WO2011160096A2 - Glial fibrillary acidic protein, autoantigens and autoantibodies thereto as biomarkers of neural injury or neurological disorder or condition - Google Patents

Glial fibrillary acidic protein, autoantigens and autoantibodies thereto as biomarkers of neural injury or neurological disorder or condition Download PDF

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WO2011160096A2
WO2011160096A2 PCT/US2011/040998 US2011040998W WO2011160096A2 WO 2011160096 A2 WO2011160096 A2 WO 2011160096A2 US 2011040998 W US2011040998 W US 2011040998W WO 2011160096 A2 WO2011160096 A2 WO 2011160096A2
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biomarker
protein
subject
injury
gfap
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PCT/US2011/040998
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French (fr)
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WO2011160096A3 (en
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Kevin Ka-Wang Wang
Zhiqun Zhang
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Banyan Biomarkers, Inc.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders

Definitions

  • the present invention in general relates to determination of neural injury or a neurological disorder or condition of an individual and in particular to measuring the quantity of a neuroprotective biomarker such as glial fibrillary acidic protein (GFAP) and other proteins present at the blood brain barrier (BBB), and antibodies such as autoantibodies directed to GFAP.
  • a neuroprotective biomarker such as glial fibrillary acidic protein (GFAP) and other proteins present at the blood brain barrier (BBB), and antibodies such as autoantibodies directed to GFAP.
  • biomarkers As biomarker detection uses a sample obtained from a subject, typically cerebrospinal fluid, blood, or plasma, and detects the biomarkers in that sample, biomarker detection holds the prospect of inexpensive, rapid, and objective measurement of neurological condition.
  • the attainment of rapid and objective indicators of neurological condition allows one to determine severity of a non-normal brain condition with a previously unrealized degree of objectivity, predict outcome, guide therapy of the condition, as well as monitor subject responsiveness and recovery. Additionally, such information as obtained from numerous subjects allows one to gain a degree of insight into the mechanism of brain injury arising from neural injury or a neurological disorder or condition.
  • biomarkers have been identified as being associated with traumatic brain injury including severe TBI as is often seen in vehicle collision and combat wounded subjects. These biomarkers included spectrin breakdown products such as SBDP150, SBDP150i, SBDP145 (calpain mediated acute neural necrosis), SBDP120 (caspase mediated delayed neural apoptosis), UCH-L1 (neuronal cell body damage marker), and MAP2 dendritic cell injury associated marker. The nature of these biomarkers is detailed in U.S. Patents 7,291,710 and 7,396,654, the contents of which are hereby incorporated by reference.
  • Glial Fibrillary Acidic Protein a member of the cytoskeletal protein family, is the principal 8-9 nanometer intermediate filament of glial cells such as mature astrocytes of the central nervous system (CNS).
  • GFAP is a monomeric molecule with a molecular mass between 40 and 53 kDa and an isoelectric point between 5.7 and 5.8.
  • GFAP is highly brain specific protein that is not found outside the CNS. GFAP is released into the blood and CSF soon after brain injury.
  • astrocytes become reactive in a way that is characterized by rapid synthesis of GFAP termed astrogliosis or gliosis.
  • GAP43 neuromodulin
  • GAD 1 GAD 1
  • GSD Gamma Enolase
  • NSE protein Gamma Enolase
  • Neurofilament Proteins NF-L, NF-M, NF-H
  • GRIA1 Protein Kinase, c AMP -dependent, regulatory, type I, alpha (PRKARIA), Paraneoplastic Antigen MA2 (PNMA2), Potassium voltage-gated channel, shaker-subfamily, beta member2 (KCNAB2) transcript variant 1 and Endophilin-Alalso known as human SH3 -domain GRB2-like 2 (SH3GL2)
  • FIG. 1 are bar graphs of GFAP and other biomarkers for human control and severe TBI subjects from CSF samples;
  • FIG. 2 are bar graphs of GFAP and other biomarkers for human control and severe TBI subjects
  • FIG. 3 are bar graphs of GFAP and other biomarkers for human control and severe TBI subjects summarizing the data of FIGs. 1 and 2;
  • FIG. 4 are plots of arterial blood pressure (MABP), intracranial pressure (ICP) and cerebral profusion pressure (CPP) for a single human subject of traumatic brain injury as a function of time;
  • MABP arterial blood pressure
  • ICP intracranial pressure
  • CPP cerebral profusion pressure
  • FIG. 5 are plots of inventive biomarkers from CSF and serum samples from the single human subject of traumatic brain injury of FIG. 4 as a function of time;
  • FIG. 6 are plots of inventive biomarkers from CSF and serum samples from another individual human subject of traumatic brain injury as a function of time;
  • FIG. 7 are plots of UCH-L1 amounts being present in CSF and serum post severe traumatic brain injury in a mouse subject;
  • FIG. 8 are bar graphs of GFAP concentration for controls, as well as individuals in the mild/moderate traumatic brain injury cohort as a function of CT scan results upon admission and 24 hours thereafter;
  • FIG. 9 are bar graphs of parallel assays for UCH-L1 biomarker from the samples used for FIG. 8;
  • FIG. 10 are bar graphs showing the concentration of UCH-L1 and GFAP as well as a biomarker not selected for diagnosis of neurological condition, SI 00 beta, provided as a function of injury magnitude between control, mild, and moderate traumatic brain injury;
  • FIG. 11 are bar graphs showing the concentration of the same markers as depicted in FIG. 10 with respect to initial evidence upon hospital admission as to lesions in tomography scans;
  • FIG. 11 represents biomarker levels in human subjects with varying types of brain injury.
  • FIG. 12 illustrates Western Blots showing the presence of human brain at least 11 different autoantigen in human subjects with TBI.
  • FIG. 13 illustrates Western Blots showing the presence of human brain at least 7 different autoantigen in human subjects with TBI.
  • FIG. 14 represents a western blot of brain-specific autoantibody response to various brain antigens, including GFAP found in stroke patients.
  • FIG. 15 represents a western blot of subacute development of blood-based autoantibody to GFAP in subset of SCI patients' serum.
  • FIG. 16 represents a western blot of brain-specific autoantibody response to various brain antigens, including GFAP, found in epilepsy patients' serum.
  • FIG. 17 represents a western blot of brain-specific Autoantibody response to various brain antigens, including GFAP, found in Parkinson's disease and migraine patients' serum.
  • FIG. 18 represents a western blot of brain-specific autoantibody response to various brain antigens, including GFAP, found in Alzheimer's disease and migraine patients' serum. DESCRIPTION OF THE INVENTION
  • the present invention has utility in the diagnosis and management of abnormal neurological condition including neural injuries or neuronal disorders.
  • a biomarker such as GFAP or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIA1, or autoantibodies to these autoantigens.
  • a subject a determination of subject neurological condition is provided with greater specificity than previously attainable.
  • the description is appreciated by one of ordinary skill in the art as fully encompassing as an inventive first biomarker as described herein.
  • Detection of these autoantigens will help diagnose brain or CNS/PNS injury (e.g. TBI) beyond 4-5 day post-initial injury event and serves to diagnose poor-outcome after brain injury or CNS/PNS (e.g. TBI) in those neural disorders and injuries that result in a change in extracellular biomarker concentration. Furthermore, monitoring of these autoantigens guides immune-modulation therapy to suppress adverse autoimmune response following brain or CNS/PNS injury.
  • Brain or CNS/PNS injury conditions that benefit from the present invention illustratively include mild to moderate brain injury, traumatic brain injury, stroke, spinal cord injury, subarachnoid hemorrhage (SAH) and peripheral nerve injury.
  • a neurological condition may be an abnormal neurological condition such as that caused by genetic disorder, injury, or disease to nervous tissue.
  • a means for detecting or diagnosing an abnormal neurological condition in a subject is provided.
  • An assay is provided for detecting or diagnosing the neurological condition of a subject.
  • the neurological condition may be the result of stress such as that from exposure to environmental, therapeutic, or investigative compounds, it is a further aspect of the present invention to provide a process and assay for screening candidate drug or other compounds or for detecting the effects of environmental contaminants.
  • a process for detecting a neurological condition optionally includes determining the neurological condition of a subject by assaying a sample derived from a subject at a first time for the presence of a first biomarker.
  • a biomarker is a cell, protein, nucleic acid, steroid, fatty acid, metabolite, or other differentiator useful for measurement of biological activity or response.
  • Illustrative biomarkers as used herein illustratively include: GFAP; or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIA1.; or antibodies directed to GFAP and/or one or more other marker.
  • the inventive process also includes assaying the sample for at least one additional neuroactive biomarker.
  • the one additional neuroactive biomarker is optionally not the same biomarker as the first biomarker.
  • a second biomarker is illustratively ubiquitin carboxyl- terminal esterase LI (UCH-L1), Neuron specific enolase (NSE), spectrin breakdown products (SBDP), preferably SBDP150, SBDP150i SBDP145, SBDP120, S100 calcium binding protein B (SI 00b), microtubule associated proteins (MAP), optionally MAP2, MAPI, MAP 3, MAP4, MAP5, myelin basic protein (MBP), Tau, Neurofilament protein (NF), Cannabinoid Receptor (CB), CAM proteins, Synaptic protein, collapsin response mediator proteins (CRMP), inducible nitric oxide synthase (iNOS), Neuronal Nuclei protein (NeuN), cysteinyl-specific peptidase (CSPas
  • SBDP120 caspase
  • SBDP150i caspase
  • MAP2-BDP1 (calpain) Cofilin ctl l-spectrin SBDP 150+145 MAP2-BDP2 (caspase) APP -BDP (Calpain) NG2; Phosphacan, neruocan; versi
  • Ach Receptor fragment Nicotinic, alpha-synuclein NSF Muscarinic
  • betall-Spectrin-BDPllO Calbindin-9K Resistin
  • betall-Spectrin-BDP85 (caspase) Tau-Total Neuropilins
  • LC3 Crerbellin 3 Beclin-1 Any number of biomarkers can be detected such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; sequentially or simultaneously from a single sample or aliquots from a sample. Detection can be either simultaneous or sequential and may be from the same biological sample or from multiple samples from the same or different subjects. Detection of multiple biomarkers is optionally in the same assay chamber.
  • the inventive process optionally further includes comparing the quantity of the first biomarker and the quantity of the at least one additional neuroactive biomarker to normal levels of each of the first biomarker and the one additional neuroactive biomarker to determine the neurological condition of the subject.
  • a biomarker is GFAP.
  • GFAP is associated with glial cells such as astrocytes.
  • a process of determining a neurological condition optionally includes detection of one or more antibodies in a biological sample.
  • An antibody is optionally an autoantibody.
  • Autoantibodies are directed to antigens released from a site of neurological trauma such as TBI, disease, injury or other abnormality.
  • neurological conditions including TBI, causes cellular damage that releases intracellular or cell membrane contents into the CSF or bloodstream.
  • the levels of many of these proteins such as those listed in Table 1 are not normally present in biological fluids other than the cytoplasm or cell membrane of neuronal tissue such as brain tissue.
  • the presence of these antigens leads to the production of autoantibodies to these antigens within a subject.
  • Detection of an autoantibody as a biomarker is optionally used to diagnose the presence of an abnormal neurological condition in a subject.
  • US Patent No. 6,010,854 describes methods of producing screening antigens and methods of screening for autoantibodies to neuronal glutamate receptors. These methods are equally applicable to the subject invention. As such, US Patent No. 6,010,854 is incorporated herein by reference for its teaching of methods of producing screening antigens that are operable for screening for autoantibodies. US Patent No. 6,010,854 is similarly incorporated herein by reference for its teaching of methods of detecting autoantibodies. It is appreciated that other methods of detecting antibodies illustratively including ELISA, western blotting, mass spectroscopy, chromatography, staining, and others known in the art are similarly operable. [0037] Several antigens have been discovered as producing autoantibodies following onset of a neurological condition. Such antigens are those illustratively listed in Table 2.
  • Neurofilament light polypeptide (NF-L)
  • Neurofilament heavy polypeptide NF-H
  • NSE Gamma-enolase
  • Table 3 Examplary autoantigens related to brain injury
  • NSE Gamma-enolase
  • MAPI A MAPI A
  • Table 4 Exemplary brain injury-induced autoantigens based on reported antigenicity.
  • Voltage-gated calcium channel VGCC P/Q-type (as in Lambert-Eaton myasthenic syndrome)
  • Voltage-gated potassium channel (VGKC) (as in Limbic encephalitis, Isaac's Syndrome.
  • Hu and Yo cerebellar Purkinje Cells (as in Paraneoplastic cerebellar syndrome)
  • Amphiphysin (as in Paraneoplastic cerebellar syndrome)
  • Glutamic acid decarboxylase (as in Diabetes mellitus type 1, Stiff person syndrome)
  • Aquaporin-4 Neuroporin-4 (Neuromyelitis optica ; evic's syndrome)
  • Basal ganglia neurons (as in Sydenham's Chorea, Pediatric Autoimmune Neuropsychiatric
  • Zic proteins (zinc finger proteins) (as in Joubert syndrome - cerebellum malformation)
  • ANNA 3 brain autoantigen
  • PCA-2 Purkinje cell antibody
  • PKC ⁇ paraneoplastic cerebellar degeneration
  • Nicotinic acetylcholine receptor (as in Myasthenia gravis) Recoverin
  • TULIP- 1 tubby-like protein 1
  • full length protein such as any protein listed in Tables 1-4, or a breakdown product thereof, is operable as a screening antigen for autoantibodies.
  • UCH-Ll is antigenic and produces autoantibodies in a subject.
  • the sequence for human UCH- Ll protein is found at NCBI accession number NP 004172.2.
  • the sequence for human GFAP is found at NCBI accession number NP 002046.1.
  • Other illustrative antigens illustratively include, alpha-spectrin or breakdown products thereof, MAP, Tau, Neurofascin, CPvMP-2, MAP2 crude sample, and human brain lysate.
  • any suitable method of producing peptides and proteins of Table 1 is operable herein.
  • cloning and protein expression systems used with or without purification tags are optionally used.
  • Illustrative methods for production of immunogenic peptides include synthetic peptide synthesis by methods known in the art. Chemical methods of peptide synthesis are known in the art and include solid phase peptide synthesis and solution phase peptide synthesis or by the method ofhackeng, TM, et al, Proc Natl Acad Sci U S A, 1997; 94(15):7845-50, the contents of which are incorporated herein by reference. Either method is operable for the production of antigens operable for screening biological samples for the presence of autoantibodies.
  • peptide means peptides of any length and includes proteins.
  • polypeptide and oligiopeptide are used herein without any particular intended size limitation, unless a particular size is otherwise stated.
  • neurological condition shall mean neural injury or neuronal disorder.
  • stroke is art recognized and is intended to include sudden diminution or loss of consciousness, sensation, and voluntary motion caused by rapture or obstruction (e.g. by a blood clot) of an artery of the brain.
  • the term "Traumatic Brain Injury” is art recognized and is intended to include the condition in which, a traumatic blow to the head causes damage to the brain, often without penetrating the skull.
  • the initial trauma can result in expanding hematoma, subarachnoid hemorrhage, cerebral edema, raised intracranial pressure (ICP), and cerebral hypoxia, which can, in turn, lead to severe secondary events due to low cerebral blood flow (CBF).
  • ICP intracranial pressure
  • CBF cerebral blood flow
  • the term "injury or neural injury” is intended to include a damage which directly or indirectly affects the normal functioning of the CNS.
  • the injury can be damage to retinal ganglion cells; a traumatic brain injury; a stroke related injury; a cerebral aneurism related injury; a spinal cord injury, including monoplegia, diplegia, paraplegia, hemiplegia and quadriplegia; a neuroproliferative disorder or neuropathic pain syndrome.
