CA2929711A1 - Detection of neurological diseases via measurement of neuromelanin in recirculating phagocytes - Google Patents

Abstract

Methods for detecting or monitoring neurological or neurodegenerative diseases such as Parkinson's disease via detection or measurement of central nervous system biomarkers within recirculating phagocytes after re-entry into the blood stream. The methods of the present invention may feature detecting neurome!anin in such recirculating phagocytes. For example, neuromelanin-binding peptides may be used to detect neuromelanin in recirculating phagocytes.

Description

DETECTION OF NEUROLOGICAL DISEASES VIA MEASUREMENT OF
NEUROMELANIN IN RECIRCULATING PHAGOCYTES
CROSS REFERENCE
[0001] This application claims priority to U.S. Provisional Patent Application No.
61/722,441, filed November 5, 2012, the specification(s) of which is/are incorporated herein in their entirety by reference.
BACKGROUND OF THE INVENTION
100021 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 cells, microglial cells) or recruited from the blood stream (e.g., monocytes). Cells 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.
100031 We have surprisingly discovered that debris-laden phagocytes may re-enter the blood stream and if sufficient numbers are present, it may be possible to measure the debris that is inside the phagocytic cells. For example, we have found that the CNS antigens Tau and Hippocalcin like-1 are present in PBMC
preparations at a frequency that is statistically different from apparently healthy controls. Examining the cargo (e.g., the debris that would only normally be found in nerve cells) of these recirculating phagocytes is termed "Window into the Brain."
This technique may provide close to real-time data on what is happening in the brain since that particular cargo may only be present in the recirculating phagocytes for a few days before it is completely digested. The present invention features methods of detecting various diseases by examining the debris present in such recirculating phagocytes.
100041 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.
10005] Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art.
Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.
SUMMARY OF THE INVENTION
100061 The present invention features a method of detecting Parkinson's disease in a mammal. In some embodiments, 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.
100071 In some embodiments, 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, vitreal fluid, urine, the like, or a combination thereof. In some embodiments, the biomarker associated with Parkinson's disease comprises neuromelanin or a fragment thereof. In some embodiments, the circulating phagocyte includes a monocyte, a macrophage, a lymphocyte, or a combination thereof. In some embodiments, detecting the biomarker comprises subjecting the first sample and the second sample each to a peptide that binds to neuromelanin. In some embodiments, the peptide that binds to neuromelanin comprises 4B4 (SEQ ID NO:1A).

2 100081 The present invention also features a kit for detecting Parkinson's disease, said kit comprising a 484 peptide (SEC) ID NO:1A), the 4B4 peptide is for detecting neuromelanin in a recirculating phagocyte. In some embodiments, the 4B4 peptide comprises a label. In some embodiments, the label comprises biotin.
[00091 The present invention also features the use of a system for detecting Parkinson's disease. In some embodiments, the system comprises a neuromelanin-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 neuromelanin-binding peptide is higher than the level of neuromelanin detected in the second sample via the neuromelanin-binding peptide then Parkinson's disease is detected. In some embodiments, the neuromelanin-binding peptide comprises 484 (SEQ ID NO:1A). In some embodiments, the first sample is derived from blood. In some embodiments, the first sample comprises PBMCs.
10010] The present invention also features a system for detecting Parkinson's disease, wherein the system comprises a neuromelanin-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 neuromelanin-binding peptide is higher than the level of neuromelanin detected in the second sample via the neuromelanin-binding peptide then Parkinson's disease is detected. In some embodiments, the neuromelanin-binding peptide comprises 484 (SEQ ID NO:1A).
In some embodiments, the first sample is derived from blood. In some embodiments, the first sample comprises PBMCs.
[0011] The present invention also features a method of determining status of Parkinson's disease. In some embodiments, 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

3 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.
[0012] In some embodiments, if 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.
100131 In some embodiments, 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, vitreal fluid, urine, the like, or a combination thereof. In some embodiments, the circulating phagocyte includes a monocyte, a macrophage, a lymphocyte, or a combination thereof. In some embodiments, the biomarker comprises neuromelanin or a fragment thereof.
10014] The present invention also features a method of detecting neuromelanin.

In some embodiments, the method comprises introducing a neuromelanin binding protein comprising a labeled 4B4 peptide (SEQ ID NO:1A) to a sample; and detecting the label on the 4B4 peptide. In some embodiments, the sample comprises a circulating phagocyte. In some embodiments, 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, vitreal fluid, or a combination thereof. In some embodiments, the label comprises an enzyme. In some embodiments, the label comprises biotin. In some embodiments, the enzyme comprises horseradish peroxidase.

