protein is indicative of multiple sclerosis or a risk thereof.
  • the multiple sclerosis-associated biomarker is a protein or a fragment thereof selected from the group consisting of neuroglobin, valosin-containing protein, brain hexokinase, hippocalcin-1 , nestin, syna ptotagm in, myelin associated glycoprotein, myelin basic protein, myelin oligodendrocyte glycoprotein, myelin proteolipid protein, transketolase, NS1 assocated protein 1 , major vault protein, synaptojanin, enolase, alpha synuclein, glial fibrillary acidic protein, S-100 proteinNeu-N, 28S proteasome subunit 9, annexin A2, annexin A3, annexin A5, annexin A6, annexin A1 1 , ubiquitin activating enzyme ZE1 , ubiquitin B precursor, vimentin, g!yceraidehyde-3- phosphate dehydrogenase
  • the multiple sclerosis-associated biomarker (e.g., Tau protein or fragment thereof) may be of various lengths.
  • the multiple scierosis-associated biomarker consists of between about 5 to 20 amino acids.
  • the multiple scierosis-associated biomarker consists of about 20 to 40 amino acids.
  • the multiple scierosis-associated biomarker consists of about 40 to 80 amino acids.
  • the multiple scierosis-associated biomarker consists of about 80 to 150 amino acids.
  • the multiple sclerosis-associated biomarker consists of about 150 to 200 amino acids.
  • the multiple sclerosis-associated biomarker consists of about 200 to 300 amino acids.
  • the multiple sclerosis-associated biomarker consists of about 300 to 400 amino acids. In some embodiments, the multiple sclerosis- associated biomarker consists of about 400 to 500 amino acids. In some embodiments, the multiple sclerosis-associated biomarker consists of about 500 to 800 amino acids.
  • the multiple sclerosis-associated biomarker may comprise various regions of the full-length protein.
  • the multiple sclerosis-associated biomarker comprises the amino-terminus (e.g., N-terminus, NH2-terminus, N-terrninal end, amine- terminus).
  • the amino-terminus refers to the amino acid at the end of a protein or polypeptide that has a free amine group (-NH2).
  • the multiple-sclerosis associated biomarker consists of about the first 15 amino acids. In some embodiments, the multiple-sclerosis associated biomarker consists of about the first 25 amino acids.
  • the multiple-sclerosis associated biomarker consists of about the first 50 amino acids. Sn some embodiments, the multiple-sclerosis associated biomarker consists of about the first 75 amino acids. In some embodiments, the multiple-sclerosis associated biomarker consists of about the first 100 amino acids. In some embodiments, the multiple-sclerosis associated biomarker consists of about the first 125 amino acids.
  • the multiple-sclerosis associated biomarker or fragment thereof comprises the carboxy-terminus (e.g., C-terminus, COOH- terminus, C-terminal end, carboxyl-terminus).
  • the carboxy-terminus refers to the amino acid at the end of a protein or polypeptide that has a free carboxylic acid group (-COOH).
  • the multiple-sclerosis associated biomarker consists of about the last 100 amino acids.
  • the present invention also features methods of determining the status of a disease or condition (e.g., a neurological condition, an inflammatory condition, multiple sclerosis, etc.) or determining the status of drug efficacy.
  • a disease or condition e.g., a neurological condition, an inflammatory condition, multiple sclerosis, etc.
  • the present invention may also feature methods of monitoring disease activity and drug efficacy.
  • biomarkers can be used to detect a disease or condition and the biomarkers may be used to determine severity of the disease or condition (e.g. relapse, remission, etc.).
  • the method comprises providing a sample (e.g., a fluid, a brain tissue), the sample comprising a circulating phagocyte.
  • the sample may be derived from a mammal (e.g., a patient, a mouse, a rat, etc.).
  • a biomarker or level thereof associated with a disease or condition e.g., a multiple sclerosis- associated biomarker
  • the biomarker detected may be compared to the level or presence of the biomarker in a second sample, the second sample having been collected prior to the first sample.
  • a biomarker may include but is not limited to Tau or a fragment thereof.
  • the monitoring of disease activity may be used to determine drug efficacy, in some embodiments, the monitoring of disease activity may be used to determine drug failure and/or breakthrough disease. In some embodiments, the monitoring of disease activity may be used to determine patient compliance with drug therapy. In some embodiments, the monitoring of disease activity may be used to determine therapeutic non-responders. In some embodiments, the monitoring of disease activity may be used to aid drug development.
  • the present invention features a method of monitoring disease activity of a neurological condition, the method comprises obtaining from a mammal a first fluid sample from outside of a brain tissue of the mammal, the first fluid sample comprises a first circulating phagocyte; detecting a level of a biomarker associated with the neurological condition in the first sample; and comparing the level of the biomarker in the first sample with a level of the biomarker in a second sample, the second sample being either a control sample or a second fluid sample from outside of a brain tissue of the mammal, the second fluid sample comprising a second circulating phagocyte, the second fluid sample having been taken prior to the first fluid sample.
  • Table 1 shows the amino acid sequence of full-length human Tau protein.
  • Table 2 shows examples of some of the possible multiple sclerosis- associated biomarkers (e.g., Tau protein or a fragment there).