  • CNS injuries or disease include TBI, stroke, concussion (including post-concussion syndrome), cerebral ischemia, neurodegenerative diseases of the brain such as Parkinson's disease, Dementia Pugilistica, Huntington's disease and Alzheimer's disease, Creutzfeldt- Jakob disease, brain injuries secondary to seizures which are induced by radiation, exposure to ionizing or iron plasma, nerve agents, cyanide, toxic concentrations of oxygen, neurotoxicity due to CNS malaria or treatment with anti-malaria agents, trypanosomes, malarial pathogens, and other CNS traumas.
  • TBI TBI
  • stroke concussion (including post-concussion syndrome)
  • cerebral ischemia neurodegenerative diseases of the brain such as Parkinson's disease, Dementia Pugilistica, Huntington's disease and Alzheimer's disease, Creutzfeldt- Jakob disease
  • brain injuries secondary to seizures which are induced by radiation, exposure to ionizing or iron plasma
  • nerve agents nerve agents
  • cyanide
  • Neurodegenerative disorders Parkinson's; Alzheimer's) or autoimmune disorders (multiple sclerosis) of the central nervous system; memory loss; long term and short term memory disorders; learning disorders; autism, depression, benign forgetfulness, childhood learning disorders, close head injury, and attention deficit disorder; autoimmune disorders of the brain, neuronal reaction to viral infection; brain damage; depression; psychiatric disorders such as bi-polarism, schizophrenia and the like; narcolepsy/sleep disorders(including circadian rhythm disorders, insomnia and narcolepsy); severance of nerves or nerve damage; severance of the cerebrospinal nerve cord (CNS) and any damage to brain or nerve cells; neurological deficits associated with AIDS; tics (e.g.
  • Giles de la Tourette's syndrome Huntington's chorea, schizophrenia, traumatic brain injury, tinnitus, neuralgia, especially trigeminal neuralgia, neuropathic pain, inappropriate neuronal activity resulting in neurodysthesias in diseases such as diabetes, MS and motor neuron disease, ataxias, muscular rigidity (spasticity) and temporomandibular joint dysfunction; Reward Deficiency Syndrome (RDS) behaviors in a subject.
  • RDS Reward Deficiency Syndrome
  • a sample is optionally a biological sample.
  • biological samples include cells, tissues, cerebral spinal fluid (CSF), artificial CSF, whole blood, serum, plasma, cytosolic fluid, urine, feces, stomach fluids, digestive fluids, saliva, nasal or other airway fluid, vaginal fluids, semen, buffered saline, saline, water, or other biological fluid recognized in the art in which a target biomarker or metabolite thereof is found.
  • a biological sample is CSF or serum. It is appreciated that two or more separate biological samples are optionally assayed to elucidate the neurological condition of the subject.
  • biomarkers In addition to increased cell expression, biomarkers also appear in biological fluids in communication with injured cells.
  • Obtaining biological fluids such as cerebrospinal fluid (CSF), blood, plasma, serum, saliva and urine, from a subject is typically much less invasive and traumatizing than obtaining a solid tissue biopsy sample.
  • CSF cerebrospinal fluid
  • samples that are biological fluids are preferred for use in the invention.
  • CSF in particular, is preferred for detecting nerve damage in a subject as it is in immediate contact with the nervous system and is readily obtainable.
  • Serum is a preferred biological sample as it is easily obtainable and presents much less risk of further injury or side-effect to a donating subject.
  • samples of CSF or serum are collected from subjects with the samples being subjected to measurement of GFAP, or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto.
  • the subjects vary in neurological condition.
  • GFAP GFAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto are optionally then correlated with CT scan results as well as GCS scoring. Based on these results, an inventive assay is developed and validated (Lee et al, Pharmacological Research 23:312-328, 2006).
  • GFAP or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto, in addition to being obtained from CSF and serum, are also readily obtained from blood, plasma, saliva, urine, as well as solid tissue biopsy.
  • CSF is a sampling fluid in many embodiments of the invention owing to direct contact with the nervous system, it is appreciated that other biological fluids have advantages in being sampled for other purposes and therefore allow for inventive determination of neurological condition as part of a battery of tests performed on a single sample such as blood, plasma, serum, saliva or urine.
  • a biological sample is obtained from a subject by conventional techniques.
  • CSF is preferably obtained by lumbar puncture.
  • Blood is preferably obtained by venipuncture, while plasma and serum are obtained by fractionating whole blood according to known methods.
  • Surgical techniques for obtaining solid tissue samples are well known in the art. For example, methods for obtaining a nervous system tissue sample are described in standard neurosurgery texts such as Atlas of Neurosurgery: Basic Approaches to Cranial and Vascular Procedures, by F. Meyer, Churchill Livingstone, 1999; Stereotactic and Image Directed Surgery of Brain Tumors, 1st ed., by David G. T.
  • nerve cells in in vitro culture or in situ in a subject express altered levels or activities of one or more proteins than do such cells not subjected to the insult.
  • samples that contain nerve cells e.g., a biopsy of a central nervous system or peripheral nervous system tissue are illustratively suitable biological samples for use in the invention.
  • other cells express illustratively GFAP including, for example, cardiomyocytes, myocytes in skeletal muscles, hepatocytes, kidney cells and cells in testis.
  • a biological sample including such cells or fluid secreted from these cells might also be used in an adaptation of the inventive methods to determine and/or characterize an injury to such non-nerve cells.
  • a subject illustratively includes a dog, a cat, a horse, a cow, a pig, a sheep, a goat, a chicken, non-human primate, a human, a rat, and a mouse.
  • Subjects who most benefit from the present invention are those suspected of having or at risk for developing abnormal neurological conditions, such as victims of brain injury caused by traumatic insults (e.g., gunshot wounds, automobile accidents, sports accidents, shaken baby syndrome), spinal cord injury, seizure, ischemic events (e.g., stroke, cerebral hemorrhage, cardiac arrest), neurodegenerative disorders (such as Alzheimer's, Huntington's, and Parkinson's diseases; prion-related disease; other forms of dementia), epilepsy, substance abuse (e.g., from amphetamines, Ecstasy/MDMA, or ethanol), and peripheral nervous system pathologies such as diabetic neuropathy, chemotherapy-induced neuropathy and neuropathic pain.
  • traumatic insults e.g., gunshot wounds, automobile accidents, sports accidents, shaken baby syndrome
  • spinal cord injury e.g., seizure, ischemic events (e.g., stroke, cerebral hemorrhage, cardiac arrest), neurodegenerative disorders (such as Alzheimer's, Hunt
  • Baseline levels of several biomarkers are those levels obtained in the target biological sample in the species of desired subject in the absence of a known neurological condition. These levels need not be expressed in hard concentrations, but may instead be known from parallel control experiments and expressed in terms of fluorescent units, density units, and the like. Typically, in the absence of a neurological condition GFAP and other or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl are present in biological samples at a negligible amount.
  • GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl are present in biological samples at a negligible amount.
  • autoantibodies to GFAP or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF- L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl are absent in a biological sample form a subject not suspected of having a neurological condition.
  • GFAP and or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl are often highly abundant in neurons.
  • Determining the baseline levels of GFAP and or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl in neurons of particular species is well within the skill of the art.
  • determining the concentration of baseline levels of autoantibodies to GFAP and or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or other biomarker is well within the skill of the art.
  • diagnosis means recognizing the presence or absence of a neurological or other condition such as an injury or disease. Diagnosing is optionally referred to as the result of an assay wherein a particular ratio or level of a biomarker is detected or is absent.
  • a "ratio" is either a positive ratio wherein the level of the target is greater than the target in a second sample or relative to a known or recognized baseline level of the same target.
  • a negative ratio describes the level of the target as lower than the target in a second sample or relative to a known or recognized baseline level of the same target.
  • a neutral ratio describes no observed change in target biomarker.
  • an injury is an alteration in cellular or molecular integrity, activity, level, robustness, state, or other alteration that is traceable to an event.
  • Injury illustratively includes a physical, mechanical, chemical, biological, functional, infectious, or other modulator of cellular or molecular characteristics.
  • An event is illustratively, a physical trauma such as an impact (percussive) or a biological abnormality such as a stroke resulting from either blockade or leakage of a blood vessel.
  • An event is optionally an infection by an infectious agent.
  • An injury is optionally a physical event such as a percussive impact.
  • An impact is the like of a percussive injury such as resulting to a blow to the head that either leaves the cranial structure intact or results in breach thereof.
  • CCI controlled cortical impact
  • TBI may also result from stroke.
  • Ischemic stroke is optionally modeled by middle cerebral artery occlusion (MCAO) in rodents.
  • MCAO middle cerebral artery occlusion
  • UCH-L1 protein levels are increased following mild MCAO which is further increased following severe MCAO challenge.
  • Mild MCAO challenge may result in an increase of protein levels within two hours that is transient and returns to control levels within 24 hours.
  • severe MCAO challenge results in an increase in protein levels within two hours following injury and may be much more persistent demonstrating statistically significant levels out to 72 hours or more.
  • Other injuries may include Severe TBI, Mild TBI, Moderate TBI, Alzheimer's Disease, Parkinson's Disease, Stroke, Migraine and Epiliepsy.
  • An exemplary process for detecting the presence or absence of one or more neuroactive biomarkers in a biological sample involves obtaining a biological sample from a subject, such as a human, contacting the biological sample with a compound or an agent capable of detecting of the marker being analyzed, illustratively including an antibody or aptamer, or an antigen in the case of detection of autoantibody biomarkers, and analyzing binding of the compound or agent to the sample after washing. Those samples having specifically bound compound or agent express the marker being analyzed.
  • GFAP or other markers such as GAP 43 , GAD 1 , Recoverin, NSE protein, NF-L, NF- H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto can be detected in a biological sample in vitro, as well as in vivo.
  • GFAP GFAP 43, GADl, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl or autoantibodies thereto in a sample is compared with appropriate controls such as a first sample known to express detectable levels of the marker being analyzed (positive control) and a second sample known to not express detectable levels of the marker being analyzed (a negative control).
  • in vitro techniques for detection of a marker illustratively include enzyme linked immunosorbent assays (ELISAs), radioimmuno assay, radioassay, western blot, Southern blot, northern blot, immunoprecipitation, immunofluorescence, mass spectrometry, RT-PCR, PCR, liquid chromatography, high performance liquid chromatography, enzyme activity assay, cellular assay, positron emission tomography, mass spectroscopy, combinations thereof, or other technique known in the art.
  • in vivo techniques for detection of a marker include introducing a labeled agent that specifically binds the marker into a biological sample or test subject.
  • the agent can be labeled with a radioactive marker whose presence and location in a biological sample or test subject can be detected by standard imaging techniques.
  • Any suitable molecule that can specifically bind GFAP, or other markers such as GAP 43, GADl, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto is operative to achieve a synergistic assay.
  • An illustrative agent for detecting GFAP or other markers such as GAP 43, GADl, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl is an antibody capable of binding to the biomarker being analyzed.
  • an antibody is conjugated with a detectable label.
  • Such antibodies can be polyclonal or monoclonal. An intact antibody, a fragment thereof (e.g., Fab or F(ab') 2 ), or an engineered variant thereof (e.g., sFv) can also be used. Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Antibodies for numerous inventive biomarkers are available from vendors known to one of skill in the art. Illustratively, antibodies directed to inventive biomarkers are available from Santa Cruz Biotechnology (Santa Cruz, CA).
  • An antibody is optionally labeled.
  • a person of ordinary skill in the art recognizes numerous labels operable herein. Labels and labeling kits are commercially available optionally from Invitrogen Corp, Carlsbad, CA. Labels illustratively include, fluorescent labels, biotin, peroxidase, radionucleotides, or other label known in the art.
  • Antibody-based assays are useful for analyzing a biological sample for the presence of GFAP or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF- M, PNMA2, Endophilin Al, KCNAB2 and GRIA1. Suitable western blotting methods are described below in the examples section. For more rapid analysis (as may be important in emergency medical situations), immunosorbent assays (e.g., ELISA and RIA) and immunoprecipitation assays may be used.
  • the biological sample or a portion thereof is immobilized on a substrate, such as a membrane made of nitrocellulose or PVDF; or a rigid substrate made of polystyrene or other plastic polymer such as a microtiter plate, and the substrate is contacted with an antibody that specifically binds GFAP, or one of the other neuroactive biomarkers under conditions that allow binding of antibody to the biomarker being analyzed. After washing, the presence of the antibody on the substrate indicates that the sample contained the marker being assessed. If the antibody is directly conjugated with a detectable label, such as an enzyme, fluorophore, or radioisotope, the presence of the label is optionally detected by examining the substrate for the detectable label. Alternatively, a detectably labeled secondary antibody that binds the marker-specific antibody is added to the substrate. The presence of detectable label on the substrate after washing indicates that the sample contained the marker.
  • a detectable label such as an enzyme, fluorophore, or radioisotope
  • any other suitable agent e.g., a peptide, an aptamer, or a small organic molecule
  • a biomarker e.g., an aptamer that specifically binds a biomarker
  • an aptamer that specifically binds GFAP and/or one or more of its GBDPs might be used.
  • Aptamers are nucleic acid-based molecules that bind specific ligands. Methods for making aptamers with a particular binding specificity are known as detailed in U.S. Patent Nos.
  • a myriad of detectable labels that are operative in a diagnostic GFAP, or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIA1, or autoantibodies thereto are optionally conjugated to a detectable label, e.g., an enzyme such as horseradish peroxidase.
  • a detectable label e.g., an enzyme such as horseradish peroxidase.
  • Agents labeled with horseradish peroxidase can be detected by adding an appropriate substrate that produces a color change in the presence of horseradish peroxidase.
  • detectable labels that may be used are known.
  • a primary/secondary antibody system is optionally used to detect one or more biomarkers.
  • a primary antibody that specifically recognizes one or more biomarkers is exposed to a biological sample that may contain the biomarker of interest.
  • a secondary antibody with an appropriate label that recognizes the species or isotype of the primary antibody is then contacted with the sample such that specific detection of the one or more biomarkers in the sample is achieved.
  • an antigen is used to detect an autoantibody.
  • an antigen such as GFAP or one or more or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIA1 are separated or placed on a substrate such as a PVDF membrane, the membrane is probed with a biological sample such as serum derived from a subject suspected of having a neurological condition, and the presence of an autoantibody is detected by contacting an autoantibody with an antibody type specific antibody such as an anti-IgG alone or combined with anti-IgM antibody that may or may not have a detectable label attached thereto.
  • an antibody type specific antibody such as an anti-IgG alone or combined with anti-IgM antibody that may or may not have a detectable label attached thereto.
  • a process optionally employs a step of correlating the presence or amount of GFAP, or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto in a biological sample with the severity and/or type of nerve cell injury.
  • the amount of GFAP, or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto in the biological sample are associated with a neurological condition such as traumatic brain injury.
  • results of an assay to measure GFAP can help a physician or veterinarian determine the type and severity of injury with implications as to the types of cells that have been compromised.
  • the present invention provides a step of comparing the quantity of GFAP, or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto to normal levels to determine the neurological condition of the subject. It is appreciated that selection of additional biomarkers allows one to identify the types of cells implicated in an abnormal neurological condition as well as the nature of cell death such as in the case of an axonal injury marker, namely an SBDP.
  • the practice of an inventive process provides a test which can help a physician determine suitable therapeutics or treatments to administer for optimal benefit of the subject.
  • An assay for analyzing cell damage in a subject includes: (a) a substrate for holding a sample isolated from a subject suspected of having a damaged nerve cell, the sample being a fluid in communication with the nervous system of the subject prior to being isolated from the subject; and (b) a GFAP (or other biomarker) specific binding agent.
  • the inventive assay can be used to detect a neurological condition for financial renumeration.
  • the assay optionally includes a detectable label such as one conjugated to the agent, or one conjugated to a substance that specifically binds to the agent, such as a secondary antibody.
  • a detectable label such as one conjugated to the agent, or one conjugated to a substance that specifically binds to the agent, such as a secondary antibody.
  • An inventive process illustratively includes diagnosing a neurological condition in a subject, treating a subject with a neurological condition, or both.