4 100151 The present invention also features a method of detecting Parkinson's disease in a patient. In some embodiments, 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 neuromelanin is detected then Parkinson's disease is detected in the patient. In some embodiments, the fluid sample comprises a circulating phagocyte. In some embodiments, the circulating phagocyte includes a monocyte, a macrophage, or a lymphocyte.
BRIEF DESCRIPTION OF THE DRAWINGS
100161 FIG. 1: Groups of plasma samples from multiple sclerosis (MS) patients that had low, medium or high levels of MSDx complex-1 were selected and then MMP-9 was measured. FIG. 1 shows the MMP-9 level was elevated in MS
patients relative to normal subjects (p=0.01-0.006) and did not vary by MSDx complex-1 level in MS subjects (p=not significant). 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 complex-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).
100171 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).
[0018] 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.
DESCRIPTION OF PREFERRED EMBODIMENTS
100191 Referring now to FIG. 1-3, the present invention features the detection and/or the monitoring of diseases, e.g., neurodegenerative diseases, Parkinson's disease, etc., by analysis of phagocylosed central nervous system (CNS) debris within phagocytes that have re-entered the blood circulation (e.g., recirculating phagocytes).
MSDX COMPLEX-I
100201 MSDx complex-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 metalloproteinases (MMPs).
10021] FIG. 1 shows that in plasma samples of Multiple Sclerosis patients, levels of MSDx complex-1 may be indirectly related to TIMP-1 levels (TIMPs are tissue inhibitors of metalloproteinases). 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 Mfv1P-9 activity and level of MSDx complex-1.
Intermediate levels of TIEV1P-1 correlate with intermediate levels of MSDx complex-1.
100221 Without wishing to limit the present invention to any theory or mechanism, it is believed that the data suggests that MSDx complex-1 may be generated by cell transmigration into tissues. For example, MSDx complex-1 may be generated by proteolytic activity of leukocytes (or other cell types) crossing the blood vessel wall and tissue barriers in order to enter the target organ.
100231 Further, 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 complex-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 mellitus, autoimmune Addison's disease, autoimmune hepatitis, celiac disease, pemphigous, chronic inflammatory demyelinating 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.
WINDOW INTO THE BRAIN
100241 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 neuroinflammatory 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 phosphorylated residue (e.g., a phosphorylated serine reside, a phosphorylated threonine reside;
e.g., serine 214, serine 235, serine 262, serine 356, serine 396, serine 404, serine 413, serine 46, serine 515, serine 516, serine 519, serine 531, serine 552, serine 610, serine 622, serine 641, 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; a protein or a fragment thereof selected from the group consisting of UCHL-1, neuromelanin, neuroglobin, valosin-containing protein, brain hexokinase, hippocalcin-1, nestin, synaptotagmin, myelin associated glycoprotein, transketolase, NS1 associated protein 1, major vault protein, synaptojanin, enolase, alpha synuclein, glial fibrillary acidic protein, S-100 protein, Neu-N, 265 proteasome subunit 9, annexin A2, annexin A3, annexin AS, annexin A6, annexin All, ubiquitin activating enzyme ZE1, ubiquitin B precursor, vimentin, glyceraldehyde-3-phosphate dehydrogenase, 13-3-3 protein, 14-3-3 protein (e.g., zeta isoform), NOGO-A.
[0025] 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. For example, neuromelanin may be detected in the debris of degenerated dopaminergic neurons (by recirculating phagocytes).
[0026] 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., biotinylated 4B4 peptide, a control peptide P601G (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, its components or adducts (both natural or synthetic); physical methods such as gas chromatography, liquid chromatography or mass spectrometry; and combinations of these methods. As shown in FIG. 2, the 4B4 peptide of sequence YERKFWHGRH (SEQ ID NO:1A) binds to neuromelanin granules in the dopaminergic neurons of the human substantia nigra.
[0027] As an example of a means of measurement of melanin within cells, 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 biotinylated 4B4 peptide. Unbound peptide was washed off and bound peptide was detected with streptavidin-HRP. In comparison, PBMCs from healthy human subjects show little binding of 4B4 peptide.
[0028] In some embodiments, more than one biomarker is detected in the sample(s). In some embodiments, the biomarker(s) is a neural¨derived biomarker.
However, the biomarker(s) is not limited to neural-derived biomarkers. In some embodiments, one or more biomarkers are detected in the sample, wherein the biomarkers are neural-derived, non-neural-derived biomarkers, or a combination thereof.
[0001] As used herein, the term "peripheral" refers to anything outside of brain tissue. For example, 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.
In some embodiments, the biomarker is an intracellular component. For example, the biomarker may be obtained from within a macrophage. In some embodiments, the macrophage sample is permeabilized. In some embodiments, the macrophage is lysed via various means, e.g., hypotonic solution treatment, detergent solution treatment, mechanical stress, etc.
DETECTABLE DISEASES AND BIOMARKERS FOR DETECTION
[0029] As previously discussed, the present invention features the detection of and/or the monitoring of various diseases via detection/measurement of various biomarkers in recirculating phagocytes.
100301 For example, in some embodiments, 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 demyelinating 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.
[0031] In some embodiments, 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 356, serine 396, serine 404, serine 413, serine 46, serine 515, serine 516, serine 519, serine 531, serine 552, serine 610, serine 622, serine 641, 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; a protein or a fragment thereof selected from the group consisting of UCHL-1, neuromelanin, neuroglobin, valosin-containing protein, brain hexokinase, hippocalcin-1, nestin, synaptotagmin, myelin associated glycoprotein, transketolase, NS1 associated protein 1, major vault protein, synaptojanin, enolase, alpha synuclein, glial fibrillary acidic protein, S-100 protein, Neu-N, 26S
proteasome subunit 9, annexin A2, annexin A3, annexin A5, annexin A6, annexin All, ubiquitin activating enzyme ZE1, ubiquitin B precursor, vimentin, glyceraldehyde-3-phosphate dehydrogenase, 13-3-3 protein, 14-3-3 protein (e.g., zeta isoform), NOGO-A, Ubiquitin carboxy-terminal hydrolase Li (UCHL1) also known as PARKS protein; neuronal-specific protein gene product 9.5; SwissProt P09936; proteolipid protein; myelin oligodendrocyte glycoprotein.
100321 Table A shows non-limiting examples of biomarkers that may be associated with disease states (e.g., degenerative disease states) with various organs. For example, 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.
100331 Table A
Damaged Organ =
=
Associated Markers Joints (eg Rheumatoid Arthritis) Citrulinated proteins Carbamylated proteins Articular cartilage degradation products Central Nervous System (eg multiple Neuromelanin (or a fragment thereof); a Tau sclerosis, Parkinsons disease, Alzheimer's protein (or fragment thereof), a Tau protein or disease etc). 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 356, serine 396, serine 404, serine 413, serine 46, serine 515, serine 516, serine 519, serine 531, serine 552, serine 610, serine 622, serine 641, 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; a protein or a fragment thereof selected from the group consisting of UCHL-1, neuromelanin. neuroglobin, valosin-containing protein, brain .hexokinase, hippocalcin-1, nestin, synaptotagmin, myelin associated glycoprotein, transketolase, NS1 associated protein 1, major vault protein, synaptojanin, enolase, alpha synuclein, glial fibrillary acidic protein, 5-100 protein, Neu-N, 265 proteasome subunit 9, annexin A2, annexin A3, annexin AS, annexin A6, annexin All, ubiquitin activating enzyme ZE1, ubiquitin B precursor, vimentin, glyceraldehyde-3-phosphate dehydrogenase, 13-3-3 protein, 14-3-3 protein (e.g., zeta isoform), NOGO-A, Ubiquitin carboxy-terminal hydrolase Li (UCHL1) also known as PARK5 protein; neuronal-specific protein gene product 9.5; SwissProt P09936; proteolipid protein;
myelin oligodendrocyte!ycoprotein.
Thyroid (eg Graves disease, Hashimotos Thyroglobulin thyroiditis) Thyroid peroxidase Retina (eg macular degeneration, retinitis Rhodopsin pigmentosa) Pancreatic islets (Diabetes) Insulin, Glucagon, somatostatin, pancreatic poiypeptide Inflammatory bowel disease Microbial glycans Lung Surfactant proteins A-C) Sjogrens Syndrome Salivary proiine rich proteins severe traumatic brain injury patients all-spectrin breakdown products (SBDPs) METHODS OF DETECTING A NEUROLOGICAL CONDITION VIA ANALYSIS
OF CIRCULATING PHAGOCYTES
[00341 Multiple Sclerosis (MS) is predominantly a disease of women of northern European origin and afflicts up to three million people worldwide. In the United States it is estimated that 400,000 people are affected. It is thought to be an autoimmune disorder and typically strikes young adults, causing a wide variety of symptoms that are often mistaken for other diseases. These symptoms stem from disruption of the central nervous system (CNS) and may include blurred or double vision; weakness in the arms or legs; changes or difficulties in balance, coordination and gait; bladder and/or bowel dysfunction; and emotional disturbances. Each patient may present a little differently and there may have been episodes in the past that were barely noticed by the patient at the time.
it is difficult to firmly diagnose MS, especially if there has been only one symptomatic episode. This leaves patients and their doctors waiting months or years for a relapse to confirm that the symptoms are due to MS.
100351 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 (MRI) 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 severity 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.
100361 The most common form of MS is relapsing-remitting multiple sclerosis (RRMS), which is characterized by symptomatic episodes separated in time, with partial or complete recovery of an apparently normal state between relapses.
It often converts to secondary progressive MS after several years, where there is a steady worsening of symptoms. A minority of patients have Primary Progressive MS which presents as a continuous slow worsening of the disease state. An even smaller minority of patients is diagnosed with Progressive-Relapsing MS, where in contrast to RRMS, there is a continuous worsening of their condition between acute episodes. 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 mRi, 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.
100371 Early diagnosis of MS is thought to be increasingly important, as much of the damage occurs early in the disease process. The earlier the diagnosis, the earlier disease-modifying treatment can begin and progression of the disease and associated disability can hopefully be slowed.
100381 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 sclerosis-associated biomarker indicates the presence of multiple sclerosis or a risk of multiple sclerosis. In some embodiments, the antigens detected in accordance with the present invention includes, for example, Ubiquitin carboxy-terminal hydrolase LI (UCHL1 ) also known as PARK5 protein; neuronal-specific protein gene product 9.5; SwissProt P09936.
Inflammatory Conditions 100391 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. For example, there may be a barrier between the fluid and the source of the biomarker. In other words, 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.
100401 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. In some embodiments, 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.
100411 As used herein, the term "peripheral" refers to anything outside of brain tissue. For example, 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.
In some embodiments, the biomarker is an intracellular component. For example, the biomarker may be obtained from within a macrophage. In some embodiments, the macrophage sample is permeabilized. In some embodiments, the macrophage is lysed via various means, e.g., hypotonic solution treatment, detergent solution treatment, mechanical stress, etc.
100421 In some embodiments, the sample is a plasma sample.
100431 Also, as used herein, "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 cells, etc.
100441 In some embodiments, one or more biomarkers are detected in the collected fluid sample. For example, 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. In some embodiments, 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. In some embodiments, 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.
[00451 In some embodiments, the inflammation condition that may be monitored or detected includes Rheumatoid Arthritis, Systemic Lupus Erythematosis, Shogren's Syndrome, and the like.
100461 In some embodiments, the biomarker(s) is a neural¨derived biomarker.
However, the biomarker(s) is not limited to neural-derived biomarkers. In some embodiments, one or more biomarkers are detected in the sample, wherein the biomarkers are neural-derived, non-neural-derived biomarkers, or a combination thereof.
100471 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. For example, in some embodiments, 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.
100481 In some embodiments, 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.
Neurological Conditions 10049J 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.
[00501 The sample (e.g., fluid) obtained does not necessarily directly come into contact with the inflamed tissue being detected. For example, in some embodiments, there may be a barrier between the fluid and the source of the biomarker. In other words, 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.

[00511 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. In some embodiments, 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.
100521 In some embodiments, detecting an increased level of the neurological 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. In some embodiments, detecting a decreased level of the neurological 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. A
control sample is discussed below.
100531 In some embodiments, 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.
100541 In some embodiments, the neurological condition-associated protein is derived from a brain source. In some embodiments, the neurological 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.
100551 The neurological 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 cells (PBMCs), 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. In some embodiments, the neurological condition-associated protein is an intracellular component. For example, the neurological condition-associated protein may be obtained from within a macrophage. In some embodiments, the macrophage sample is permeabilized. In some embodiments, the macrophage is lysed via various means, e.g., hypotonic solution treatment, detergent solution treatment, mechanical stress, etc.
100561 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.
100571 In some embodiments, 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.
Multiple Sclerosis 100581 The present invention also features methods of detecting multiple sclerosis or a risk of multiple sclerosis. In some embodiments, the methods of the present invention may allow for monitoring, detecting and/or predicting a relapse or a remission of multiple sclerosis. In some embodiments, the method of detecting multiple sclerosis is used in combination with one or more methods of detecting multiple sclerosis. For example, the present methods may be used in conjunction with other modalities to monitor, detect or predicting a relapse or a remission of multiple sclerosis.
100591 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 biamarker in the phagocyte. The sample may be provided from a mammal (e.g., a patient, a mouse, a rat, etc.).
In some embodiments, one or more multiple sclerosis-associated biomarkers is detected in the sample. The multiple sclerosis-associated biomarkers are associated with multiple sclerosis.
100601 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. In some embodiments, 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.
10061j In some embodiments, detecting an increased 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. 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. In some embodiments, 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.
100621 In some embodiments, 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. In some embodiments, the sample is obtained from the mammal before a relapse. In some embodiments, the sample is obtained during the course of the drug (e.g., steroid) treatment.
[00631 The multiple sclerosis-associated biomarker may be present in a circulating phagocyte. Phagocytes may include monocytes, macrophages, and/or lymphocytes. For example, macrophages are a type of monocyte and are phagocytic cells important in both specific cell-mediated immunity and non-specific innate immunity. Circulating phagocytes may be found in tissues, cells, 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, vitreal fluid, urine, the like, or a combination thereof. In some embodiments, the neurological condition-associated protein is an intracellular component.
For example, the neurological condition-associated protein may be obtained from within a macrophage. In some embodiments, the macrophage sample is permeabilized.
[00641 As used herein, 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.
10065] In some embodiments, 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 phosphorylated threonine reside). In some embodiments, the phosphorylated residue is serine 214, serine 235, serine 262, serine 356, serine 396, serine 404, serine 413, serine 46, serine 515, serine 516, serine 519, serine 531, serine 552, serine 610, serine 622, serine 641, 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.
100661 In some embodiments, phosphorylation of Tau can decrease its solubility.
In some embodiments, the method of detecting multiple sclerosis comprises detecting a level of insoluble Tau protein in the sample. In some embodiments, 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.