  • 3GMPGAPLLP EGPREATR.QP SGTGPEDTEG GRHAPELLKH QLLGDLHQEG PPLKGAGGKE RPGSKEEVDE DRDVDESSPQ DSPPSKASPA QDGRPPQTAA REATSIPGFP AEGAIPLPVD FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE FTFHVEITPN VQKEOAHSEE HLGRAAFPGA PGEGPEARGP SLGEDTKEAD LPEPSEKQPA AAPRGKPVSR VPQLKARMVS KSKDGTGSDD KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS DPLIQPSSPA
  • SGMPGAPLLP EGPREATRQP SGTGPEDTEG GRHAPELLKH QLLGDLHQEG PPLKGAGGKE RPGSKEEVDE DRDVDESSPQ DSPPSKASPA QDGRPPQTAA REATSIPGFP AEGAIPLPVD FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE FTFHVEITPN VQKEQAH3EE HLGRAAFPGA
  • 3GMPGAPLLP EGPREATRQP SGTGPEDTEG GRKAPELLKH QLLGDLHQEG PPLKGAGGKE RPGSKEEVDE DRDVDESSPQ DSPPSKASPA QDGRPPQTAA REATSIPGFP AEGAIPLPVD FL3KVSTEIP ASEPDGP3VG RAKGQDAPLE FTFHVEITPN VQKEQAHSEE HLGRAAFPGA PGEGPEARGP SLGEDTKEAD LPEPSEKQPA AAPRGKPV3R VPQLKARMVS KSKDGTGSDD KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG SSGAKEMKLK GADGKTKIAT PRGAAPPGQK GQANATRIPA KTPPAPKTPP SSGEPPKSGD
  • 3GMPGAPLLP EGPREATR.QP SGTGPEDTEG GRHAPELLKH QLLGDLHQEG PPLKGAGGKE RPGSKEEVDE DRDVDESSPQ DSPPSKASPA QDGRPPQTAA REATSIPGFP AEGAIPLPVD FLSKVSTEIP ASEPDGP3VG RAKGQDAPLE FTFHVEITPN VQKEQAHSEE HLGRAAFPGA PGEGPEARGP SLGEDTKEAD LPEP3EKQPA
  • AAPRGKPVSR VPQLKARMVS KS DGTGSDD KKAKTSTR33 A TLKNRPCL SPKLPTPGSS DPLIQPSSPA VCPEPP33P HVSSVTSRTG SSGAKE KLK GADGKTKIAT PRGAAPPGQK GQA ATRI A KTPPAPKTPP SSGEPPKSGD RSGYSSPG3P GTPGSRSRTP SLPTPPTREP
  • 3GMPGAPLLP EGPREATRQP SGTGPSDTEG GRHAPELLKH QLLGDLHQEG PPLKGAGGKE RPG3KEEVDE DRDVDE33PQ DSPPSKASPA
  • 3GMPGAPLLP EGPREATRQP SGTGPEDTEG GR.KAPELLKH QLLGDLHQEG PPLKGAGGKE RPGSKEEVDE DRDVDESSPQ DSPP3KASPA
  • 3GMPGAPLLP EGPREATRQP SGTGPEDTEG GRHAPELLKH QLLGDLHQEG PPL GAGGKE RPGSKEEVDE DRDVDE33PQ DSPPSKASPA
  • 3GMPGAPLLP EGPREATRQP SGTGPEDTEG GRHAPELLKH QLLGDLHQEG PPLKGAGGKE RPGSKEEVDE DRDVDESSPQ DSPP3KASPA
  • AAPRGKPVSR VPQLKARMV3 KSKDGTGSDD KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS DPLIQPSSPA VCPEPPSSPK HVS3VT3RTG SSGAKEMKLK GADGKTKTAT PRGAAPPGQK GQAMATRIPA KTPPAPKTPP SSGEPPKSGD
  • AAPRGKPVSR VPQLKAR.MV3 KSKDGTGSDD KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG SSGAKEMKLK GA.DGKTKIAT PRGAAPPGQK GQA.NATRIPA KTPPAPKTPP SSGEPPKSGD RSGYSSPGSP GTPGSRSRTP SLPTPPTREP KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL KNVKSKIGST ENLKHQPGGG KVQI INKKLD LSNVQSKCGS KDKIKHVPGG GSVQIVYKPV DLSKVTSKCG SLGNIHHKPG GGQVEVKSEK LDFKDRVQSK IGSLDNITHV PGGGNKKIET HKLTFRENAK AKTDHGAEIV YKSPVVSGDT SPRHLSNVS3 TGSIDMVDSP
  • 3GMPGAPLLP EGPREATRQP SGTGPEDTEG GRHAPELLKH QLLGDLHQEG PPLKGAGGKE RPGSKEEVDE DRDVDESSPQ DSPPSKASPA QDGRPPQTAA RSATSIPGFP AEGAIPLPVD FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE FTFHVEITPN VQKEOAHSEE HLGRAA.FPGA
  • the step of detecting the multiple sclerosis- associated biomarker in the sample may comprise introducing an antibody to the sample, wherein the antibody binds to the multiple sclerosis-associated biomarker.
  • the step of detecting the multiple sclerosis- associated biomarker in the sample comprises subjecting the sample to a western blot, an enzyme-linked immunosorbent assay (ELISA), a lateral flow assay, a radioimmunoassay, an immunohistochemistry assay, a bioluminescent assay, a chemiluminescent assay, a mass spectrometry assay, a flow cytometry assay (e.g., florescence-activated ceil sorting (FACS)), or a combination thereof and the like.
  • ELISA enzyme-linked immunosorbent assay
  • a lateral flow assay e.g., a radioimmunoassay, an immunohistochemistry assay, a bioluminescent assay, a chemiluminescent assay, a mass spectrometry assay, a flow cytometry assay (e.g., florescence-activated ceil sorting (FACS)), or a combination thereof and the
  • the step of detecting the multiple sclerosis- associated biomarker further comprises contacting the sample with an antibody that binds to the multiple sclerosis-associated biomarker and detecting an antibody-biomarker complex.
  • the step of detecting an antibody-biomarker complex may comprise subjecting the sample to a micro array, western blot, an enzyme-linked immunosorbent assay (ELISA), a lateral flow assay, a radioimmunoassay, an immunohistochemistry assay, a bioluminescent assay, a chemiluminescent assay, a flow cytometry assay (e.g., florescence-activated cell sorting (FACS)), or a combination thereof and the like.
  • detecting the antibody-biomarker complex indicates the presence of multiple sclerosis or a risk of multiple sclerosis.
  • the step of detecting the multiple sclerosis-associated biomarker may comprise subjecting the sample florescence-activated cell sorting (FACS).
  • FACS Fluorescence-activated cell sorting
  • FACS Fluorescence-activated cell sorting
  • a current of a rapidly flowing stream of liquid carries a suspension of cells through a nozzle. The flow is selected such that there is a large separation between cells relative to their diameter.
  • Vibrations at the tip of the nozzle cause the stream of ceils to break into individual droplets, and the system is adjusted so that there is a low probability of more than one ceil being in a droplet.
  • a monochromatic laser beam illuminates the droplets, which are electronically monitored by fluorescent detectors.
  • the droplets that emit the proper fluorescent wavelengths are electrically charged between deflection plates in order to be sorted into collection tubes.
  • the step of detecting the multiple sclerosis-associated biomarker may comprise subjecting the sample to an enzyme-linked immunosorbent assay (ELISA).
