Biomarkers and methods for distinguishing mild traumatic brain injury from ultra-mild traumatic brain injury

Abstract

Disclosed herein are methods for aiding the diagnosis and evaluation of subjects (e.g., human subjects) who have suffered or may have suffered from head injury, such as to determine whether the subject suffers from mild traumatic brain injury or ultra-mild traumatic brain injury.

Description

[0001] Related Application Information

[0002] This application claims priority to U.S. Application No. 63 / 406,828, filed on September 15, 2022, and U.S. Application No. 63 / 426,964, filed on November 21, 2022, the respective contents of which are incorporated herein by reference.

[0003] Sequence Listing Submission

[0004] The content of the electronic sequence listing entitled 40844-601-SQL-ST26.xml (size: 8,192 bytes; and creation date: September 14, 2023) is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates to methods for assisting in the diagnosis and evaluation of a subject (e.g., a human subject) who has suffered or may have suffered a head injury. In some aspects, the present disclosure relates to methods for assisting in the diagnosis or determination of whether a human subject who has suffered an actual or suspected head injury has mild traumatic brain injury (TBI) or ultra-mild TBI by detecting the levels of biomarkers (such as ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) and glial fibrillary acidic protein (GFAP) or a combination thereof) in a sample obtained from the subject. Background Art

[0006] In the United States alone, there are over 5 million mild traumatic brain injuries (TBIs) each year. Currently, there is no simple, objective, and accurate measurement available to assist in patient evaluation. In fact, most of the evaluations and diagnoses of TBI are based on subjective data. Unfortunately, objective measurements such as head CT and Glasgow Coma Scale (GCS) scores are not very comprehensive or sensitive in evaluating mild TBI. In addition, for mild TBI, head CT is not visible most of the time, is expensive, and exposes the patient to unnecessary radiation. Additionally, a negative head CT does not mean that the patient has been ruled out for concussion; rather, it may only mean that certain interventions, such as surgery, are not warranted. Clinicians and patients need objective and reliable information to accurately assess the situation in order to facilitate appropriate triage and rehabilitation.

[0007] Mild TBI or concussion is more difficult to objectively detect and represents an everyday challenge for emergency centers worldwide. Concussions generally do not result in gross pathology, such as bleeding, and do not appear abnormal on routine computed tomography scans of the brain, but rather result in a rapid-onset neuronal dysfunction that resolves spontaneously within days to weeks. Approximately 15% of mild TBI patients develop persistent cognitive dysfunction. There is an unmet need for detecting and evaluating mild TBI victims at the scene, in the emergency room and clinics, in the sports arena, and in military activities (e.g., combat).

[0008] Current algorithms for assessing the severity of brain injury include the Glasgow Coma Scale score and other measures. These measures may sometimes be sufficient to correlate acute severity, but are not sensitive enough to subtle pathologies that may lead to permanent deficits. The GCS and other measures also fail to distinguish between injury types and may be inadequate. Thus, patients entering clinical trials grouped to a single GCS level may have very different severities and types of injury. Since outcomes vary accordingly, inappropriate classification undermines the integrity of clinical trials. Improved injury classification will enable more precise characterization of the disease severity and type of TBI patients in clinical trials.

[0009] In addition, current brain injury trials rely on outcome measures, such as the Extended Glasgow Outcome Scale, which capture global phenomena but fail to assess subtle differences in outcomes. As a result, 30 consecutive trials of brain injury therapeutics have failed. Sensitive outcome measures are needed to determine how patients recover from brain injury in order to test therapeutics and prophylactics. SUMMARY OF THE INVENTION

[0010] In some aspects, the present disclosure relates to methods of assisting in diagnosing or determining whether a subject who has suffered an actual or suspected head injury has a mild traumatic brain injury (TBI) or ultra-mild TBI. In some embodiments, provided herein is a method that includes performing at least one assay for ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), at least one assay for glial fibrillary acidic protein (GFAP), or at least one assay for UCH-L1 and GFAP in at least one sample obtained from a human subject. In some embodiments, provided herein is a method that includes performing at least one assay on a sample obtained from a subject after an actual or suspected head injury to measure the level of GFAP and / or the level of UCH-L1 in the sample. In some embodiments, the method includes performing at least one assay on the sample to measure the level of GFAP in the sample. In some embodiments, the method includes performing at least one assay on the sample to measure the level of UCH-L1 in the sample. In some embodiments, the method includes performing at least one assay on the sample to measure the levels of GFAP and UCH-L1 in the sample. In some embodiments, the levels of GFAP and UCH-L1 are measured in the same assay. In some embodiments, the levels of GFAP and UCH-L1 are measured in separate assays. In some embodiments, the sample is obtained from the subject within about 48 hours after an actual or suspected head injury. For example, in some embodiments, the sample is obtained within about 24 hours after an actual or suspected head injury. In some embodiments, the sample is obtained within about 12 hours after an actual or suspected head injury. In some embodiments, the sample is obtained within about 8 hours after an actual or suspected head injury. In some embodiments, the sample is obtained within about 4 hours after an actual or suspected head injury.

