WO2015066211A1 - Uch-l1 isoforms, assays and devices for detection of a neurological condition - Google Patents

Abstract

An in vitro diagnostic (IVD) device used to detect the presence of and/or severity of neural injuries or neuronal disorders in a subject. The IVD device relies on an immunoassay which identifies biomarkers that are diagnostic of neural injury and/or neuronal disorders in a biological sample, such as whole blood, plasma, serum, cerebrospinal fluid (CSF), sweat, saliva or urine. The IVD device may measure one or more of several neural specific markers in a biological sample and output the results to a machine readable format, whether to a display device or to a storage device internal or external to the IVD.

Description

UCH-L1 ISOFORMS, ASSAYS AND DEVICES FOR DETECTION OF A NEUROLOGICAL

CONDITION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 61/896,779, filed Oct. 29, 2013, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention provides for compositions of matter, methods, processes, kits and in vitro diagnostic devices which enables the reliable detection of damage to the nervous system (central nervous system (CNS) and peripheral nervous system (PNS)), brain injury and neural disorders of an individual through biomarker identification. These compositions of matter, methods, processes, kits and in vitro diagnostic devices allow for a simple yet sensitive clinical approach to diagnosing damage to the central nervous system, neurotoxicity, brain injury and neuronal disorders using biological fluid particularly measuring for one or more of a biomarker classified as a UCH-L1 protein isoform, namely UCH-Llb, UCH-Llc, UCH-Lld, and UCH- Lie. In addition the foregoing also allows for the synergistic combination of such iso forms with other clinically relevant neural markers such as glial fibrillary acidic protein (GFAP) or one of its breakdown products, neuron specific enolase (NSE), a SI 00 calcium binding protein β (SIOOP) including its neural specific iso forms, such as SlOOpi- S100p6, Microtubule-associated protein 2 (MAP2), myelin basic protein (MBP), a-II spectrin breakdown product (SBDP) preferably SBDP150, SBDP150i, SBDP 145, SBDP120, vesicular membrane protein neurensin-1 (p24), Tau, TDP-43 (Trans-activation response DNA-binding protein of 43 kDa), NF-H (Neurofilament protein H (heavy chain), collapsin response mediated proteins (CRMP's) and breakdown products thereof, or synaptotagmin and breakdown products thereof. Inventive markers include proteins; or protein fragments; auto-antibodies; DNA; RNA; or miRNA.

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 genetic disorders, all present the prospect of brain damage. Traumatic, ischemic, and neurotoxic chemical insult also present the prospect of other neurological damage.

[0004] One of many neurological conditions, Traumatic brain injury (TBI), occurs when external forces, through direct impact or acceleration, traumatically injure the brain often through falls, vehicle accidents, and violence. TBI can be characterized by its severity, from mild to severe, and the effects of such injury can be physical, cognitive, social, emotional, or behavioral and clinical outcomes range from complete recovery to permanent disability and death. Post-traumatic stress disorder (PTSD) is a psychological trauma that mirrors the symptoms of more moderate TBI making injuries both hard to distinguish and diagnose. Moreover, because of the similar symptoms of several other neurological conditions, such as stroke, subarachnoid hemorrhage, or a neurodegenerative disease, distinguishing one injury type from another has frustrated clinicians for years. Thus there is a need to identify the underlying molecular pathology in order to classify and distinguish these traumas.

[0005] More common methods and areas of continuing research, for diagnosing a neurological condition usually involve a neurological examination and assigning a Glasgow Coma Score to an individual. These methods are of limited value and often preclude a nuanced diagnosis due to the subjectivity of the testing, and the ability of a patient to knowingly alter their true response to achieve a desired result. Neuroimaging, such as computed tomography (CT) and magnetic resonance imaging (MRI) are also widely used to help determine the scope of injury and potential for intervention. However, these tests are both costly and time consuming, as well as are frustrated by the same problems of having an inability to distinguish one injury type from another. In addition, individuals with only mild or moderate TBI may be unaware damage has occurred and fail to seek treatment. It should be appreciated that repeated mild to moderate injuries can have a cumulative effect and result overall in a poor clinical diagnosis.

[0006] Early clinical diagnosis of a neurological condition continues to be an area requiring further development. Early diagnosis can limit the damage caused by facilitating earlier intervention. Much emphasis is being placed on developing biomarkers as early indicators of central nervous system damage. Upon injury to neural cells, neural proteins are released in to the interstitial fluid of the brain and eventually cross the blood brain barrier where they can be easily measured. Identifying specific proteins and measuring the levels that enter circulation upon the onset of a neurological condition can provide an effective early means of detecting the severity and type of the injury. [0007] A number of biomarkers have been identified as being associated with diagnosing several neurological conditions or nerve cell damage. 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 MAP-2 (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. Other biomarkers may be used to detect for neural injury, neuronal disease, or neural disorders, detailed in the disclosures presented in US 2007/0003982 Al, US 2005/0260697 Al , US 2009/0317805 Al, US 2005/0260654 Al , US 2009/0087868 Al, US 2010/0317041 Al , US 2011/0177974 Al, US 2010/0047817 Al , US 2012/0196307 Al, US 2011/0082203 Al , US 2011/0097392 Al, US 2011/0143375 Al , US 2013/0029859 Al, US 2012/0202231 Al, and US 2013/0022982 Al , the contents of which are also hereby incorporated by reference.

[0008] UCH-L1 is present in neurons and is commonly used as a biomarker in studies of the mammalian CNS. As such, UCH-L1 has been widely used as a biomarker of a variety of neurological conditions and it is elevated upon damage to neurons making it a highly neural specific assay. Conventional UCH-L1 is known to be a ~25 kDa. However, elevated levels of UCH-Llis difficult to detect reliably due to the complex structure. This is a result of the knotted tertiary structure folding upon itself and obscuring the binding sites from detection antibodies raised against that protein.

[0009] Thus, the reliability of UCH-L1 as a measure of a neurological condition depends upon the ability of an antibody to bind to the protein to enable its reliable detection. Thus, there exists the need to identify an isoform of UCH-L1 that is equally released as a result of neuron damage, without having the structural characteristics which frustrate its detection and to allow for new compositions of matter, such as antigens or antibodies, to support processes and assays for providing improved measurement of the neuron-specific biomarker of a neurological condition, either alone or in synergistic combination, with the specificity and sensitivity necessary to distinguish a neurological condition and to detect mild and moderate forms of brain injury. There also remains an unmet need for clinical intervention through the use of an in vitro diagnostic device to identify these neurochemical markers so that subject results may be obtained rapidly in any medical setting to direct the proper course of treatment for subjects suffering from a neural injury or neuronal disorder. SUMMARY OF THE INVENTION

[0010] A composition of matter, antibodies and antigens related thereto, methods, processes, kits and in vitro diagnostic devices specifically designed and calibrated to detect protein markers that are differentially present in the samples of patients suffering from neural injury and/or neuronal disorders including neurotoxicity and nerve cell damage. These devices present a sensitive, quick, and non-invasive method to aid in diagnosis of neural injury and/or neuronal disorders by detecting and determining the amount of biomarkers that are indicative to the respective injury type. The measurement of these markers, alone or in combination of other markers for the injury type, in patient samples provides information that a diagnostician can correlate with a probable diagnosis of the extent of an injury such as sports concussion, traumatic brain injury (TBI) and stroke.

[0011] In certain embodiments, an in vitro diagnostic device is provided to measure biomarkers that are indicative of traumatic brain injury, stroke, Alzheimer's disease, epilepsy, hypoxic ischemic encephalopathy (HIE), chronic traumatic encephalopathy (CTE), neural disorders, brain damage, neural damage due to drug or alcohol addiction, or other diseases and disorders associated with the brain or nervous system, such as the central nervous system or peripheral nervous system. In other embodiments, the biomarkers are proteins, fragments or derivatives thereof, and are associated with neuronal cells, brain cells or any cell that is present in the brain, central nervous system, and peripheral nervous system.

[0012] In other embodiments, the biomarkers are neural proteins, peptides, fragments or derivatives thereof which are detected by an assay. An in vitro diagnostic device is provided that further includes a process for determining the neurological condition of a subject or cells from the subject, that includes measuring a sample obtained from the subject or cells from the subject at a first time for a quantity of a first biomarker which represents a neural specific or brain specific isoform of UCH-L1 , namely UCH-Llb, UCH-Llc, UCH-Lld and UCH-Lle. These markers may be measured alone or in combination with other markers such as GFAP, SlOOp including its neural specific isoforms, such as S lOOpi- ΞΙΟΟββ, NSE, conventional UCH-L1 (UCH-Lla), MBP, MAP2, SBDP, CRMP, synaptotagmin, or neurensin-1 (p24). Through comparison of the quantity of the first biomarker and the quantity of at least one additional neuroactive biomarker to normal levels for each biomarker, the neurological condition of the subject is determined over one or more possible injury indicators that only one biomarker may represent. An in vitro diagnostic device is also provided that necessarily incorporates an assay for determining the neurological condition of a subject or neural cells from the subject is also provided. The assay includes at least a first biomarker specifically binding agent wherein a first biomarker is a neural specific or brain specific isoform of UCH-Ll , namely UCH-Llb, UCH- Llc, UCH-Lld and UCH-Lle. In other embodiments, an assay is incorporated which may detect one or more additional markers selected from the group of GFAP, SlOOp including its neural specific isoforms, such as SlOOpi- ΞΙΟΟββ, NSE, UCH-Lla, MBP, MAP2, SBDP, CRMP, synaptotagmin, or neurensin-1 (p24).