  • a process illustratively includes obtaining a biological sample from a subject.
  • the biological sample is assayed by mechanisms known in the art for detecting or identifying the presence of one or more biomarkers present in the biological sample. Based on the amount or presence of a target biomarker in a biological sample, a ratio of one or more biomarkers is optionally calculated.
  • the ratio is optionally the level of one or more biomarkers relative to the level of another biomarker in the same or a parallel sample, or the ratio of the quantity of the biomarker to a measured or previously established baseline level of the same biomarker in a subject known to be free of a pathological neurological condition.
  • the ratio allows for the diagnosis of a neurological condition in the subject.
  • An inventive process also optionally administers a therapeutic to the subject that will either directly or indirectly alter the ratio of one or more biomarkers.
  • a therapeutic is optionally designed to modulate the immune response in a subject.
  • the levels, production of, breakdown of, or other related parameters of autoantibodies are altered by immunomodulatory therapy.
  • immunomodulatory therapies are known in the art that are applicable to the presence of autoantibodies to GFAP or one or more other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl such as therapies used for multiple sclerosis.
  • Such therapies illustratively include administration of glatiramer acetate (GA), beta-interferons, laquinimod, or other therapeutics known in the art.
  • combinations of therapeutics are administered as a form of immunomodulatory therapy.
  • Illustrative combinations include IFN -la and methotrexate, IFN -la and azathioprine, and mitoxantrone plus methylprednisolone.
  • Other suitable combinations are known in the art.
  • An inventive process is also provided for diagnosing and optionally treating a multiple-organ injury. Multiple organs illustratively include subsets of neurological tissue such as brain, spinal cord and the like, or specific regions of the brain such as cortex, hippocampus and the like. .
  • the inventive process illustratively includes assaying for a plurality of biomarkers in a biological sample obtained from a subject wherein the biological was optionally in fluidic contact with an organ suspected of having undergone injury or control organ when the biological sample was obtained from the subject.
  • the inventive process determines a first subtype of organ injury based on a first ratio of a plurality of biomarkers.
  • the inventive process also determines a second subtype of a second organ injury based on a second ratio of the plurality of biomarkers in the biological sample.
  • the ratios are illustratively determined by processes described herein or known in the art.
  • the subject invention illustratively includes a composition for distinguishing the magnitude of a neurological condition in a subject.
  • An inventive composition is either an agent entity or a mixture of multiple agents.
  • a composition is a mixture.
  • the mixture optionally contains a biological sample derived from a subject.
  • the subject is optionally suspected of having a neurological condition.
  • the biological sample in communication with the nervous system of the subject prior to being isolated from the subject.
  • inventive composition also optionally contains at least two primary agents, optionally antibodies that specifically and independently bind to at least two biomarkers that may be present in the biological sample.
  • the first primary agent is in antibody that specifically binds GFAP or one or more or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIA1.
  • a second primary agent is optionally an antibody that specifically binds an ubiquitin carboxyl -terminal hydrolase, preferably UCH-L1, or a spectrin breakdown product.
  • the agents of the inventive composition are optionally immobilized or otherwise in contact with a substrate.
  • the inventive agents are also optionally labeled with at least one detectable label.
  • the detectable label on each agent is unique and independently detectable in either the same assay chamber or alternate chambers.
  • a secondary agent specific for detecting or binding to the primary agent is labeled with at least one detectable label.
  • the primary agent is a rabbit derived antibody.
  • a secondary agent is optionally an antibody specific for a rabbit derived primary antibody.
  • the invention optionally employs a step of correlating the presence or amount of a biomarker in a biological sample with the severity and/or type of nerve cell (or other biomarker- expressing cell) injury.
  • the amount of biomarker(s) in the biological sample directly relates to severity of nerve tissue injury as a more severe injury damages a greater number of nerve cells which in turn causes a larger amount of biomarker(s) to accumulate in the biological sample (e.g., CSF; serum).
  • Whether a nerve cell injury triggers an apoptotic and/or necrotic type of cell death can also be determined by examining the other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIA1 present in the biological sample.
  • GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIA1 present in the biological sample.
  • Necrotic cell death preferentially activates calpain
  • apoptotic cell death preferentially activates caspase-3. Because calpain and caspase-3 GBDPs can be distinguished, measurement of these markers indicates the type of cell damage in the subject.
  • the level of or kinetic extent of UCH-L1, and or GFAP present in a biological sample may optionally distinguish mild injury from a more severe injury.
  • severe MCAO (2h) produces increased UCH-L1 in both CSF and serum relative to mild challenge (30 min) while both produce UCH-L1 levels in excess of uninjured subjects.
  • persistence or kinetic extent of the markers in a biological sample is indicative of the severity of the injury with greater injury indicating increases persistence of illustratively GFAP, UCH-L1, or SBDP in the subject that is measured by an inventive process in biological samples taken at several time points following injury.
  • results of such a test can help a physician determine whether the administration a particular therapeutic such as calpain and/or caspase inhibitors or muscarinic cholinergic receptor antagonists might be of benefit to a patient.
  • This method may be especially important in detecting age and gender difference in cell death mechanism.
  • assay grade water such as assay grade water, buffering agents, membranes, assay plates, secondary antibodies, salts, and other ancillary reagents are available from vendors known to those of skill in the art.
  • assay plates are available from Corning, Inc. (Corning, NY) and reagents are available from Sigma-Aldrich Co. (St. Louis, MO).
  • Illustrative reagents used in performing the subject invention include Sodium bicarbonate (Sigma Cat #: C-3041), blocking buffer (Startingblock T20-TBS) (Pierce Cat#: 37543), Tris buffered saline with Tween 20 (TBST; Sigma Cat #: T-9039). Phosphate buffered saline (PBS; Sigma Cat #: P-3813); Tween 20 (Sigma Cat #: P5927); Ultra TMB ELISA (Pierce Cat #: 34028); and Nunc maxisorp ELISA plates (Fisher).
  • Monoclonal and polyclonal GFAP and UCH-Ll antibodies are made in-house or are obtained from Santa Cruz Biotechnology, Santa Cruz, CA. Antibodies directed to a-II spectrin and breakdown products as well as to MAP2 are available from Santa Cruz Biotechnology, Santa Cruz, CA. Labels for antibodies of numerous subtypes are available from Invitrogen, Corp., Carlsbad, CA. Protein concentrations in biological samples are determined using bicinchoninic acid microprotein assays (Pierce Inc., Rockford, IL, USA) with albumin standards. All other necessary reagents and materials are known to those of skill in the art and are readily ascertainable.
  • Anti-biomarker specific rabbit polyclonal antibody and monoclonal antibodies are produced in the laboratory. To determine reactivity specificity of the antibodies to detect a target biomarker a known quantity of isolated or partially isolated biomarker is analyzed or a tissue panel is probed by western blot. An indirect ELISA is used with the recombinant biomarker protein attached to the ELISA plate to determine optimal concentration of the antibodies used in the assay. Microplate wells are coated with rabbit polyclonal anti-human biomarker antibody. After determining the concentration of rabbit anti-human biomarker antibody for a maximum signal, the lower detection limit of the indirect ELISA for each antibody is determined.
  • Example 3 In vivo model of TBI injury model:
  • a controlled cortical impact (CCI) device is used to model TBI on rats as previously described (Pike et al, 1998).
  • Adult male (280-300 g) Sprague-Dawley rats (Harlan: Indianapolis, IN) are anesthetized with 4% isoflurane in a carrier gas of 1 : 1 0 2 / ⁇ 2 0 (4 min.) and maintained in 2.5% isoflurane in the same carrier gas.
  • Core body temperature is monitored continuously by a rectal thermistor probe and maintained at 37 ⁇ 1°C by placing an adjustable temperature controlled heating pad beneath the rats.
  • Animals are mounted in a stereotactic frame in a prone position and secured by ear and incisor bars.
  • a unilateral (ipsilateral to site of impact) craniotomy (7 mm diameter) is performed adjacent to the central suture, midway between bregma and lambda.
  • the dura mater is kept intact over the cortex.
  • Brain trauma is produced by impacting the right (ipsilateral) cortex with a 5 mm diameter aluminum impactor tip (housed in a pneumatic cylinder) at a velocity of 3.5 m/s with a 1.6 mm compression and 150 ms dwell time. Sham-injured control animals are subjected to identical surgical procedures but do not receive the impact injury.
  • Example 4 Middle cerebral artery occlusion (MCAO) injury model:
  • Rats are incubated under isoflurane anesthesia (5% isoflurane via induction chamber followed by 2% isoflurane via nose cone), the right common carotid artery (CCA) of the rat is exposed at the external and internal carotid artery (ECA and ICA) bifurcation level with a midline neck incision.
  • the ICA is followed rostrally to the pterygopalatine branch and the ECA is ligated and cut at its lingual and maxillary branches.
  • a 3-0 nylon suture is then introduced into the ICA via an incision on the ECA stump (the suture's path was visually monitored through the vessel wall) and advanced through the carotid canal approximately 20 mm from the carotid bifurcation until it becomes lodged in the narrowing of the anterior cerebral artery blocking the origin of the middle cerebral artery.
  • the skin incision is then closed and the endovascular suture left in place for 30 minutes or 2 hours.
  • the rat is briefly re-anesthetized and the suture filament is retracted to allow reperfusion.
  • the filament is advanced only 10 mm beyond the internal-external carotid bifurcation and is left in place until the rat is sacrificed.
  • the brain samples are pulverized with a small mortar and pestle set over dry ice to a fine powder.
  • the pulverized brain tissue powder is then lysed for 90 min at 4°C in a buffer of 50 mM Tris (pH 7.4), 5 mM EDTA, 1% (v/v) Triton X-100, 1 mM DTT, lx protease inhibitor cocktail (Roche Biochemicals).
  • the brain lysates are then centrifuged at 15,000xg for 5 min at 4°C to clear and remove insoluble debris, snap-frozen, and stored at - 80°C until used.
  • cleared CSF samples (7 ⁇ ) are prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 2X loading buffer containing 0.25 M Tris (pH 6.8), 0.2 M DTT, 8% SDS, 0.02% bromophenol blue, and 20% glycerol in distilled H 2 0. Twenty micrograms (20 ⁇ g) of protein per lane are routinely resolved by SDS-PAGE on 10-20% Tris/glycine gels (Invitrogen, Cat #EC61352) at 130 V for 2 hours.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • PVDF polyvinylidene fluoride
  • a control group A synonymously detailed as CSF controls, included 10 individuals also being over the age of 18 or older and no injuries. Samples are obtained during spinal anesthesia for routine surgical procedures or access to CSF associated with treatment of hydrocephalus or meningitis.
  • a control group B synonymously described as normal controls, totaled 64 individuals, each age 18 or older and experiencing multiple injuries without brain injury. Further details with respect to the demographics of the study are provided in Table 5.
  • FIGs. 1 and 2 The level of biomarkers found in the first available CSF and serum samples obtained in the study are provided in FIGs. 1 and 2, respectively.
  • the average first CSF sample collected as detailed in FIG. 1 was 11.2 hours while the average time for collection of a serum sample subsequent to injury event as per FIG. 2 is 10.1 hours.
  • the quantity of each of the biomarkers of UCH-Ll, MAP2, SBDP145, SBDP120, and GFAP are provided for each sample for the cohort of traumatic brain injury sufferers as compared to a control group.
  • the diagnostic utility of the various biomarkers within the first 12 hours subsequent to injury based on a compilation of CSF and serum data is provided in Fig.3 and indicates in particular the value of GFAP as well as that of additional markers UCH-Ll and the spectrin breakdown products. Elevated levels of UCH-Ll are indicative of the compromise of neuronal cell body damage while an increase in SPDP145 with a corresponding decrease in SPDP120 is suggestive of acute axonal necrosis.
  • One subject from the traumatic brain injury cohort was a 52 year old Caucasian woman who had been involved in a motorcycle accident while not wearing a helmet.
  • her GCS was 3 and during the first 24 hours subsequent to trauma her best GCS was 8.
  • her GCS was 11.
  • CT scanning revealed SAH and facial fractures with a Marshall score of 11 and a Rotterdam score of 2.
  • Ventriculostomy was removed after 5 years and an overall good outcome was obtained.
  • Arterial blood pressure (MABP), intracranial pressure (ICP) and cerebral profusion pressure (CPP) for this sufferer of traumatic brain injury as a function of time is depicted in FIG. 4.
  • a possible secondary insult is noted at approximately 40 hours subsequent to the injury as noted by a drop in MABP and CPP.
  • the changes in concentration of inventive biomarkers per CSF and serum samples from this individual are noted in FIG. 5. These results include a sharp increase in GFAP in both the CSF and serum as well as the changes in the other biomarkers depicted in FIG. 5 and provide important clinical information as to the nature of the injury and the types of cells involved, as well as modes of cell death associated with the spectrin breakdown products.
  • Another individual of the severe traumatic brain injury cohort included a 51 year old Caucasian woman who suffered a crush injury associated with a horse falling on the individual.
  • GCS on admission to emergency room was 3 with imaging analysis initially being unremarkable with minor cortical and subcortical contusions.
  • MRI on day 5 revealed significant contusions in posterior fossa.
  • the Marshall scale at that point was indicated to be 11 with a Rotterdam scale score of 3.
  • the CSF and serum values for this individual during a period of time are provided in FIG. 6.
  • GFAP values as a function of time are noted to be markedly elevated relative to normal controls (control group B) as a function of time.
  • Stepwise regression analysis was the statistical method used to evaluate each of the biomarkers as an independent predictive factor, along with the demographic factors of age and gender, and also interactions between pairs of factors. Interactions determine important predictive potential between related factors, such as when the relationship between a biomarker and outcome may be different for men and women, such a relationship would be defined as a gender by biomarker interaction.
  • Stepwise Regression Analysis 2 - Cohort includes:
  • Stepwise Regression Analysis 1 - Cohort includes:
  • Stepwise Regression Analysis 2 - Cohort includes:
  • Example 6 The study of Example 6 was repeated with a moderate traumatic brain injury cohort characterized by GCS scores of between 9 and 11, as well as a mild traumatic brain injury cohort characterized by GCS scores of 12-15. Blood samples were obtained from each patient on arrival to the emergency department of a hospital within 2 hours of injury and measured by ELISA for levels of GFAP in nanograms per milliliter. The results were compared to those of a control group who had not experienced any form of injury. Secondary outcomes included the presence of intracranial lesions in head CT scans.
  • FIG. 8 shows GFAP concentration for controls as well as individuals in the mild/moderate traumatic brain injury cohort as a function of CT scan results upon admission and 24 hours thereafter. Simultaneous assays were performed in the course of this study for UCH-Ll biomarker.
  • the UCH-L1 concentration derived from the same samples as those used to determine GFAP is provided FIG. 9.
  • the concentration of UCH-L1 and GFAP as well as a biomarker not selected for diagnosis of neurological condition, SI 00b, is provided as a function of injury magnitude between control, mild, and moderate traumatic brain injury as shown in FIG. 10.
  • FIG. 11 shows the concentration of the same markers as depicted in FIG. 10 with respect to initial evidence upon hospital admission as to lesions in tomography scans illustrating the high confidence in predictive outcome of the inventive process.
  • FIG. 12 shows that both NSE and MAP2 are elevated in subjects with MTBI in serum both at admission and at 24 hours of follow up.
  • biomarkers such as UCH-L1, GFAP, NSE, and MAP2
  • rapid and quantifiable determination as to the severity of the brain injury is obtained consistent with GSC scoring and CT scanning yet in a surprisingly more quantifiable, expeditious and economic process.
  • biomarkers indicative of neurological condition the nature of the neurological abnormality is assessed and in this particular study suggestive of neuronal cell body damage.
  • gender variations are noted suggesting a role for hormonal anti-inflammatories as therapeutic candidates.
  • paraneoplastic antigens such as PNMA2, recoverin, Endophilin Al, KCNAB2, GRIA1, GAD1.