100671 In some embodiments, 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, synaptotagmin, 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, 26S proteasome subunit 9, annexin A2, annexin A3, annexin A5, annexin A6, annexin All, ubiquitin activating enzyme ZE1, ubiquitin B precursor, vimentin, glyceraldehyde-3-phosphate dehydrogenase, 13-3-3 protein, 14-4-4 protein, neurofilament heavy chain and neurofilament light chain.
100681 The multiple sclerosis-associated biomarker (e.g., Tau protein or fragment thereof) may be of various lengths. For example, in some embodiments, the multiple sclerosis-associated biomarker consists of between about 5 to 20 amino acids. In some embodiments, the multiple sclerosis-associated biomarker consists of about 20 to 40 amino acids. In some embodiments, the multiple sclerosis-associated biomarker consists of about 40 to 80 amino acids. In some embodiments, the multiple sclerosis-associated biomarker consists of about 80 to 150 amino acids. In some embodiments, the multiple sclerosis-associated biomarker consists of about 150 to 200 amino acids. In some embodiments, the multiple sclerosis-associated biomarker consists of about 200 to 300 amino acids.
In some embodiments, 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 600 amino acids.
100691 The multiple sclerosis-associated biomarker (e.g., Tau protein or fragment thereof) may comprise various regions of the full-length protein. For example, in some embodiments, the multiple sclerosis-associated biomarker comprises the amino-terminus (e.g., N-terminus, NH2-terminus, N-terminal 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). In some embodiments, 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. In some embodiments, the multiple-sclerosis associated biomarker consists of about the first 50 amino acids. In 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.
100701 In some embodiments, 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). In some embodiments, the multiple-sclerosis associated biomarker consists of about the last 100 amino acids.
Monitoring Disease Activity and Drug Efficacy 100711 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. The present invention may also feature methods of monitoring disease activity and drug efficacy. For example, 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.).
[0072j in some embodiments, 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) may be detected in the sample (e.g., in the phagocyte) and compared to the level or presence of the biomarker in control samples. In some embodiments, 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. By comparing the level or presence of the biomarker in the sample to either a control sample or a patient's previous sample, disease activity may be determined. A biomarker may include but is not limited to Tau or a fragment thereof.
100731 In some embodiments, 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.
100741 In some embodiments, 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.
10075] Table 1 shows the amino acid sequence of full-length human Tau protein.

Length (#
MW Amino SEQ
of Amino Amino Acid Sequence (Daltons) Acids ID
NO
Acids) 758 AA 78,878 Da MA??.Qv AZDHAGn:GL C.D?.KDQGC7YT
07his is M7his is the MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA 1_758 the length MW of the AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
of the unprocessed HVTQEPESGK VVQEM,REP GPPGLSHQLM
unprocessed precursor] SGMPGA1;=

precursor] GizjiA2LLKH QLLGULUQG 2Z.LKAGGi<:6 RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VOKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL
KNVKSKIGST ENLKHQPGGG KVQIINKKLD
LSNVOKCGS KDNIKHVPGG GSVQIVYKPV
DLSKVTSKCG SLGNIHHKPG GGQVEVKSEK
LDFKDRVQSK IGSLDNITHV PGGGNKKIET
HKLTFRENAK AKTDHGAEIV YKSPVVSGDT
SPRHLSNVSS TGSIDMVDSP QLATLADEVS
ASLAKQGL
[00771 Table 2 shows examples of some of the possible multiple sclerosis-associated biomarkers (e.g., Tau protein or a fragment there).
[00781 TABLE 2 Length (# MW Amino Acid Sequence Amino SEQ ID
of Amino (Daltons) Acids NO
Acids) 1235.4 MAEPRQEFEV 1-10 2 2310.56 MAEPRQEFEV MEDHAGTYGL 1-20 3 3388.69 MAEPRQEFEV MEDHAGTYGL

4545.84 MAEPRQEFEV MEDHAGTYGL

MHQDQEGDTD
5571.02 MAEPRQEFEV MEDHAGTYGL

MHQDQEGDTD AGLKESPLQT

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
7574.04 MAEPRQEFEV MEDHAGTYGL

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP
8571.17 MAEPRQEFEV MEDHAGTYGL

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV
9496.14 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 1-MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
100 10556.29 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEI PEG
110 11501.26 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT ------------------------------------------------SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD
120 12472.27 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
130 13565.44 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK
140 14720.77 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP
150 15738.98 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
160 16660.12 MAEPRQEFEV

GDRKDQGGYT MHQDQEGDTD AGLKESPLQT
PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP
170 17782.35 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP
180 18713.25 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
190 19822.58 1-190 20 200 20879.8 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG
210 21847.88 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM

SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
220 23050.05 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA

HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE
230 24062.07 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRBAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ
240 25070.18 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHOLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH OLLGDLHOG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
250 26125.36 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGOLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA
260 27062.44 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGOLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP
270 28148.74 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGOLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA

280 29145.79 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG

HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP
290 30141.88 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG
300 31313.22 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
310 32422.47 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN
320 33510.63 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHOLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE
330 34449.64 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHOLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE

ETTHVEI:PN VQKEQAHSEE HLGRAAFPGA

340 35433.69 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH OLLGDLHOEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG PAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP
350 36529.87 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD
360 37535.06 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
370 38611.37 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKOPA
AAPRGKPVSR
380 39701.64 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 1-380 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA

QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS
390 40719.73 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 1-390 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGOLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA

FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
400 41806.98 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA

HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS
410 42900.36 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH OLLGDLHOEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL
420 43892.47 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA

HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA

AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
430 44857.59 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA
440 45867.71 'MAEPROEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPOLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK
450 46828.72 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHOLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH OLLGDLHOEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG
460 47858.97 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGOLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKOPA

AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS

SSGAKEMKLK
470 48822.13 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQ.DAPLE
FTFHVEITPN VQKEQAH SEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKOPA
AAPPGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT
480 49775.15 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHOLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH OLLGDLHOEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA PEATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGODAr'LE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKOPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
490 50804.42 MAEPRQEFEV
MEDHAGTYGL GDRKDQGGYT i-490 SU
MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPPEATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DPDVDESSPQ DSPPSKASPA
QDGPPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSPTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA
500 51782.51 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 1-500 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHOLM

SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP
410 52807.56 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHOLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHOEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKOPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP SSGEPPKSGD
520 53701.53 MAEPRQEFEV MEDHAGTYGL

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG

SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKOPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP
530 54735.74 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGOLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKOPA
---------------------------------------------------- AAPRGKPVSR VPQLKARMVS
KSKDGTGSDD

KKAK2S21.33 A17LKNRPCL SPKLPTPGSS

SSGAKEMKLY GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPYTPP SSGEPPKSGU
RSGYSS.7'GST, GTPGSRSRTP
540 55677.7 MAEF7,0,-_v M_J¨A¨,YGL GDRKDQGGY7 1-540 55 MHQDQEGUTD AGLKESPLQT PZEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPH2EiPEG TTAEEAGIGD 2PSLEDEAAG

SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH OLLGDI_HQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA

FTFHVEL:PN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LREPSEKQ?A
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD

SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP SSGEPPKSGD

56295.63 MAEPROEFEV MEDHAGTYGL

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM

GRHAPELLKH QLLGULHQEG PRLKGAGGKE
RPGSKEEVDE DPDVDESSPQ DSPPSKASPA

FTFHVEITPN VOKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD

GOANA2RiPA KTPPARKTPP SSGEPPKSGD

KKVAVVR_PP
360 57337.84 MAEPRQEFEV
MEDHAGTYGL GDRKDQGGY_ 57 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA

HVTQEPESGK VVQEGFLREP GPPGLSHQLM

GRHAPELLYH QLLGOLHOEG PPLYGAGGKE
RPGSKEEVDE DRENUESSPQ DSRPSKASPA
QDGRPPQ_AA REATSIPGFP AEGA1PLPVD

FTFHVEi_PN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKOPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD

DPLIQPSSPA VCPEPP33F1c HVSSVTSRTG
SSGAKEMKLY GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPYTPP SSGEPPKSGU

RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL
570 58388.1 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA

HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL
580 59431.32 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHOLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA

FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL
KNVKSKIGST
590 60449.41 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG

GQANATRIPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP

KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL
KNVKSKIGST ENLKHQPGGG
600 61629.85 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS

SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL
KNVKSKIGST ENLKHQPGGG KVQIINKKLD
610 62633.98 MAEPRQEFEV
MEDHAGTYGL GDRKDQGGYT 1-610 r, MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA

HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKOPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL
KNVKSKIGST ENLKHQPGGG KVQIINKKLD
LSNVQSKCGS
620 63680.16 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHOLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHOEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA

FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKOPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK

GQANATRIPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL, KNVKSKIGST ENLKHQPGGG KVQIINKKLD
LSNVOSKCGS KDNIKHVPGG
630 64751.44 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 1-630 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGOLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL
KNVKSKIGST ENLKHQPGGG KVQIINKKLD
LSNVQSKCGS KDNIKHVPGG GSVQIVYKPV
640 65770.63 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VOKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG

GQANATRIPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL
KNVKSKIGST ENLKHQPGGG KVQIINKKLD
LSNVOKCGS KDNIKHVPGG GSVOIVYKPV
DLSKVTSKCG
650 66811.8 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VOKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA --------------------AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS

GQANATRiPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL
KNVKSKIGST ENLKHQPGGG KVQIINKKLD
LSNVOKCGS KDNIKHVPGG GSVQIVYKPV
DLSKVTSKCG SLGNiHHKPG
660 67853.95 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GREAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA

FTFHVEITPN VOKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSPTG

RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL
KNVKSKIGST ENLKHQPGGG KVQIINKKLD
LSNVOKCGS KDNIKHVPGG GSVQIVYKPV
DLSKVTSKCG SLGNiHHKPG GGQVEVKSEK
670 69071.34 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPPEATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DPDVDESSPQ DSPPSKASPA

FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPOLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSPTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRiPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL
KNVKSKIGST ENLKHQPGGG KVQIINKKLD

DLSKVTSKCG SLGNIHHKPG GGQVEVKSEK
LDFKDRVQSK
680 70121.52 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 1-680 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG

HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGOLHOEG PPLKGAGGKF
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKOPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL
KNVKSKIGST ENLKHQPGGG KVQIINKKLD
LSNVQSKCGS KDNIKHVPGG GSVQIVYKPV
DLSKVTSKCG SLGNIHHKPG GGQVEVKSEK
LDFKDRVQSK IGSLDNITHV
690 71103.62 MAEPPQEFEV MEDHAGTYGL GDRKDQGGYT 1-690 70 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPFGLSHOLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH OLLGDLHOEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG PAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPOLKARMVS KSKDGTGSDD
KKAKTSTRSS Ar< '1KNRPCL SPKLPTPGSS
DPLIQPSSPA VCz',L22SSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL
KNVKSKIGST ENLKHQPGGG KVQIINKKLD
LSNVQSKCGS KDNIKHVPGG GSVQIVYKPV
DLSKVTSKCG SLGNIHHKPG GGQVEVKSEK
LDFKDRVQSK IGSLDNITHV PGGGNKKII:

.

SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPPEATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPOLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSPTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL

DLSKVTSKCG SLGNIHHKPG GGQVEVKSEK

HKLTFRENAK
73351.17 MAEPPQEFEV MEDHAGTYGL

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH OLLGDLHOEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD

FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS

SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP SSGEPPKSGD

DLSKVTSKCG SLGNIHHKPG GGQVEVKSEK
LDFKDRVQSK 1GSLDNITHV PGGGNKK1Er HKLTFRENAK AKTDHGAEIV
720 74385.31 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH OLLGDLHOEG PPLKGAGGKE

QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP SSGEPPKSGD

KNVKSKIGST ENLKHQPGGG KVQIINKKLD

DLSKVTSKCG SLGNIHHKPG GGQVEVKSEK
LDFKDRVQSK 1GSLDNITHV PGGGNKK1E_ 730 75450.47 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 1-730 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM

SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL
KNVKSKIGST ENLKHQPGGG KVQIINKKLD
LSNVOSKCGS KDNIKHVPGG GSVQIVYKPV
DLSKVTSKCG SLGNIHHKPG GGQVEVKSEK
LDFKDRVQSK IGSLDNITHV PGGGNKKIET
HKLTFRENAK AKTDHGAEIV YKSPVVSGDT
SPRHLSNVSS
740 76804.91 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHQLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKOPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIQPSSPA VCPEPPSSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK
GQANATRIPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL
KNVKSKIGST ENLKHQPGGG KVQIINKKLD
LSNVQSKCGS KDNIKHVPGG GSVQIVYKPV
DLSKVTSKCG SLGNIHHKPG GGQVEVKSEK
LDFKDRVQSK IGSLDNITHV PGGGNKKIET
HKLTFRENAK AKTDHGAEIV YKSPVVSGDT
SPRHLSNVSS TGSIDMVDSP
750 78109.39 MAEPRQEFEV

MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQEPESGK VVQEGFLREP GPPGLSHOLM
SGMPGAPLLP EGPREATRQP SGTGPEDTEG
GRHAPELLKH QLLGDLHQEG PPLKGAGGKE
RPGSKEEVDE DRDVDESSPQ DSPPSKASPA
QDGRPPQTAA REATSIPGFP AEGAIPLPVD
FLSKVSTEIP ASEPDGPSVG RAKGQDAPLE
FTFHVEITPN VQKEQAHSEE HLGRAAFPGA
PGEGPEARGP SLGEDTKEAD LPEPSEKQPA
AAPRGKPVSR VPQLKARMVS KSKDGTGSDD
KKAKTSTRSS AKTLKNRPCL SPKLPTPGSS
DPLIUSSPA VCPEPPSSPK HVSSVTSRTG
SSGAKEMKLK GADGKTKIAT PRGAAPPGQK

GQANATRIPA KTPPAPKTPP SSGEPPKSGD
RSGYSSPGSP GTPGSRSRTP SLPTPPTREP
KKVAVVRTPP KSPSSAKSRL QTAPVPMPDL
KNVKSKIGST ENLKHQPGGG KVQIINKKLD
LSNVOSKCGS KDNIKHVPGG GSVQIVYKPV
DLSKVTSKCG SLGNIHHKPG GGQVEVKSEK
LDFKDRVQSK IGSLDNITHV PGGGNKKIET
HKLTFRENAK AKTDHGAEIV YKSPVVSGDT
SPRHLSNVSS TGSIDMVDSP QLATLADEVS
1815.06 QLATLADEVS ASLAKQGL 749-758 77 2818.17 TGSIDMVDSP QLATLADEVS ASLAKQGL 739-758 78 2992.33 SS TGSIDMVDSP QLATLADEVS 729-758 79 ASLAKQGL
4099.53 DT SPRHLSNVSS TGSIDMVDSP 719-758 80 QLATLADEVS ASLAKQGL
100791 In some embodiments, 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.
100801 In some embodiments, 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 cell sorting (FACS)); or a combination thereof and the like. Such assays are well known in the art.
[00811 In some embodiments, 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 (FAGS)), or a combination thereof and the like. In some embodiments, detecting the antibody-biomarker complex indicates the presence of multiple sclerosis or a risk of multiple sclerosis.
10082] As described above, in some embodiments, the step of detecting the multiple sclerosis-associated biomarker may comprise subjecting the sample florescence-activated cell sorting (FAGS). Fluorescence-activated cell sorting (FAGS) is a type of flow cytometry that sorts a mixture of biological cells, one at a time, into separate containers based upon the specific light scattering and fluorescent characteristics of each cell. It provides quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. Generally, 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 cells to break into individual droplets, and the system is adjusted so that there is a low probability of more than one cell 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.
10083] As described above, in some embodiments, 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. Generally, in ELISA, a sample containing an unknown amount of biomarker, e.g., an antigen, is affixed/immobilized to a surface (e.g., a polystyrene microtiter plate). Then, 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. In some cases, this antibody is covalently linked to an enzyme. In some cases, the antibody is not covalently linked to an enzyme but can be detected by a secondary antibody that is linked to an enzyme. In the final step, a substance (e.g., substrate) that the enzyme is capable of converting to a detectable visible signal (e.g., color signal) is added to the reaction. Thus, if the antibody/antigen complex is present, the substrate will be converted to the detectable visible signal, and then amount of antigen in the sample can be measured.
100841 As mentioned above, in some embodiments, 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.
In some embodiments, the antibody is a monoclonal or a polyclonal antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a chimera. In some embodiments, 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. In some embodiments, the antibody is synthetic. In some embodiments, the antibody is a recombinant antibody.
100851 Frequently, antibodies are labelled either covalently or non-covalently by combining the antibody with a second substance that provides for detectable signal. A wide variety of 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, fluorescers, chemiluminescers, magnetic particles, and the like. In some embodiments of the present invention, the antibody comprises a label.
100861 In some embodiments, the present invention is used to detect the presence of multiple sclerosis. For example, a patient may present with symptoms of a demyelinating disease. A sample (e.g., derived from the paitent) may be tested for an elevated level of a multiple sclerosis-associated biamarker.
If, according to the present invention, the level of a multiple sclerosis-associated biomarker is elevated and the patient presents symptoms of a demyelinating disease, then the patient is diagnosed as having multiple sclerosis.
100871 In some embodiments, the present invention is used to detect a risk of multiple sclerosis. For example, a patient may present with no symptoms of a demyelinating 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.
100881 As used herein, 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). To identify the level of the multiple sclerosis-associated biomarker that is the normal level, 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. From those pooled samples, 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 units, about 500 units, about 1000 units, about 10,000 units, about 100,000 units, about 1,000,000 units. In some embodiments, a unit may be an absorbance unit (e.g., from an ELISA), a percent positive (e.g., from a flow cytometry or FACS

assay), or a fluorescence unit.
10089] If the normal level of the multiple sclerosis-associated biomarker is some positive value (e.g., 5 units, 10 units, 50 units, 100 units, 500 units), then an elevated level refers to any level that is higher than the normal level. In some embodiments, 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. In some embodiments, 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.
In some embodiments, 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.
In 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.
In some embodiments, 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.
In some embodiments, 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.
In 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-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 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.