[0013] A device also provides a process for determining if a subject has suffered mild traumatic brain injury, moderate traumatic brain injury, or severe traumatic brain injury in an event, which includes measuring a sample obtained from the subject or cells from the subject at a first time after the event for a quantity of a neural specific or brain specific isoform of UCH-Ll , namely UCH-Llb, UCH-Llc, UCH-Lld and UCH-Lle have a particular synergy being measured with GFAP and SlOOp including its neural specific isoforms, such as SlOOpi- ΞΙΟΟββ, for diagnosing different severities of brain injury, as well as, distinguishing injury types, such as determining whether a subject is suffering from traumatic brain injury, stroke, subarachnoid hemorrhage, or other neural disorders and neural degenerative diseases, thus by comparing the quantity of the neural specific isoforms of SlOOp, GFAP and isoforms of UCH-Ll to each other and to a metric of what level is expected in a non-injured subject, using an algorithm of the assay output and a pre-programmed comparison metric, which has been clinically validated, a device interpolates the data to determine if the subject has suffered an injury (TBI, stroke, SAH, etc.), determine the severity of injury (mild, moderate, severe) and by including even more markers, may determine the time after injury and predict a recovery outcome. A comparison of these markers may also be used to determine other neurological disorders such as Alzheimer's, Parkinson's disease, and may predict other neural injuries using this or any number of additional biomarkers, such as neurotoxicity such as is disclosed in WO/2011/123844 and whose disclosure is incorporated herein by reference.

[0014] A kit is also provided for analyzing cell damage in a subject. The kit, in some embodiments includes: (a) a substrate for holding a biological sample isolated from a human subject suspected of having a damaged nerve cell, (c) an agent that specifically binds at least one or more of the neural proteins; and (c) printed instructions for reacting the agent with the biological sample or a portion of the biological sample to detect the presence or amount of at least one marker in the biological sample. The agents that accompany such kit are those reagents, antigens, antibodies and/or recombinant proteins for the brain specific isoform of UCH-Ll , namely UCH-Llb, UCH-Llc, UCH-Lld and UCH-Lle. The kits may further include reagents, antigens, antibodies and/or recombinant proteins for an additional one or more neuroactive biomarkers, such as glial fibrillary acidic protein (GFAP), SI 00 calcium binding protein β (SIOOP) including its neural specific iso forms, such as SlOOpi- S100p6, Neuron specific enolase (NSE), spectrin breakdown products (SBDP), preferably SBDP150, SBDP150i SBDP 145, SBDP120, UCH-Lla), microtubule associated proteins (MAP), preferably MAP2, MAPI , MAP3, 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), 2',3'- cyclic nucleotide-3'-phosp ho hydrolase (CNPase), Neuroserpin, alpha-internexin, microtubule- associated protein 1 light chain 3 (LC3), Neurofascin, the glutamate transporters (EAAT), Nestin, Cortin-1 , 2', and BIII-Tubulin

[0015] A biological sample is a fluid in communication with the nervous system of the subject prior to being isolated from the subject; for example, CSF or blood, and the agent can be an antibody, aptamer, or other molecule that specifically binds at least one or more of the neural proteins. The kit can also include a detectable label such as one conjugated to the agent, or one conjugated to a substance that specifically binds to the agent (e.g., a secondary antibody).

[0016] In certain embodiments, compositions of matter, proteins, antigens, and antibodies raised against a peptide having the sequence of SEQ ID NO 2; SEQ ID NO 3; SEQ ID NO 4; or SEQ ID NO 5 are provided which represent the UCH-L1 isoforms, UCH-Llb , UCH-Llc, UCH-L 1 d, and UCH-L 1 e, respectively.

[0017] In certain embodiments, diagnostic processes and methods are provided in order to diagnose a neural injury or neuronal disorder. The diagnostic methods make use of the proteins, antigens, and antibodies raised against a peptide having the sequence of SEQ ID NO 2; SEQ ID NO 3; SEQ ID NO 4; and SEQ ID NO 5. In addition, one or more additional biomarkers may be measured. These methods or process may be incorporated into an assay, or into a cartridge containing an assay and reagents to be used to detect the markers herein described, alone, or in combination.

[0018] Other aspects of the invention are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 are bar graphs showing the concentration of UCH-L1 , GFAP and S100p, provided as a function of injury magnitude between control, mild, and moderate traumatic brain injury; [0020] FIG. 2 are bar graphs showing the concentration of the same markers as depicted in FIG. 1 with respect to initial evidence upon hospital admission as to lesions in tomography scans;

[0021] FIG. 3 represents biomarker levels in human subjects with varying types of brain injury;

[0022] FIG. 4 represents an ROC curve showing the improved specificity and synergistic effects for a neurological condition by using a combination of biomarkers;

[0023] FIG. 5 illustrates the level of GFAP, UCH-Ll and SlOOp in serum from TBI human subjects with mild and moderate injury magnitude;

[0024] FIG. 6 is a schematic view of the in vitro diagnostic device.

[0025] FIG. 7 represents UCH-Ll , GFAP, SlOOp, NSE, MBP, and MAP2 amounts present in serum post severe traumatic brain injury in human subjects as a function of CT scan results;

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention has utility in the diagnosis and management of abnormal neurological condition. Through the measurement of biomarkers in a biological sample from a subject, such as the neural specific UCH-Ll iso forms, UCH-Llb through UCH-Ll e, alone or in combination with each other or in combination with one or more additional neuroactive markers selected from GFAP, SIOOP including its neural specific iso forms, such as S100p2- S100p6, NSE, MBP, MAP2, SBDP, CRMP, CNPase, NRP-2 synaptotagmin, or neurensin-1 (p24) from a subject, a determination of subjects neurological condition is provided with greater specificity than previously attainable. The present description is directed toward a first iso form of UCH-Ll , selected from UCH-Llb through UCH-Ll e, each separately as a biomarker for brain injury of various origins and severity, as detailed herein below. It is appreciated that the invention encompasses several other additional biomarkers illustratively including GFAP, SlOOp including its neural specific iso forms, such as S100p2 - S100p6, NSE, MAP2, and SBDP. Surprisingly, by combining the detection of more than one biomarker, a synergistic result is achieved. Illustratively, combining the detection of two neuroactive biomarkers such as S100p2 and UCH- Llb or GFAP provides sensitive detection that is unexpectedly able to discern the level and severity of an abnormal neurological condition in a subject. It should also be appreciated that other neural specific forms of SlOOp or UCH-Ll, represented by any of SEQ. ID. NO. 2-5, provides this synergistic effect when measured in combination with GFAP. An even greater synergy and assay specificity is achieved by combining any neural specific isoform of UCH-L1 in an assay with GFAP and a neural specific isoform of SlOOp.

[0027] 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 used in some instances herein referred to as the result of an assay wherein a particular ratio or level of a biomarker is detected or is absent.

[0028] 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.

[0029] As used herein a "neural 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.

[0030] A neural 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.

[0031] As used herein "coma" shall mean the initial stage after a severe brain injury, a state of unconsciousness. People in a coma are unaware and unresponsive, but not asleep as there is no sleep-wake cycle. While in a coma, people are unable to speak, follow commands or open their eyes. As a person's GCS score improves, he or she is considered to be emerging from the coma. These changes usually take place gradually. For instance, eyes may open or there may be evidence of sleep cycles, but still no ability to speak or follow commands. As these abilities appear, most rehabilitation centers will use the Rancho Los Amigos Cognitive Scale (see separate document) to describe progress after this point.

[0032] As used herein "vegetative state" shall mean the period where emergence from coma can seem to stop before the person becomes conscious. People in a vegetative state may open their eyes and have sleep-wake cycles, but are still unconscious. Although not considered to be in a coma, the patients remain totally unaware. In a vegetative state, any apparent signs of responding to surroundings are reflexes and not indications of awareness. The term permanent vegetative state is used only when a person is determined to be in a vegetative state for twelve months after trauma or three months after a brain injury that caused oxygen insufficiency. Always discuss with the physician questions about responses and awareness.

[0033] As used herein "minimally conscious state" refers to people, who demonstrate some, but very little, awareness and responsiveness to their surroundings. Responses are typically inconsistent and thus not considered comatose or vegetative. As the name suggests, a person is considered conscious in this state. Occasionally, physicians may prescribe medicines that help stimulate the brain, especially if a person is not becoming more responsive with time. Some people do not progress beyond this stage in their recovery process. 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.