  • ELISA is an assay used to detect the presence of an antibody or a biomarker in a sample.
  • a sample containing an unknown amount of biomarker e.g., an antigen
  • a surface e.g., a polystyrene microtiter plate.
  • an antibody that binds to the antigen of interest is washed over the surface so that it can bind the antigen and form an antibody/antigen complex.
  • this antibody is covalently linked to an enzyme.
  • the antibody is not covalently linked to an enzyme but can be detected by a secondary antibody that is linked to an enzyme.
  • a substance e.g., substrate
  • a detectable visible signal e.g., color signal
  • an antibody is used to detect the presence of the multiple sclerosis-associated biomarker.
  • the multiple sclerosis-associated biomarker may be detected with a variety of antibodies.
  • the antibody is a monoclonal or a polyclonal antibody.
  • the antibody is a humanized antibody.
  • the antibody is a chimera.
  • the antibody is derived from a human, a mouse, a rat, a llama, a rabbit, a dog, a primate, a guinea pig, a cat, a hamster, a pig, a chicken, a goat, a horse, or a cow.
  • the antibody is synthetic.
  • the antibody is a recombinant antibody.
  • antibodies are labelled either covendingiy or non-covalently by combining the antibody with a second substance that provides for detectable signal.
  • labels and conjugation techniques are known in the art and are reported extensively in both the scientific and patent literature. Examples of labels include but are not limited to radioisotopes, enzymes, substrates, cofactors, inhibitors, fiuorescers, chemiluminescers, magnetic particles, and the like.
  • the antibody comprises a label.
  • the present invention is used to detect the presence of multiple sclerosis.
  • a patient may present with symptoms of a demyeiinating disease.
  • a sample e.g., derived from the paitent
  • a sample may be tested for an elevated level of a multiple sclerosis-associated biomarker. If, according to the present invention, the level of a multiple sclerosis-associated biomarker is elevated and the patient presents symptoms of a demyeiinating disease, then the patient is diagnosed as having multiple sclerosis.
  • the present invention is used to detect a risk of multiple sclerosis.
  • a patient may present with no symptoms of a demyeiinating disease, but he or she wishes to be tested for a risk of multiple sclerosis. If, according to the present invention, the level of a multiple sclerosis- associated biomarker is elevated and the patient does not present symptoms of a demyelinating disease, then the patient is diagnosed as having a risk of multiple sclerosis.
  • the term "elevated level” refers to a level that is higher than the normal level of the multiple sclerosis-associated biomarker (e.g., the level that would be detected in a person who does not have multiple sclerosis).
  • samples are pooled from about, for example, 500 patients (or an appropriate number of patients that would be statistically meaningful) who do not experience any symptoms of multiple sclerosis (or other demyelinating diseases) and who do not test positive for multiple sclerosis as detected by MRI.
  • the average level of the multiple sclerosis-associated biomarker can be quantified and then defined as being the normal level of the multiple sclerosis- associated biomarker. If the normal level of the multiple sclerosis-associated biomarker is about zero, then an elevated level refers to any level that is greater than zero, for example, about 5 units, about 25 units, about 50 units, about 100 units, about 500 units, about 1000 units, about 10,000 units, about 100,000 units, about 1 ,000,000 units.
  • a unit may be an absorbance unit (e.g., from an EL!SA), a percent positive (e.g., from a flow cytometry or FACS assay), or a fluorescence unit.
  • an elevated level refers to any level that is higher than the normal level.
  • an elevated level of the multiple sclerosis-associated biomarker may be a level that is about 10-20% higher than the normal level of the multiple sclerosis-associated biomarker.
  • an elevated level of the multiple sclerosis-associated biomarker may be a level that is about 20-30% higher than the normal level of the multiple sclerosis-associated biomarker.
  • an elevated level of the multiple sclerosis-associated biomarker may be a level that is about 30-40% higher than the normal level of the multiple sclerosis-associated biomarker. In some embodiments, an elevated level of the multiple sclerosis-associated biomarker may be a level that is about 40-50% higher than the normal level of the multiple sclerosis-associated biomarker, Sn some embodiments, an elevated level of the multiple sclerosis-associated biomarker may be a level that is about 50-60% higher than the normal level of the multiple sclerosis-associated biomarker. In some embodiments, an elevated level of the multiple sclerosis-associated biomarker may be a level that is about 60-70% higher than the normal level of the multiple sclerosis-associated biomarker.
  • an elevated level of the multiple sclerosis-associated biomarker may be a level that is about 70-80% higher than the normal level of the multiple sclerosis-associated biomarker. In some embodiments, an elevated level of the multiple sclerosis-associated biomarker may be a level that is about 80-90% higher than the normal level of the multiple sclerosis-associated biomarker. In some embodiments, an elevated level of the multiple sclerosis-associated biomarker may be a level that is about 90-100% higher than the normal level of the multiple sclerosis-associated biomarker. In some embodiments, an elevated level of the multiple sclerosis-associated biomarker may be a level that is about 1 - 2 fold higher than the normal level of the multiple sclerosis-associated biomarker.
  • an elevated level of the multiple sclerosis-associated biomarker may be a level that is about 2-3 fold higher than the normal level of the multiple sclerosis-associated biomarker. In some embodiments, an elevated level of the multiple sclerosis-associated biomarker may be a level that is about 3-4 fold higher than the normal level of the multiple sclerosis-associated biomarker. Sn some embodiments, an elevated level of the multiple sclerosis-associated biomarker may be a level that is about 4-5 fold higher than the normal level of the multiple sclerosis-associated biomarker. In some embodiments, an elevated level of the multiple sclerosis-associated biomarker may be a level that is about 5-10 fold higher than the normal level of the multiple sclerosis-associated biomarker.
  • an elevated level of the multiple sclerosis-associated biomarker may be a level that is about 10-20 fold higher than the normal level of the multiple sclerosis-associated biomarker. In some embodiments, an elevated level of the multiple sclerosis-associated biomarker may be a level that is about 20-50 fold higher than the normal level of the multiple sclerosis-associated biomarker. [0090] The present invention also provides a method of monitoring the progression of multiple sclerosis and/or monitoring the treatment of multiple sderosis.
  • the present invention may be used to measure the level of the multiple sclerosis-associated biomarker in order to detect a change in the level (e.g., an increase in the level, a decrease in the level, a maintaining of the level).