[0011] In some embodiments, the method further includes determining that the subject has suffered or may have suffered ultra-mild TBI when the level of GFAP in the sample is below the reference level of GFAP and / or the level of UCH-L1 in the sample is below the reference level of UCH-L1.

[0012] In some embodiments, the method includes differentiating mild TBI from ultra - mild TBI based on the GFAP and / or UCH - L1 levels in a sample. In some embodiments, the method further includes differentiating mild TBI from ultra - mild TBI based on whether the GFAP level in the sample is equal to or less than the reference level of GFAP, and / or based on whether the UCH - L1 level in the sample is equal to or less than the reference level of UCH - L1. In some embodiments, differentiating mild TBI from ultra - mild TBI includes determining that the subject has suffered or may have suffered mild traumatic brain injury (TBI) when the GFAP level in the sample is equal to the reference level of GFAP, and / or when the UCH - L1 level in the sample is equal to the reference level of UCH - L1. For example, in some embodiments, the method includes determining that the subject has suffered or may have suffered mild TBI when the GFAP level in the sample is equal to the reference level of GFAP. As another example, in some embodiments, the method includes determining that the subject has suffered or may have suffered mild TBI when the UCH - L1 level in the sample is equal to the reference level of UCH - L1. In some embodiments, the method includes determining that the subject has suffered or may have suffered mild TBI when the GFAP level in the sample is equal to the reference level of GFAP and when the UCH - L1 level in the sample is equal to the reference level of UCH - L1.

[0013] In some embodiments, the method includes determining that the subject has suffered or may have suffered ultra - mild TBI when the GFAP level in the sample is below the reference level of GFAP and / or when the UCH - L1 level in the sample is below the reference level of UCH - L1. In some embodiments, the method includes determining that the subject has suffered or may have suffered ultra - mild TBI when the GFAP level in the sample is below the reference level of GFAP. As another example, in some embodiments, the method includes determining that the subject has suffered or may have suffered ultra - mild TBI when the UCH - L1 level in the sample is below the reference level of UCH - L1. In some embodiments, the method includes determining that the subject has suffered or may have suffered ultra - mild TBI when the GFAP level in the sample is below the reference level of GFAP and when the UCH - L1 level in the sample is below the reference level of UCH - L1.

[0014] In some embodiments, the method further comprises treating and / or monitoring a subject. In some embodiments, the method further comprises treating a subject determined to have or likely have mild TBI. In some embodiments, the method further comprises treating a subject determined to have or likely have ultra-mild TBI. In some embodiments, the method further comprises monitoring a subject determined to have or likely have mild TBI. In some embodiments, the method further comprises monitoring a subject determined to have or likely have ultra-mild TBI.

[0015] In some embodiments, the sample is obtained within about 5 minutes, about 10 minutes, about 12 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours or about 48 hours after an actual or suspected head injury.

[0016] The at least one determination for UCH-L1 and / or the at least one determination for GFAP can be performed simultaneously or sequentially in any order.

[0017] In some embodiments, a sample is obtained after the subject has suffered a head injury caused by blunt impact, one or more falls, an explosion or shock wave, or other types of blunt force trauma resulting from body shaking, external mechanical force, or other force that causes closed or open head trauma. In some embodiments, a sample is obtained after the subject has ingested or been exposed to a chemical, toxin, or combination of chemicals and toxins. In some embodiments, the chemical or toxin is fire, mold, asbestos, pesticide, insecticide, organic solvent, paint, glue, gas, organometal, abused drug, or a combination of one or more thereof. In some embodiments, the sample is obtained from a subject suffering from an autoimmune disease, metabolic disorder, brain tumor, hypoxia, viral infection, fungal infection (e.g., SARS-CoV-2), bacterial infection, meningitis, hydrocephalus, or any combination thereof.

[0018] In some embodiments, the assay (e.g., the assay for GFAP and / or the assay for UCH-L1) is an immunoassay or a clinical chemistry assay. In some embodiments, the assay is a single molecule detection assay or a point-of-care assay. In some embodiments, the amount of the at least one sample is from about 10 μL to about 30 μL. For example, in some embodiments, the amount of the at least one sample is about 20 μL.

[0019] In some embodiments, the at least one assay for UCH-L1, the at least one assay for GFAP, or the at least one assay for UCH-L1 and the at least one assay for GFAP are performed in about 10 to about 20 minutes. For example, in some embodiments, the at least one assay for UCH-L1, the at least one assay for GFAP, or the at least one assay for UCH-L1 and the at least one assay for GFAP are performed in about 15 minutes.