10090j The present invention also provides a method of monitoring the progression of multiple sclerosis and/or monitoring the treatment of multiple sclerosis. For example, in some embodiments, 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). Without wishing to limit the present invention to any theory or mechanism, 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). For example, a decrease in the level of the multiple sclerosis-associated biomarker may indicate the patient has entered or will enter a remission period. In some embodiments, 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). Without wishing to limit the present invention to any theory or mechanism, a treatment regimen (e.g., a drug) 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.
100911 As mentioned above, in some embodiments, 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 (MRI) findings are used to diagnose multiple sclerosis. Currently, 1V1R1 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 callosum, corona radiate, internal capsule, and centrum semiovale. In some embodiments, 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 (MRI) measurement. Without wishing to limit the present invention to any theory or mechanism, it is believed that elevated levels of the multiple sclerosis-associated biomarker correlate with a 1V1R1 scan showing the presence of multiple sclerosis plaques in the brain.

100921 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. For example, the method of the present invention may also help to distinguish multiple sclerosis from other diseases with similar clinical manifestations. For example, neuromyelitis optica (NMO), also known as Devic'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) 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. In some embodiments, 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.
100931 In some embodiments, 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.
100941 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.
100951 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, neuroglobin, valosin-containing protein, brain hexokinase, hippocalcin-1, nestin, synaptotagmin, myelin associated glycoprotein, Pv1yelin Basic Protein (MBP), Proteolipid Protein, Pv1yelin Oligodendrocyte Glycoprotein, transketolase, NS1 assocated protein 1, major vault protein, synaptojanin, enolase, alpha synuclein, glial fibrillary acidic protein, S-100 proteinNeu-N, 26S proteasome subunit 9, annexin A2, annexin A3, annexin A5, annexin A6, annexin All, ubiquitin activating enzyme ZE1, ubiguitin B
precursor, vimentin, glyceraldehyde-3-phosphate dehydrogenase, 13-3-3 protein, or fragments thereof. In some embodiments, the kit further comprises a means for detecting the binding of the antibody to the multiple sclerosis-associated biomarker. In some embodiments, the antibody is a monoclonal or a polyclonal antibody.
100961 In some embodiments, the detection of perforin is used in combination with detection of a marker (e.g., MBP) in phagocyltes. For example, it has been surprisingly discovered that perforin levels can decline in CD16 cells as MBP
levels increase.
Kit for Detecting Multiple Sclerosis 100971 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. In some embodiments, 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).
In some embodiments, the detecting of an elevated level of an antibody-antigen complex indicates presence of multiple sclerosis or a risk of multiple sclerosis.
100981 In some embodiments, the kit comprises an antibody, wherein the antibody is a monoclonal or a polyclonal antibody. In some embodiments, 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.
In some embodiments, the antibody is humanized. In some embodiments, the antibody is a chimera. In some embodiments, the antibody is specific for the multiple sclerosis-associated biomarker.
EXAMPLE I - Detecting Multiple Sclerosis in a Patient 100991 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 central 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.
1001001 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 1X PBS. After the final wash, extracts of the PBMCs are prepared by lysing with a hypotonic solution or other method. Then 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. Thus, the results of the assay indicate that the patient has multiple sclerosis. The physician notifies the patient, who then begins treatment immediately.
1001011 The laboratory receives the patient's blood sample collected in a CPT
tube. PBMCs are obtained from a BD Vacutainerrm 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 lx PBS. The cells are then subjected to assay involving an antibody that binds to Tau protein fragment comprising the phosphorylated serine residue Ser-404. The assay is the assay of example 4.
EXAMPLE 2 ¨ Direct ELISA Assay Protocol 1001021 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.
MicroELISA plates are coated by addition of 100 pL of a 5- 20 pgimL solution of the sample, which is then incubated for 1 hour at 210. The wells are washed out with phosphate buffered solution (PBS) with 0.05% polysorbate (Tween 20Tm).
The wells are then filled with 0.1 M glycine in PBS and incubated for 1 hour at 21 C to block unoccupied binding sites. After rewashing the wells, 100 pL of an appropriate dilution of antibody in PBS-0.05% Tween TM 20 with 1% bovine serum albumin (BSA) is added and incubated for 1 hour at 21C. The unbound antibody is then washed out with three exchanges of PBS-0.05% TweenTm 20. One hundred pL of an appropriately diluted horse radish peroxidase conjugated anti-immunoglobulin G (IgG) in PBS-0.05% TweenT" 20-1% 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% TweenT"" 20 and finally with PBS. One hundred pL of soluble MTB
substrate solution is added to each well and incubated for 30 minutes at 21'C
after which 100 pL of 1V1TB stop reagent is added and the color intensity is measured at 450nm using an ELISA plate reader.
1001031 Appropriate dilutions of the antigen and antibody are established by performing checkerboard titrations. Antigen concentrations in samples are interpolated from standard curves.
EXAMPLE 3 ¨ Indirect ELISA Assay Protocol 1001041 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% TweenT"' 20. The monoclonal antibody is then added at an appropriate dilution in PBS-0.05% Tweenrm20 - 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 Igrv1 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 pL of soluble MTB substrate solution is added to each well and incubated for 30 minutes at 21 C after which 100 pL of MTB stop reagent is added and the color intensity is measured at 450nm using an ELISA plate reader.
1001051 Appropriate dilutions of antigen and antibody are established by performing checkerboard titrations. Antigen concentrations in samples are interpolated from standard curves.
EXAMPLE 4 ¨ Flow Cytometry Protocol 1001061 The following example describes a flow cytometry assay used for detecting a multiple sclerosis-associated antigen in various samples. PBrv1Cs 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-lymphocyte marker or CD 19 B-Lymphocyte marker, CD68 intracellular monocyte marker and CD14 monocytei macrophage cell surface marker. The labeled cells are analyzed by flow cytometry for qualitative or quantitative differences.
1001071 PBMCs are obtained from a BD Vacutainerry 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 lx PBS. After the final wash, the cells are resuspended to approximately 4.0 mL in 1X PBS. Approximately 50 pL of the cell suspension to be analyzed is transferred into tubes for double staining with selected antibody pairs. Ten pL of 40mgimL normal human IgG (Sigma-Aldrich) for a total of 400 pg is added to each tube to block FC binding. The appropriate cell surface monoclonal antibodies CD3 PE, CD19 PE or CD14 PE are added at this time and incubated for 20 minutes at room temperature.
1001081 One hundred pi of Dako Intrastain TM Reagent A (fixative) is added to each tube and then mixed gently with a vortex mixer to ensure that the cells are in suspension. Cells are incubated at room temperature for 15 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. Supernatant is aspirated leaving about 50 pl of fluid. The fluid is mixed thoroughly to ensure that the cells are in suspension.
1001091 One hundred pL of Dako intrastain Ty 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 pl of fluid. The fluid is mixed thoroughly to ensure that the cells are in suspension.
1001101 One hundred pL of Dako lntrastainTM Reagent B (permeabilization) is added to each tube. The appropriate volume of the 2nd step antibody conjugated to FITC (specific to 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. To each tube, 2.0 mLs of 1XPBS working solution is added. The tubes are mixed gently then centrifuged at 300 X g for 5 minutes. The supernatant is aspirated, leaving approximately 50 pl of fluid. The tubes are mixed thoroughly to ensure that the cells are in suspension.
1001111 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. For analysis, 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 permeabilization time.
METHODS FOR MEASURING HIGH MOLECULAR WEIGHT COMPLEXES OF
FIBRINOGEN WITH FIBRONECTIN AND FIBULIN-1 (MSDX COMPLEX-I) [00112l It has been surprisingly discovered that the expression of a protein complex (e.g. an aggregate, a complex) termed "MSDX Complex-1" is elevated in multiple sclerosis patients as compared to healthy controls. MSDX Complex-1 is a high molecule weight complex comprising fibrinogen, fibronectin, and fibulin-1.
MSDX Complex-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. rvlSDx 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.
100113j 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 mellitus. The present invention is not limited to the aforementioned diseases and conditions or the aforementioned applications.
1001141 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. As used herein, the term "MSDx Complex-1" refers to a high molecular weight complex of fibrinogen, fibronectin, and fibulin-1. The detection of MSDX Complex-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.
1001151 A circulating high molecular weight protein complex has been found to bind certain small peptides selectively. For example, by sephacryl 5200 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 Pv15Dx Complex-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., biotinylated peptide). In some embodiments, 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.
[00116I In some embodiments, the method further comprises providing a well (e.g., Et.ISA 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 Complex-1 is not detected. If the level of the signal is lower than that of the control then MSDX Complex-1 is detected.
1001171 In some embodiments, the labeled peptide is or comprises SEQ ID
NO:3A. In some embodiments, the labeled peptide is or comprises SEC) ID NO:
4A. In some embodiments, the labeled peptide is or comprises SEC) ID NO: 5A.
1001181 In some embodiments, 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 pl of about 6.1. In some embodiments, the labeled peptide has a pl 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 7Ø In some embodiments, the labeled peptide comprises an epitope tag disposed at the C-terminus, the N-terminus, or at both termini.
100119] 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 fibulin-1. The first antibody comprises a label molecule (e.g., HRP). A well (e.g., ELISA well) is provided. The well is coated with a second antibody, wherein the second antibody is specific for one of fibrinogen, fibronectin, or fibulin-1. In some embodiments, 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). When the signal is detected then MSDX Complex-1 is detected.
1001201 In some embodiments, the first antibody is an anti-fibulin-1 antibody and the second antibody is an anti-fibrinogen antibody. In some embodiments, the first antibody is an anti-fibronectin antibody and the second antibody is an anti-fibrinogen antibody. In some embodiments, the first antibody is an anti-fibrinogen antibody and the second antibody is an anti-fibrinogen antibody. In some embodiments, the first antibody is an anti-fibulin-1 antibody and the second antibody is an anti-fibronectin antibody. In some embodiments, the first antibody is an anti-fibronectin antibody and the second antibody is an anti-fibronectin antibody. In some embodiments, the first antibody is an anti-fibrinogen antibody and the second antibody is an anti-fibronectin antibody. In some embodiments, 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-fibrinogen antibody and the second antibody is an anti-fibulin-1 antibody.
[00121] In some embodiments, 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 fibrinogen, wherein the third antibody has a different specificity than the first antibody. In some embodiments, 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 fibrinogen, wherein the third antibody has a different specificity than the first antibody and a different specificity than the third antibody.
[00122] In some embodiments, the label molecule comprises an enzyme. In some embodiments, the enzyme comprises horseradish peroxidase.
[00123] In some embodiments, 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.). In some embodiments, 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.
[00124] As used herein, the term "about" refers to plus or minus 10% of the referenced number.