  • a change in the level of the multiple sclerosis-associated biomarker may correlate with a change in the patient's status (e.g., remission, progression, worsening).
  • a decrease in the level of the multiple sclerosis-associated biomarker may indicate the patient has entered or will enter a remission period.
  • the present invention may be used to monitor the level of the multiple sclerosis-associated biomarker in a patient while the patient is on a treatment regimen (e.g., a drug).
  • a treatment regimen e.g., a drug
  • a treatment regimen that is effective at inhibiting the progression of multiple sclerosis and/or reducing the symptoms of multiple sclerosis may decrease the level of the multiple sclerosis-associated biomarker in the patient.
  • the method of the present invention for detecting multiple sclerosis is used in combination with one or more different methods for detecting multiple sclerosis. For example, in some cases, a combination of family history, a physical exam, and magnetic resonance imaging (MR! findings are used to diagnose multiple sclerosis.
  • MR! magnetic resonance imaging
  • MR! is the most sensitive radiographic technique for the imaging of multiple sclerosis.
  • Multiple sclerosis plaques are commonly seen as round or void discrete lesions in the periventricular white matter. Other common locations for multiple sclerosis plaques include the corpus caliosum, corona radiate, internal capsule, and centrum semiova!e.
  • the present invention is used to measure a multiple sclerosis-associated biomarker, and the level of the multiple sclerosis-associated biomarker is correlated with a magnetic resonance imaging (MR! measurement.
  • MR magnetic resonance imaging
  • elevated levels of the multiple sclerosis-associated biomarker correlate with a MR! scan showing the presence of multiple sclerosis plaques in the brain.
  • the method of the present invention for detecting multiple sclerosis may be used in combination with one or more methods for detecting a different condition.
  • the method of the present invention may also help to distinguish multiple sclerosis from other diseases with similar clinical manifestations.
  • NMO neuromyelitis optica
  • Devie's syndrome is a neurological disorder regarded as a severe variant of multiple sclerosis.
  • the characteristic inflammatory demyelinating lesions of NMO selectively and repeatedly affect the optic nerves and the spinal cord, causing blindness and paralysis.
  • a marker e.g., aquaporin-4 antibodies
  • a marker has been identified in serum and cerebrospinal fluid of patients with NMO, and the presence of a NMO marker (e.g., aquaporin-4 antibodies) may be used to distinguish NMO from multiple sclerosis.
  • the method of detecting the presence of multiple sclerosis or a risk of multiple sclerosis comprises detecting the presence or absence of at least two biomarkers (e.g., proteins, antigens, or the like) wherein at least one biomarker is detected in order to distinguish multiple sclerosis from a disease with similar clinical manifestations.
  • biomarkers e.g., proteins, antigens, or the like
  • the method of detecting the presence of multiple sclerosis or a risk of multiple sclerosis comprises detecting an elevated level of two or more multiple sclerosis-associated biomarkers. In some embodiments, the method of detecting the presence of multiple sclerosis or a risk of multiple sclerosis comprises detecting an elevated level of three or more multiple sclerosis-associated biomarkers.
  • the present invention also provides a method of diagnosing multiple sclerosis at an early stage of the disease before all clinical criteria are fulfilled, thus justifying early initiation of a multiple sclerosis-appropriate therapy.
  • the present invention also features a kit for detecting the status of a disease or condition (e.g., an inflammatory condition, a neurological condition, multiple sclerosis, etc.).
  • the kit may comprise an antibody specific for a biomarker (e.g., a multiple sclerosis-associated biomarker), wherein the biomarker is a protein selected from the group consisting of Tau or a fragment thereof, phosphorylated Tau or a fragment thereof, neurog!obin, vaiosin-containing protein, brain hexokinase, hippocaicin-1 , nestin, synaptotagmin, myelin associated glycoprotein, Myelin Basic Protein (MBP), Proteoiipid Protein, Myelin Oligodendrocyte Glycoprotein, transketoiase, NS1 assocated protein 1 , major vault protein, synaptojanin, enoiase, alpha synuclein, glial fibrillary acidic protein, 8-100 protein
  • the detection of perforin is used in combination with detection of a marker (e.g., MBP) in phagocyites.
  • a marker e.g., MBP
  • perforin levels can decline in CD16 cells as MBP levels increase.
  • the present invention also features a kit for detecting the presence of multiple sclerosis or a risk of multiple sclerosis in a circulating phagocyte sample derived from a mammal.
  • the kit comprises an antibody that binds to a multiple sclerosis-associated biomarker.
  • the kit further comprises a means for detecting the binding of the antibody to the multiple sclerosis- associated biomarker/antigen in the sample (e.g., an antibody-antigen complex).
  • the detecting of an elevated level of an antibody-antigen complex indicates presence of multiple sclerosis or a risk of muitiple sclerosis.
  • the kit comprises an antibody, wherein the antibody is a monoclonal or a polyclonal antibody.
  • the antibody is derived from a human, a mouse, a rat, a llama, a rabbit, a dog, a primate, a guinea pig, a cat, a hamster, a pig, a chicken, a goat, a horse, or a cow.
  • the antibody is humanized.
  • the antibody is a chimera.
  • the antibody is specific for the multiple sclerosis-associated biomarker.
  • the following example describes the detection of multiple sclerosis in a patient according to two methods disclosed in the present invention.
  • a 24-year- old male patient presents to his primary care physician complaining of changes in vision, limb weakness, and extreme fatigue. He mentions his symptoms have been recurring over the last 3 months.
  • the physician suspects the possibility of a tumor in the centra! nervous system (CNS) or a CNS disease, as well as multiple sclerosis.
  • the physician obtains a blood sample to be sent to a diagnostic laboratory for multiple sclerosis testing, and also refers the patient to a neurologist.
  • PBMCs are obtained from a BD VacutainerTM CPT tube using a cell separation procedure.
  • the cells are washed three times in 1X PBS and centrifuged in a horizontal rotor (swing-out head) for a minimum of 5 minutes at 1200 to 1500 RCF (Relative Centrifugal force).
  • the supernatant is removed and the cells are resuspended in 1X PBS.
  • extracts of the PBMCs are prepared by iysing with a hypotonic solution or other method.
  • the lysate is subjected to assay involving an antibody that binds to Tau protein fragment comprising the phosphorylated serine residue Ser-404.