[0034] The present invention provides for the detection of a neurological condition in a subject. A neurological condition may be an abnormal neurological condition such as that caused by genetic disorder, injury, or disease to nervous tissue. As such, it is a further object of the present invention to provide a means for detecting or diagnosing an abnormal neurological condition in a subject.

[0035] The present invention also provides an assay 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 drugs or other compounds or for detecting the effects of environmental contaminants regardless of whether the subject itself or cells derived there from are exposed to the drug candidate or other possible stressors.

[0036] For purposes of the subject invention, brain injury is divided into two levels, mild traumatic brain injury (MTBI), and traumatic brain injury (TBI). An intermediate level of moderate TBI is also referred to herein. The spectrum between MTBI and extending through moderate TBI is also referred to synonymously as mild to moderate TBI or by the abbreviation MMTBI.

In Vitro Diagnostic Device

[0037] FIG.6 schematically illustrates an inventive in vitro diagnostic device. An inventive in vitro diagnostic device includes at least a sample collection chamber 603, an assay module 602 used to detect biomarkers of neural injury or neuronal disorder, and a user interface that relates the amount of the measured biomarker measured in the assay module. The in vitro diagnostic device in various embodiments is a handheld device, a bench top device, or a point of care device.

[0038] The sample chamber 603 can be of any sample collection apparatus known in the art for holding a biological fluid. In one embodiment, the sample collection chamber can accommodate any one of the biological fluids herein contemplated, such as whole blood, plasma, serum, urine, sweat or saliva.

[0039] The assay module 602 in some embodiments includes an assay which may be used for detecting a protein antigen in a biological sample, for instance, through the use of antibodies in an immunoassay. The assay module 602 may include any assay currently known in the art; however the assay should be optimized for the detection of neural biomarkers used for detecting neural injury or neuronal disorder in a subject. The assay module 602 is in fluid communication with the sample collection chamber 603. In one embodiment, the assay module 602 includes of an immunoassay where the immunoassay may be any one of a radioimmunoassay, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assay, fluorescent immunoassay, chemiluminescent immunoassay, phosphorescent immunoassay, or an anodic stripping voltammetry immunoassay. In one embodiment a colorimetric assay may be used which may include only of a sample collection chamber 603 and an assay module 602 of the assay. Although not specifically shown these components are preferably housed in one assembly 607. In one embodiment the assay module 602 contains an agent specific for detecting one or more of the biomarkers of glial fibrillary acidic protein (GFAP) or one of its breakdown products, Ubiquitin carboxyl-terminal hydrolase LI (UCH-Lla-UCH-Lle), neuron specific enolase (NSE), Microtubule-associated protein 2 (MAP2), myelin basic protein (MBP), a-II spectrin breakdown product (SBDP) preferably SBDP150, SBDP150i, SBDP145, SBDP120, SI 00 calcium binding protein B (S100bl-S100b6), collapsin response mediated proteins (CRMP's) and breakdown products thereof, or synaptotagmin and breakdown products thereof. The assay module 602 may contain additional agents to detect additional biomarkers, as is described herein.

[0040] In one embodiment, the inventive in vitro diagnostic device contains a power supply 601, an assay module 602, a sample chamber 603, and a data processing module 605. The power supply 601 is electrically connected to the assay module and the data processing module. The assay module 602 and the data processing module 605 are in electrical communication with each other. As described above, the assay module 602 may include any assay currently known in the art; however the assay should be optimized for the detection of neural biomarkers used for detecting neural injury or neuronal disorder in a subject. The assay module 602 is in fluid communication with the sample collection chamber 603. The assay module 602 includes of an immunoassay where the immunoassay may be any one of a radioimmunoassay, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assay, fluorescent immunoassay, chemiluminescent immunoassay, phosphorescent immunoassay, or an anodic stripping voltammetry immunoassay. A biological sample is placed in the sample chamber 603 and assayed by the assay module 602 detecting for a biomarker of neural injury or neuronal disorder. The measured amount of the biomarker by the assay module 602 is then electrically communicated to the data processing module 604. The data processing 604 module may include of any known data processing element known in the art, and may include a chip, a central processing unit (CPU), or a software package which processes the information supplied from the assay module 602.

[0041] In one embodiment, the data processing module 604 is in electrical communication with a display 605, a memory device 606, or an external device 608 or software package (such as laboratory and information management software (LIMS)). In one embodiment, the data processing module 604 is used to process the data into a user defined usable format. This format includes of the measured amount of neural biomarkers detected in the sample, indication that a neural injury or neuronal disorder is present, or indication of the severity of the neural injury or neuronal disorder. The information from the data processing module 604 may be illustrated on the display 605, saved in machine readable format to a memory device, or electrically communicated to an external device 608 for additional processing or display. Although not specifically shown these components are preferably housed in one assembly 607. In one embodiment, the data processing module 604 may be programmed to compare the detected amount of the biomarker transmitted from the assay module 602, to a comparator algorithm. The comparator algorithm may compare the measured amount to the user defined threshold which may be any limit useful by the user. In one embodiment, the user defined threshold is set to the amount of the biomarker measured in control subject, or a statistically significant average of a control population.

[0042] In one embodiment, an in vitro diagnostic device may include one or more devices, tools, and equipment configured to hold or collect a biological sample from an individual. In one embodiment of an in vitro diagnostic device, tools to collect a biological sample may include one or more of a swab, a scalpel, a syringe, a scraper, a container, and other devices and reagents designed to facilitate the collection, storage, and transport of a biological sample. In one embodiment, an in vitro diagnostic test may include reagents or solutions for collecting, stabilizing, storing, and processing a biological sample. These reagents include those peptides represented by SEQ. ID No. 1-5, or antibodies raised against said SEQ ID NO. Such reagents and solutions for nucleotide collecting, stabilizing, storing, and processing are well known by those of skill in the art and may be indicated by specific methods used by an in vitro diagnostic test as described herein. In one embodiment, an in vitro diagnostic device, as disclosed herein, may include a micro array apparatus and reagents, a flow cell apparatus and reagents, a multiplex nucleotide sequencer and reagents, and additional hardware and software necessary to assay a genetic sample for certain genetic markers and to detect and visualize certain biological markers.

KITS

[0043] In yet another aspect, the invention provides kits for aiding a diagnosis of neural injury, degree of severity of injury, subcellular localization and/or neuronal disorders, wherein the kits can be used to detect the markers of the present invention. For example, the kits can be used to detect any one or more of the markers described herein, which markers are differentially present in samples of a patient and normal subjects. The kits of the invention have many applications. For example, the kits can be used to differentiate if a subject has axonal injury versus, for example, dendritic, or has a negative diagnosis, thus aiding neuronal injury diagnosis. In another example, the kits can be used to identify compounds that modulate expression of one or more of the markers in in vitro or in vivo animal models to determine the effects of treatment.

[0044] In one embodiment, a kit includes (a) an antibody that specifically binds to a marker; and (b) a detection reagent. Such kits can be prepared from the materials described above, and the previous discussion regarding the materials {e.g., antibodies, detection reagents, immobilized supports, etc.) is fully applicable to this section and will not be repeated. In some specific inventive embodiments, the kit includes pre-fractionation spin columns. In one embodiment, the kit may further include instructions for suitable operation parameters in the form of a label or a separate insert.

[0045] In another embodiment, the kit includes (a) a panel or composition of detecting agent to detect a panel or composition of biomarkers. The panel or composition of reagents included in a kit provide for the ability to detect at least one biomarker and/or a plurality of biomarkers in order to diagnose a neural injury. These biomarkers shall first detect for at least one neural specific or brain specific isoform of UCH-L1 , namely UCH-Llb, UCH-Llc, UCH-Lld, and UCH-Lle. The kit may additionally include reagents to test for other biomarkers such as GFAP, ubiquitin carboxyl-terminal esterase LI a (UCH-Lla), Neuron specific enolase (NSE), spectrin breakdown products (SBDP), preferably SBDP150, SBDP150i, SBDP145, SBDP120, S100 βΐ - β6, microtubule associated proteins (MAP), preferably MAP2, MAPI, MAP3, 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), 2',3 '-cyclic nucleotide-3'- phosphohydrolase (CNPase), Neuroserpin, alpha-internexin, microtubule-associated protein 1 light chain 3 (LC3), Neurofascin, the glutamate transporters (EAAT), Nestin, Cortin-1 , 2', TDP- 43 (Trans-activation response DNA-binding protein of 43 kDa), NF-H (Neurofilament protein H (heavy chain) and BIII-Tubulin

[0046] In one embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the peptide of the invention. These antigens may be one of any of the peptides represented by SEQ ID NO. 1-5. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with peptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or peptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. Antibodies used in the inventive kit are those raised against any one of the peptides represented in SEQ ID NO's 1-5. In one embodiment, the antibody is a monoclonal or polyclonal antibody raised against the rat, rabbit or human forms of the brain or neural specific isoforms of UCH-L1 described herein. The detecting means of the kit includes a second, labeled monoclonal or polyclonal antibody. Alternatively, or in addition thereto, the detecting means includes a labeled, competing antigen.