[00125] The following example describes an example of a method of detecting MSDX Complex-1. 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) is then contacted with the immobilized antibody and unbound material is washed off. Then antibodies to fibronectin and/or Fibulin-1 are contacted with the immobilized material and unbound antibodies are washed off.

The bound antibodies are then detected with a labelled anti-immunoglobulin of the appropriate specificity to generate a measurable signal (the signal may be chemiluminescent, fluorescent, colorimetric, potentiometric, amperometric etc).

1001261 The following example describes an example of a method of detecting MSDX Complex-1. In some embodiments, method is a competitive ELISA assay format. In some embodiments, the competitive ELISA assay used for detecting MDSX Complex-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. In this assay the labeled analyte is bound by an unrelated binding protein that prevents ifs binding to antibody and is washed off before the detection step. A standard curve to quantify binding by mosx Complex-1 is generated by competition with "cold" peptide.
1001271 ELISA wells coated with goat anti-rabbit IgG(Fc) trap immune complexes formed between a rabbit anti-peptide antibody and biotinylated peptide. MSDX
Complex-1 in added plasma competes with the rabbit anti-peptide antibody for binding to biotinylated peptide. The more MSDX Complex-1 that is present in the plasma the more biotinylated peptide it binds leaving less available to bind to antibody. Thus high levels of MSDX Complex-1 result in low optical density and vice versa.
1001281 In some embodiments, the peptide is labeled at the C-terminal with biotin or another detection agent. In some embodiments, the N-terminal of the peptide may be amine or amide. In some embodiments, the peptide is 24 amino acids long. In some embodiments, the peptide sequence is:
CQYRCFQVITNGIGLNLFKDPVAD (SEQ ID NO: 3A). In some embodiments, the peptide has a pl of 6.1. In some embodiments, the peptide has a net charge of -0.1 at pH 7Ø 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%. In some embodiments, an epitope tag is attached to a terminus, e.g., the N-terminus, to enable the use of other capture antibodies, for example a polyHistidine tag (HisTag).

1001291 In some embodiments, 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.
1001301 In some embodiments, the peptide sequence is CSFKCYSVVINGLGINVFKDPVAD (SEQ ID NO: 4A). In some embodiments, the peptide has a pl of 6.1. In some embodiments, the peptide has a net charge of -0.1 at pH 7Ø 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%.
1001311 In some embodiments, the peptide sequence is CQYRCFQIITNGIGLNLFKDPVAD (SEQ ID NO: 5A). In some embodiments, the peptide has a pl of 6.1. In some embodiments, the peptide has a net charge of -0.1 at pH 7Ø 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%.
1001321 The various peptides described bind selectively to a macromolecular complex consisting of Fibrinogen B, Fibronectin and Fibulin 1. The levels of this complex have surprisingly been found to be associated with neuroinflammatory 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.

APPENDIX
ABSTRACT
Several factors currently impede therapy development and clinical study design for neuroprotective agents for Parkinson's disease. These include the limited ability to detect early stage PD prior to the onset of motor signs, inadequate measures of processes or pathways related to disease pathogenesis, and the lack of biomarkers that define disease progression.
We propose to develop a new source of circulating biomarkers for use in diagnosis and monitoring of disease activity by exploiting the ability of monocytesimacrophages to enter the brain and engulf debris coupled with the paradigm shifting hypothesis that at least some of these cells re-enter the circulation. Thus we may exploit these re-circulating cells as a shuttle vector that retrieves antigen from the brain so that we may probe these cells for new and established CNS tissue markers. Unlike circulating antibody markers, which can remain in the circulation for years after the tissue insult, antigens engulfed by phagocytes are degraded within a few days. This means that if these CNS
antigens are detected, they are reflective of recent tissue injury. Given that 80% of striato-nigral neurons are lost before clinical symptoms arise, the clearance of the dead neurons by recruited and re-circulating mononuclear phagocytes should be an early indicator of neuronal damage. If we are successful in validating this concept, we will be in a position to rapidly identify and develop potential biomarkers to improve the efficiency and outcome of Phase H clinical trials and advance therapeutic development for PD.
PROJECT NARRATIVE:
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 H drug trials as no such biomarkers are currently available for this purpose. We will develop a novel strategy exploiting re-circulating phagocytes to retrieve biomarkers from the brain for this purpose.
SPECIFIC AIMS
We have postulated, counter to immunological dogma, that phagocytes entering the brain may re-enter the blood circulation. We have generated preliminary data that is consistent with this hypothesis and propose that these phagocytes may be exploited as a novel source of biomarkers because they carry CNS debris.
Furthermore, as the debris is degraded within days, this approach may be a means of monitoring "active" neurodegenerative processes. The goal of this U18 proposal is to apply this innovative concept to monitoring disease activity in Parkinson's disease by pursuing the following specific aims:
Specific Aim 1; To discover relevant CNS proteins in re-circulating phagocytes from Parkinson's Disease (PD) Subjects:
Our preliminary studies in multiple sclerosis and Cuprizone fed mice have shown that Tau, Hippocalcin like 1, myelin basic protein and proteolipid protein can be found by ELISA assay in lysates of peripheral blood mononuclear cells (PBMCs).

Consequently we will probe for these proteins in PBMCs from PD subjects.

Additionally we will probe lysates for alpha-synuclein, glutamic acid decarboxylase (GAD) and ubiquitin carboxyl-terminal esterase Li (UCHL1) proteins. We will also attempt to discover potentially informative CNS antigens in PD PBMCs using a shotgun proteomic analysis described in the approach section of the research plan. Hippocalcin like 1 was discovered to be a phagocytosed biomarker using this approach.
Specific Aim 2: To determine marker prevalence in various clinical phenotypes of Parkinson's disease:
Using an ELISA employing specific antibodies to identified candidate CNS
antigens in PD PBMC lysates we will determine the prevalence of each marker in 50 recently diagnosed (< 3 years) PD subjects and 20 apparently healthy subjects.
Specific Aim 3; To determine the phenotype of the PBMCs that contain the neural antigens:
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. This may be achieved by flow cytometry, immunofluorescent microscopy and/or cell type specific enrichment/depletion using magnetic beads.
We expect the results to identify known phagocytic cell types (CD14+ monocytes and/or macrophages (CD 68/CD11b)) to be the source of the neural antigens in P B MCs RESEARCH STRATEGY
(a) Significance As stated in the request for applications, several factors currently impede therapy development and clinical study design for neuroprotective agents for Parkinson's disease. These include the limited ability to detect early stage PD prior to the onset of motor signs, inadequate measures of processes or pathways related to disease pathogenesis, and the lack of biomarkers that define disease progression.
We propose to develop a new source of circulating biomarkers for use in diagnosis and monitoring of disease activity by exploiting the ability of monocytesimacrophages to enter the brain and engulf debris coupled with the paradigm shifting hypothesis that at least some of these cells re-enter the circulation. Thus we may exploit these re-circulating cells as a shuttle vector that retrieves antigen from the brain so that we may probe these cells for new and established CNS tissue markers. Unlike circulating antibody markers, which can remain in the circulation for years after the tissue insult, antigens engulfed by phagocytes are degraded within a few days. This means that if these CNS
antigens are detected, they are reflective of recent tissue injury. Given that 80% of striato-nigral neurons are lost before clinical symptoms arise, the clearance of the dead neurons by recruited and re-circulating mononuclear phagocytes should be an early indicator of neuronal damage. If we are successful in validating this concept, we will be in a position to rapidly identify and develop potential biomarkers to improve the efficiency and outcome of Phase H clinical trials and advance therapeutic development for PD.
(b) Innovation 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. We have postulated, counter to immunological dogma, that phagocytes entering the brain may re-enter the blood circulation, and we have generated preliminary data in multiple sclerosis (MS) subjects and Cuprizone fed mice that is consistent with this hypothesis. We propose that these phagocytes may be exploited as a novel source of biomarkers because they carry CNS
debris.
Furthermore, as the debris is degraded within days, this approach is a means of monitoring "active" neurodegenerative processes. The goal of this U18 proposal is to validate this innovative concept for monitoring disease activity in Parkinson's disease.
(c) Approach Preliminary Studies To obtain pilot data to support this concept, we obtained peripheral blood from 18 subjects with MS and 12 apparently healthy individuals. PBMCs were isolated and osmotically lysed. The lysates were coated onto ELISA wells at 5ug/mL and probed for Tau antigen or Hippocalcin like-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. Hippocalcin like-1 is a neuron specific calcium sequestering protein that is most abundant in the hippocampus. The ELISA assay results for these 2 antigens in the pilot population are shown below (Figure ).