[0020] In some embodiments, the sample is selected from the group consisting of: whole blood sample, capillary blood sample, serum sample, cerebrospinal fluid sample, mixed sample of venous blood and capillary blood, mixed sample of capillary blood and interstitial fluid, tissue sample, body fluid, and plasma sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1A Shows a comparison of receiver operating characteristic (ROC) analyses of GFAP levels in mild TBI samples and ultra-mild TBI samples obtained within 4 hours after injury, as shown in Example 3. Figure 1B Shows a comparison of ROC analyses of UCH-L1 levels in mild TBI samples and ultra-mild TBI samples obtained within 4 hours after injury, as shown in Example 3.

[0022] Figure 2A Shows a comparison of ROC analyses of GFAP levels in mild TBI samples and ultra - mild TBI samples obtained within 8 hours after injury, as shown in Example 3. Figure 2B Shows a comparison of ROC analyses of UCH - L1 levels in mild TBI samples and ultra - mild TBI samples obtained within 8 hours after injury, as shown in Example 3.

[0023] Figure 3A Shows a comparison of ROC analyses of GFAP levels in mild TBI samples and ultra - mild TBI samples obtained within 12 hours after injury, as shown in Example 3. Figure 3B Shows a comparison of ROC analyses of UCH - L1 levels in mild TBI samples and ultra - mild TBI samples obtained within 12 hours after injury, as shown in Example 3.

[0024] Figure 4A Shows a comparison of ROC analyses of GFAP levels in mild TBI samples and ultra - mild TBI samples obtained within 24 hours after injury, as shown in Example 3. Figure 4B Shows a comparison of ROC analyses of UCH - L1 levels in mild TBI samples and ultra - mild TBI samples obtained within 24 hours after injury, as shown in Example 3. Detailed Description

[0025] The present disclosure relates to methods for assisting in the diagnosis and evaluation of a subject (e.g., a human subject) who has or may have suffered a head injury. In some aspects, the present disclosure provides methods for using one or more biomarkers, such as ubiquitin carboxyl - terminal hydrolase L1 (UCH - L1), glial fibrillary acidic protein (GFAP), or a combination thereof, to assist in the diagnosis and evaluation of a subject (e.g., a human subject) who has or may have suffered a head injury such as mild or ultra - mild traumatic brain injury (TBI). In some embodiments, these methods involve detecting the levels of one or more biomarkers in one or more samples obtained from a subject (e.g., a human subject) at a time point within 24 hours of an actual or suspected head injury. In some embodiments, these methods involve detecting the levels of one or more biomarkers in one or more samples obtained from a subject (e.g., a human subject) at about 48 hours after an actual or suspected head injury. In some embodiments, these methods involve detecting the levels of one or more biomarkers in one or more samples obtained from a subject (e.g., a human subject) at about 2 weeks after an actual or suspected head injury.

[0026] As used in this section and throughout the present disclosure, the section headings are for organizational purposes only and are not intended to be limiting.

[0027] 1. Definitions

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. The following describes preferred methods and materials, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. The materials, methods, and examples disclosed herein are merely exemplary and not intended to be limiting.

[0029] As used herein, the terms "comprising," "including," "having," "has," "can," "containing," and variations thereof are open-ended conjunctions, terms, or words that are intended not to exclude the possibility of additional acts or structures. Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include plural referents. The present disclosure also encompasses other embodiments "comprising the embodiments or elements presented herein," "consisting of the embodiments or elements presented herein," and "consisting essentially of the embodiments or elements presented herein," whether or not explicitly stated.

[0030] For the recitation of numerical ranges herein, every intermediate number having the same degree of precision is explicitly covered. For example, for the range of 6 - 9, the numbers 7 and 8 are covered in addition to 6 and 9, and for the range of 6.0 - 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly covered.

[0031] "Affinity matured antibody" is used herein to refer to an antibody having one or more changes in one or more CDRs, said changes resulting in an increase in the affinity (i.e., K D 、k d or k a)Improved. Exemplary affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. A variety of procedures are known in the art for generating affinity matured antibodies, including screening of combinatorial antibody libraries prepared by biopanning. For example, Marks et al., BioTechnology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described in Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-3319 (1995); and Hawkins et al., J. Mol. Biol., 226:889-896 (1992). Selective mutagenesis at selected mutagenesis positions and at contact or hypermutation positions with activity enhancing amino acid residues is described in U.S. Patent No. 6,914,128 B1.

[0032] As used herein, "an antibody" and "antibodies" refer to monoclonal antibodies, monospecific antibodies (e.g., which may be monoclonal or produced by other means in addition to from a common germ cell), multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as but not limited to birds (e.g., duck or goose), sharks, whales, and mammals (including non-primate animals (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, etc.) or non-human primates (e.g., monkey, chimpanzee, etc.)), recombinant antibodies, chimeric antibodies, single-chain Fv ("scFv"), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fv ("sdFv"), and anti-idiotypic ("anti-Id") antibodies, dual-domain antibodies, dual-variable domain (DVD) or triple-variable domain (TVD) antibodies (dual-variable domain immunoglobulins and methods for their preparation are described in Wu, C et al., Nature Biotechnology, 25(11):1290-1297 (2007) and PCT international application WO 2001 / 058956, the contents of each document are incorporated herein by reference), and functional active epitope-binding fragments of any of the foregoing. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. For simplicity, an antibody against an analyte is generally referred to herein as an "anti-analyte antibody" or simply an "analyte antibody" (e.g., anti-UCH-L1 antibody or UCH-L1 antibody).