  • paraneoplastic brain antigens are exemplary targets of autoimmune response after TBI or other neural injuries or neuronal disorders.
  • Recoverin is a neurologically important calcium binding protein, involved in tumor pathology. It is conserved across species and there are indications of several isoforms of this molecule. It is especially enriched in retina, pineal gland-derived tissue and the surrounding tissue of the brain. Autoantibodies could launch an attack of retina and pineal gland, leading to functional deficit.
  • the autoantibody response is not only limited to TBI, but also other CNS injury conditions such as stroke and spinal cord injury (SCI).
  • Figure 15 shows that serum samples from stroke subjects shows brain specific antigen autoimmune response, including the GFAP.
  • human brain lysate on western blot are probed with individual serum samples (7day) at 1/100 dilution.
  • Figure 24 shows that in certain post-SCI patients (e.g. SCI patient 1), after 5-6 days, there is development of autoantibody, including response to GFAP as autoantigen.
  • GFAP novel brain antigen
  • Figures 17, 18 and 19 further shows that sera from epilepsy, Parkinson's disease, Alzheimer's disease and migraine subjects show brain specific antigen autoimmune response, including GFAP.
  • Figure 17 illustrates Brain- specific Autoantibody response to various brain antigens, including GFAP are found in epilepsy patients serum, Human brain lysate (Sample: Human brain lysate (loading: 300ug) on western blot are probed with individual serum samples at 1/100 dilution. Secondary antibody: AP-conjugated Donkey Anti-human IgGH+L); Dilution: 1 : 10,000. GFAP as indicated and other brain autoantigens are also indicated by asterisks.
  • Figure 18 shows Brain-specific Autoantibody response to various brain antigens, including GFAP was found in Parkinson's disease and migraine patients' serum.
  • Human brain lysate (Sample: Human brain lysate (loading: 300ug) on western blot were probed with individual serum samples at 1/100 dilution.
  • Secondary antibody AP-conjugated Donkey Anti-human IgGH+L); Dilution: 1 : 10,000.
  • GFAP as indicated and other brain autoantigens are also indicated by asterisks.
  • FIG 19 shows Brain-specific Autoantibody response to various brain antigens, including GFAP-BDP are found in Alzheimer's disease and migraine patients' serum.
  • Human brain lysate (Sample: Human brain lysate (loading: 300ug) on western blot are probed with individual serum samples at 1/100 dilution.
  • Secondary antibody AP-conjugated Donkey Anti-human IgGH+L); Dilution: 1 : 10,000.
  • GFAP as indicated and other brain autoantigens are also indicated by asterisks.
  • Patent documents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. These documents and publications are incorporated herein by reference to the same extent as if each individual document or publication was specifically and individually incorporated herein by reference.

Abstract

The subject invention provides a robust, quantitative, and reproducible process and assay for diagnosis of a neurological condition in a subject. The invention provides measurement of one or more autoantibodies to biomarkers in a biological fluid such as CSF or serum for determining the extent of neurological damage in a subject with an abnormal neurological condition and for discerning subtypes thereof or tissue types subjected to damage.

Description

GLIAL FIBRILLARY ACIDIC PROTEIN, AUTOANTIGENS AND AUTOANTIBODIES THERETO AS BIOMARKERS OF NEURAL INJURY OR NEUROLOGICAL DISORDER
OR CONDITION
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application Serial No. 61/355,779, filed June 17, 2010 and U.S. Provisional Application Serial No. 61/476,158, filed April 15, 2011 the disclosures of which are hereby incorporated by reference in their entirety, including all figures, tables and amino acid or nucleic acid sequences.
FIELD OF THE INVENTION
[0002] The present invention in general relates to determination of neural injury or a neurological disorder or condition of an individual and in particular to measuring the quantity of a neuroprotective biomarker such as glial fibrillary acidic protein (GFAP) and other proteins present at the blood brain barrier (BBB), and antibodies such as autoantibodies directed to GFAP.
BACKGROUND OF THE INVENTION
[0003] The field of clinical neurology remains frustrated by the recognition that secondary injury to a central nervous system tissue associated with physiologic response to the initial insult could be lessened if only the initial insult could be rapidly diagnosed or in the case of a progressive disorder before stress on central nervous system tissues reached a preselected threshold. Traumatic, ischemic, and neurotoxic chemical insult, along with generic disorders, all present the prospect of brain damage. While the diagnosis of severe forms of each of these causes of brain damage is straightforward through clinical response testing, computed tomography (CT), and magnetic resonance imaging (MRI), the imaging diagnostics are limited by both the high cost of spectroscopic imaging and long diagnostic time. The clinical response testing of incapacitated individuals is of limited value and often precludes a nuanced diagnosis. Additionally, owing to the limitations of existing diagnostics, situations arise wherein a subject experiences a stress to their neurological condition but are often unaware that damage has occurred or fail seek treatment as the subtle symptoms often quickly resolve. The lack of treatment of these mild to moderate challenges to neurologic condition of a subject can have a cumulative effect or otherwise result in a severe brain damage event, either of which have a poor clinical prognosis.
[0004] In order to overcome the limitations associated with spectroscopic and clinical response diagnosis of neurological condition, there is increasing attention on the use of biomarkers as internal indicators of change to molecular or cellular level health condition of a subject. As biomarker detection uses a sample obtained from a subject, typically cerebrospinal fluid, blood, or plasma, and detects the biomarkers in that sample, biomarker detection holds the prospect of inexpensive, rapid, and objective measurement of neurological condition. The attainment of rapid and objective indicators of neurological condition allows one to determine severity of a non-normal brain condition with a previously unrealized degree of objectivity, predict outcome, guide therapy of the condition, as well as monitor subject responsiveness and recovery. Additionally, such information as obtained from numerous subjects allows one to gain a degree of insight into the mechanism of brain injury arising from neural injury or a neurological disorder or condition.
[0005] A number of biomarkers have been identified as being associated with traumatic brain injury including severe TBI as is often seen in vehicle collision and combat wounded subjects. These biomarkers included spectrin breakdown products such as SBDP150, SBDP150i, SBDP145 (calpain mediated acute neural necrosis), SBDP120 (caspase mediated delayed neural apoptosis), UCH-L1 (neuronal cell body damage marker), and MAP2 dendritic cell injury associated marker. The nature of these biomarkers is detailed in U.S. Patents 7,291,710 and 7,396,654, the contents of which are hereby incorporated by reference.
[0006] Glial Fibrillary Acidic Protein (GFAP), a member of the cytoskeletal protein family, is the principal 8-9 nanometer intermediate filament of glial cells such as mature astrocytes of the central nervous system (CNS). GFAP is a monomeric molecule with a molecular mass between 40 and 53 kDa and an isoelectric point between 5.7 and 5.8. GFAP is highly brain specific protein that is not found outside the CNS. GFAP is released into the blood and CSF soon after brain injury. In the CNS following injury, either as a result of trauma, disease, genetic disorders, or chemical insult, astrocytes become reactive in a way that is characterized by rapid synthesis of GFAP termed astrogliosis or gliosis. A similar pathology is noted for other brain autoantigens such as: GAP43(neuromodulin), GAD 1 (GAD -67K), Recoverin, Gamma Enolase (NSE protein), Neurofilament Proteins (NF-L, NF-M, NF-H), mGLuRl (GRIA1), Protein Kinase, c AMP -dependent, regulatory, type I, alpha (PRKARIA), Paraneoplastic Antigen MA2 (PNMA2), Potassium voltage-gated channel, shaker-subfamily, beta member2 (KCNAB2) transcript variant 1 and Endophilin-Alalso known as human SH3 -domain GRB2-like 2 (SH3GL2)
[0007] Thus, there exists a need for a process and an assay for providing improved measurement of neurological condition through the quantification of specific biomarkers for neural injury or neural disorders such as GFAP, or antibodies directed to GFAP. Furthermore, there exists a need to identify certain autoantigens that are specific to neural injury and neuronal disorders which stimulates the production of autoantibodies such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIA1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 are bar graphs of GFAP and other biomarkers for human control and severe TBI subjects from CSF samples;
[0009] FIG. 2 are bar graphs of GFAP and other biomarkers for human control and severe TBI subjects;
[0010] FIG. 3 are bar graphs of GFAP and other biomarkers for human control and severe TBI subjects summarizing the data of FIGs. 1 and 2;
[0011] FIG. 4 are plots of arterial blood pressure (MABP), intracranial pressure (ICP) and cerebral profusion pressure (CPP) for a single human subject of traumatic brain injury as a function of time;
[0012] FIG. 5 are plots of inventive biomarkers from CSF and serum samples from the single human subject of traumatic brain injury of FIG. 4 as a function of time;
[0013] FIG. 6 are plots of inventive biomarkers from CSF and serum samples from another individual human subject of traumatic brain injury as a function of time;
[0014] FIG. 7 are plots of UCH-L1 amounts being present in CSF and serum post severe traumatic brain injury in a mouse subject; [0015] FIG. 8 are bar graphs of GFAP concentration for controls, as well as individuals in the mild/moderate traumatic brain injury cohort as a function of CT scan results upon admission and 24 hours thereafter;
[0016] FIG. 9 are bar graphs of parallel assays for UCH-L1 biomarker from the samples used for FIG. 8;
[0017] FIG. 10 are bar graphs showing the concentration of UCH-L1 and GFAP as well as a biomarker not selected for diagnosis of neurological condition, SI 00 beta, provided as a function of injury magnitude between control, mild, and moderate traumatic brain injury;
[0018] FIG. 11 are bar graphs showing the concentration of the same markers as depicted in FIG. 10 with respect to initial evidence upon hospital admission as to lesions in tomography scans; and
[0019] FIG. 11 represents biomarker levels in human subjects with varying types of brain injury.
[0020] FIG. 12 illustrates Western Blots showing the presence of human brain at least 11 different autoantigen in human subjects with TBI.
[0021] FIG. 13 illustrates Western Blots showing the presence of human brain at least 7 different autoantigen in human subjects with TBI.
[0022] FIG. 14 represents a western blot of brain-specific autoantibody response to various brain antigens, including GFAP found in stroke patients.
[0023] FIG. 15 represents a western blot of subacute development of blood-based autoantibody to GFAP in subset of SCI patients' serum.
[0024] FIG. 16 represents a western blot of brain-specific autoantibody response to various brain antigens, including GFAP, found in epilepsy patients' serum.
[0025] FIG. 17 represents a western blot of brain-specific Autoantibody response to various brain antigens, including GFAP, found in Parkinson's disease and migraine patients' serum.
[0026] FIG. 18 represents a western blot of brain-specific autoantibody response to various brain antigens, including GFAP, found in Alzheimer's disease and migraine patients' serum. DESCRIPTION OF THE INVENTION
[0027] The present invention has utility in the diagnosis and management of abnormal neurological condition including neural injuries or neuronal disorders. Through the measurement of a biomarker such as GFAP or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIA1, or autoantibodies to these autoantigens, a subject a determination of subject neurological condition is provided with greater specificity than previously attainable. The description is appreciated by one of ordinary skill in the art as fully encompassing as an inventive first biomarker as described herein.
[0028] Detection of these autoantigens will help diagnose brain or CNS/PNS injury (e.g. TBI) beyond 4-5 day post-initial injury event and serves to diagnose poor-outcome after brain injury or CNS/PNS (e.g. TBI) in those neural disorders and injuries that result in a change in extracellular biomarker concentration. Furthermore, monitoring of these autoantigens guides immune-modulation therapy to suppress adverse autoimmune response following brain or CNS/PNS injury. Brain or CNS/PNS injury conditions that benefit from the present invention illustratively include mild to moderate brain injury, traumatic brain injury, stroke, spinal cord injury, subarachnoid hemorrhage (SAH) and peripheral nerve injury.
[0029] As such, through the detection of GFAP or other markers, or autoantibodies thereto, a means of detecting a neurological condition in a subject is provided. A neurological condition may be an abnormal neurological condition such as that caused by genetic disorder, injury, or disease to nervous tissue. As such, a means for detecting or diagnosing an abnormal neurological condition in a subject is provided.
[0030] An assay is provided for detecting or diagnosing the neurological condition of a subject. As the neurological condition may be the result of stress such as that from exposure to environmental, therapeutic, or investigative compounds, it is a further aspect of the present invention to provide a process and assay for screening candidate drug or other compounds or for detecting the effects of environmental contaminants.
[0031] A process for detecting a neurological condition optionally includes determining the neurological condition of a subject by assaying a sample derived from a subject at a first time for the presence of a first biomarker. A biomarker is a cell, protein, nucleic acid, steroid, fatty acid, metabolite, or other differentiator useful for measurement of biological activity or response. Illustrative biomarkers as used herein illustratively include: GFAP; or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIA1.; or antibodies directed to GFAP and/or one or more other marker.
[0032] The inventive process also includes assaying the sample for at least one additional neuroactive biomarker. The one additional neuroactive biomarker is optionally not the same biomarker as the first biomarker. A second biomarker is illustratively ubiquitin carboxyl- terminal esterase LI (UCH-L1), Neuron specific enolase (NSE), spectrin breakdown products (SBDP), preferably SBDP150, SBDP150i SBDP145, SBDP120, S100 calcium binding protein B (SI 00b), microtubule associated proteins (MAP), optionally MAP2, MAPI, MAP 3, MAP4, MAP5, myelin basic protein (MBP), Tau, Neurofilament protein (NF), Cannabinoid Receptor (CB), CAM proteins, Synaptic protein, collapsin response mediator proteins (CRMP), inducible nitric oxide synthase (iNOS), Neuronal Nuclei protein (NeuN), cysteinyl-specific peptidase (CSPase), Neuroserpin, alpha-internexin, light chain 3 protein (LC3), Neurofascin, the glutamate transporters (EAAT), Nestin, Cortin-1, 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), and BIII-Tubulin, or any biomarker listed in Table 1 or breakdown product (BDP) thereof.
Table 1:
Glycogen phosphorylase, (BB-form)GP-
UCH-L1 BB Precerebellin
MBP isoforms CRMP-2 Cortexin
SBDP150 (calpain) N P25, N P22; Transgelin-3 EMAP-II
SBDP120 (caspase) SBDP150i (caspase) Calcineurin-BDP
MBP— fragment (10/8K) Ca MPK-lla MAP2
SBDP145 MOG N-Cadherin
Synaptophysin PLP N-CAM
βΙ ΙΙ-Tubulin PTPase (CD45) Synaptobrevin
Tau-BDP-35K (calpain) Nesprin-BDP MAP1A (MAPI)
N F-L-BDP1 OX-42 MAP1B (MAP5)
N F-M-BDP1 OX-8 Prion-protein
N F-H-BDP1 OX-6 PEP19; PCP4
Synaptotagmin Ca MPKIV Synaptotagmin-BDPl PSD93-BDP1 Dynamin BDN F
AM PA-R-BDP1 Clathrin HC Nestin
N MDA-R-BDP SNAP25 IL-6
SBDP150i (caspase) Profilin IL-10
MAP2-BDP1 (calpain) Cofilin ctl l-spectrin SBDP 150+145 MAP2-BDP2 (caspase) APP -BDP (Calpain) NG2; Phosphacan, neruocan; versi
Ach Receptor fragment (Nicotinic, alpha-synuclein NSF Muscarinic)
Synapsin 1 IL-6 I -CAM
Synapsin 2-BDP MMP-9 V-CAM
NeuN sioo AL-CAM
GFAP Neuroglobin CN Pase
p24; VM P UCH-L1 autoantibody Neurofascins
PSD95 Tau-BDP-35K (calpain) Neuroserpin
ctl,2-Tubulin Tau-BDP-45K (caspase) EAAT(1 and 2)
Pl,2-Tubulin Huntingtin-BDP-1 (calpain) Nestin
Stathmin-2,3,4 (Dendritic) Huntingtin-BDP-2 (caspase) Synaptopodin
Striatin-BDPl Prion-protein BDP
Snaptojanin-l,2-BDPl MBP (N-term half)
betall l-Spectrin β-synuclein
betall-Spectrin-BDPllO (calpain) Calbindin-9K Resistin
betall-Spectrin-BDP85 (caspase) Tau-Total Neuropilins
Cannabinoid-receptorl(CBl) NSE Orexin
Cannabinoid-receptor2(CB2) CRMP-1 Fracktalkine
MBP isoforms 14K+17K CRMP-3 β-NGF
Neurocalcin-delta (Glia) CRMP-4 L-selectin
Ibal (Microglia) CRMP-5 iNOS
DAT
Peripherin (PNS) Vimentin
LC3 Crerbellin 3 Beclin-1 [0033] Any number of biomarkers can be detected such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; sequentially or simultaneously from a single sample or aliquots from a sample. Detection can be either simultaneous or sequential and may be from the same biological sample or from multiple samples from the same or different subjects. Detection of multiple biomarkers is optionally in the same assay chamber. The inventive process optionally further includes comparing the quantity of the first biomarker and the quantity of the at least one additional neuroactive biomarker to normal levels of each of the first biomarker and the one additional neuroactive biomarker to determine the neurological condition of the subject.