[0047] In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, Mo.).

[0048] The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydro xyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).

[0049] In some embodiments, the kit may include a standard or control information so that the test sample can be compared with the control information standard to determine if the test amount of a marker detected in a sample is a diagnostic amount consistent with a diagnosis of neural injury, degree of severity of the injury, subcellular localization, neuronal disorder and/or effect of treatment on the patient.

[0050] In one embodiment, a kit includes: (a) a substrate including an adsorbent thereon, wherein the adsorbent is suitable for binding a marker, and (b) instructions to detect the marker or markers by contacting a sample with the adsorbent and detecting the marker or markers retained by the adsorbent. In some embodiments, the kit may include an eluant (as an alternative or in combination with instructions) or instructions for making an eluant, wherein the combination of the adsorbent and the eluant allows detection of the markers using gas phase ion spectrometry. Such kits can be prepared from the materials described above, and the previous discussion of these materials (e.g., probe substrates, adsorbents, washing solutions, etc.) is fully applicable to this section and will not be repeated. [0051] In one embodiment, the kit includes a first substrate including an adsorbent thereon (e.g., a particle functionalized with an adsorbent) and a second substrate onto which the first substrate can be positioned to form a probe which is removably insertable into a gas phase ion spectrometer. Alternatively, the kit may include a single substrate which is in the form of a removably insertable probe with adsorbents on the substrate. The kit may further include a pre- fractionation spin column (e.g., Cibacron blue agarose column, anti-HSA agarose column, size exclusion column, Q-anion exchange spin column, single stranded DNA column, lectin column, etc.).

[0052] In certain embodiments, the kit further includes instructions for suitable operational parameters in the form of a label or a separate insert. For example, the kit may have standard instructions informing a consumer how to wash the probe after a sample is contacted on the probe. In another example, the kit may have instructions for pre-fractionating a sample to reduce complexity of proteins in the sample. In another example, the kit may have instructions for automating the fractionation or other processes.

Biological Fluids

[0053] The inventive compositions of matter, processes, methods and in vitro diagnostic devices provide the ability to detect and monitor levels of those neural proteins or autoantibodies which are released into the body after neurotoxicity or CNS injury to provide enhanced diagnostic capability by allowing clinicians (1) to determine the level of injury severity in patients with various CNS injuries, (2) to monitor patients for signs of secondary CNS injuries that may elicit these cellular changes and (3) to continually monitor the progress of the injury and the effects of therapy by examination of these proteins in biological fluids, such as blood, plasma, serum, CSF, urine, saliva or sweat. Unlike other organ-based diseases where rapid diagnostics for surrogate biomarkers prove invaluable to the course of action taken to treat the disease, no such rapid, definitive diagnostic tests exist for traumatic or ischemic brain injury that might provide physicians with quantifiable neurochemical markers to help determine the seriousness of the injury, the anatomical and cellular pathology of the injury, and the implementation of appropriate medical management and treatment.

[0054] A sample is preferably a biological sample. Preferred examples of biological samples are illustratively 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. The biological samples illustratively include CSF, blood, serum, plasma, sweat, saliva or urine. It should be appreciated that after injury to the nervous system (such as brain injury), the neural cell membrane is compromised, leading to the efflux of neural proteins first into the extracellular fluid or space and to the cerebrospinal fluid. Eventually the neural proteins efflux to the circulating blood (as assisted by the compromised blood brain barrier) and, through normal bodily function (such as impurity removal from the kidneys), the neural proteins migrate to other biological fluids such as urine, sweat, and saliva. Thus, other suitable biological samples include, but are not limited to such cells or fluid secreted from these cells. It should also be appreciated that obtaining biological fluids such as cerebrospinal fluid, 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, which are biological fluids, are preferred for use in the invention.

[0055] Biological samples of CSF, blood, urine and saliva are collected using normal collection techniques. For example, and not to limit the sample collection to the procedures contained herein, CSF Lumbar Puncture (LP) a 20-gauge introducer needle is inserted and an amount of CSF is withdrawn. For blood, the samples may be collected by venipuncture in Vacutainer tubes, and if preferred spun down and separated into serum and plasma. For urine and saliva, samples that are collected avoiding the introduction of contaminants into the specimen are preferred. All biological samples may be stored in aliquots at -80 °C for later assay. 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). Any suitable biological samples can be obtained from a subject to detect markers. It should be appreciated that the methods employed herein may be identically reproduced for any biological fluid to detect a marker or markers in a sample.

[0056] After insult, the damaged tissue, organs, or 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.

[0057] 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), 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.

[0058] 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, baselines are determined from subjects where there is an absence of a biomarker or present in biological samples at a negligible amount. However, some proteins may be expressed less in an injured patient. Determining the baseline levels of protein biomarkers in a particular species is well within the skill of the art. Similarly, determining the concentration of baseline levels of neural or liver injury biomarkers is well within the skill of the art.

Neural Biomarkers

[0059] The inventive processes, methods, compositions of matter and devices preferably include determining the neurological condition of a subject by assaying a sample derived from a subject at a first time for the presence of at least one biomarker wherein said at least one biomarker is a neural specific or brain specific isoform of UCH-L1 , namely UCH-Llb, UCH- Llc, UCH-Lld, and UCH-Lle. A biomarker is a cell, protein, nucleic acid, steroid, fatty acid, metabolite, or other differentiator useful for measurement of biological activity or response. Additional biomarkers operable herein illustratively include: ubiquitin carboxyl-terminal esterase, ubiquitin carboxy-terminal hydrolase, spectrin breakdown product(s), a neuronally- localized intracellular protein, MAP-tau, C-tau, MAP2, poly (ADP-ribose) polymerase (PARP), collapsin response mediator protein, Annexin Al 1, Aldehyde dehydrogenase family 7, Cofilin 1 , Profilin 1 , α-Enolase (non-neural enolase), Enolase 1 protein, Glyceraldehyde-3 -phosphate dehydrogenase, Hexokinase 1 , Aconitase 2, Acetyl-CoA synthetase 2, Neuronal protein 22, Phosphoglycerate kinase 2, Phosphoglycerate kinase 1 , Hsc70-psl , Glutamate dehydrogenase 1 , Aldolase A, Aldolase C, fructose-biphosphate, Dimethylarginine dimethylaminohydrolase 1 , Microtubule-associated protein 2, Carbonic anhydrase, ADP-ribosylation factor 3, Transferrin, Liver regeneration-related protein, Hemoglobin a-chain, Hemoglobin β chain, Liver regeneration-related protein, Fetuin β, 3-Oxoacid-CoA transferase, Malate dehydrogenase 1 , NAD (soluble), Lactate dehydrogenase B, Malate dehydrogenase, Carboxylesterase El precursor, Serine protease inhibitor al , Haptoglobin, Ubiquitin carboxyl-terminal hydrolase LI , Serine protease inhibitor 2a, T-kininogen, al major acute phase protein, Albumin, al major acute phase protein prepeptide, Murino globulin 1 homolog, Group-specific component protein, Guanosine diphosphate dissociation inhibitor 1, Collapsin response mediator protein 2, Murino globulin 1 homolog, Ferroxidase, Ceruloplasmin, Spectrin a-chain, brain, C-reactive protein, Brain creatine kinase, Proteasome subunit a-type 7, 14-3-3 protein, Synaptotagmin, subtypes thereof, fragments thereof, breakdown products thereof, or combinations thereof. Other potential biomarkers illustratively include those identified by Kobeissy, FH, et al, Molecular & Cellular Proteomics, 2006; 5 :1887-1898, the contents of which are incorporated herein by reference, or others known in the art. However is should be appreciated that UCH-Llb, UCH- Ll c, UCH-Lld, and UCH-Ll e may be used in an assay alone, in combination with each other, or alternatively combined or multiplexed with other markers, preferably UCH-Ll a, GFAP, spectrin breakdown products (SBDP), preferably SBDP150, SBDP150i, SBDP145, SBDP120, SlOOpi - β6, CRMP's, Synaptotagmin, Cannabinoid Receptor (CB), TDP-43, MAP-2 or neurofilament proteins (NF-H. NF-M, NF-L).

[0060] It is appreciated however, that multiple biomarkers may be predictors of different modes or types of damage to the same cell type. Through the use of an inventive assay inclusive of biomarkers associated with glial cells as well as at least one other type of neural cell, the type of neural cells being stressed or killed as well as quantification of neurological condition results provides rapid and robust diagnosis of traumatic brain injury type. Measuring a neural or brain specific isoform of UCH-Ll along with at least one additional neuroactive biomarker and comparing the quantity of the UCH-Ll isoform and the additional biomarker to normal levels of the markers provides a determination of subject neurological condition.