[0033] As used herein, "antibody fragment" refers to a portion of a whole antibody that contains an antigen-binding site or variable region. The portion does not include the constant heavy chain domains of the complete antibody Fc region (i.e., CH2, CH3, or CH4, depending on the antibody isotype). Examples of antibody fragments include but are not limited to Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing three CDRs of the light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing three CDRs of the heavy chain variable region.

[0034] "Area under the curve" or "AUC" refers to the area under the ROC curve. The AUC under the ROC curve is a measure of accuracy. An AUC of 1 represents a perfect test, while an AUC of 0.5 represents a meaningless test. Preferred AUCs can be at least about 0.700, at least about 0.750, at least about 0.800, at least about 0.850, at least about 0.900, at least about 0.910, at least about 0.920, at least about 0.930, at least about 0.940, at least about 0.950, at least about 0.960, at least about 0.970, at least about 0.980, at least about 0.990 or at least about 0.995.

[0035] "Bead" and "particle" are used interchangeably herein and refer to a substantially spherical solid support. An example of a bead or particle is a microparticle. The microparticles that can be used herein can be any type known in the art. For example, the bead or particle can be a magnetic bead or magnetic particle. The magnetic bead / particle can be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic or ferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO2, MnAs, MnBi, EuO and NiO / Fe. Examples of ferrimagnetic materials include NiFe2O4, CoFe2O4, Fe3O4 (or FeO . Fe2O3). The bead can have a magnetic solid core portion and be surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion can be a layer surrounding a non-magnetic core. The microparticle can have any size that functions in the methods described herein, such as about 0.75 to about 5 nm, or about 1 to about 5 nm, or about 1 to about 3 nm.

[0036] "Binding protein" is used herein to refer to a monomeric or multimeric protein that binds to a binding partner and forms a complex therewith, such as a polypeptide, antigen, chemical compound or other molecule or any kind of substrate. The binding protein specifically binds to the binding partner. Binding proteins include antibodies, as well as antigen-binding fragments thereof and various other forms and derivatives known in the art and described hereinafter, and other molecules containing an antigen-binding domain that binds to one or more antigen molecules or specific sites (epitopes) on the antigen molecule. Thus, binding proteins include, but are not limited to, antibodies, tetrameric immunoglobulins, IgG molecules, IgG1 molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, affinity matured antibodies and fragments of any such antibodies that retain the ability to bind antigen.

[0037] "Bispecific antibody" is used herein to refer to full-length antibodies produced by the following techniques: the tetraploid hybridoma technique (see Milstein et al., Nature, 305(5934):537-540(1983)); chemical conjugation of two different monoclonal antibodies (see Staerz et al., Nature, 314(6012):628-631(1985)); or the diabody or similar methods by introducing mutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14):6444-6448(1993)), which methods produce a variety of different immunoglobulin substances, only one of which is a functional bispecific antibody. A bispecific antibody binds one antigen (or epitope) on one of its two binding arms (a pair of HC / LC), and binds a different antigen (or epitope) on its second arm (the other pair of HC / LC). According to this definition, a bispecific antibody has two different antigen-binding arms (both in terms of specificity and CDR sequence), and is monovalent for each antigen it binds.

[0038] "CDR" is used herein to refer to the "complementary determining region" within the variable sequence of an antibody. There are three CDRs in each variable region of the heavy and light chains. For each variable region, starting from the N-terminus of the heavy or light chain, these regions are designated "CDR1", "CDR2", and "CDR3". The term "CDR set" as used herein refers to the set of three antigen-binding CDRs present in a single variable region. Thus, an antigen-binding site can include six CDRs, which comprise the CDR sets from each of the heavy and light chain variable regions. A polypeptide containing a single CDR (e.g., CDR1, CDR2, or CDR3) can be referred to as a "molecular recognition unit". Crystallographic analysis of antigen-antibody complexes has demonstrated extensive contacts between the amino acid residues of the CDRs and the bound antigen, with the most extensive antigen contacts being with the heavy chain CDR3. Thus, the molecular recognition unit may be primarily responsible for the specificity of the antigen-binding site. Generally speaking, CDR residues are directly and most substantially involved in influencing antigen binding.