[0034] In some embodiments a biomarker is GFAP. GFAP is associated with glial cells such as astrocytes.
[0035] A process of determining a neurological condition optionally includes detection of one or more antibodies in a biological sample. An antibody is optionally an autoantibody. Autoantibodies are directed to antigens released from a site of neurological trauma such as TBI, disease, injury or other abnormality. Without being limited to a particular theory, neurological conditions, including TBI, causes cellular damage that releases intracellular or cell membrane contents into the CSF or bloodstream. The levels of many of these proteins such as those listed in Table 1 are not normally present in biological fluids other than the cytoplasm or cell membrane of neuronal tissue such as brain tissue. The presence of these antigens leads to the production of autoantibodies to these antigens within a subject. Detection of an autoantibody as a biomarker is optionally used to diagnose the presence of an abnormal neurological condition in a subject.
[0036] US Patent No. 6,010,854 describes methods of producing screening antigens and methods of screening for autoantibodies to neuronal glutamate receptors. These methods are equally applicable to the subject invention. As such, US Patent No. 6,010,854 is incorporated herein by reference for its teaching of methods of producing screening antigens that are operable for screening for autoantibodies. US Patent No. 6,010,854 is similarly incorporated herein by reference for its teaching of methods of detecting autoantibodies. It is appreciated that other methods of detecting antibodies illustratively including ELISA, western blotting, mass spectroscopy, chromatography, staining, and others known in the art are similarly operable. [0037] Several antigens have been discovered as producing autoantibodies following onset of a neurological condition. Such antigens are those illustratively listed in Table 2.
Table 2: Exemplary autoantigens
GFAP
Neurofilament light polypeptide (NF-L)
Neurofilament Medium polypeptide (NF- M)
Neurofilament heavy polypeptide (NF-H)
V-type proton ATPase
Endophilin-Al
Vimentin
Gamma-enolase (NSE)
Microtubule-associated protein 2
Dihydropyrimidinase-related protein 2
Alpha-internexin
Neuroserpin
Neuromodulin
Synaptotagmin- 1
Voltage-gated potassium channel
[0038] In addition several of these and other antigens are associated with brain injury. Illustrative specific examples of autoantigens related to brain injury are listed in Table 3.
Table 3: Examplary autoantigens related to brain injury
UCH-L1
MBP and MBP-BDP
alpha-spectrin
Beta-spectrin
GFAP and GFAP-BDP CNPase
βΙΙΙ-Tubulin
Tau-BDP-35K (calpain)
NF-L-BDP1
NF-M-BDP1
NF-H-BDP1
sioop
Synaptotagmin
PSD93-BDP1
AMPA-Receptor
NMDA-Receptor
MAP2 and MAP2-BDP alpha-synuclein
Synapsin and synapsin-BDP
NeuN
p24; VMP
PSD95
l,2-Tubulin
Pl,2-Tubulin
Stathmin-2,3,4 (Dendritic) Striatin-BDPl
Snaptoj anin- 1 ,2-BDP 1 Cannabinoid-receptor 1 (CB 1 ) Cannabinoid-receptor2(CB2) Neurocalcin-delta (Glia) Ibal (Microglia)
Peripherin (PNS)
Glycogen phosphorylase, form)GP-BB CRMPs (1-5)
NP25, NP22; Transgelin-3
Synaptopodin
CaMPK-IIa
MOG
PLP
PTPase (CD45)
Nesprin-BDP
OX-42
OX-8
OX-6
CaMPKrV
Dynamin
Clathrin HC
SNAP25
Profilin
Cofilin
APP -BDP (Calpain)
NSF
IL-6
MMP-9
sioop
Neuroglobin
UCH-L1 autoantibody Tau-BDP-35 (calpain) Tau-BDP-45K (caspase) Huntingtin-BDP-1 (calpain) Huntingtin-BDP-2 (caspase) Prion-protein BDP MBP-Total (N-term half) β-synuclein
Calbindin-9K
Tau-Total
Gamma-enolase (NSE)
Crerbellin 3
Precerebellin
Cortexin
EMAP-II
Calcineurin and calcineurin-BDP
MAP2
N-Cadherin
N-CAM
Synaptobrevin
MAPI A (MAPI)
MAP IB (MAP5)
Prion-protein
PEP 19; PCP4
Synaptotagmin-BDP 1
BDNF
Nestin
IL-6
IL-10
Total all-spectrin
all-spectrin SBDP 150+145
NG2; Phosphacan, neurocan; versican Ach Receptor fragment Nicotinic, Muscarinic)
I-CAM V-CAM
AL-CAM
CNPase
Synaptophysin
[0039] Table 4: Exemplary brain injury-induced autoantigens based on reported antigenicity.
Voltage-gated calcium channel VGCC (P/Q-type) (as in Lambert-Eaton myasthenic syndrome) Voltage-gated potassium channel (VGKC) (as in Limbic encephalitis, Isaac's Syndrome.
Autoimmune Neuromyotonia)
Ri (Anti-neuronal nuclear antibody-2) (as in Opsoclonus)
Hu and Yo (cerebellar Purkinje Cells) (as in Paraneoplastic cerebellar syndrome)
Amphiphysin (as in Paraneoplastic cerebellar syndrome)
Glutamic acid decarboxylase (GAD) (as in Diabetes mellitus type 1, Stiff person syndrome) Aquaporin-4 (Neuromyelitis optica ; evic's syndrome)
Basal ganglia neurons (as in Sydenham's Chorea, Pediatric Autoimmune Neuropsychiatric
Disease Associated with Streptococcus (PANDAS)
Homer 3 (subacute idiopathic cerebellar ataxia )
Zic proteins (zinc finger proteins) (as in Joubert syndrome - cerebellum malformation)
ANNA 3 ( brain autoantigen)
Purkinje cell antibody (PCA-2)
PKC γ (paraneoplastic cerebellar degeneration)
SOX1 (Myasthenic Syndrome Lambert-Eaton (LEMS))
Gephyrin (Stiff Man Syndrome)
Ma2
CV2 (= CRMP5)
N-methyl-D-aspartate (NMD A) - develop memory impairment
mGluRl (Cerebellar ataxia)
Nicotinic acetylcholine receptor (as in Myasthenia gravis) Recoverin
Enolase
TULIP- 1 (tubby-like protein 1)
[0040] In some embodiments, full length protein such as any protein listed in Tables 1-4, or a breakdown product thereof, is operable as a screening antigen for autoantibodies. For example, UCH-Ll is antigenic and produces autoantibodies in a subject. The sequence for human UCH- Ll protein is found at NCBI accession number NP 004172.2. Similarly, the sequence for human GFAP is found at NCBI accession number NP 002046.1. Other illustrative antigens illustratively include, alpha-spectrin or breakdown products thereof, MAP, Tau, Neurofascin, CPvMP-2, MAP2 crude sample, and human brain lysate.
[0041] Any suitable method of producing peptides and proteins of Table 1 is operable herein. Illustratively, cloning and protein expression systems used with or without purification tags are optionally used. Illustrative methods for production of immunogenic peptides include synthetic peptide synthesis by methods known in the art. Chemical methods of peptide synthesis are known in the art and include solid phase peptide synthesis and solution phase peptide synthesis or by the method of Hackeng, TM, et al, Proc Natl Acad Sci U S A, 1997; 94(15):7845-50, the contents of which are incorporated herein by reference. Either method is operable for the production of antigens operable for screening biological samples for the presence of autoantibodies.
[0042] As used herein, "peptide" means peptides of any length and includes proteins. The terms "polypeptide" and "oligiopeptide" are used herein without any particular intended size limitation, unless a particular size is otherwise stated.
[0043] As used herein the term "neurological condition" shall mean neural injury or neuronal disorder.
[0044] As used herein, the term "stroke" is art recognized and is intended to include sudden diminution or loss of consciousness, sensation, and voluntary motion caused by rapture or obstruction (e.g. by a blood clot) of an artery of the brain.
[0045] As used herein, the term "Traumatic Brain Injury" is art recognized and is intended to include the condition in which, a traumatic blow to the head causes damage to the brain, often without penetrating the skull. Usually, the initial trauma can result in expanding hematoma, subarachnoid hemorrhage, cerebral edema, raised intracranial pressure (ICP), and cerebral hypoxia, which can, in turn, lead to severe secondary events due to low cerebral blood flow (CBF).
[0046] As used herein, the term "injury or neural injury" is intended to include a damage which directly or indirectly affects the normal functioning of the CNS. For example, the injury can be damage to retinal ganglion cells; a traumatic brain injury; a stroke related injury; a cerebral aneurism related injury; a spinal cord injury, including monoplegia, diplegia, paraplegia, hemiplegia and quadriplegia; a neuroproliferative disorder or neuropathic pain syndrome. Examples of CNS injuries or disease include TBI, stroke, concussion (including post-concussion syndrome), cerebral ischemia, neurodegenerative diseases of the brain such as Parkinson's disease, Dementia Pugilistica, Huntington's disease and Alzheimer's disease, Creutzfeldt- Jakob disease, brain injuries secondary to seizures which are induced by radiation, exposure to ionizing or iron plasma, nerve agents, cyanide, toxic concentrations of oxygen, neurotoxicity due to CNS malaria or treatment with anti-malaria agents, trypanosomes, malarial pathogens, and other CNS traumas.
[0047] "Neural (neuronal) defects, disorders or diseases" as used herein refers to any neurological disorder, including but not limited to neurodegenerative disorders (Parkinson's; Alzheimer's) or autoimmune disorders (multiple sclerosis) of the central nervous system; memory loss; long term and short term memory disorders; learning disorders; autism, depression, benign forgetfulness, childhood learning disorders, close head injury, and attention deficit disorder; autoimmune disorders of the brain, neuronal reaction to viral infection; brain damage; depression; psychiatric disorders such as bi-polarism, schizophrenia and the like; narcolepsy/sleep disorders(including circadian rhythm disorders, insomnia and narcolepsy); severance of nerves or nerve damage; severance of the cerebrospinal nerve cord (CNS) and any damage to brain or nerve cells; neurological deficits associated with AIDS; tics (e.g. Giles de la Tourette's syndrome); Huntington's chorea, schizophrenia, traumatic brain injury, tinnitus, neuralgia, especially trigeminal neuralgia, neuropathic pain, inappropriate neuronal activity resulting in neurodysthesias in diseases such as diabetes, MS and motor neuron disease, ataxias, muscular rigidity (spasticity) and temporomandibular joint dysfunction; Reward Deficiency Syndrome (RDS) behaviors in a subject.
[0048] A sample is optionally a biological sample. Illustrative examples of biological samples include cells, tissues, cerebral spinal fluid (CSF), artificial CSF, whole blood, serum, plasma, cytosolic fluid, urine, feces, stomach fluids, digestive fluids, saliva, nasal or other airway fluid, vaginal fluids, semen, buffered saline, saline, water, or other biological fluid recognized in the art in which a target biomarker or metabolite thereof is found. In some embodiments, a biological sample is CSF or serum. It is appreciated that two or more separate biological samples are optionally assayed to elucidate the neurological condition of the subject.
[0049] In addition to increased cell expression, biomarkers also appear in biological fluids in communication with injured cells. Obtaining biological fluids such as cerebrospinal fluid (CSF), blood, plasma, serum, saliva and urine, from a subject is typically much less invasive and traumatizing than obtaining a solid tissue biopsy sample. Thus, samples that are biological fluids are preferred for use in the invention. CSF, in particular, is preferred for detecting nerve damage in a subject as it is in immediate contact with the nervous system and is readily obtainable. Serum is a preferred biological sample as it is easily obtainable and presents much less risk of further injury or side-effect to a donating subject.
[0050] To provide correlations between neurological condition and measured quantities of GFAP, or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto, samples of CSF or serum are collected from subjects with the samples being subjected to measurement of GFAP, or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto. The subjects vary in neurological condition. Detected levels of GFAP, or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto are optionally then correlated with CT scan results as well as GCS scoring. Based on these results, an inventive assay is developed and validated (Lee et al, Pharmacological Research 23:312-328, 2006). It is appreciated that GFAP, or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto, in addition to being obtained from CSF and serum, are also readily obtained from blood, plasma, saliva, urine, as well as solid tissue biopsy. While CSF is a sampling fluid in many embodiments of the invention owing to direct contact with the nervous system, it is appreciated that other biological fluids have advantages in being sampled for other purposes and therefore allow for inventive determination of neurological condition as part of a battery of tests performed on a single sample such as blood, plasma, serum, saliva or urine.
[0051] A biological sample is obtained from a subject by conventional techniques. For example, CSF is preferably obtained by lumbar puncture. Blood is preferably obtained by venipuncture, while plasma and serum are obtained by fractionating whole blood according to known methods. Surgical techniques for obtaining solid tissue samples are well known in the art. For example, methods for obtaining a nervous system tissue sample are described in standard neurosurgery texts such as Atlas of Neurosurgery: Basic Approaches to Cranial and Vascular Procedures, by F. Meyer, Churchill Livingstone, 1999; Stereotactic and Image Directed Surgery of Brain Tumors, 1st ed., by David G. T. Thomas, WB Saunders Co., 1993; and Cranial Microsurgery: Approaches and Techniques, by L. N. Sekhar and E. De Oliveira, 1st ed., Thieme Medical Publishing, 1999. Methods for obtaining and analyzing brain tissue are also described in Belay et al, Arch. Neurol. 58: 1673-1678 (2001); and Seijo et al, J. Clin. Microbiol. 38: 3892- 3895 (2000).
[0052] After insult, nerve cells in in vitro culture or in situ in a subject express altered levels or activities of one or more proteins than do such cells not subjected to the insult. Thus, samples that contain nerve cells, e.g., a biopsy of a central nervous system or peripheral nervous system tissue are illustratively suitable biological samples for use in the invention. In addition to nerve cells, however, other cells express illustratively GFAP including, for example, cardiomyocytes, myocytes in skeletal muscles, hepatocytes, kidney cells and cells in testis. A biological sample including such cells or fluid secreted from these cells might also be used in an adaptation of the inventive methods to determine and/or characterize an injury to such non-nerve cells.
[0053] A subject illustratively includes a dog, a cat, a horse, a cow, a pig, a sheep, a goat, a chicken, non-human primate, a human, a rat, and a mouse. Subjects who most benefit from the present invention are those suspected of having or at risk for developing abnormal neurological conditions, such as victims of brain injury caused by traumatic insults (e.g., gunshot wounds, automobile accidents, sports accidents, shaken baby syndrome), spinal cord injury, seizure, ischemic events (e.g., stroke, cerebral hemorrhage, cardiac arrest), neurodegenerative disorders (such as Alzheimer's, Huntington's, and Parkinson's diseases; prion-related disease; other forms of dementia), epilepsy, substance abuse (e.g., from amphetamines, Ecstasy/MDMA, or ethanol), and peripheral nervous system pathologies such as diabetic neuropathy, chemotherapy-induced neuropathy and neuropathic pain.