  • the assay indicates that an elevated level of said Tau protein fragment is present in the PBMCs.
  • the assay is the assay of example 2 or example 3.
  • the results of the assay indicate that the patient has multiple sclerosis. The physician notifies the patient, who then begins treatment immediately.
  • the laboratory receives the patient's blood sample collected in a CPT tube.
  • PBMCs are obtained from a BD VacutainerTM CPT tube using a cell separation procedure.
  • the cells are washed three times in 1X PBS and centrifuged in a horizontal rotor (swing-out head) for a minimum of 5 minutes at 1200 to 1500 RCF (Relative Centrifugal force).
  • the supernatant is removed and the cells are resuspended in 1 X PBS.
  • the cells are then subjected to assay involving an antibody that binds to Tau protein fragment comprising the phosphory!ated serine residue Ser-404.
  • the assay is the assay of example 4.
  • the following example describes a direct ELISA assay used for detecting a multiple sclerosis-associated antigen in a sample.
  • the protein concentration of the sample is determined using the BioRadTM (Bradford method) assay.
  • MicroELJSA plates are coated by addition of 100 ⁇ _ of a 5- 20 pg/mL solution of the sample, which is then incubated for 1 hour at 21 ° C.
  • the wells are washed out with phosphate buffered solution (PBS) with 0.05% poiysorbate (Tween 20TM).
  • PBS phosphate buffered solution
  • Tween 20TM poiysorbate
  • the following example describes an indirect ELISA assay used for detecting a multiple sclerosis-associated antigen in various samples.
  • This assay is constructed using polyclonal and monoclonal antibodies.
  • ELISA wells are coated with polyclonal antibody at an appropriate concentration and the wells are washed and blocked as described above.
  • Various dilutions of antigen containing samples are added to the wells and incubated for 1 hour at 21 ° C, after which the wells are washed 3 times with PBS-0.05% TweenTM 20.
  • the monoclonal antibody is then added at an appropriate dilution in PBS-0.05% TweenTM20 - 1 % BSA and incubated for 1 hour at 21 ° C.
  • the wells are then washed 3 times and an appropriately diluted horse radish peroxidase conjugated anti-mouse Ig in PBS- 0.05% TweenTM20-1 % BSA is then added to each well and incubated for 1 hour at 21 ° C.
  • the wells are then washed twice with PBS-0.05% TweenTM20 and finally with PBS.
  • One hundred ⁇ _ of soluble MTB substrate solution is added to each well and incubated for 30 minutes at 21 ° C after which 100 ⁇ _ of MTB stop reagent is added and the color intensity is measured at 450nm using an EL!SA plate reader.
  • the following example describes a flow cytometry assay used for detecting a multiple sclerosis-associated antigen in various samples.
  • PBMCs from multiple sclerosis (MS) subjects and control subjects are stained with fluorescent antibodies to the multiple sclerosis-associated antigen (e.g., Tau protein) and also with fluorescent labeled antibodies to cluster designation (CD) 3 T-!ymphocyte marker or CD 19 B-Lymphocyte marker, CD68 intracellular monocyte marker and CD14 monocyte/ macrophage cell surface marker.
  • CD cluster designation
  • CD68 intracellular monocyte marker CD14 monocyte/ macrophage cell surface marker.
  • PBMCs are obtained from a BD VacutainerTM CPT tube using a cell separation procedure.
  • the cells are washed three times in 1X PBS and centrifuged in a horizontal rotor (swing-out head) for a minimum of 5 minutes at 1200 to 1500 RCF (Relative Centrifugal force).
  • the supernatant is removed and the cells are resuspended in 1X PBS.
  • the cells are resuspended to approximately 4.0 mL in 1X PBS.
  • Approximately 50 ⁇ _ of the cell suspension to be analyzed is transferred into tubes for double staining with selected antibody pairs.
  • Dako IntrastainTM Reagent A fixative
  • 1 X PBS working solution is added to each test tube and mixed gently.
  • the tubes are centrifuged at 300 X g for 5 minutes. Supernatant is aspirated leaving about 50 ⁇ ! of fluid. The fluid is mixed thoroughly to ensure that the ceils are in suspension.
  • One hundred ⁇ _ of Dako IntrastainTM Reagent B (permeabilization) is added to each tube.
  • the appropriate amount of the antibody specific for the multiple sclerosis-associated antigen is added to the appropriate tubes.
  • the tubes are mixed gently to ensure that the cells are in suspension and incubated at room temperature for 15-60 minutes.
  • Two mL of 1X PBS working solution is added to each test tube and mixed gently.
  • the tubes are centrifuged at 300 X g for 5 minutes, and then the supernatant is aspirated, leaving approximately 50 ⁇ of fluid.
  • the fluid is mixed thoroughly to ensure that the cells are in suspension.
  • the pellet is resuspended in an appropriate volume of fluid for flow cytometry analysis.
  • the sample is analyzed on a flow cytometer within 24 -48 hours.
  • the gate is on the monocyte population and the data is collected in list mode.
  • Qualitative and or quantitative differences are determined between normal and MS patients using the analysis software. Optimization steps include varying incubation time with antibodies, fixation time and permeabi!ization time.
  • MSDX Compiex-1 is a protein complex (e.g. an aggregate, a complex) termed "MSDX Compiex-1 " is elevated in multiple sclerosis patients as compared to healthy controls.
  • MSDX Compiex-1 is a high molecule weight complex comprising fibrinogen, fibronectin, and fibulin-1.
  • MSDX Compiex-1 alone or in combination with other markers may be useful as an indicator of multiple sclerosis or other diseases or conditions, for example for an inflammatory condition, a neurodegenerative disease or condition, cancer, stroke, or other diseases.
  • MSDx complex-1 alone or in combination with one or more other biomarkers may help monitor disease activity (e.g., relapse, remission, etc.). Monitoring disease activity may be useful for detecting a response (e.g., positive response, negative response, lack of response) to a therapy, for detecting patient compliance with a therapy, or for providing useful clinical information for disease management.
  • the present invention features methods for measuring high molecular weight complexes of fibrinogen with fibronectin and fibulin-1 ("MSDx Complex-1 ”) and applications thereof.
  • the methods may be used to monitor disease activity and therapeutic efficacy in diseases or conditions that have an inflammatory component, for example autoimmune diseases, neurodegenerative diseases, cancers and metabolic diseases such as type 2 diabetes mei!itus.