[0039] The precise boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991))) not only provides a well-defined residue numbering system applicable to any variable region of an antibody, but also provides the precise residue boundaries that define the three CDRs. These CDRs can be referred to as "Kabat CDRs". Chothia and colleagues (Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that although there is great diversity at the level of amino acid sequences, certain subportions within the Kabat CDRs adopt nearly identical peptide backbone conformations. These subportions are designated as "L1", "L2", and "L3" or "H1", "H2", and "H3", where "L" and "H" denote the light chain region and the heavy chain region, respectively. These regions can be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries that define CDRs overlapping with the Kabat CDRs have been described by Padlan, FASEB J., 9:133-139 (1995) and MacCallum, J. Mol. Biol, 262(5):732-745 (1996). There are still other CDR boundary definitions that may not strictly follow one of the systems in this article, but will still overlap with the Kabat CDRs, and they can be shortened or lengthened given that a particular residue or group of residues or even an entire CDR does not significantly affect the prediction or experimental finding of antigen binding. The methods used in this article can utilize CDRs defined according to any of these systems, but certain embodiments use CDRs defined by Kabat or Chothia.

[0040] As used herein, "communicated" or "communicating" means transmitting, transferring, and / or reporting an item of information. In some aspects, the information communicated is an item of information obtained by performing an assay, such as the amount or presence of a biomarker in a sample (e.g., a result). The information obtained by performing an assay can be communicated by a computer, in a document and / or spreadsheet, on a mobile device (e.g., a smartphone), on a website, in an email, or any combination thereof. In some other aspects, the information is communicated on or from an instrument or device. In other aspects, the information is communicated by, for example, being displayed on an instrument or device.

[0041] "Component", "multiple components", or "at least one component" generally refers to a capture antibody, analyte, or conjugate, calibrator, control, sensitivity set, container, buffer, diluent, salt, enzyme, cofactor of an enzyme, detection reagent, pretreatment reagent / solution, substrate (e.g., as a solution), stop solution, etc. that can be included in a kit for assaying a test sample (such as a patient urine, whole blood, serum, or plasma sample) according to the methods described herein and other methods known in the art. Some components may be in solution or lyophilized for reconstitution for use in the assay.

[0042] "Associated with" as used herein means compared to.

[0043] "CT scan" as used herein refers to computed tomography (CT) scan. A CT scan combines a series of X-ray images taken from different angles and uses computer processing to create cross-sectional images or slices of the bones, blood vessels, and soft tissues inside your body. CT scans can use X-ray CT, positron emission tomography (PET), single photon emission computed tomography (SPECT), computed axial tomography (CAT scan), or computer-assisted tomography. CT scans can be conventional CT scans or spiral / helical CT scans. In a conventional CT scan, the scan is done slice by slice, and after each slice, the scan stops and moves down to the next slice, e.g., from the top of the abdomen down to the pelvis. Conventional CT scans require the patient to hold their breath to avoid motion artifacts. Spiral CT scans are continuous scans that are taken in a spiral pattern and are a faster process where the scan images are continuous.

[0044] A "derivative" of an antibody as used herein can refer to an antibody that has one or more modifications to its amino acid sequence compared to the native or parental antibody and exhibits a modified domain structure. The derivative can still be capable of adopting the typical domain configuration found in native antibodies, as well as the amino acid sequence capable of specifically binding to a target (antigen). Typical examples of antibody derivatives are antibodies conjugated to other polypeptides, rearranged antibody domains, or antibody fragments. The derivative can also contain at least one additional compound, such as a protein domain, which is linked by covalent or non-covalent bonds. According to methods known in the art, the linkage can be based on gene fusion. The additional domain present in a fusion protein containing an antibody can preferably be linked by a flexible linker, advantageously a peptide linker, where the peptide linker contains multiple hydrophilic peptide-bonded amino acids, the length of which is sufficient to span the distance between the C-terminus of the additional protein domain and the N-terminus of the antibody, and vice versa. The antibody can be linked to an effector molecule having a conformation suitable for biological activity or selective binding to, for example, a solid support, a bioactive substance (such as a cytokine or growth hormone), a chemical reagent, a peptide, a protein, or a drug.

[0045] "Determined by assay" as used herein refers to determining a reference level by any suitable assay. In some embodiments, determination of the reference level can be achieved by the same type of assay to be applied to a sample from a subject (e.g., by immunoassay, clinical chemistry assay, single molecule detection assay, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, or protein immunostaining, electrophoretic analysis, protein assay, competitive binding assay, functional protein assay, or chromatographic or spectroscopic methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS)). In some embodiments, determination of the reference level can be achieved by the same type of assay under the same assay conditions to be applied to a sample from a subject. As noted herein, the present disclosure provides exemplary reference levels (e.g., calculated by comparing reference levels at different time points). Based on the description provided in the present disclosure, it is entirely within the ability of a person of ordinary skill in the art to adapt the disclosure herein to other assays to obtain assay-specific reference levels for those other assays. For example, a set of training samples can be used to obtain assay-specific reference levels, the samples including: samples from subjects known to have suffered a head injury (e.g., samples obtained from human subjects), where the subjects are known to have suffered (i) mild TBI and / or (ii) ultra-mild TBI; and samples obtained from subjects known not to have suffered a head injury (e.g., human subjects). It should be understood that a reference level "determined by assay" and having the listed "sensitivity" and / or "specificity" levels as used herein refers to such a reference level that has been determined to provide the listed sensitivity and / or specificity when employed in the methods of the present disclosure. For example, it is entirely within the ability of a person of ordinary skill in the art to determine the sensitivity and specificity associated with a given reference level in the methods of the present disclosure by repeated statistical analysis of assay data using multiple different possible reference levels.