[0054] Baseline levels of several biomarkers are those levels obtained in the target biological sample in the species of desired subject in the absence of a known neurological condition. These levels need not be expressed in hard concentrations, but may instead be known from parallel control experiments and expressed in terms of fluorescent units, density units, and the like. Typically, in the absence of a neurological condition GFAP and other or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl are present in biological samples at a negligible amount. Illustratively, autoantibodies to GFAP or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF- L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl are absent in a biological sample form a subject not suspected of having a neurological condition. However, GFAP and or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl are often highly abundant in neurons. Determining the baseline levels of GFAP and or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl in neurons of particular species is well within the skill of the art. Similarly, determining the concentration of baseline levels of autoantibodies to GFAP and or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or other biomarker is well within the skill of the art.
[0055] As used herein the term "diagnosing" means recognizing the presence or absence of a neurological or other condition such as an injury or disease. Diagnosing is optionally referred to as the result of an assay wherein a particular ratio or level of a biomarker is detected or is absent.
[0056] As used herein a "ratio" is either a positive ratio wherein the level of the target is greater than the target in a second sample or relative to a known or recognized baseline level of the same target. A negative ratio describes the level of the target as lower than the target in a second sample or relative to a known or recognized baseline level of the same target. A neutral ratio describes no observed change in target biomarker.
[0057] As used herein an injury is an alteration in cellular or molecular integrity, activity, level, robustness, state, or other alteration that is traceable to an event. Injury illustratively includes a physical, mechanical, chemical, biological, functional, infectious, or other modulator of cellular or molecular characteristics. An event is illustratively, a physical trauma such as an impact (percussive) or a biological abnormality such as a stroke resulting from either blockade or leakage of a blood vessel. An event is optionally an infection by an infectious agent. A person of skill in the art recognizes numerous equivalent events that are encompassed by the terms injury or event.
[0058] An injury is optionally a physical event such as a percussive impact. An impact is the like of a percussive injury such as resulting to a blow to the head that either leaves the cranial structure intact or results in breach thereof. Experimentally, several impact methods are used illustratively including controlled cortical impact (CCI) at a 1.6 mm depression depth, equivalent to severe TBI in human. This method is described in detail by Cox, CD, et al, J Neurotrauma, 2008; 25(11): 1355-65. It is appreciated that other experimental methods producing impact trauma are similarly operable.
[0059] TBI may also result from stroke. Ischemic stroke is optionally modeled by middle cerebral artery occlusion (MCAO) in rodents. UCH-L1 protein levels, for example, are increased following mild MCAO which is further increased following severe MCAO challenge. Mild MCAO challenge may result in an increase of protein levels within two hours that is transient and returns to control levels within 24 hours. In contrast, severe MCAO challenge results in an increase in protein levels within two hours following injury and may be much more persistent demonstrating statistically significant levels out to 72 hours or more.
[0060] Other injuries may include Severe TBI, Mild TBI, Moderate TBI, Alzheimer's Disease, Parkinson's Disease, Stroke, Migraine and Epiliepsy.
[0061] An exemplary process for detecting the presence or absence of one or more neuroactive biomarkers in a biological sample involves obtaining a biological sample from a subject, such as a human, contacting the biological sample with a compound or an agent capable of detecting of the marker being analyzed, illustratively including an antibody or aptamer, or an antigen in the case of detection of autoantibody biomarkers, and analyzing binding of the compound or agent to the sample after washing. Those samples having specifically bound compound or agent express the marker being analyzed.
[0062] GFAP or other markers such as GAP 43 , GAD 1 , Recoverin, NSE protein, NF-L, NF- H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto can be detected in a biological sample in vitro, as well as in vivo. The quantity of GFAP, or other markers such as GAP 43, GADl, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl or autoantibodies thereto in a sample is compared with appropriate controls such as a first sample known to express detectable levels of the marker being analyzed (positive control) and a second sample known to not express detectable levels of the marker being analyzed (a negative control). For example, in vitro techniques for detection of a marker illustratively include enzyme linked immunosorbent assays (ELISAs), radioimmuno assay, radioassay, western blot, Southern blot, northern blot, immunoprecipitation, immunofluorescence, mass spectrometry, RT-PCR, PCR, liquid chromatography, high performance liquid chromatography, enzyme activity assay, cellular assay, positron emission tomography, mass spectroscopy, combinations thereof, or other technique known in the art.. Furthermore, in vivo techniques for detection of a marker include introducing a labeled agent that specifically binds the marker into a biological sample or test subject. For example, the agent can be labeled with a radioactive marker whose presence and location in a biological sample or test subject can be detected by standard imaging techniques.
[0063] Any suitable molecule that can specifically bind GFAP, or other markers such as GAP 43, GADl, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto is operative to achieve a synergistic assay. An illustrative agent for detecting GFAP or other markers such as GAP 43, GADl, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl is an antibody capable of binding to the biomarker being analyzed. Optionally, an antibody is conjugated with a detectable label. Such antibodies can be polyclonal or monoclonal. An intact antibody, a fragment thereof (e.g., Fab or F(ab')2), or an engineered variant thereof (e.g., sFv) can also be used. Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Antibodies for numerous inventive biomarkers are available from vendors known to one of skill in the art. Illustratively, antibodies directed to inventive biomarkers are available from Santa Cruz Biotechnology (Santa Cruz, CA).
[0064] An antibody is optionally labeled. A person of ordinary skill in the art recognizes numerous labels operable herein. Labels and labeling kits are commercially available optionally from Invitrogen Corp, Carlsbad, CA. Labels illustratively include, fluorescent labels, biotin, peroxidase, radionucleotides, or other label known in the art.
[0065] Antibody-based assays are useful for analyzing a biological sample for the presence of GFAP or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF- M, PNMA2, Endophilin Al, KCNAB2 and GRIA1. Suitable western blotting methods are described below in the examples section. For more rapid analysis (as may be important in emergency medical situations), immunosorbent assays (e.g., ELISA and RIA) and immunoprecipitation assays may be used. As one example, the biological sample or a portion thereof is immobilized on a substrate, such as a membrane made of nitrocellulose or PVDF; or a rigid substrate made of polystyrene or other plastic polymer such as a microtiter plate, and the substrate is contacted with an antibody that specifically binds GFAP, or one of the other neuroactive biomarkers under conditions that allow binding of antibody to the biomarker being analyzed. After washing, the presence of the antibody on the substrate indicates that the sample contained the marker being assessed. If the antibody is directly conjugated with a detectable label, such as an enzyme, fluorophore, or radioisotope, the presence of the label is optionally detected by examining the substrate for the detectable label. Alternatively, a detectably labeled secondary antibody that binds the marker-specific antibody is added to the substrate. The presence of detectable label on the substrate after washing indicates that the sample contained the marker.
[0066] Numerous permutations of these basic immunoassays are also operative in the invention. These include the biomarker-specific antibody, as opposed to the sample being immobilized on a substrate, and the substrate is contacted with GFAP or another neuroactive biomarker conjugated with a detectable label under conditions that cause binding of antibody to the labeled marker. The substrate is then contacted with a sample under conditions that allow binding of the marker being analyzed to the antibody. A reduction in the amount of detectable label on the substrate after washing indicates that the sample contained the marker.
[0067] Although antibodies are useful in the invention because of their extensive characterization, any other suitable agent (e.g., a peptide, an aptamer, or a small organic molecule) that specifically binds a biomarker is optionally used in place of the antibody in the above described immunoassays. For example, an aptamer that specifically binds GFAP and/or one or more of its GBDPs might be used. Aptamers are nucleic acid-based molecules that bind specific ligands. Methods for making aptamers with a particular binding specificity are known as detailed in U.S. Patent Nos. 5,475,096; 5,670,637; 5,696,249; 5,270,163; 5,707,796; 5,595,877; 5,660,985; 5,567,588; 5,683,867; 5,637,459; and 6,011,020.
[0068] A myriad of detectable labels that are operative in a diagnostic GFAP, or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIA1, or autoantibodies thereto are optionally conjugated to a detectable label, e.g., an enzyme such as horseradish peroxidase. Agents labeled with horseradish peroxidase can be detected by adding an appropriate substrate that produces a color change in the presence of horseradish peroxidase. Several other detectable labels that may be used are known. Common examples of these include alkaline phosphatase, horseradish peroxidase, fluorescent compounds, luminescent compounds, colloidal gold, magnetic particles, biotin, radioisotopes, and other enzymes. It is appreciated that a primary/secondary antibody system is optionally used to detect one or more biomarkers. A primary antibody that specifically recognizes one or more biomarkers is exposed to a biological sample that may contain the biomarker of interest. A secondary antibody with an appropriate label that recognizes the species or isotype of the primary antibody is then contacted with the sample such that specific detection of the one or more biomarkers in the sample is achieved.
[0069] In some embodiments an antigen is used to detect an autoantibody. Illustratively, an antigen such as GFAP or one or more or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIA1 are separated or placed on a substrate such as a PVDF membrane, the membrane is probed with a biological sample such as serum derived from a subject suspected of having a neurological condition, and the presence of an autoantibody is detected by contacting an autoantibody with an antibody type specific antibody such as an anti-IgG alone or combined with anti-IgM antibody that may or may not have a detectable label attached thereto.
[0070] A process optionally employs a step of correlating the presence or amount of GFAP, or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto in a biological sample with the severity and/or type of nerve cell injury. The amount of GFAP, or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto in the biological sample are associated with a neurological condition such as traumatic brain injury. The results of an assay to measure GFAP, or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto can help a physician or veterinarian determine the type and severity of injury with implications as to the types of cells that have been compromised. These results are in agreement with CT scan and GCS results, yet are quantitative, obtained more rapidly, and at far lower cost.
[0071] The present invention provides a step of comparing the quantity of GFAP, or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl, or autoantibodies thereto to normal levels to determine the neurological condition of the subject. It is appreciated that selection of additional biomarkers allows one to identify the types of cells implicated in an abnormal neurological condition as well as the nature of cell death such as in the case of an axonal injury marker, namely an SBDP. The practice of an inventive process provides a test which can help a physician determine suitable therapeutics or treatments to administer for optimal benefit of the subject. While the data provided in the examples herein are provided with respect to a full spectrum of traumatic brain injury, it is appreciated that these results are applicable to ischemic events, neurodegenerative disorders, prion related disease, epilepsy, chemical etiology and peripheral nervous system pathologies. A gender difference is optionally considered.
[0072] An assay for analyzing cell damage in a subject is also provided. The assay includes: (a) a substrate for holding a sample isolated from a subject suspected of having a damaged nerve cell, the sample being a fluid in communication with the nervous system of the subject prior to being isolated from the subject; and (b) a GFAP (or other biomarker) specific binding agent. The inventive assay can be used to detect a neurological condition for financial renumeration.
[0073] The assay optionally includes a detectable label such as one conjugated to the agent, or one conjugated to a substance that specifically binds to the agent, such as a secondary antibody.
[0074] An inventive process illustratively includes diagnosing a neurological condition in a subject, treating a subject with a neurological condition, or both. In some embodiments a process illustratively includes obtaining a biological sample from a subject. The biological sample is assayed by mechanisms known in the art for detecting or identifying the presence of one or more biomarkers present in the biological sample. Based on the amount or presence of a target biomarker in a biological sample, a ratio of one or more biomarkers is optionally calculated. The ratio is optionally the level of one or more biomarkers relative to the level of another biomarker in the same or a parallel sample, or the ratio of the quantity of the biomarker to a measured or previously established baseline level of the same biomarker in a subject known to be free of a pathological neurological condition. The ratio allows for the diagnosis of a neurological condition in the subject. An inventive process also optionally administers a therapeutic to the subject that will either directly or indirectly alter the ratio of one or more biomarkers.
[0075] A therapeutic is optionally designed to modulate the immune response in a subject. Illustratively, the levels, production of, breakdown of, or other related parameters of autoantibodies are altered by immunomodulatory therapy. Illustrative examples of immunomodulatory therapies are known in the art that are applicable to the presence of autoantibodies to GFAP or one or more other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIAl such as therapies used for multiple sclerosis. Such therapies illustratively include administration of glatiramer acetate (GA), beta-interferons, laquinimod, or other therapeutics known in the art. Optionally, combinations of therapeutics are administered as a form of immunomodulatory therapy. Illustrative combinations include IFN -la and methotrexate, IFN -la and azathioprine, and mitoxantrone plus methylprednisolone. Other suitable combinations are known in the art. [0076] An inventive process is also provided for diagnosing and optionally treating a multiple-organ injury. Multiple organs illustratively include subsets of neurological tissue such as brain, spinal cord and the like, or specific regions of the brain such as cortex, hippocampus and the like. . The inventive process illustratively includes assaying for a plurality of biomarkers in a biological sample obtained from a subject wherein the biological was optionally in fluidic contact with an organ suspected of having undergone injury or control organ when the biological sample was obtained from the subject. The inventive process determines a first subtype of organ injury based on a first ratio of a plurality of biomarkers. The inventive process also determines a second subtype of a second organ injury based on a second ratio of the plurality of biomarkers in the biological sample. The ratios are illustratively determined by processes described herein or known in the art.
[0077] The subject invention illustratively includes a composition for distinguishing the magnitude of a neurological condition in a subject. An inventive composition is either an agent entity or a mixture of multiple agents. In some embodiments a composition is a mixture. The mixture optionally contains a biological sample derived from a subject. The subject is optionally suspected of having a neurological condition. The biological sample in communication with the nervous system of the subject prior to being isolated from the subject. In inventive composition also optionally contains at least two primary agents, optionally antibodies that specifically and independently bind to at least two biomarkers that may be present in the biological sample. In some embodiments the first primary agent is in antibody that specifically binds GFAP or one or more or other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIA1. A second primary agent is optionally an antibody that specifically binds an ubiquitin carboxyl -terminal hydrolase, preferably UCH-L1, or a spectrin breakdown product.
[0078] The agents of the inventive composition are optionally immobilized or otherwise in contact with a substrate. The inventive agents are also optionally labeled with at least one detectable label. In some embodiments the detectable label on each agent is unique and independently detectable in either the same assay chamber or alternate chambers. Optionally, a secondary agent specific for detecting or binding to the primary agent is labeled with at least one detectable label. In the nonlimiting example the primary agent is a rabbit derived antibody. A secondary agent is optionally an antibody specific for a rabbit derived primary antibody. Mechanisms of detecting antibody binding to an antigen are well known in the art, and a person of ordinary skill in the art readily envisions numerous methods and agents suitable for detecting antigens or biomarkers in a biological sample.
[0079] The invention optionally employs a step of correlating the presence or amount of a biomarker in a biological sample with the severity and/or type of nerve cell (or other biomarker- expressing cell) injury. The amount of biomarker(s) in the biological sample directly relates to severity of nerve tissue injury as a more severe injury damages a greater number of nerve cells which in turn causes a larger amount of biomarker(s) to accumulate in the biological sample (e.g., CSF; serum). Whether a nerve cell injury triggers an apoptotic and/or necrotic type of cell death can also be determined by examining the other markers such as GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 and GRIA1 present in the biological sample. Necrotic cell death preferentially activates calpain, whereas apoptotic cell death preferentially activates caspase-3. Because calpain and caspase-3 GBDPs can be distinguished, measurement of these markers indicates the type of cell damage in the subject. Also, the level of or kinetic extent of UCH-L1, and or GFAP present in a biological sample may optionally distinguish mild injury from a more severe injury. In an illustrative example, severe MCAO (2h) produces increased UCH-L1 in both CSF and serum relative to mild challenge (30 min) while both produce UCH-L1 levels in excess of uninjured subjects. Moreover, the persistence or kinetic extent of the markers in a biological sample is indicative of the severity of the injury with greater injury indicating increases persistence of illustratively GFAP, UCH-L1, or SBDP in the subject that is measured by an inventive process in biological samples taken at several time points following injury.