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in the control group one individual gave anomalously high results in both the Tau assay and the Hic)pecalciri Like-1 assay: Consequently the second highest control was art)itrarily selected as a cut-off point for each assay. For each assay this resulteti in 7/18 positive MS subjects and 1/12 i..)ositive controls. The results were not statistically significant for either antigen alone. However, analyzing the results for positivity for either of the two antigei)s resulted in 9/1f3 positive MS
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Tablet 2x2 contingency table of positivity for Tau or Hippocalcin Like-1.
We also examined PBMC from normal C5761/6 mice and cuprizone fed C5761/6 mice for CNS antigens. Cuprizone causes Demyelination at 3-4 weeks and also neurodegeneration at 4-5 weeks. Discontinuation of Cuprizone feeding at 6 weeks or earlier results in repair of the CNS damage. Five mice were fed with regular lab chow, two groups of five mice were fed with Cuprizone (0.2%) chow for 3 weeks and 4 weeks and one group of 4 mice were fed with Cuprizone (0.2%) chow for 5 weeks. The mice were bled and PBMCs isolated by Ficoll density gradient centrifugation. The PBMCs were lysed and the ysates were coated onto EL1SA wells and assayed with antibodies to CNS antigens as with the human samples. The mouse lysake.s 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.
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Comparison of grouped assay results by unmatched 2 tailed I.-test confirmed the visual conclusion that only the mice fed Cuprizone for 5 weeks were different from the control mice (Table 2).
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*** statistically significant difference Table 2: Comparison of Cuprizone fed mice with control mice using the t-test (2 tailed P values) These results are consistent with the hypothesis that some phagocytes that have engulfed debris re-enter the blood circulation and suggest that measurement of the phagocyte's cargo may be informative in measuring disease activity in the CNS, The year after we filed our patent application, Joy et al reported that resident and recruited phagocytes remove dead photoreceptor cells from the retina in a rat model of retinal degeneration. They published the electron micrographs shown below in figure 5 showing a macrophage loaded with photoreceptor cell debris re-entering the blood circulation via a capillary. This cell has to be entering the capillary rather than leaving it as photoreceptor cells are unique to the retina and therefore cannot have been acquired elsewhere. This study provides incontrovertible evidence that phagocytes do indeed re-enter the blood circulation but did not consider the diagnostic implications.
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Figure 5. From Joy S et al. Resident Microglia and Bone Marrow immigrants Remove Dead Photoreceptors in Retinal Lesions. The American Journal of Pathology, Vol.
174, No, 6, June 2009.
Macrophage with engulfed photoreceptor debris in the process of diapedesis through an optic nerve head capillary the cell nucleus is inside the capillary whereas parts of the debris-containing cytoplasm is still externally located (arrow).
These studies provide "proof of principle".
Our strategy to achieve the specific aims of this proposal is discussed below.
Specific Aim 1; To discover relevant CNS proteins in re-circulating phagocytes from Parkinson's Disease MD) Subjects.

While we have generated proof of principle data we do not yet know which antigens will be relevant to Parkinson's disease (PD). Consequently, our strategy is to obtain PBMCs from Parkinson's disease subjects and test them for the antigens that we have found to be relevant in other CNS diseases, namely Tau, Hippocalcin like-1 and myelin basic protein. We will also probe for alpha synuclein, glutamic acid decarboxylase (GAD) and ubiquitin carboxyl-terminal esterase Li. Alpha synuclein is associated with PD
pathology and GAD is associated with "stiff man" syndrome which may have some overlap with symptoms of PD. Ubiquitin carboxyl-terminal esterase Li (UCHL1) is found in all neurons of the brain and should be present in recirculating phagocytes when there is active neurodegeneration present. Thus UCHL1 represents a safety net biomarker. UCHL1 polymorphisms have been reported to influence risk of development of PD.
We will also employ a shotgun proteomic approach to discovering new antigens that may associate with PD. We have used this method to identify Hippocalcin like-1 as an antigen of interest. 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 by 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.
By using both a candidate antigen approach and a proteomic discovery approach we reduce the risk of not having potentially useful biomarkers for validation in PD.
Specific Aim 2; To determine marker prevalence in various clinical phenotypes of Parkinson's disease:
Candidate biomarkers will be screened on lysates from fifty recently diagnosed (< 3 years) PD subjects to determine prevalence. Comparisons will also be made to apparently healthy control subjects. A single high prevalence (-80%) antigen or a combination of antigens providing high prevalence in aggregate will be sought.
As there is no pre-existing data on which to base power calculations, 50 subjects for each group has been selected as it will enable the detection of a minimum marker prevalence of 2% (1/50) Specific Aim 3; To determine the phenotype of the PBMCs that contain the neural anticiens:
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.
This may be achieved by flow cytometry, immunoffuorescent microscopy and/or cell type specific enrichment/depletion using magnetic beads. We expect the results to identify known phagocytic cell types (CD14+ monocytes and/or macrophages (CD 68/CD11 b)) to be the source of the neural antigens in PBMCs. Identifying the cell type carrying neural antigens will enable additional sample preparation steps that will enrich the relevant cells and will be expected to increase sensitivity of the assays.
We will collect 2 x 8 mL tubes of blood from PD subjects and prepare PBMCs.
Half of the PBMC will be used to make a lysates and the other half temporarily cryopreserved. The lysates will be assayed to determine which neural biomarkers are present and then the cryopreserved cells will be resuscitated for analysis with CD markers for leukocyte sub-populations and the relevant neural antigens.
Statistical Analysis:
Statistical analysis will be required to interpret the results of specific aim 2. The unmatched t-test will be used to determine whether the means of each group are significantly different from each other. A p-value of 0.05 or less will indicate statistical significance.
Apparently healthy controls will be used to determine a cut off value (mean +2SD) in order to create categorical data that can be analyzed with 2 x 2 contingency tables and the chi squared test. This will also enable expression of the results as positive and negative predictive values.
Assay performance characteristics:
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.
We have recently launched a research use only assay kit for multiple sclerosis and have experience in making these determinations.
Expected results, potential pitfalls and alternative approaches:
The potential pitfall in specific aim one is that the markers we have worked with to date have not yet been evaluated in PD and may not be found in phagocytes in PD. To reduce the risk of not having appropriate antigens to probe for in PD PBMCs we will also perform proteomic studies on PD PBMCs to detect potentially relevant CNS
antigens for PD. We also have included the UCHL1 antigen which is expressed in all neurons and should be present in engulfed debris. The tau antigen has also been implicated in PD
and we expect to find it in PO PBMCs. Some antigens may have regional differences in abundance that may affect detect ability and may therefore only be seen in particular clinical phenotypes. Given the combination of approaches we expect to have several promising candidates.
A potential pitfall in specific aim 2 is that all potential markers will be found to have a low prevalence. This could be due to inadequate sensitivity of the assay. Specific aim three will provide the solution to this problem should it arise. Identifying the phenotype of the antigen laden cells will enable their enrichment prior to preparing a lysates.
This will increase the amount of specific protein in the lysates to be assayed. We do not anticipate difficulties with specific aim three.
Alignment with PDBP goals:
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.
We have developed proof of principle data for a novel approach for antigen retrieval from the brain utilizing re-circulating phagocytes as a shuttle vector. Because these antigens are short lived in phagocytes, the detection of their presence will indicate current active neurodegeneration. This approach is in alignment with PDBP goals as we propose to develop this concept beyond the "proof of principle" stage to create a validated assay for monitoring disease activity in Parkinson's disease. As drugs that decrease disease activity will be expected to decrease CNS antigen laden phagocytes in the circulation, this approach will facilitate evaluation of response to therapy and early evaluation of likelihood of success of phase II trials.
Usefulness to other PD biomarker researchers:
Progress in the development and use of biomarkers for PD has been limited by inherent barriers associated with studying the brain. Blood based biomarkers for PD has been unsatisfactory, perhaps because in transport from the brain to blood they are degraded or otherwise made inaccessible in many patients. Cerebrospinal fluid (CSF) is preferred by many PD biomarker researchers as it is felt that molecules entering the CSF
have not been as heavily processed as those moving to the blood supply. However, a simple, inexpensive and reliable blood based biomarker assay would greatly facilitate both research and clinical care. The novel use of 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 neurodegeneration.
Data Sharing Plan:
Data will be deposited into the PDBP DMR following completion of data analysis, application for provisional patents and/or within one week of publication.
BioSpecimen Requirement:
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. The corresponding patient demographics collected in the Case Report Forms will also be supplied.
Proposed Annual Milestones:
Specific AIM 1 Milestones- Candidate biomarkers will be screened on 10 lysates from short duration PD subjects. Biomarkers found to be positive in at least 2 subjects will be selected for further analysis. A minimum of three biomarkers will be selected for further study.