[0046] In fact, when differentiating subjects with mild traumatic brain injury from those with ultra - mild traumatic brain injury, one skilled in the art will balance the effect of raising the cut - off value on sensitivity and specificity. Raising or lowering the cut - off value will have a definite and predictable effect on sensitivity, specificity, and other standard statistical measures. It is well known that raising the cut - off value will increase specificity, but may decrease sensitivity (the proportion of patients with the disease who test positive). In contrast, lowering the cut - off value will increase sensitivity, but will decrease specificity (the proportion of non - diseased individuals who test negative). Determining the consequences of mild traumatic brain injury compared to ultra - mild traumatic brain injury is obvious to one skilled in the art. In differentiating whether a subject has mild traumatic brain injury or ultra - mild traumatic brain injury, the higher the cut - off value, the more specific it becomes as more true negatives (i.e., subjects without mild traumatic brain injury) are distinguished from subjects with mild traumatic brain injury. But at the same time, raising the cut - off value reduces the number of cases identified as positive overall, as well as the number of true positives, so sensitivity must decrease. Conversely, the lower the cut - off value, the more sensitive it becomes as more true positives (i.e., subjects with mild traumatic brain injury) are distinguished from subjects with ultra - mild TBI. But at the same time, lowering the cut - off value increases the number of cases identified as positive overall, as well as the number of false positives, so specificity must decrease.

[0047] Generally, a high sensitivity value helps one skilled in the art to rule out a disease or condition (such as mild TBI), and a high specificity value helps one skilled in the art to rule in a disease or condition. Whether the skilled person wants to rule out or rule in a disease depends on the consequences of each type of error for the patient. Thus, without fully disclosing the underlying information on how to choose the value, it is not possible to know or predict the exact balance used to derive the test cut - off value. The balance of sensitivity with specificity and other factors will depend on the circumstances. This is why it is sometimes preferred to provide alternative cut - off values (e.g., reference values) so that a physician or doctor can choose.

[0048] The term "bispecific antibody" is used herein to refer to a full - length antibody that can bind two different antigens (or epitopes) in each of its two binding arms (a pair of HC / LC) (see PCT Publication WO 02 / 02773). Thus, a bispecific binding protein has two identical antigen - binding arms with the same specificity and the same CDR sequences, and is bivalent for each antigen to which it binds.

[0049] "Dual variable domain" is used herein to refer to two or more antigen-binding sites on a binding protein, which can be a bivalent (two antigen-binding sites), tetravalent (four antigen-binding sites) or multivalent binding protein. The DVD can be monospecific, i.e., capable of binding one antigen (or one specific epitope), or multispecific, i.e., capable of binding two or more antigens (i.e., two or more epitopes of the same target antigen molecule or two or more epitopes of different target antigens). Preferred DVD binding proteins comprise two heavy chain DVD polypeptides and two light chain DVD polypeptides and are referred to as "DVD immunoglobulin" or "DVD-Ig". Such a DVD-Ig binding protein is thus tetrameric and resembles an IgG molecule, but provides more antigen-binding sites than an IgG molecule. Thus, each half of the tetrameric DVD-Ig molecule resembles half of an IgG molecule and comprises a heavy chain DVD polypeptide and a light chain DVD polypeptide, but unlike the pair of heavy and light chains of an IgG molecule that provide a single antigen-binding domain, the pair of heavy and light chains of a DVD-Ig provide two or more antigen-binding sites.

[0050] Each antigen-binding site of a DVD-Ig binding protein can be derived from a donor ("parent") monoclonal antibody and thus comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) having a total of six CDRs with each antigen-binding site participating in antigen binding. Thus, a DVD-Ig binding protein that binds two different epitopes (i.e., two different epitopes of two different antigen molecules or two different epitopes of the same antigen molecule) comprises an antigen-binding site derived from a first parent monoclonal antibody and an antigen-binding site derived from a second parent monoclonal antibody.

[0051] Descriptions of the design, expression, and characterization of DVD-Ig binding molecules are provided in PCT Publication No. WO 2007 / 024715, U.S. Patent No. 7,612,181, and Wu et al., Nature Biotech., 25:1290-1297 (2007). Preferred examples of such DVD-Ig molecules include: a heavy chain comprising the structural formula VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker (provided that it is not CH1), X2 is an Fc region, and n is 0 or 1, but preferably 1; and a light chain comprising VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker (provided that it is not CH1), and X2 does not contain an Fc region, and n is 0 or 1, but preferably 1. Such a DVD-Ig can comprise two such heavy chains and two such light chains, where each chain comprises tandemly linked variable domains with no intervening constant regions between the variable regions, where the heavy and light chains associate to form tandem functional antigen-binding sites, and where a pair of heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein with four functional antigen-binding sites. In another example, a DVD-Ig molecule can comprise such heavy and light chains, each of the heavy and light chains comprising three tandemly linked variable domains (VD1, VD2, VD3) with no intervening constant regions between the variable domains, where a pair of heavy and light chains can associate to form three antigen-binding sites, and where a pair of heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein with six antigen-binding sites.