[0080] The results of such a test can help a physician determine whether the administration a particular therapeutic such as calpain and/or caspase inhibitors or muscarinic cholinergic receptor antagonists might be of benefit to a patient. This method may be especially important in detecting age and gender difference in cell death mechanism.
[0081] It is appreciated that other reagents such as assay grade water, buffering agents, membranes, assay plates, secondary antibodies, salts, and other ancillary reagents are available from vendors known to those of skill in the art. Illustratively, assay plates are available from Corning, Inc. (Corning, NY) and reagents are available from Sigma-Aldrich Co. (St. Louis, MO).
[0082] Methods involving conventional biological techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Current Protocols in Molecular Biology, ed. Ausubel et al, Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Immunological methods (e.g., preparation of antigen-specific antibodies, immunoprecipitation, and immunoblotting) are described, e.g., in Current Protocols in Immunology, ed. Coligan et al, John Wiley & Sons, New York, 1991; and Methods of Immunological Analysis, ed. Masseyeff et al, John Wiley & Sons, New York, 1992.
[0083] Various aspects of the present invention are illustrated by the following non-limiting examples. The examples are for illustrative purposes and are not a limitation on any practice of the present invention. It will be understood that variations and modifications can be made without departing from the spirit and scope of the invention. While the examples are generally directed to mammalian tissue, specifically, analyses of mouse tissue, a person having ordinary skill in the art recognizes that similar techniques and other techniques known in the art readily translate the examples to other mammals such as humans. Reagents illustrated herein are commonly cross reactive between mammalian species or alternative reagents with similar properties are commercially available, and a person of ordinary skill in the art readily understands where such reagents may be obtained. Variations within the concepts of the invention are apparent to those skilled in the art.
Example 1: Materials for Biomarker Analyses.
[0084] Illustrative reagents used in performing the subject invention include Sodium bicarbonate (Sigma Cat #: C-3041), blocking buffer (Startingblock T20-TBS) (Pierce Cat#: 37543), Tris buffered saline with Tween 20 (TBST; Sigma Cat #: T-9039). Phosphate buffered saline (PBS; Sigma Cat #: P-3813); Tween 20 (Sigma Cat #: P5927); Ultra TMB ELISA (Pierce Cat #: 34028); and Nunc maxisorp ELISA plates (Fisher). Monoclonal and polyclonal GFAP and UCH-Ll antibodies are made in-house or are obtained from Santa Cruz Biotechnology, Santa Cruz, CA. Antibodies directed to a-II spectrin and breakdown products as well as to MAP2 are available from Santa Cruz Biotechnology, Santa Cruz, CA. Labels for antibodies of numerous subtypes are available from Invitrogen, Corp., Carlsbad, CA. Protein concentrations in biological samples are determined using bicinchoninic acid microprotein assays (Pierce Inc., Rockford, IL, USA) with albumin standards. All other necessary reagents and materials are known to those of skill in the art and are readily ascertainable.
Example 2: Biomarker Assay Development
[0085] Anti-biomarker specific rabbit polyclonal antibody and monoclonal antibodies are produced in the laboratory. To determine reactivity specificity of the antibodies to detect a target biomarker a known quantity of isolated or partially isolated biomarker is analyzed or a tissue panel is probed by western blot. An indirect ELISA is used with the recombinant biomarker protein attached to the ELISA plate to determine optimal concentration of the antibodies used in the assay. Microplate wells are coated with rabbit polyclonal anti-human biomarker antibody. After determining the concentration of rabbit anti-human biomarker antibody for a maximum signal, the lower detection limit of the indirect ELISA for each antibody is determined. An appropriate diluted sample is incubated with a rabbit polyclonal antihuman biomarker antibody for 2 hours and then washed. Biotin labeled monoclonal anti-human biomarker antibody is then added and incubated with captured biomarker. After thorough wash, streptavidin horseradish peroxidase conjugate is added. After 1 hour incubation and the last washing step, the remaining conjugate is allowed to react with substrate of hydrogen peroxide tetramethyl benzadine. The reaction is stopped by addition of the acidic solution and absorbance of the resulting yellow reaction product is measured at 450 nanometers. The absorbance is proportional to the concentration of the biomarker. A standard curve is constructed by plotting absorbance values as a function of biomarker concentration using calibrator samples and concentrations of unknown samples are determined using the standard curve.
Example 3: In vivo model of TBI injury model:
[0086] A controlled cortical impact (CCI) device is used to model TBI on rats as previously described (Pike et al, 1998). Adult male (280-300 g) Sprague-Dawley rats (Harlan: Indianapolis, IN) are anesthetized with 4% isoflurane in a carrier gas of 1 : 1 02/Ν20 (4 min.) and maintained in 2.5% isoflurane in the same carrier gas. Core body temperature is monitored continuously by a rectal thermistor probe and maintained at 37±1°C by placing an adjustable temperature controlled heating pad beneath the rats. Animals are mounted in a stereotactic frame in a prone position and secured by ear and incisor bars. Following a midline cranial incision and reflection of the soft tissues, a unilateral (ipsilateral to site of impact) craniotomy (7 mm diameter) is performed adjacent to the central suture, midway between bregma and lambda. The dura mater is kept intact over the cortex. Brain trauma is produced by impacting the right (ipsilateral) cortex with a 5 mm diameter aluminum impactor tip (housed in a pneumatic cylinder) at a velocity of 3.5 m/s with a 1.6 mm compression and 150 ms dwell time. Sham-injured control animals are subjected to identical surgical procedures but do not receive the impact injury. Appropriate pre- and post-injury management is preformed to insure compliance with guidelines set forth by the University of Florida Institutional Animal Care and Use Committee and the National Institutes of Health guidelines detailed in the Guide for the Care and Use of Laboratory Animals. In addition, research is conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhered to principles stated in the "Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 edition."
Example 4: Middle cerebral artery occlusion (MCAO) injury model:
[0087] Rats are incubated under isoflurane anesthesia (5% isoflurane via induction chamber followed by 2% isoflurane via nose cone), the right common carotid artery (CCA) of the rat is exposed at the external and internal carotid artery (ECA and ICA) bifurcation level with a midline neck incision. The ICA is followed rostrally to the pterygopalatine branch and the ECA is ligated and cut at its lingual and maxillary branches. A 3-0 nylon suture is then introduced into the ICA via an incision on the ECA stump (the suture's path was visually monitored through the vessel wall) and advanced through the carotid canal approximately 20 mm from the carotid bifurcation until it becomes lodged in the narrowing of the anterior cerebral artery blocking the origin of the middle cerebral artery. The skin incision is then closed and the endovascular suture left in place for 30 minutes or 2 hours. Afterwards the rat is briefly re-anesthetized and the suture filament is retracted to allow reperfusion. For sham MCAO surgeries, the same procedure is followed, but the filament is advanced only 10 mm beyond the internal-external carotid bifurcation and is left in place until the rat is sacrificed. During all surgical procedures, animals are maintained at 37 ± 1°C by a homoeothermic heating blanket (Harvard Apparatus, Holliston, MA, U.S.A.). It is important to note that at the conclusion of each experiment, if the rat brains show pathologic evidence of subarachnoid hemorrhage upon necropsy they are excluded from the study. Appropriate pre- and post-injury management is preformed to insure compliance with all animal care and use guidelines.
Example 5: Tissue and Sample Preparation:
[0088] At the appropriate time points (2, 6, 24 hours and 2, 3, 5 days) after injury, animals are anesthetized and immediately sacrificed by decapitation. Brains are quickly removed, rinsed with ice cold PBS and halved. The right hemisphere (cerebrocortex around the impact area and hippocampus) is rapidly dissected, rinsed in ice cold PBS, snap-frozen in liquid nitrogen, and stored at -80°C until used. For immunohistochemistry, brains are quick frozen in dry ice slurry, sectioned via cryostat (20 μπι) onto SUPERFROST PLUS GOLD® (Fisher Scientific) slides, and then stored at -80°C until used. For the left hemisphere, the same tissue as the right side is collected. For Western blot analysis, the brain samples are pulverized with a small mortar and pestle set over dry ice to a fine powder. The pulverized brain tissue powder is then lysed for 90 min at 4°C in a buffer of 50 mM Tris (pH 7.4), 5 mM EDTA, 1% (v/v) Triton X-100, 1 mM DTT, lx protease inhibitor cocktail (Roche Biochemicals). The brain lysates are then centrifuged at 15,000xg for 5 min at 4°C to clear and remove insoluble debris, snap-frozen, and stored at - 80°C until used.
[0089] For gel electrophoresis and electroblotting, cleared CSF samples (7 μΐ) are prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 2X loading buffer containing 0.25 M Tris (pH 6.8), 0.2 M DTT, 8% SDS, 0.02% bromophenol blue, and 20% glycerol in distilled H20. Twenty micrograms (20 μg) of protein per lane are routinely resolved by SDS-PAGE on 10-20% Tris/glycine gels (Invitrogen, Cat #EC61352) at 130 V for 2 hours. Following electrophoresis, separated proteins are laterally transferred to polyvinylidene fluoride (PVDF) membranes in a transfer buffer containing 39 mM glycine, 48 mM Tris-HCl ( H 8.3), and 5% methanol at a constant voltage of 20 V for 2 hours at ambient temperature in a semi-dry transfer unit (Bio-Rad). After electro-transfer, the membranes are blocked for 1 hour at ambient temperature in 5% non-fat milk in TBS and 0.05% Tween-2 (TBST) then are incubated with the primary monoclonal GFAP antibody in TBST with 5% non-fat milk at 1 :2000 dilution as recommended by the manufacturer at 4°C overnight. This is followed by three washes with TBST, a 2 hour incubation at ambient temperature with a biotinylated linked secondary antibody (Amersham, Cat # RPN1177vl), and a 30 min incubation with Streptavidin-conjugated alkaline phosphatase (BCIP/NBT reagent: KPL, Cat # 50-81-08). Molecular weights of intact biomarker proteins are assessed using rainbow colored molecular weight standards (Amersham, Cat # RPN800V). Semi-quantitative evaluation of intact GFAP, UCH-L1, or SBDP protein levels is performed via computer-assisted densitometric scanning (Epson XL3500 scanner) and image analysis with ImageJ software (NIH).
Example 6: Severe Traumatic Brain Injury Study
[0090] A study was conducted that included 46 human subjects suffering severe traumatic brain injury. Each of these subjects is characterized by being over age 18, having a GCS of less than or equal to 8 and required ventriculostomy and neuromonitoring as part of routine care. A control group A, synonymously detailed as CSF controls, included 10 individuals also being over the age of 18 or older and no injuries. Samples are obtained during spinal anesthesia for routine surgical procedures or access to CSF associated with treatment of hydrocephalus or meningitis. A control group B, synonymously described as normal controls, totaled 64 individuals, each age 18 or older and experiencing multiple injuries without brain injury. Further details with respect to the demographics of the study are provided in Table 5.
Table 5. Subject Demographics for Severe Traumatic Brain Injury Study
TBI CSF Controls Normal Controls
Number 46 10 64
Males 34 (73.9%) 29 (65.9%) 26 (40.6%)
Females 12 (26.1%) 15 (34.1%) 38 (59.4% Average 50.2 58.2 1, 2 30.09 2, 3
Std Dev 19.54 20.52 15.42
Minimum 19 23 18
Maximum 88 82 74
Caucasian
Black 45 38 (86.4%) 52 (81.2%)
Asian 1 6 (13.6) 4 (6.3%)
Other 7 (10.9%)
1 (1.6%)
GCS in Emergency Department
Average 5.3
Std Dev 1.9
[0091] The level of biomarkers found in the first available CSF and serum samples obtained in the study are provided in FIGs. 1 and 2, respectively. The average first CSF sample collected as detailed in FIG. 1 was 11.2 hours while the average time for collection of a serum sample subsequent to injury event as per FIG. 2 is 10.1 hours. The quantity of each of the biomarkers of UCH-Ll, MAP2, SBDP145, SBDP120, and GFAP are provided for each sample for the cohort of traumatic brain injury sufferers as compared to a control group. The diagnostic utility of the various biomarkers within the first 12 hours subsequent to injury based on a compilation of CSF and serum data is provided in Fig.3 and indicates in particular the value of GFAP as well as that of additional markers UCH-Ll and the spectrin breakdown products. Elevated levels of UCH-Ll are indicative of the compromise of neuronal cell body damage while an increase in SPDP145 with a corresponding decrease in SPDP120 is suggestive of acute axonal necrosis.
[0092] One subject from the traumatic brain injury cohort was a 52 year old Caucasian woman who had been involved in a motorcycle accident while not wearing a helmet. Upon admission to an emergency room her GCS was 3 and during the first 24 hours subsequent to trauma her best GCS was 8. After 10 days her GCS was 11. CT scanning revealed SAH and facial fractures with a Marshall score of 11 and a Rotterdam score of 2. Ventriculostomy was removed after 5 years and an overall good outcome was obtained. Arterial blood pressure (MABP), intracranial pressure (ICP) and cerebral profusion pressure (CPP) for this sufferer of traumatic brain injury as a function of time is depicted in FIG. 4. A possible secondary insult is noted at approximately 40 hours subsequent to the injury as noted by a drop in MABP and CPP. The changes in concentration of inventive biomarkers per CSF and serum samples from this individual are noted in FIG. 5. These results include a sharp increase in GFAP in both the CSF and serum as well as the changes in the other biomarkers depicted in FIG. 5 and provide important clinical information as to the nature of the injury and the types of cells involved, as well as modes of cell death associated with the spectrin breakdown products.
[0093] Another individual of the severe traumatic brain injury cohort included a 51 year old Caucasian woman who suffered a crush injury associated with a horse falling on the individual. GCS on admission to emergency room was 3 with imaging analysis initially being unremarkable with minor cortical and subcortical contusions. MRI on day 5 revealed significant contusions in posterior fossa. The Marshall scale at that point was indicated to be 11 with a Rotterdam scale score of 3. The subject deteriorated and care was withdrawn 10 days after injury. The CSF and serum values for this individual during a period of time are provided in FIG. 6.
[0094] Based on the sandwich ELISA testing, GFAP values as a function of time are noted to be markedly elevated relative to normal controls (control group B) as a function of time.
[0095] The concentration of spectrin breakdown products, MAP2 and UCH-L1 as a function of time subsequent to traumatic brain injury has been reported elsewhere as exemplified in U.S. Patents 7,291,710 and 7,396,654 each of which is incorporated herein by reference.
[0096] An analysis was performed to evaluate the ability of biomarkers measured in serum to predict TBI outcome, specifically GCS. Stepwise regression analysis was the statistical method used to evaluate each of the biomarkers as an independent predictive factor, along with the demographic factors of age and gender, and also interactions between pairs of factors. Interactions determine important predictive potential between related factors, such as when the relationship between a biomarker and outcome may be different for men and women, such a relationship would be defined as a gender by biomarker interaction.