[0061] Preferably, specific biomarker levels that when measured in concert with UCH-Ll iso forms afford superior evaluation of subject neurological condition include GFAP, SBDP 150, SBDP150i, a combination of SBDP145 (calpain mediated acute neural necrosis) and SBDP120 (caspase mediated delayed neural apoptosis), SIOOP and its isoforms, and MAP2. This is noted to be of particular value in measuring MMTBI, predicting abnormal CT scans, determining neurotoxicity, distinguishing stroke from TBI or subarachnoid hemorrhage.

[0062] To provide correlations between a neurological condition and measured quantities of a neural specific isoform of UCH-Ll alone or in combination with each other or other neuroactive biomarkers, biological samples are collected from subjects with the samples being subjected to measurement for these biomarkers. The subjects vary in neurological condition. Detected levels of a neural specific isoform of UCH-Ll alone or in combination with each other or other neuroactive biomarkers 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 the aforementioned biomarkers, in addition to being obtained from CSF and serum, are also readily obtained from blood, plasma, saliva, urine, sweat, buccal swab, as well as solid tissue biopsy.

Neurotoxicity Markers

[0063] In a preferred embodiment, the described compositions of matter, methods, processes, kits and in vitro diagnostic devices provide the ability to detect and monitor levels of proteins detecting a neurotoxic insult. Several biomarkers are used, each of which an assay is developed and incorporated for use with the described methods, processes, kits and in vitro diagnostic devices, or incorporated to use the described compositions of matter. Biomarkers of neurotoxicity include neural specific isoforms of UCH-Ll (UCH-Llb, UCH-Llc, UCH-Lld, or UCH-Lle) a S100 calcium binding protein β (SIOOP) including its neural specific isoforms, such as SlOOpi- S100P6 spectrin; a spectrin breakdown product (SBDP); MAPI , MAP2; GFAP, ubiquitin carboxyl-terminal esterase; ubiquitin carboxyl-terminal hydrolase; a neuronally- localized intracellular protein; MAP-tau; C-tau; Poly (ADP-ribose) polymerase (PARP); a collapsin response mediator protein, synaptotagmin, βΙΙΙ-tubulin; neuron-specific enolase, neurofilament protein light chain, nestin, a-internexin; breakdown products thereof, post- translationally modified forms thereof, derivatives thereof, and combinations thereof.

Immunoassays

[0064] The detection methods, process may be implemented into assays or into kits for performing assays. These kits or assays may alternatively be packaged into a cartridge to be used with an inventive in vitro diagnostic device. Such a device makes use of these cartridges, kits or assay in an assay module 602, which may be one of many types of assays. The biomarkers of the invention can be detected in a sample by any means. Methods for detecting the biomarkers are described in detail in the materials and methods and examples which follow. For example, immunoassays, include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, magnetic immunoassays, radioisotope immunoassay, fluorescent immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, fluorescent immunoassays, chemiluminescent immunoassays, phosphorescent immunoassays, anodic stripping voltammetry immunoassay and the like. Inventive in vitro diagnostic devices may also include any know devices currently available that utilize ion-selective electrode potentiometry, microfluids technology, fluorescence or chemiluminescence, or reflection technology that optically interprets color changes on a protein test strip. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1 , John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation). It should be appreciated, that at present, none of the existing technologies present a method of detecting or measuring any of the ailments disclosed herein, nor does there exist any methods of using such in vitro diagnostic devices to detect any of the disclosed biomarkers to detect their associated injuries.

Compositions of Matter

[0065] An exemplary process for detecting the presence or absence of a biomarker, alone or in combination, 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, 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.

[0066] For example, in vitro techniques for detection of a marker illustratively include enzyme linked immunosorbent assays (ELISAs), radioimmunoassay, 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. Optionally, the first biomarker specifically binding agent and the agent specifically binding at least one additional neuroactive biomarker are both bound to a substrate. It is appreciated that a bound agent assay is readily formed with the agents bound with spatial overlap, with detection occurring through discernibly different detection for first biomarker and each of at least one additional neuroactive biomarkers. A color intensity based quantification of each of the spatially overlapping bound biomarkers is representative of such techniques.

[0067] Any suitable molecule that can specifically bind to a biomarker and any suitable molecule that specifically binds one or more other biomarkers of a particular condition are operative in the invention to achieve a synergistic assay. A preferred agent for detecting the one or more biomarkers of a condition is an antibody capable of binding to the biomarker being analyzed. Preferably, 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). Exemplary antibodies operative herein are used to detect a biomarker of the disclosed conditions. In addition antigens to detect autoantibodies may also be used to detect chronic injury of the stated injuries and disorders.

[0068] 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. Alternatively, a detection species of another antibody or other compound known to the art is used as form detection of a biomarker bound by an antibody.

[0069] Antibody-based assays are preferred for analyzing a biological sample for the presence of a biomarker and one or more other biomarkers of a particular injury or condition. 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.

[0070] Reagents as described herein may be any antibody to a protein or peptide sequence or any antigen to detect an antibody formed as part of an autoimmune response in a subject. In particular for the antigens used for these detection methods and devices, they may be those proteins or peptides generated to detect an antibody that has been produced by a subject's own tissues as an autoimmune response to cells, tissues or native proteins of the organism in which it was formed. Preferably these antigens are peptides having the following sequence for each of the defined isoforms from which antibodies are, have been, or will be generated: SEQ ID NO. 1 is the publicly recognized generic sequence commonly referred to or linked to all UCH-L1 studies currently recognized in the art. Because of the recent discoveries of the several isoforms, not previously known in the art, the conventional name UCH-L1 necessarily needs to be changed to UCH-Lla as is the convention within in scientific research community. This peptide, or antibodies raised against this peptide, is recognized in the art as not being neural specific. This protein is known to have the following sequence as noted is the result of splicing exons 1 through 9:

MQLKPMEINPEMLNK.VLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFPLTAQ HENFRK QIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGSVLKQF LSETEKMSPEDRAKCFEK E AIQ AAHD AVAQEGQCRVDDKVNFHFILF N VDGHLYEL DGRMPFP VNHGAS SEDTLLKD AA VCREFTEREQGEVRFSA VAL CKAA SEQ ID NO. 2 is the first of the several newly discovered compositions of matter formed as a result of the splicing exons 1- 8, exon 9 (truncated) and the newly discovered exon 10 of the human peptide sequence, thus named UCH-Llb. The neural specific peptide UCH-Llb, has been found to have the following sequence: MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFPLTAQ HENFRK QIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGSVLKQF LSETEKMSPEDRAKCFEK EAIQAAHDAVAQEGQCRVDDKVNFHFILF NVDGHLYEL DGRMPFP VNHGAS SEDTLLKT3AAK^L H O&4 QLSTGPL WCEL QMVKHSPQCMSCIR Y LTL SEQ ID NO. 3 is the second of the several newly discovered compositions of matter formed as a result of the combination of exons 1-6, exon 7 (truncated) and exons 8-9 of the human peptide sequence, thus named UCH-Llc. The neural specific peptide UCH-Llc, has been found to have the following sequence:

MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFPLTAQ HENFRK QIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGS VLKQF LSETEKMSPEDPvAKCFEKNEAIOAAHDAVAQEGOCRVDDK ZXECL R

SEQ ID NO. 4 is the third of the several newly discovered compositions of matter formed as a result of exons 1-6, exon 7 (truncated), and exon 9 of the human peptide sequence, thus named UCH-Lld. The neural specific peptide UCH-Lld, has been found to have the following sequence: MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFPLTAQ HENFRK QIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGSVLKQF LSETEKMSPEDPvA CFEK EAIQAAHDAVAQEGQCRVDD rL RS^NS SKS Eja^S LPWLSARQPNAL WEGLC SEQ ID NO. 5 is the fourth of the several newly discovered compositions of matter formed as a result of exons 1-6, exon 7 (truncated), exon 9 (truncated) and newly discovered exon 10 of the human peptide sequence, thus named UCH-Lle. The neural specific peptide UCH-Lle, has been found to have the following sequence: MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFP LTAQHENFRKKQIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGSV L QFLSETEKMSPEDRA CFEK EAIQAAHDAVAQEGQCRVDD rL RHLSrSRKHSC PL GHCG VSFR W

[0071] It should be appreciated that while the sequences described above are those UCH-L1 isoforms found for the human peptide sequence, that similar isoforms are available in rat, mouse, rabbit, bovine, Chinese hamster, Rhesus monkey, zebrafish, goat, chicken, dog, domestic cat, and pig and other species having a similar sequence alignment to the UCH-L1 for humans. These alternative sequences may be substituted in the inventive processes, methods, assays, and in vitro diagnostic devices described herein. In addition, while discovery of these proteins are a landmark discovery in diagnostics of neural injuries, other compositions of matter of importance are those antibodies raised against any of the peptide sequences represented by SEQ ID NO's 1- 5, or antibodies raised against the rat, mouse, rabbit, bovine, Chinese hamster, Rhesus monkey, zebrafish, goat, chicken, dog, domestic cat, and pig and other similar species formulations of the similar sequence alignment.