[0052] In a preferred embodiment, the DVD-Ig binding protein not only binds the same target molecule as its parental monoclonal antibody binds, but also has one or more of the desired properties of one or more of its parental monoclonal antibodies. Preferably, such additional property is an antibody parameter of one or more of the parental monoclonal antibodies. Antibody parameters that may contribute to the DVD-Ig binding protein from one or more of its parental monoclonal antibodies include, but are not limited to, antigen specificity, antigen affinity, potency, biological function, epitope recognition, protein stability, protein solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross-reactivity, and orthologous antigen binding.

[0053] The DVD-Ig binding protein binds to at least one epitope of UCH-L1, GFAP, or UCH-L1 and GFAP. Non-limiting examples of the DVD-Ig binding protein include: (1) a DVD-Ig binding protein that binds to one or more epitopes of UCH-L1, a DVD-Ig binding protein that binds to an epitope of human UCH-L1 and an epitope of UCH-L1 of another species (e.g., mouse), and a DVD-Ig binding protein that binds to an epitope of human UCH-L1 and an epitope of another target molecule; (2) a DVD-Ig binding protein that binds to one or more epitopes of GFAP, a DVD-Ig binding protein that binds to an epitope of human GFAP and an epitope of GFAP of another species (e.g., mouse), and a DVD-Ig binding protein that binds to an epitope of human GFAP and an epitope of another target molecule; or (3) a DVD-Ig binding protein that binds to one or more epitopes of UCH-L1 and GFAP, a DVD-Ig binding protein that binds to an epitope of human UCH-L1, human GFAP, and an epitope of UCH-L1 of another species (e.g., mouse), and a DVD-Ig binding protein that binds to an epitope of human UCH-L1, human GFAP, and an epitope of another target molecule.

[0054] As used herein, "dynamic range" refers to the range in which the assay reading is proportional to the amount of the target molecule or analyte in the sample being analyzed.

[0055] "Epitope" or "epitopes" or "epitopes of interest" refers to a site on any molecule that is recognized and can bind to a complementary site on its specific binding partner. The molecule and the specific binding partner are part of a specific binding pair. For example, an epitope can be on a polypeptide, protein, hapten, carbohydrate antigen (such as but not limited to glycolipid, glycoprotein, or lipopolysaccharide), or polysaccharide. Its specific binding partner can be but is not limited to an antibody.

[0056] As used herein, "fragment antigen-binding fragment" or "Fab fragment" refers to an antibody fragment that binds to an antigen and contains one antigen-binding site, a complete light chain, and a portion of a heavy chain. Fab is a monovalent fragment composed of the VL, VH, CL, and CH1 domains. Fab is composed of one constant domain and one variable domain each of the heavy chain and the light chain. The variable domain contains a paratope (antigen-binding site) at the amino terminus of the monomer, which contains a set of complementarity-determining regions. Each arm of the Y thus binds to an epitope on the antigen. Fab fragments can be produced as described in the art, for example, using the enzyme papain, which can be used to cleave an immunoglobulin monomer into two Fab fragments and an Fc fragment, or can be produced by recombinant methods.

[0057] As used herein, "F(ab')2 fragment" refers to an antibody produced by pepsin digestion of whole IgG antibody to remove most of the Fc region while leaving some hinge region intact. The F(ab')2 fragment has two antigen-binding F(ab) portions linked together by disulfide bonds and is thus divalent, with a molecular weight of approximately 110 kDa. The divalent antibody fragment (F(ab')2 fragment) is smaller than the whole IgG molecule and can penetrate tissues better, thus facilitating better antigen recognition in immunohistochemistry. The use of F(ab')2 fragments also avoids non-specific binding to Fc receptors or protein A / G on live cells. F(ab')2 fragments can bind and precipitate antigens.

[0058] As used herein, "framework" (FR) or "framework sequence" can mean the remaining sequence of the variable region minus the CDRs. Since the exact definition of CDR sequences can be determined by different systems (e.g., see above), the meaning of framework sequences is correspondingly subject to different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) also divide the framework regions on the light and heavy chains into four sub-regions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. Without designating a particular sub-region as FR1, FR2, FR3, or FR4, as otherwise mentioned, the framework region represents the combined FR within the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR represents one of the four sub-regions, and multiple FRs represent two or more of the four sub-regions that make up the framework region.