  • the present invention is not limited to the aforementioned diseases and conditions or the aforementioned applications.
  • the present invention features methods for measuring high molecular weight complexes of MSDX Complex-1 , e.g., fibrinogen with fibronectin and fibulin-1 , in a sample.
  • MSDx Complex-1 refers to a high molecular weight complex of fibrinogen, fibronectin, and fibulin-1.
  • the detection of MSDX Compiex-1 may be used for a variety of purposes, for example for detecting a disease or condition, for monitoring a disease or condition, for monitoring a therapy, etc.
  • a circulating high molecular weight protein complex has been found to bind certain small peptides selectively. For example, by sephacryi S200 gel filtration chromatography, the binding activity was found in a broad peak of 400,000-900,000 kD. This peak was collected and shown by LC/MS, after in solution protease digestion, to consist of Fibrinogen, Fibronectin and Fibulin-1.
  • the present invention features a unique competitive ELISA assay format to measure the amount of MSDx Compiex-1 in a sample, e.g., plasma, by its ability to compete with an anti-peptide antibody for binding of the labeled peptide (e.g., biofinylated peptide).
  • the method comprises introducing a labeled peptide and an anti-peptide antibody to a sample to create an antibody- sample mixture.
  • the anti-peptide antibody can bind to at least the labeled peptide and MSDX Complex-1.
  • the labeled peptide comprises a label molecule (e.g., biotin).
  • the label molecule is not limited to biotin but may include any appropriate label. Labels are well known to one of ordinary skill in the art.
  • the method further comprises providing a well (e.g., EL!SA well) coated with a "well antibody".
  • the well antibody is specific for a complex of labeled peptide and anti-peptide antibody.
  • the method further comprises introducing the antibody-sample mixture to the well and introducing a substrate to the antibody-sample mixture in the well.
  • the label molecule of the labeled peptide and the substrate interact to provide a signal.
  • the level of the signal is compared to a control. If the level of the signal is higher than that of the control, then MSDX Compiex-1 is not detected. If the level of the signal is lower than that of the control then MSDX Complex-1 is detected.
  • the labeled peptide is or comprises SEQ ID NO:3A. In some embodiments, the labeled peptide is or comprises SEQ ID NO: 4A. In some embodiments, the labeled peptide is or comprises SEQ ID NO: 5A.
  • the label of the labeled peptide is located at the C- terminus, the N-terminus or at both termini. In some embodiments, the labeled peptide is between about 15 to 50 amino acids in length, e.g., 24 amino acids, between about 15 to 40 amino acids, between about 15 to 30 amino acids, between about 20 to 30 amino acids, etc. In some embodiments, the labeled peptide has a pi of about 6.1. In some embodiments, the labeled peptide has a pi between about 6 and 7.0, between about 5.5 and 6.5, between about 5.8 and 6.4, etc. In some embodiments, the labeled peptide has a net charge of about -0.1 at pH 7.0. In some embodiments, the labeled peptide comprises an epitope tag disposed at the C-terminus, the N-terminus, or at both termini.
  • the present invention also features a method of detecting MSDX
  • Complex-1 comprising introducing a first antibody to a sample to create an antibody-sample mixture, wherein the first antibody is specific for one of fibrinogen, fibronectin, or fibuiin-1 .
  • the first antibody comprises a label molecule (e.g., HRP).
  • a well e.g., ELISA well
  • the well is coated with a second antibody, wherein the second antibody is specific for one of fibrinogen, fibronectin, or fibuiin-1.
  • the method further comprises introducing the antibody-sample mixture to the well and introducing a substrate to the antibody- sample mixture in the well.
  • the label molecule and the substrate interact to provide a signal (e.g., a chemiluminescent signal, a fluorescent signal, a colorimetric signal, a potentiometric signal, an amperometric signal, or a combination thereof).
  • a signal e.g., a chemiluminescent signal, a fluorescent signal, a colorimetric signal, a potentiometric signal, an amperometric signal, or a combination thereof.
  • the first antibody is an anti-fibuiin-1 antibody and the second antibody is an anti-fibrinogen antibody.
  • the first antibody is an anti-fibronectin antibody and the second antibody is an anti- fibrinogen antibody.
  • the first antibody is an anti-fibrinogen antibody and the second antibody is an anti-fibrinogen antibody.
  • the first antibody is an anti-fibulin-1 antibody and the second antibody is an anti-fibronectin antibody.
  • the first antibody is an anti-fibronectin antibody and the second antibody is an anti-fibronectin antibody.
  • the first antibody is an anti-fibrinogen antibody and the second antibody is an anti-fibronectin antibody.
  • the first antibody is an anti-fibulin-1 antibody and the second antibody is an anti- fibulin-1 antibody. In some embodiments, the first antibody is an anti-fibronectin antibody and the second antibody is an anti-fibulin-1 antibody. In some embodiments, the first antibody is an anti-fibronectin antibody and the second antibody is an anti-fibulin-1 antibody. In some embodiments,
  • the first antibody is an anti-fibrinogen antibody and the second antibody is an anti-fibulin-1 antibody.
  • the method further comprises introducing a third antibody to the antibody-sample mixture prior to introduction to the well, the third antibody is specific for one of fibulin-1 , fibronectin, or fibnnogen, wherein the third antibody has a different specificity than the first antibody.
  • the method further comprises introducing a fourth antibody to the antibody-sample mixture prior to introduction to the well, the third antibody is specific for one of fibulin-1 , fibronectin, or fibnnogen, wherein the third antibody has a different specificity than the first antibody and a different specificity than the third antibody.
  • the label molecule comprises an enzyme.
  • the enzyme comprises horseradish peroxidase.
  • the first antibody is a rabbit antibody.
  • the first antibody is not limited to rabbit and may be any other appropriate antibody (e.g., mouse, human, etc.).
  • the second antibody comprises an anti-rabbit antibody, e.g., a goat anti-rabbit antibody, a mouse anti-rabbit antibody, a human anti-rabbit antibody, etc.
  • Anti-Fibrinogen antibodies are immobilized onto an assay surface (e.g., ELISA well, glass slide, magnetic particle, antibody array matrix) and blocked using conventional methods.