[0072] 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 a 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.

[0073] Although antibodies are preferred for use 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 all spectrin and/or one or more of its SBDPs 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.

[0074] A myriad of detectable labels that are operative in a diagnostic assay for biomarker expression are known in the art. Agents used in methods for detecting a biomarker are 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.

[0075] The present invention provides a step of comparing the quantity of one or more biomarkers 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 organ or physical condition as well as the nature of cell death 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 to administer for optimal benefit of the subject. While the neural data provided in the examples herein are provided with respect to a full spectrum of traumatic brain injury, neurotoxicity, and neuronal cell death, it is appreciated that these results are applicable to other ischemic events, neurodegenerative disorders, prion related disease, epilepsy, chemical etiology and peripheral nervous system pathologies. As is shown in the subsequently provided example data, a gender difference is unexpectedly noted in abnormal subject neurological condition.

[0076] The results of such a test using an in vitro diagnostic device can help a physician determine whether the administration of a particular therapeutic or treatment regimen may be effective, and provide a rapid clinical intervention to the injury or disorder to enhance a patient's recovery. [0077] 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).

[0078] 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 immunob lotting) 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.

EXAMPLES

[0079] 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.

[0080] Illustrative reagents to use in performing the subject invention include Sodium bicarbonate (Sigma Cat #: C-3041), blocking buffer (Starting block 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 antibodies, as well as recombinant proteins and calibrator to the several neural specific isoforms of UCH-L1 represented by SEQ ID NO's 1-5 are made in-house. Monoclonal and polyclonal GFAP and SIOOP 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

[0081] 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 then concentrations of unknown samples are determined using the standard curve.

Example 3: TBI Patient Samples

[0082] Subjects with suspected TBI are enrolled at several investigational sites globally. All Subjects receive standard of care treatment when presenting to the investigational site. Biological samples of blood, urine, saliva and CSF are collected from the Subjects at specified time points. Inclusion criteria for the subjects include 1) The Subject is at least 18 years of age at screening (has had their 18th birthday) and no more than 80 years of age (did not have their 81st birthday); 2) the Subject received an accelerated or decelerated closed injury to the head (this includes head injuries inflicted by blunt force mechanism) self-reported or witnessed; 3) the biological samples of blood, urine and saliva are able to be collected within four (4) hours after injury; 4) the Subject is admitted with an initial Glasgow Coma Scale score of 3-8 (severe TBI), or from 5-15 (mild or moderate TBI); 5) the Subject is willing to undergo a computerized tomography (CT) of the head; 6) proper informed consent from patient or guardian. Severe TBI patients may be admitted if in a coma or a vegetative state, while mild to moderate patients may be admitted in a minimally conscious state or suffering from post-traumatic amnesia, retrograde or otherwise. Notwithstanding, the Glasgow coma score upon admission to an investigational site shall control which severity of injury the Subject is included in. Follow up visits at 7 and 35 days after injury are included in the sample cohort, where again biological samples are drawn. Upon enrollment into the study, further neuro cognitive tests such as RBANS, King Devic, GOAT, BESS and other tests measuring the neuro cognitive abilities of a Subject are employed. These tests are also administered during patient follow-up visits to track a patient's recovery and correlate with chronic biomarker measurement. Example 4: Stroke Patient Samples

[0083] Subjects with suspected stroke are enrolled at several investigational sites globally. All Subjects receive standard of care treatment when presenting to the investigational site. Biological samples of blood, urine, saliva and CSF are collected from the Subjects at specified time points. Inclusion criteria for the Subjects include 1) The Subject is at least 18 years of age at screening (has had their 18th birthday) and no more than 80 years of age (did not have their 81st birthday); 2) the Subjects primary diagnosis is ischemic or hemorrhagic stroke, self- reported or witnessed; 3) the biological samples of blood, urine and saliva are able to be collected within four (4) hours after injury; 4) the Subject is willing to undergo a computerized tomography (CT) of the head; 5) proper informed consent from patient or guardian.

Example 5: Normal Patient Samples

[0084] Normal Subjects without any known or suspected TBI, liver damage, stroke or other conditions which may alter protein biomarker levels are enrolled at several investigational sites globally. All Subjects receive standard screening to ensure that no medications or ailments are experienced by the patients prior to enrollment into the study. Biological samples of blood, urine, saliva and CSF are collected from the Subjects upon enrollment. Inclusion criteria for the Subjects include 1) The Subject is at least 18 years of age at screening (has had their 18th birthday) and no more than 80 years of age (did not have their 81st birthday); 2) the Subject is screened and found to not be taking medications or suffering from any neurological injury, neurological disorder, neurotoxicity, or liver injury; 3) proper informed consent from patient or guardian Example 6: Analysis of Mild, Moderate and Severe TBI Markers

[0085] Accumulation of the novel neural specific isoforms of UCH-Ll , namely UCH-Llb- e, are analyzed in the biological samples, using the processes described herein, through the use of an assay which includes antibodies raised against the peptides of SEQ ID NO 2-5. These assays are then incorporated into the in vitro diagnostic devices where the methods of detection of the neurological condition are performed and the results are illustrated. The assays are for each UCH-Ll isoform, but other assays are multiplexed to include one or more combination markers. A portion of the samples are tested using on UCH-Ll isoform assays, while a portion tests for a neural specific UCH-Ll isoform in combination with glial fibrillary acidic protein (GFAP), a portion tests for a neural specific UCH-Ll isoform, a portion tests for a neural specific UCH-Ll isoform in combination with Microtubule-associated protein 2 (MAP2), a portion tests for a neural specific UCH-Ll isoform in combination with myelin basic protein (MBP), a portion tests for a neural specific UCH-Ll isoform in combination with a-II spectrin breakdown product (SBDP) preferably SBDP150, SBDP150i, SBDP145, SBDP120, a portion tests for a neural specific UCH-Ll isoform in combination with neurensin-1 (p24), a portion tests for a neural specific UCH-Ll isoform in combination with collapsin response mediated proteins (CRMP's), TDP-43 (Trans-activation response DNA-binding protein of 43 kDa), NF-H (Neurofilament protein H (heavy chain) and breakdown products thereof, a portion tests for a neural specific UCH-Ll isoform in combination with synaptotagmin and breakdown products thereof. Normal patient samples are also analyzed for the same biomarkers, and a normal metric is calculated to indicate a non-injury state. The metric is then incorporated into the in vitro diagnostic device either through a computer algorithm, or where a calorimetric indication is provided; the dyes are activated indicating injury when the level of the measured biomarker is higher than what is determined in the normal metric. [0086] Prior to analysis, an assay is developed using a detection and capture antibody, each antibody being specific to the biomarker intended to be measured. For example, for neural specific UCH-Llb a monoclonal/monoclonal pair (capture/detection) is used to detect the level of biomarkers. Notwithstanding, similar results are achieved through the use of a monoclonal/polyclonal pair, a polyclonal monoclonal pair, and a polyclonal/polyclonal pair. The assay is optimized and tested using a calibrator and spiked serum to ensure that assay can measure known positive and known negative controls and detect the levels of known proteins within 1 picogram/mL detection sensitivity. The assay is incorporated into an in vitro diagnostic device using a cartridge or other disposable, whereby the cartridge contains the assay and a biological sample collection chamber for receiving the biological sample. The present invention further incorporates by reference the antibody and detection methods for the claimed biomarkers being used in the device for the specific indication disclosed therein presented in US 2007/0003982 Al , US 2005/0260697 Al, US 2009/0317805 Al , US 2005/0260654 Al, US 2009/0087868 Al , US 2010/0317041 Al, US 2011/0177974 Al , US 2010/0047817 Al, US 2012/0196307 Al , US 2011/0082203 Al, US 2011/0097392 Al , US 2011/0143375 Al, US 2013/0029859 Al, US 2012/0202231 Al , and US 2013/0022982 Al and application 13/470,079. The in vitro diagnostic devices used in this example have incorporated assays contained therein, which assays may be substituted herein using the methods therein contained. Example 7: Analysis of Neurotoxic Marker

[0087] Accumulation of novel markers indicating neurotoxic insult such as neural specific isoforms of UCH-Ll , namely UCH-Llb-e, are analyzed in the biological samples, using the processes described herein, through the use of an assay which includes antibodies raised against the peptides of SEQ ID NO 2-5. These assays are then incorporated into the in vitro diagnostic devices where the methods of detection of the neurological condition are performed and the results are illustrated. The assays are for each UCH-Ll isoform, but other assays are multiplexed to include one or more combination markers. A portion of the samples are tested using on UCH- Ll isoform assays, while a portion tests for a neural specific UCH-Ll isoform in combination with spectrin; a spectrin breakdown product (SBDP); MAPI, MAP2; GFAP, ubiquitin carboxyl- terminal esterase; ubiquitin carboxyl-terminal hydrolase; a neuronally- localized intracellular protein; MAP-tau; C-tau; Poly (ADP-ribose) polymerase (PARP); a collapsin response mediator protein, synaptotagmin, βΙΙΙ-tubulin, UCH-Ll ; neuron-specific enolase, neurofilament protein light chain, nestin, a-internexin; breakdown products thereof, post-translationally modified forms thereof, derivatives thereof, and combinations thereof, are analyzed in the biological samples taken after TBI using the inventive in vitro diagnostic devices.