[0097] The resulting analysis identified biomarkers UCH-L1, MAP2, and GFAP as being statistically significant predictors of GCS (Table 6, 7). Furthermore, GFAP was shown to have improved predictability when evaluated in interaction with UCH-L1 and gender (Table 8, 9). Table 6. Stepwise Regression Analysis 1 - Cohort includes:
All Subjects >= 18 Years Old
Summary of Stepwise Selection - 48 Subjects
Variable Parameter Model
Step Entered Estimate R-Square F Value p-value
Intercept 13.02579
2 SEXCD -2.99242 0.1580 7.29 0.0098
1 CSF UCH Ll -0.01164 0.2519 11.54 0.0015
3 Serum_MAP_2 0.96055 0.3226 4.59 0.0377
Table 7. Stepwise Regression Analysis 2 - Cohort includes:
TBI Subjects >= 18 Years Old
Summary of Stepwise Selection - 39 Subjects
Variable Parameter Model
Step Entered Estimate R-Square F Value p-value
Intercept 5.73685
1 Serum UCH Ll -0.30025 0.0821 8.82 0.0053
2 Serum GFAP 0.12083 0.1973 5.16 0.0291
Table 8. Stepwise Regression Analysis 1 - Cohort includes:
TBI and A Subjects >= 18 Years Old
Summary of Stepwise Selection - 57 Subjects
Variable Parameter Model
Step Entered Estimate R-Square F Value p-value
Intercept 8.04382
1 Serum UCH L -0.92556 0.1126 12.90 0.0007
2 Serum_MAP_2 1.07573 0.2061 5.79 0.0197
3 Serum UCH-L1 0.01643 0.2663 4.35 0.0419
+Serum GFAP Table 9. Stepwise Regression Analysis 2 - Cohort includes:
TBI Subjects >= 18 Years Old
Summary of Stepwise Selection - 44 Subjects
Variable Parameter Model
Step Entered Estimate R-Square F Value p-value
Intercept 5.50479
1 Serum UCH Ll -0.36311 0.0737 11.95 0.0013
2 SEX Serum GFAP 0.05922 0.1840 5.09 0.0296
3 Serum MAP 2 0.63072 0.2336 2.59 0.1157
Example 7:
[0098] The study of Example 6 was repeated with a moderate traumatic brain injury cohort characterized by GCS scores of between 9 and 11, as well as a mild traumatic brain injury cohort characterized by GCS scores of 12-15. Blood samples were obtained from each patient on arrival to the emergency department of a hospital within 2 hours of injury and measured by ELISA for levels of GFAP in nanograms per milliliter. The results were compared to those of a control group who had not experienced any form of injury. Secondary outcomes included the presence of intracranial lesions in head CT scans.
[0099] Over 3 months 53 patients were enrolled: 35 with GCS 13-15, 4 with GCS 9-12 and 14 controls. The mean age was 37 years (range 18-69) and 66% were male. The mean GFAP serum level was 0 in control patients, 0.107 (0.012) in patients with GCS 13-15 and 0.366 (0.126) in GCS 9-12 (P<0.001). The difference between GCS 13-15 and controls was significant at P<0.001. In patients with intracranial lesions on CT GFAP levels were 0.234 (0.055) compared to 0.085 (0.003) in patients without lesions (P<0.001). There is a significant increase in GFAP in serum following a MTBI compared to uninjured controls in both the mild and moderate groups. GFAP was also significantly associated with the presence of intracranial lesions on CT.
[00100] FIG. 8 shows GFAP concentration for controls as well as individuals in the mild/moderate traumatic brain injury cohort as a function of CT scan results upon admission and 24 hours thereafter. Simultaneous assays were performed in the course of this study for UCH-Ll biomarker. The UCH-L1 concentration derived from the same samples as those used to determine GFAP is provided FIG. 9. The concentration of UCH-L1 and GFAP as well as a biomarker not selected for diagnosis of neurological condition, SI 00b, is provided as a function of injury magnitude between control, mild, and moderate traumatic brain injury as shown in FIG. 10. The simultaneous analyses of UCH-L1 and GFAP from these patients illustrates the synergistic effect of the inventive process in allowing an investigator to simultaneously diagnose traumatic brain injury as well as discern the level of traumatic brain injury between mild and moderate levels of severity. FIG. 11 shows the concentration of the same markers as depicted in FIG. 10 with respect to initial evidence upon hospital admission as to lesions in tomography scans illustrating the high confidence in predictive outcome of the inventive process. FIG. 12 shows that both NSE and MAP2 are elevated in subjects with MTBI in serum both at admission and at 24 hours of follow up. These data demonstrate a synergistic diagnostic effect of measuring multiple biomarkers such as GFAP, UCH-L1, NSE, and MAP2 in a subject.
[00101] Through the simultaneous measurement of multiple biomarkers such as UCH-L1, GFAP, NSE, and MAP2, rapid and quantifiable determination as to the severity of the brain injury is obtained consistent with GSC scoring and CT scanning yet in a surprisingly more quantifiable, expeditious and economic process. Additionally, with a coupled assay for biomarkers indicative of neurological condition, the nature of the neurological abnormality is assessed and in this particular study suggestive of neuronal cell body damage. As with severe traumatic brain injury, gender variations are noted suggesting a role for hormonal anti-inflammatories as therapeutic candidates.
Example 8:
[00102] 12 human brain autoantigens (purified recombinant protein) purified recombinant or native proteins are run on western blot and probed against pooled normal (n=5) and post-TBI serum (day 5-10) (n=5) at 1/100 dilution. Blots are developed with antihuman IgG coupled with alkaline phosphatase and BCIP- NBT (5-Bromo-4-chloro-3-indolyl phosphate / Nitro blue tetrazolium substrate. 7 of these antigens then are found to have protein bands with increased autoimmune reactivity when probed with TBI sera. These include GAP43, GAD1, recoverin, NF-L, NF-M, PNMA2, Endophilin Al . Furthermore, when 7 selected strong autoantigen (included GFAP) two individual normal serum and two post-TBI (day 5-10) serum samples. It is confirmed that GAP43, GAD1, recoverin, NF-L, NF-M, Endophilin Al and GFAP are selectively elevated in TBI subjects. See Figure 14. Also, certain autoantigens (e.g. NF-M, Endophilin Al) are only readily detected in one TBI subject but not the other. This indicates that individual subjects have different autoantigen and that a panel of autoantigen approach is an exemplary screening method. Many of these proteins (e.g. NF-M) are presented as multiple bands, some with molecular weight lower than calculated full length molecular weight, proving that breakdown products exist as autoantigens as well.
[00103] A number of these autoantigens tested are in fact paraneoplastic antigens (such as PNMA2, recoverin, Endophilin Al, KCNAB2, GRIA1, GAD1). Thus paraneoplastic brain antigens are exemplary targets of autoimmune response after TBI or other neural injuries or neuronal disorders.
[00104] Important note is that these brain proteins are under attack by immune system after TBI, and resulting in neurological syndromes. Example of that is Recoverin. Recoverin is a neurologically important calcium binding protein, involved in tumor pathology. It is conserved across species and there are indications of several isoforms of this molecule. It is especially enriched in retina, pineal gland-derived tissue and the surrounding tissue of the brain. Autoantibodies could launch an attack of retina and pineal gland, leading to functional deficit.
Example 8:
[00105] In another example, the autoantibody response is not only limited to TBI, but also other CNS injury conditions such as stroke and spinal cord injury (SCI). Figure 15 shows that serum samples from stroke subjects shows brain specific antigen autoimmune response, including the GFAP. Here human brain lysate on western blot are probed with individual serum samples (7day) at 1/100 dilution. Similarly, Figure 24 shows that in certain post-SCI patients (e.g. SCI patient 1), after 5-6 days, there is development of autoantibody, including response to GFAP as autoantigen. Here subacute development of blood-based autoantibody to a novel brain antigen (GFAP) in subset of SCI patients' serum. Human spinal cord lysate on western blot are probed with individual serum samples at 1/100 dilution.
[00106] Example 9:
[00107] Figures 17, 18 and 19 further shows that sera from epilepsy, Parkinson's disease, Alzheimer's disease and migraine subjects show brain specific antigen autoimmune response, including GFAP. Figure 17 illustrates Brain- specific Autoantibody response to various brain antigens, including GFAP are found in epilepsy patients serum, Human brain lysate (Sample: Human brain lysate (loading: 300ug) on western blot are probed with individual serum samples at 1/100 dilution. Secondary antibody: AP-conjugated Donkey Anti-human IgGH+L); Dilution: 1 : 10,000. GFAP as indicated and other brain autoantigens are also indicated by asterisks.
[00108] Figure 18 shows Brain-specific Autoantibody response to various brain antigens, including GFAP was found in Parkinson's disease and migraine patients' serum. Human brain lysate (Sample: Human brain lysate (loading: 300ug) on western blot were probed with individual serum samples at 1/100 dilution. Secondary antibody: AP-conjugated Donkey Anti-human IgGH+L); Dilution: 1 : 10,000. GFAP as indicated and other brain autoantigens are also indicated by asterisks.
[00109] Figure 19 shows Brain-specific Autoantibody response to various brain antigens, including GFAP-BDP are found in Alzheimer's disease and migraine patients' serum. Human brain lysate (Sample: Human brain lysate (loading: 300ug) on western blot are probed with individual serum samples at 1/100 dilution. Secondary antibody: AP-conjugated Donkey Anti-human IgGH+L); Dilution: 1 : 10,000. GFAP as indicated and other brain autoantigens are also indicated by asterisks.
[00110] Patent documents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. These documents and publications are incorporated herein by reference to the same extent as if each individual document or publication was specifically and individually incorporated herein by reference.
[00111] The foregoing description is illustrative of particular embodiments of the invention, but is not meant to be a limitation upon the practice thereof. The following claims, including all equivalents thereof, are intended to define the scope of the invention.

Claims

1. A method of detecting a neural injury or neuronal disorder in a subject comprising:
collecting a biological sample from a subject suspected of having a neural injury or neuronal disorder;
measuring said sample or a fraction thereof for an amount of a first biomarker that is a first autoantibody to one or more autoantigen of GFAP, GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 or GRIA1;
comparing said amount of the measured biomarker with the amount of the respective biomarker in an uninjured subject;
wherein an increased amount of said first biomarker in said injured subject compared with the amount of respective biomarker in the uninjured subject is indicative of a neural injury or neurological disorder.
2. The method of claim 1 further comprising measuring a second biomarker from the subject that is a second autoantibody to one or more of the nervous system (CNS,PNS) autoantigens of GFAP, GBDP UCH-L1, neuron specific enolase (NSE), a all-spectrin breakdown product, SlOOp, MAP (MAP2, MAPI, MAP3, MAP4, MAP5), myelin basic protein (MBP), MBP-fragments, Vimentin and Vimentin breakdown products, Tau, Tau breakdown products, a-internexin, a Neurofilament protein, Peripherin, CAM of N-CAM, I-CAM, V-CAM, or AL-CAM), GAD65, synaptic proteins of synaptotagmin, synaptojanin, synapsin, or synaptophysin, amphiphysin synucleins, neuroresin of p24 or vesicular membrane protein, Postsnayptic proteins of PSD95, PSD93, SAP-97, or SAP-102), CRMP, NOS of iNOS, eNOS, or n-NOS, NeuN, CNPase, Neuroserpin, Neurofascin, LC3, autophagy-linked p65, Nestin, doublecortin (DCx) Cortin-1, βΙΙΙ-Tubulin, (S100A2 (pl l)), Calmodulin dependent kinases (CAMPKs), CAMPK II-alpha, beta, gamma, Canabionoid Receptors (CB), ionotropic glutamate receptors (NMDA/AMPA/Kainate receptors), metabotropic glutamate receptors (mGluRs), Cholinergic receptor, GABA receptor, serotonin, Dopamine Receptors or receptor fragments thereof, Myelin proteolipid protein (PLP), Myelin Oligodendrocyte specific protein (MOSP), protein disulfide isomerase (PDI), PEBP, βΙΙ-spectrin breakdown products (PSBDPS), EAAT, serotonin-transporter, dopamine transporter (DAT), GABA transporter.
3. The method of claim 2 wherein said second biomarker that is said second autoantibody is of the same or different physical conformation as said first biomarker.
4. The method of claim 1 further comprising measuring a second quantity of said first biomarker at a second time to yield a kinetic profile for said first biomarker.
5. The method of claim 1 further comprising comparing the quantity of said first biomarker to normal levels of said first biomarker in the subject to other individuals of the same gender as the subject.
6. The method of claim 1 further comprising administering to said subject immunomodulatory therapy in response to said measuring.
7. The method of claim 1 wherein the neural injury or neuronal disorder is a trauma indiced brain injury, stroke, spinal cord injury, epilepsy, seizures, intracerebral hemorrhage, subarachnoid hemorrhage, migraine headache, or brain tumor.
8. The method of claim 1 wherein the biological sample is cerebrospinal fluid, serum, plasma, blood, urine or another biofluid containing said first biomarker.
9. The method of claim 1 , wherein said marker is detected using an immunoassay.
10. The method of claim 9 wherein the immunoassay is an ELISA, immunoblot or other immunoassays.
1 1. The method of claim 10 further comprising immobilizing said first biomarker on a biochip array.
12. The method of claim 11 further comprising and laser ionizing said first biomarker to detect a biomarker molecular weight a nd comparing the biomarker molecular weight against a threshold intensity that is normalized against total ion current.
13. The method of claim 11 wherein the biochip array surface comprises a substance selected from the group consisting of: an antibody, nucleic acid, protein, peptides, amino acid probes, and a phage display library.
14. A kit using the method of claim 13, the kit comprising:
a substrate for holding a biological sample isolated from a subject;
an agent that specifically interacts with one or more autoantigen of GFAP, GAP 43, GAD1, Recoverin, NSE protein, NF-L, NF-H, NF-M, PNMA2, Endophilin Al, KCNAB2 or GRIAl;
an optional additional agent that specifically interacts with at least one additional autoantigen upon contact with said sample; and
printed instructions for reacting the agent and the optional additional agent with the sample or a portion of the sample for diagnosing a neural injury or neuronal disorder in the subject.
15. The kit of claim 14, wherein said optionally additional agent is present and said optional additional agents interacts with said one additional autoantigen upon contact with said sample, wherein said additional autoantigen is one or more of GFAP, GBDP UCH-L1, neuron specific enolase (NSE), a αΙΙ-spectrin breakdown product, SIOOP, MAP (MAP2, MAPI, MAP3, MAP4, MAP5), myelin basic protein (MBP), MBP-fragments, Vimentin and Vimentin breakdown products, Tau, Tau breakdown products, a-internexin, a Neurofilament protein, Peripherin, CAM of N-CAM, I-CAM, V-CAM, or AL-CAM), GAD65, synaptic proteins of synaptotagmin, synaptojanin, synapsin, or synaptophysin, amphiphysin synucleins, neuroresin ofp24, or vesicular membrane protein, Postsnayptic proteins of PSD95, PSD93, SAP-97, or SAP- 102), CRMP, NOS of iNOS, eNOS, or n-NOS, NeuN, CNPase, Neuroserpin, Neurofascin, LC3, autophagy-linked p65, Nestin, doublecortin (DCx) Cortin-1, βΙΙΙ-Tubulin, (S100A2 (pl l)), Calmodulin dependent kinases (CAMPKs), CAMPK II-alpha, beta, gamma, Canabionoid Receptors (CB), ionotropic glutamate receptors (NMDA/AMPA/Kainate receptors), metabotropic glutamate receptors (mGluRs), Cholinergic receptor, GABA receptor, serotonin, Dopamine Receptors or receptor fragments thereof, Myelin proteolipid protein (PLP), Myelin Oligodendrocyte specific protein (MOSP), protein disulfide isomerase (PDI), PEBP, βΙΙ-spectrin breakdown products (PSBDPs), EAAT, serotonin-transporter, dopamine transporter (DAT), GABA transporter or combinations thereof.
16. The kit of claim 15 wherein said optional additional agent is an autoantibody of the same or different physical conformation as said first agent, said first agent being an autoantibody.
17. The kit of claim 14 wherein the neural injury or neuronal disorder is trauma indiced brain injury, stroke, spinal cord injury, epilepsy, seizures, intracerebral hemorrhage, subarachnoid hemorrhage, migraine headache, brain tumor.
18. The kit of claim 14 wherein the biological sample is cerebrospinal fluid, serum, plasma, blood, urine or other bio fluids.
19. The kit of claim 14 wherein said substrate is a biochip array having a surface comprising a substance selected from the group consisting of: an antibody, nucleic acid, protein, peptides, amino acid probes, and a phage display library wherein the biochip array is a protein chip array or a nucleic acid array.
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