  • a biological fluid e.g., serum, plasma, cerebrospinal fluid
  • a biological fluid e.g., serum, plasma, cerebrospinal fluid
  • antibodies to fibronectin and/or Fibuiin ⁇ 1 are contacted with the immobilized material and unbound antibodies are washed off.
  • the bound antibodies are then detected with a labelled anti-immunog!obulin of the appropriate specificity to generate a measurable signal (the signal may be chemiluminescent, fluorescent, colorimetric, potentiometric, amperometric etc).
  • method is a competitive ELISA assay format.
  • the competitive ELISA assay used for detecting MDSX Compiex-1 utilizes a labeled analyte and measures the ability of an unlabelled native analyte in a biological fluid to compete with the labeled analyte for binding to the antibody.
  • the labeled analyte is bound by an unrelated binding protein that prevents it's binding to antibody and is washed off before the detection step.
  • MSDX Complex-1 in added plasma competes with the rabbit anti-peptide antibody for binding to biotinyiated peptide.
  • MSDX Compiex-1 The more MSDX Compiex-1 that is present in the plasma the more biotinyiated peptide it binds leaving less available to bind to antibody.
  • high levels of MSDX Compiex-1 result in low optical density and vice versa.
  • the peptide is labeled at the C-terminai with biotin or another detection agent.
  • the N-terminal of the peptide may be amine or amide.
  • the peptide is 24 amino acids long.
  • the peptide sequence is:
  • the peptide has a pi of 6.1. In some embodiments, the peptide has a net charge of - 0.1 at pH 7.0. In some embodiments, the peptide has an average hydrophilicity (Hopp & Woods method) of -0.3. In some embodiments, the peptide has a ratio of hydrophilic residues to total residues of 33%.
  • an epitope tag is attached to a terminus, e.g., the N-terminus, to enable the use of other capture antibodies, for example a poiyHistidine tag (HisTag).
  • any peptide sequence derived by conservative amino acid substitution rules such as the Dayhoff matrix and the like of SEQ ID NO: 3A may be used. In some embodiments, alternative peptides may be used.
  • the peptide has a pi of 6.1. In some embodiments, the peptide has a net charge of - 0.1 at pH 7.0. In some embodiments, the peptide has an average hydrophilicity (Hopp & Woods method) of -0.3. In some embodiments, the peptide has a ratio of hydrophilic residues to total residues of 33%.
  • the peptide has a pi of 6.1. In some embodiments, the peptide has a net charge of - 0.1 at pH 7.0. In some embodiments, the peptide has an average hydrophilicity (Hopp & Woods method) of -0.3. In some embodiments, the peptide has a ratio of hydrophilic residues to total residues of 33%.
  • the various peptides described bind selectively to a macromo!ecu!ar complex consisting of Fibrinogen B, Fibronectin and Fibulin 1.
  • the levels of this complex have surprisingly been found to be associated with neuroinf!ammatory diseases including multiple sclerosis.
  • Addition of the peptide to plasma or serum causes the peptide to bind to the complex of Fibrinogen B, Fibronectin and Fibulin 1 effecting a transformation of matter that results in the formation of the aggrefatin complex.
  • the work proposed in this application intends to develop biomarkers that enable rapid, reproducible and cost effective monitoring of disease activity and response to therapy to facilitate phase II drug trials as no such biomarkers are currently available for this purpose.
  • the cells containing the neural markers will be characterized by immunophenotyping PBMCs. Coexistence of neural antigens with antigens specific for leukocyte subpopu!ations will be determined by use of specific antibodies. This may be achieved by flow cytometry, immunofiuorescent microscopy and/or cell type specific enrichment/depletion using magnetic beads. We expect the results to identify known phagocytic ceil types (CD14+ monocytes and/or macrophages (CD 88/CD1 1 b)) to be the source of the neural antigens in PBMCs. cotArt n 3 ⁇ 4 s ft ! fcisiT
  • Immunological dogma recognizes that mononuclear phagocytes are recruited to sites of tissue injury where they perform a number of functions including clearance of debris. It is also a matter of dogma that if these debris laden phagocytes egress from the damaged tissue, they do so into the draining lymph nodes of the lymphatic system.
  • phagocytes entering the brain may re-enter the biood circulation, and we have generated preliminary data in multiple sclerosis (MS) subjects and Cuprizone fed mice that is consistent with this hypothesis.
  • MS multiple sclerosis
  • PBMCs peripheral biood from 18 subjects with MS and 12 apparently healthy individuals.
  • PBMCs peripheral biood from 18 subjects with MS and 12 apparently healthy individuals.
  • PBMCs peripheral biood from 18 subjects with MS and 12 apparently healthy individuals.
  • PBMCs peripheral biood from 18 subjects with MS and 12 apparently healthy individuals.
  • PBMCs peripheral biood from 18 subjects with MS and 12 apparently healthy individuals.
  • PBMCs peripheral biood from 18 subjects with MS and 12 apparently healthy individuals.
  • the lysates were coated onto ELISA wells at 5ug/mL and probed for Tau antigen or Hippocaicin iike-1 antigen using standard protocols.
  • Tau is a neuron specific microtubule associated protein that is best known as the substrate of neurofibrillary tangles in Alzheimer's disease.
  • Hippocaicin like-1 is a neuron specific calcium sequestering protein that is most abundant in the hippocampus.
  • the PBMCs were lysed and the lysates were coated onto ELISA wells and assayed with antibodies to CNS antigens as with the human samples.
  • the mouse lysates were probed for Tau, Hippocalcin like-1 and myelin basic protein (MBP). Simply by viewing figures 2 to4 it is apparent that there is no difference in control and Cuprizone treated mice until 5 weeks of Cuprizone feeding, a time point when there is extensive histological evidence of demyelination and neurodegeneration.
  • Tabie 2 Comparison of Cuprizone fed mice with control mice using the t-tesi
  • Macrophage with engulfed photoreceptor debris in the process of diapedesis through an optic nerve head capillary the ceil nucleus is inside the capillary whereas parts of the debris-containing cytoplasm is still externally located (arrow).
  • UCHL1 libiquitin carboxyl-terminai esterase L1
  • UCHL1 libiquitin carboxyl-terminai esterase L1
  • PBMCs from 10 short duration PD subjects will be utilized for new phagocytosed antigen discovery. Briefly, a lysate of PD PBMCs will be subjected to 1 -D PAGE and the gel will be coomassie stained. Coomassie stained protein bands are cut out and subjected to in gel trypsin digestion followed by protein identification by LC-MS- MS. The list of proteins present will be examined for the presence of CNS proteins. Candidate biomarkers found will be studied using either commercial antibodies if available or b contracted production of anti peptide antibodies in rabbits by Genscript Inc.