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:a PROTECTiON OF HUMAN SUBJECTS: RISK TO HUMAN SUBJECTS
a. i-tuniatl Subjects Involvement end Characteristics 100 subjects will be enrolled in this study. In this application we propose three specific aims. The first specific aim will study 20 short duration PD subjects. Ten subjects will be analysed by a shot gun proteomic approach for biomarker discovery. These biomarkers along with other candidate markers will be screened by ELBA assay on lysates from a further 10 short duration PD subjects To be eligible for entry into this study, candidates must meet all of the following eligibility criteria at the time of entry:
1. Diagnosis of Parkinson's disease within the last 3 years.
2. Male or female, age 50-80 years inclusive.
3. Willing and able to provide written informed consent in compliance with the regulatory requirements. If a subject is unable to provide written informed consent, written informed consent may be obtained from the subject's legal representative.
Candidates will be excluded from study entry if any of the following exclusion criteria exist at the time of entry:
1. Any clinically significant disease other than PD.
2. Unwilling or unable to comply with the requirements of this protocol, including the presence of any condition (physical, mental, or social) that is likely to affect the subject's ability to comply with the protocol.
3. Any other reasons that, in the opinion of the Investigator, the candidate is determined to be unsuitable for entry into the study.
b. Sources of Materials The clinical PI or authorized staff member will screen patients coming to the clinic for entry into the study. Prior to any study-related activity subjects will sign an informed consent document that complies with the requirements of 21 CFR Part 50, HIPAA
and all local regulatory requirements and laws. The following activities will then be performed:
= Obtain written informed consent.
= Review of inclusion/exclusion criteria.
= Demographics (e.g., age, sex, race).
= Review of medical history.
= Review of concomitant and past medications and therapies.
= Obtain two tubes of blood (total maximum volume approximately 20 mL drawn in a BD CPT Tube).
Only the staff at the recruiting Institutions will have access to individually identifiable private information about human subjects. All samples and associated data will have individually identifiable private information removed and replaced with a code number before transmittal to MSDX, Inc.
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.
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 EUSA, 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 any subject as well as multi-subject reports based on data across all subjects in the study. Furthermore, since the data is all readily available in the database, the data can be directly accessed and analyzed by many third party statistical analysis software packages (such as sIMP, 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.
Protection of Human subjects: Adequacy of Protection Against Risks a. Recruitment and Informed Consent 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 by 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.
b. Protection against Risk Only the staff at the recruiting institutions will have access to individually identifiable private information about human subjects enrolled into this study by them. All samples and associated data will have individually identifiable private information removed and replaced with a code number before transmittal to MSDX, Inc. The electronic database we use is password protected and the computer that the database resides on is in a locked room. Hard copies of data will be kept in locked filing cabinets in a locked room.
All personnel with access to the data have been trained and certified in protection of human subjects from research risks. While absolute guarantees of security of these data are not possible it is highly unlikely that a breach of privacy will occur.
Protection of Human subjects: Potential Benefits of the Proposed Research to Human Subjects and Others There will not be any direct benefit to the subject by taking part in this research study.
Knowledge gained from the study may benefit others in the future by leading to the development of a simple, inexpensive blood test to monitor new drug effectiveness in PD.
The general results of the study may be published in scientific journals that may be beneficial to further research into PD. The likely benefits of this research far outweigh the risks as only a low risk blood draw is involved and adequate measures to protect subject confidentiality have been adopted.

Protection of Human subjects: Importance of the Knowledge to be gained Knowledge gained from the study may benefit others in the future by leading to the development of a simple, inexpensive blood test that facilitate phase II
trials of drugs that would preserve neurological function by limiting damage to the central nervous system, slow or prevent disease progression and limit disability thus improving quality of life for the patient and reducing the health care costs of PD.
Targeted/Planned Enrollment Table Study Title: Recirculating Phagocytes: A Shuttle Vector for Retrieval of Biomarkers from the Brain.
Total Planned Enrollment 100 Targeted/Planned Enrollment: Number of Subjects Ethnic Category Sex/Gender Male Female Total Hispanic or Latino Not Hispanic or Latino 50 50 Ethnic Category: Total of all Subjects 50 50 Racial Categories American Indian/ Alaska Native Asian Native Hawaiian or other Pacific Islander Black or African American White 50 50 Racial Categories: Total of All Subjects 50 50 Inclusion of Women and Minorities:
A. Gender Subjects of both genders will be recruited for this study. Parkinson's disease affects slightly more men than women.
B. Minority Groups or subaroups Whether PD is less common in Blacks and other ethnic minorities has been a matter of debate. Several epidemiological studies have suggested that Blacks may be less likely to develop PD than Whites, but other studies have reported similar disease rates.
In addition, PD in certain non-White populations may be clinically different.
Neuroclegenerative diseases, PD among them, are clinico-pathological entities almost exclusively defined in White populations. It is difficult to determine then whether the disease is similar across different ethnic groups, because of a paucity of studies in non-Whites. Race and ethnicity modulate diseases via genetic background, therefore, PD
symptoms and signs may be expected to differ across different ethnicities. In PD, two studies from Nigeria observed more frequent atypical features in African Black PD
patients, but data from the US is lacking. Until the issue of ethnic effects in PD is resolved, it is reasonable to assume that all people have a similar probability of developing the disease and that the results gained will be applicable to all ethnicities.

From the table below it can be seen that Phoenix and the state of Arizona have a comparable ethnic and racial distribution of population. The vast majority of the population is non-hispanic white and the minority populations are largely composed of Hispanic, non-Hispanic black, non-Hispanic Native American and non-Hispanic Asian.
From the information cited above it is clear that the minority population available in Phoenix and the state of Arizona consists of populations at low risk for given the time frame of the study and the scarcity of patients of these ethnicities the study is likely to be predominantly recruiting patients of white European origin. Data collection includes ethnicity/race and where sufficient numbers are available, statistical analysis by ethnicity will be performed.
The ethnic and racial breakdown for Arizona and the city of Phoenix Total Non- Non- Non- Non- Non-Hispanic Hispanic Hispanic Hispanic Hispanic Hispanic White Black Native Asian other American Arizona 4,961,953 916,017 3,575,201 139,650 255,619 69,749 5,718 City of 1,289,125 258,379 925,079 63,743 20,581 19,566 1,778 Phoenix Source: Arizona Department of Economic Security, Research Administration Inclusion of children Juvenile 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 [001331 As used herein, the term "about" refers to plus or minus 10% of the referenced number.
[001341 Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description.
Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.
[001351 Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. Reference numbers recited in the claims are exemplary and for ease of review by the patent office only, and are not limiting in any way. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of 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.
1001361 The reference numbers recited in the below claims are solely for ease of examination of this patent application, and are exemplary, and are not intended in any way to limit the scope of the claims to the particular features having the corresponding reference numbers in the drawings.

Claims (31)

WHAT IS CLAIMED IS:

1. A method of detecting Parkinson's disease in a mammal, said method comprising:
(a) 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 (b) 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.

2. The method of claim 1, wherein 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, vitreal fluid, urine, the like, or a combination thereof.

3. The method of claim 1, wherein the biomarker associated with Parkinson's disease comprises neuromelanin or a fragment thereof.

4. The method of claim 1, wherein the circulating phagocyte includes a monocyte, a macrophage, a lymphocyte, or a combination thereof.

5. The method claim 3, wherein detecting the biomarker comprises subjecting the first sample and the second sample each to a peptide that binds to neuromelanin.

6. The method of claim 5, wherein the peptide that binds to neuromelanin comprises 4B4 (SEQ ID NO:1A).

7. A kit for detecting Parkinson's disease, said kit comprising a 4B4 peptide (SEQ ID NO:1A), the 4B4 peptide is for detecting neuromelanin in a recirculating phagocyte.
7.2 The kit of claim 7, wherein the 4B4 peptide comprises a label.
7.3 The method of claim 7.2, wherein the label comprises biotin.

8. The use of a system for detecting Parkinson's disease, the system comprises a neuromelanin-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 neuromelanin-binding peptide is higher than the level of neuromelanin detected in the second sample via the neuromelanin-binding peptide then Parkinson's disease is detected.

9. The use of claim 8, wherein the neuromelanin-binding peptide comprises 4B4 (SEQ ID NO:1A).

10. The use of claim 8, wherein the first sample is derived from blood.

11. The use of claim 10, wherein the first sample comprises PBMCs.

12. A system for detecting Parkinson's disease, wherein the system comprises a neuromelanin-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 neuromelanin-binding peptide is higher than the level of neuromelanin detected in the second sample via the neuromelanin-binding peptide then Parkinson's disease is detected.

13. The use of claim 12, wherein the neuromelanin-binding peptide comprises 4B4 (SEQ ID NO:1A).

14. The use of claim 12, wherein the first sample is derived from blood.

15. The use of claim 14, wherein the first sample comprises PBMCs.

16. A method of determining status of Parkinson's disease, the method comprises:
(a) 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;
(b) 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.

17. The method of claim 16, wherein if 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.

18. The method of claim 16, wherein 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.

19. The method of claim 16, wherein 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.

20. The method of claim 16, wherein 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, vitreal fluid, urine, the like, or a combination thereof.

21. The method of claim 16, wherein the circulating phagocyte includes a monocyte, a macrophage, a lymphocyte, or a combination thereof.

22. The method of claim 16, wherein the biomarker comprises neuromelanin or a fragment thereof.

23. A method of detecting neuromelanin, said method comprising:
(a) introducing a neuromelanin binding protein comprising a labeled 484 peptide (SEQ ID NO:1A) to a sample; and (b) detecting the label on the 4E34 peptide.

24. The method of claim 23, wherein the sample comprises a circulating phagocyte.

25. The method of claim 23, wherein 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, vitreal fluid, or a combination thereof.

26. The method of claim 23, wherein the label comprises an enzyme.

27. The method of claim 23, wherein the label comprises biotin.

28. The method of claim 26, wherein the enzyme comprises horseradish peroxidase.

29. A method of detecting Parkinson's disease in a patient, the method comprises:
(a) 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 (b) detecting neuromelanin in the fluid sample, wherein when neuromelanin is detected then Parkinson's disease is detected in the patient.

30. The method of claim 29, wherein the fluid sample comprises a circulating phagocyte.

31. The method of claim 30, wherein the circulating phagocyte includes a monocyte, a macrophage, or a lymphocyte.