[0088] Normal patient samples are also analyzed for the same biomarkers, and a normal metric is calculated to indicate a non-injury state. The metric is then incorporated into the in vitro diagnostic device either through a computer algorithm, or in the event of a calorimetric indication, the dyes are activated indicating injury when the level of the measured biomarker is higher than what is determined in the normal metric.

[0089] Prior to analysis, an assay is developed using a detection and capture antibody, each antibody being specific to the biomarker intended to be measured. For example, for UCH-Llb a monoclonal/monoclonal pair (capture/detection) is used to detect the level of biomarkers. Notwithstanding, similar results are achieved through the use of a monoclonal/polyclonal pair, a polyclonal monoclonal pair, and a polyclonal/polyclonal pair. The assay is optimized and tested using a calibrator and spiked serum to ensure that assay can measure known positive and known negative controls and detect the levels of known proteins within 1 picogram/mL detection sensitivity. The assay is incorporated into an in vitro diagnostic device using a cartridge or other disposable, whereby the cartridge contains the assay and a biological sample collection chamber for receiving the biological sample. The present invention further incorporates by reference the antibody and detection methods for the claimed biomarkers being used in the device for the specific indication disclosed therein presented in US 2013/0029362 Al . The in vitro diagnostic devices used in this example have incorporated assays contained therein, which assays may be substituted herein using the methods therein contained.

Example 8: 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 1.

Table 1. 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%

Age: 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

Race: Caucasian ???? ANY???

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 the Figures. The average first CSF sample collected as detailed in the Figures was 11.2 hours while the average time for collection of a serum sample subsequent to injury event as per the Figures is 10.1 hours. The quantity of each of the biomarkers of UCH-L 1 , 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 the Figures and indicates in particular the value of GFAP as well as that of additional markers UCH-L 1 and the spectrin breakdown products. Elevated levels of UCH-L 1 are indicative of the compromise of neuronal cell body damage while an increase in SPDP145 with a corresponding decrease in SBDP120 is suggestive of acute axonal necrosis. The sample analysis was later performed on the cryogenically stored samples obtained for this study and tested for the neural specific iso forms of UCH-L 1 , namely UCH-L lb-e, where elevated levels were also noted to the same degree.

[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 1 1. 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 the Figures. 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 the Figures. 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 the Figures 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 the Figures.

[0094] Based on the sandwich ELISA testing, UCH-Llb-e values as a function of time are noted to be markedly elevated relative to normal controls (control group B) as a function of time similar to what is noted for other neuroactive biomarkers such as GFAP and UCH-L1.

[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. In addition the kinetic profiles of the other stated markers presented herein as one or more additional biomarkers are presented in US 2007/0003982 Al, US 2005/0260697 Al , US 2009/0317805 Al, US 2005/0260654 Al, US 2009/0087868 Al, US 2010/0317041 Al, US 2011/0177974 Al , US 2010/0047817 Al , US 2012/0196307 Al, US 2011/0082203 Al , US 2011/0097392 Al, US 2011/0143375 Al, US 2013/0029859 Al, US 2012/0202231 Al, and US 2013/0022982 Al and application 13/470,079, 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-Llb-e, MAP2, and GFAP as being statistically significant predictors of GCS. Furthermore, GFAP was shown to have improved predictability when evaluated in interaction with UCH-L1 and gender. Example 9:

[0098] The study of Example 8 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 (PO.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] The sample analysis was later performed on the cryogenically stored samples obtained for this study and tested for the neural specific iso forms of UCH-L1 , namely UCH-Llb- e where elevated levels were also noted to the same degree

[00101] The figures show 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-L1 biomarker. The UCH-L1 concentration derived from the same samples as those used to determine GFAP is provided in the figures. The concentration of GFAP as well as UCH-Llb-e is provided as a function of injury magnitude between control, mild, and moderate traumatic brain injury as shown in the figures. The analyses of any of the identified neural specific iso forms, UCH-Llb-e alone or in combination with one another, or the simultaneous analyses of any neural specific UCH-L1 iso form along with 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. The figures show the concentration of the same markers 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. The figures show that both NSE and MAP2 are also elevated in subjects with MTBI in serum both at admission and at 24 hours of follow up. This data demonstrates a synergistic diagnostic effect of measuring multiple biomarkers such as UCH-Llb-e, GFAP, NSE, and MAP2 in a subject.

[00102] Through the simultaneous measurement of multiple biomarkers such as UCH-Llb-e, 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. A receiver operating characteristic (ROC) modeling of UCH-Llb-e, GFAP and SBDP145 post TBI further supports the value of simultaneous measurement of these biomarkers, as shown in the Figures.

[00103] 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.

[00104] 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. Sequence Listing

SEQ ID NO 1 :

MQLKPMEINPEMLNK.VLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFPLTAQ HENFRK QIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGSVLKQF LSETEKMSPEDRAKCFEKNEAIQAAHDAVAQEGQCRVDDKVNFHFILFNNVDGHLYEL DGRMPFPVNHGASSEDTLLKDAA VCREFTEREQGEVRFSAVALCKAA

SEQ ID NO 2:

MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFPLTAQ HENFRK QIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGSVLKQF LSETEKMSPEDRAKCFEKNEAIQAAHDAVAQEGQCRVDDKVNFHFILFNNVDGHLYEL DGR PFP VNHGAS SEDTLLKD AAIC4LA¾i(g&4 QLSTGPL WCEL QMVKHSPQCMSCIR Y LTL

SEQ ID NO 3 :

MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFPLTAQ HENFRK QIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGSVLKQF LSETEKMSPEDRAKCFEKNEAIQAAHDAVAQEGQCRVDDKMD^CL R

SEQ ID NO 4:

MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFPLTAQ HENFRK QIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGSVLKQF LSETEKMSPEDPvAKCFEKNEAIQAAHDAVAQEGQCRVDDKrL RS^ VS SKS E^T&4S LPWLSARQPNALWEGLC SEQ ID NO 5 :

MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFPLTAQ HENFRK QIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGSVLKQF LSETEKMSPEDRAKCFEKNE AIQ AAHD AVAQEGQCRVDDKJL RHLSrSR VHSCPL GH CGVSFRW

Claims

1. An in vitro diagnostic device for detecting a neural injury or neuronal disorder in a subject, the device comprising:

a sample chamber for holding a first biological sample collected from the subject;

an assay module in fluid communication with said sample chamber, said assay module containing an agent for detecting one or more biomarkers of a neural injury or neuronal disorder selected from the group consisting of UCH-Llb, UCH-Llc, UCH-Lld, or UCH-Lle;

wherein said assay module analyzes said first biological sample to detect the amount of the one or more biomarkers present in said sample;

a user interface, wherein said user interface relates the amount of the one or more biomarkers measured in said assay module to detecting a neural injury or neuronal disorder in said subject or the severity of neural injury or neuronal disorder in said subject.

2. The device of claim 1 further comprising a power supply and a data processing module in operable communication with said power supply and said assay module wherein said data processing module has an output that relates to detecting the neural injury or neuronal disorder in said subject, said output displaying the amount of the one or more biomarker measured in said sample, said output displaying the presence or absence of a neural injury or neuronal disorder, or said output displaying the severity of neural injury or neuronal disorder.

3. The device of claim 2 further comprising analyzing a second biological sample obtained from said subject, at some time after the first sample is collected , wherein if the device detects a decreased amount of the one or more biomarker in the second sample relative to said first sample a recovery output is provided by the data processing module.

4. The device of claim 2 further comprising a display in electrical communication with said data processing module and displaying the output as at least one of an amount of the one or more biomarker, a comparison between the amount of the one or more biomarker and a control, presence of the neural injury or neuronal disorder, or severity of the neural injury or neuronal disorder.

5. The device of claim 2 further comprising a transponder for communication with a remote device.

6. The device of claim 2 wherein the communication is digital.

7. The device of any one of claims 1 to 6 wherein the neural injury or neuronal disorder is one of: stroke, epilepsy, hypoxic ischemic encephalopathy (HIE), chronic traumatic encephalopathy (CTE), Alzheimer's disease (AD), Parkinson's disease (PD), traumatic brain injury (TBI), neurotoxicity, spinal cord injury (SCI) or neural cell damage.