  • the potential biomarkers selected for initial testing will be screened on lysates from a further 10, short duration PD, PBMC lysates by ELISA assay. Any potential biomarker that is positive on two lysates will be a candidate for further screening in specific aim 2. A minimum of three such candidate antigens will be screened in specific aim 2.
  • Candidate biomarkers will be screened on lysates from fifty recently diagnosed ( ⁇ 3 years) PD subjects to determine prevalence. Comparisons will also be made to 20 apparently healthy control subjects. A single high prevalence (-80%) antigen or a combination of antigens providing high prevalence in aggregate will be sought.
  • the cell type(s) carrying neural antigens in the PBMCs have not been specifically identified but it is assumed that they are bone marrow derived mononuclear cells.
  • the cells containing the neural markers will be characterized by immunophenotyping PBMCs. Coexistence of neural antigens with antigens specific for leukocyte subpopulations will be determined by use of specific antibodies.
  • PBMCs PBMCs
  • Half of the PBMC will be used to make a lysates and the other half temporarily cryo preserved.
  • the lysates will be assayed to determine which neural biomarkers are present and then the eryopreserved ceils will be resuscitated for analysis with CD markers for leukocyte sub- populations and the relevant neural antigens.
  • kits Prior to marketing research use only kits, we will perform reagent stability studies to determine a minimum shelf life of 6 months. Furthermore, we will provide kits and PD blood sample to 3 alpha testing sites to determine inter lab reproducibility and coefficients of variation. We will also ship kits to our self to determine transportation effects on stability at ambient temperature and on ice.
  • the goal of the PDBP is to develop new and/or improved PD biomarker methodologies and technologies that can help inform Go/NoGo decisions in phase 2 clinical trials this includes studies required for moving an assay or method from an exploratory stage towards a validated approach for PD biomarker assessment.
  • CSF Cerebrospinal fluid
  • recirculating phagocytes as an antigen retrieval system coupled with its function in degrading tissue debris may be developed into just such a simple, inexpensive and reliable blood based biomarker assay of active n e u rod eg e n e rat io n .
  • MSDx will collect sufficient prospective samples from subject that will allow MSDx to send a sample (Serum, plasma, PBMC prep) to the NIH PDBP repository for use by other researchers.
  • a sample serum, plasma, PBMC prep
  • the corresponding patient demographics collected in the Case Report Forms will also be supplied.
  • Demographics e.g., age, sex, race.
  • Blood will be drawn by qualified personnel and associated data will be extracted from medical records and recorded on IRB approved data collection forms. The data on the collection forms will then be entered into the database created for this study.
  • subject-specific information For each subject visit, we enter subject-specific information into the database including demographics, medical history, drug therapy history, and symptom severities. Specimen samples collected from the subject during each visit are also tracked in the database, along with any ELISA, Western blot or Flow cytometry analysis data collected on the specimens over time.
  • the database provides detailed single subject reports that display the data available for an subject as well as multi-subject reports based on data across ail subjects in the study. Furthermore, since the data is ail readily available in the database, the data can be directly accessed and analyzed by many third party statistical analysis software packages (such as JMP, etc). The data can also be exported from the database into a format compatible with any software statistical analysis package. c. Potential Risks
  • Drawing blood involves temporary discomfort (from the needle stick), and may lead to bruising at the puncture site, fainting or, very rarely, infection.
  • a blood draw is considered to be a low risk procedure. This study involves little to no physical, psychological, financial, legal or other risk to the subject.
  • the clinical Pi or authorized staff member will obtain informed consent. Prior to any study-related activity (including screening assessments), all subjects will sign an informed consent document that has been approved b the IRB and complies with the requirements of 21 CFR Part 50, HIPAA, and all local regulatory requirements and laws. If a subject is unable to provide written informed consent, written informed consent may be obtained from the subject ' s legal representative.
  • the Investigator will provide in writing and explain the nature, purpose, and potential risks and benefits of the study and provide the subject with a copy of the informed consent document. The subject will be given sufficient time to consider the study's implications before deciding to participate. Subjects will be informed that they may withdraw from this study at any time at their own request without jeopardizing in any way their access to and quality of treatment they receive. A copy of the informed consent document signed by the subject will be given to the subject.
  • the clinical PI will retain the original of each subject's signed informed consent document.
  • Parkinson's disease affects slightly more men than women.
  • Parkinson's disease is very rare. Parkinson's disease is more common in older people with a prevalence of approximately 1 % in people over 60. Consequently this proposal will focus on Parkinson's disease in adults
  • references to the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting of”, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting of is met.

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Abstract

L'invention concerne des procédés de détection ou de surveillance de maladies neurologiques ou neurodégénératives comme la maladie de Parkinson par détection ou mesure de marqueurs biologiques du système nerveux central dans des phagocytes en recirculation après leur rentrée dans la circulation sanguine. Les procédés de la présente invention peuvent comprendre la détection de la neuromélanine dans ces phagocytes en recirculation. Par exemple, des peptides liant la neuromélanine peuvent être utilisés pour détecter la neuromélanine dans les phagocytes en recirculation.
PCT/US2013/068465 2007-11-30 2013-11-05 Détection de maladies neurologiques par mesure de la neuromélanine dans des phagocytes en recirculation Ceased WO2014071359A1 (fr)

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CA2929711A CA2929711A1 (fr) 2012-11-05 2013-11-05 Detection de maladies neurologiques par mesure de la neuromelanine dans des phagocytes en recirculation
US14/704,791 US20150233904A1 (en) 2009-11-27 2015-05-05 Detection of neurological diseases via measurement of neuromelanin in recirculating phagocytes
US17/228,416 US20220236288A1 (en) 2007-11-30 2021-04-12 Detection of neural-derived debris in recirculating phagocytes
US17/719,177 US20220236291A1 (en) 2007-11-30 2022-04-12 Detection of neural-derived debris in recirculating phagocytes
US18/379,431 US20240036065A1 (en) 2007-11-30 2023-10-12 Detection of debris in recirculating phagocytes

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