8. The device of any one of claims 1 to 6 wherein said assay module further comprises at least one additional agent selective to measure for at least one additional biomarker selected from the group consisting of: (GFAP) or one of its breakdown products, Ubiquitin carboxyl-terminal hydrolase LI (UCH-Lla), neuron specific enolase (NSE), Microtubule- associated protein 2 (MAP2), myelin basic protein (MBP), Tau, TDP-43 (Trans-activation response DNA-binding protein of 43 kDa), NF-H (Neurofilament protein H (heavy chain), a-II spectrin breakdown product (SBDP) preferably SBDP150, SBDP150i SBDP145, SBDP120, S100pi-6, vesicular membrane protein neurensin-1 (p24), collapsin response mediated proteins (CRMP's) and breakdown products thereof, or synaptotagmin and breakdown products thereof.

9. The device of any one of claims 1 to 6 wherein the first biological sample is selected from the group consisting of blood, blood plasma, serum, sweat, saliva, cerebrospinal fluid (CSF) and urine.

10. The device of any one of claims 1 to 6 wherein said assay further comprises a dye providing a colorimetric change in response to the one or more biomarker present in the first biological sample.

11. The device of any one of claims 1 to 6 wherein said assay module is an immunoassay.

12. The device of any one of claims 1 to 6 wherein assay module is an ELISA.

13. The device of any one of claims 1 to 6 wherein said agent is an antibody or a protein.

14. A method for using an in vitro diagnostic device for detecting a neural injury or neuronal disorder in a subject, the method comprising:

calibrating an in vitro diagnostic device of claim 1 , incorporating an assay for measuring one or more biomarkers of a neural injury or neuronal disorder in a biological sample, the one or more biomarkers selected from the group consisting of UCH-Llb, UCH-Llc, UCH-Lld, or UCH-Lle or breakdown products thereof;

obtaining a biological sample from a subject; applying said sample to said in vitro diagnostic device wherein said assay includes reagents to determine the amount of the one or more biomarker present in said sample, wherein said device provides an output which relates the amount of the one or more biomarker detected to a neural injury or neuronal disorder, or lack thereof, in the subject.

15. The method of claim 14 further comprising:

calibrating an in vitro diagnostic device incorporating an assay for additionally measuring at least one additional biomarker selected from the group consisting of: (GFAP) or one of its breakdown products, Ubiquitin carboxyl-terminal hydrolase LI (UCH-Lla), neuron specific enolase (NSE), Microtubule-associated protein 2 (MAP2), myelin basic protein (MBP), a-II spectrin breakdown product (SBDP) preferably SBDP150, SBDP150i, SBDP145, SBDP120, a S100 calcium binding protein β (SlOOpi) including its neural specific isoforms, such as S100p2- S100p6, vesicular membrane protein neurensin-1 (p24), Tau, TDP-43 (Trans-activation response DNA-binding protein of 43 kDa), NF-H (Neurofilament protein H (heavy chain), collapsin response mediated proteins (CRMP's) and breakdown products thereof, or synaptotagmin and breakdown products thereof; and

applying said sample to said in vitro diagnostic device wherein said assay includes reagents to determine the amount of the additional biomarker present in said sample, wherein said device provides an output which relates the amount of the additional biomarker detected, alone or in synergistic combination with the one or more biomarker, to a neural injury or neuronal disorder, or lack thereof, in the subject.

16. A method of treating a neural injury or neuronal disorder in a subject:

calibrating an in vitro diagnostic device of claim 1 incorporating an assay for measuring for one or more biomarkers in a biological sample, the one or more biomarkers selected from the group consisting of UCH-Llb, UCH-Llc, UCH-Lld, or UCH-Lle and breakdown products thereof;

obtaining a biological sample from a subject;

applying said sample to said in vitro diagnostic device wherein said assay includes reagents to determine the amount of the one or more biomarker present in said sample, wherein said device provides an output which relates the amount of the one or more biomarker detected to a neural injury or neuronal disorder, or lack thereof, in the subject, wherein if said output of said in vitro diagnostic device relates the amount of the one or more biomarker to a neuronal injury or neuronal disorder a therapeutic intervention is employed to treat injury and/or inhibit injury progression.

17. A process for determining the neurological condition of a subject comprising: measuring a sample obtained from the subject or cells from the subject at a first time for a quantity of one or more biomarker selected from the group of UCH-Llb, UCH-Llc, UCH-Lld, or UCH-L 1 e and breakdown products thereof; and

comparing the quantity of said biomarker to normal levels of said biomarker to determine the neurological condition of the subject.

18. The process of claim 17 wherein the sample is selected from the group consisting of blood, blood plasma, serum, sweat, saliva, cerebrospinal fluid (CSF) and urine.

19. The process of claim 17 wherein the sample is a culture of the cells exposed to a drug candidate or an environmental contaminant.

20. The process of claims 17 to 19 further comprising measuring for at least one or more additional neuroactive biomarkers wherein said at least one additional neuroactive biomarker is GFAP and breakdown products thereof, UCH-Lla and breakdown products thereof, NSE and breakdown products thereof, SBDP150 and breakdown products thereof, SBDP145, SBDP120, SlOOpi and isoforms, such as S100p2- ΞΙΟΟββ, and breakdown products thereof, MAP2 and breakdown products thereof, MAPI and breakdown products thereof, MAP3 and breakdown products thereof, MAP4 and breakdown products thereof, MAP5 and breakdown products thereof, MBP and breakdown products thereof, Tau and breakdown products thereof, Neurofilament protein (NF) and breakdown products thereof, Cannabinoid Receptor CB and breakdown products thereof, CAM and breakdown products thereof, Synaptic protein and breakdown products thereof, CRMP and breakdown products thereof, iNOS and breakdown products thereof, NeuN and breakdown products thereof, CNPase and breakdown products thereof, Neuroserpin and breakdown products thereof, alpha-internexin and breakdown products thereof, LC3 and breakdown products thereof, Neurofascin and breakdown products thereof, EAAT and breakdown products thereof, Nestin and breakdown products thereof, Cortin-1 and breakdown products thereof, or BIII-Tubulin and breakdown products thereof.

21. The process of claims 17 to 19 further comprising measuring a second quantity of said biomarker at a second time to yield a kinetic profile for said biomarker.

22. The process of claims 17 to 19 further comprising predicting mortality based on the quantity of the one or more biomarkers of UCH-Llb, UCH-Llc, UCH-Lld, or UCH-Lle and the one or more additional neuroactive biomarkers UCH-L1 or GFAP.

23. A kit using the method of claims 17 to 19, the kit comprising: (a) a substrate for holding a sample isolated from a subject;

(b) one or more agents that specifically interact with one or more biomarkers selected from the group consisting of: UCH-Llb, UCH-Llc, UCH-Lld, or UCH-Lle, or breakdown products thereof or antibodies thereto; and

(c) printed instructions for reacting the agent sample or a portion of the sample for diagnosing a neurological condition in the subject.

24. The kit of claim 23, wherein the one or more agent is an antibody raised against a peptide having a sequence of one of SEQ ID NO's 2, 3, 4, or 5.

25. The kit of claim 23, further comprising one or more additional agents for detecting one or more additional neuroactive biomarkers, wherein the one or more additional agents is an antibody that binds with said at least one additional protein biomarker, said at least one additional protein biomarker selected from the group consisting of: GFAP, UCH-Lla, UCH-Llb, UCH-Llc, UCH-Lld, UCH-Lle, SlOOP-l , S100p-2, S100p-3, S100p-4, S100p-5, S100P-6, vesicular membrane protein p-24, synuclein, microtubule-associated protein, synaptophysin, Vimentin, Synaptotagmin, Synaptojanin-2, Synapsin2, CRMP1 , 2, Amphiphysin- 1, PSD95, PSD-93, Calmodulin dependent protein kinase II (CAMPK)-alpha, beta, gamma, Myelin basic protein (MBP), Myelin proteolipid protein (PLP), Tau, TDP-43 (Trans-activation response DNA-binding protein of 43 kDa), Myelin Oligodendrocyte specific protein (MOSP), Myelin Oligodendrocyte glycoprotein (MOG), myelin associated protein (MAG), NF-H, NF-L, NF-M, BIII-tubulin-1 and combinations thereof.

26. A composition of matter comprising the amino acid sequence as listed in SEQ ID NO. 2.

27. A composition of matter comprising an antibody raised against the peptide of SEQ ID NO. 2.

28. A composition of matter comprising the amino acid sequence as listed in SEQ ID

NO. 3.

29. A composition of matter comprising an antibody raised against the peptide of SEQ ID NO. 3.

30. A composition of matter comprising the amino acid sequence as listed in SEQ ID

NO. 4.

31. A composition of matter comprising an antibody raised against the peptide of SEQ ID NO. 4.

32. A composition of matter comprising the amino acid sequence as listed in SEQ ID

NO. 5.

33. A composition of matter comprising an antibody raised against the peptide of

SEQ ID NO. 5.