[0059] Human heavy and light chain FR sequences are known in the art and can be used as heavy and light chain "recipient" framework sequences (or simply "recipient" sequences) to humanize non-human antibodies by using techniques known in the art. In one embodiment, the human heavy and light chain recipient sequences are selected from framework sequences listed in publicly available databases such as V-base (http: / / vbase.mrc-cpe.cam.ac.uk / ) or the International ImMunoGeneTics Information System (http: / / imgt.cines.fr / texts / IMGTrepertoire / LocusGenes / ).

[0060] As used herein, "functional antigen-binding site" can mean a site on a binding protein (e.g., an antibody) that is capable of binding a target antigen. The antigen-binding affinity of the antigen-binding site may not be as strong as that of the parental binding protein (e.g., parental antibody) from which the antigen-binding site is derived, but the ability to bind the antigen must be measurable using any of a variety of methods known for evaluating the binding of a protein (e.g., an antibody) to an antigen. In addition, the antigen-binding affinities of each antigen-binding site of a multivalent protein (e.g., a multivalent antibody) herein need not be the same in number.

[0061] "GFAP" is used herein to describe glial fibrillary acidic protein. GFAP is a protein encoded by the human GFAP gene and GFAP gene counterparts of other species, and it can be produced (e.g., recombinantly, in other species).

[0062] "GFAP status" can mean the level or amount of GFAP at a given point in time (such as, using a single measurement of GFAP), the level or amount of GFAP associated with monitoring (such as, repeated testing of a subject to identify an increase or decrease in the amount of GFAP), the level or amount of GFAP associated with the treatment of traumatic brain injury (whether primary brain injury and / or secondary brain injury), or a combination thereof.

[0063] As used herein, "Glasgow Coma Scale" or "GCS" refers to a 15-point scale (e.g., described by Graham Teasdale and Bryan Jennett, Lancet 1974; 2:81-4 in 1974), which provides a practical method for assessing the level of impairment of consciousness in patients suffering from brain injury. The test measures the best motor response, verbal response, and eye opening response using the following values: I. Best motor response (6 - obeys two-part command; 5 - places hand above clavicle in response to stimulation of head and neck; 4 - bends arm at elbow quickly, but features not predominantly abnormal; 3 - bends arm at elbow, features clearly predominantly abnormal; 2 - extends arm at elbow; 1 - no movement of arm / leg, no interfering factors; NT - paralysis or other restrictive factors); II. Verbal response (5 - correctly states name, location, and date; 4 - not oriented, but communication coherent; 3 - single understandable words; 2 - only groans / sighs; 1 - no audible response, no interfering factors; NT - factors interfering with communication); and III. Eye opening (4 - eyes open before stimulation; 3 - after speech or shouted command; 2 - after fingertip stimulation; 1 - eyes never open, no interfering factors; NT - eyes closed due to local factors). The final score is determined by adding the values of I + II + III. If the GCS score is 13 - 15, the subject is considered to have suffered a mild TBI. Ultra-mild TBI is a subclass of mild TBI. If the GCS score is 15, the subject is considered to have suffered an ultra-mild TBI. If the GCS score is 9 - 12, the subject is considered to have suffered a moderate TBI. If the GCS score is 8 or lower, typically 3 - 8, the subject is considered to have suffered a severe TBI.

[0064] As used herein, "Glasgow Outcome Scale" refers to a global scale for functional outcome that rates the patient's status into one of the following five categories: death, vegetative state, severe disability, moderate disability, or good recovery. The "Extended Glasgow Outcome Scale" or "GOSE", which can be used interchangeably herein, provides a more detailed eight-category classification by subdividing the categories of severe disability, moderate disability, and good recovery into lower and higher levels, as shown in Table 1.

[0065] Table 1

[0066]

[0067]

[0068] The term "humanized antibody" is used herein to describe an antibody that contains heavy and light chain variable region sequences from a non-human species (e.g., mouse) but in which at least a portion of the VH and / or VL sequences have been made more "human-like", i.e., more similar to human germline variable sequences. A "humanized antibody" is such an antibody or its variant, derivative, analogue or fragment that immunospecifically binds to an antigen of interest and contains a framework (FR) region that substantially has the amino acid sequence of a human antibody and a complementarity determining region (CDR) that substantially has the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR means a CDR in which the amino acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to the amino acid sequence of a non-human antibody CDR. A humanized antibody contains substantially all of at least one and usually two variable domains (Fab, Fab', F(ab')2, FabC, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., the donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In one embodiment, the humanized antibody also contains at least a portion of the immunoglobulin constant region (Fc), usually at least a portion of a human immunoglobulin. In some embodiments, the humanized antibody contains a light chain and at least the variable domain of the heavy chain. The antibody may also include the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only a humanized light chain. In some embodiments, the humanized antibody contains only a humanized heavy chain. In a particular embodiment, the humanized antibody contains only the humanized variable domains of the light chain and / or the humanized heavy chain.

[0069] Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including but not limited to IgG1, IgG2, IgG3 and IgG4. Humanized antibodies can contain sequences from more than one class or isotype, and specific constant domains can be selected using techniques well known in the art to optimize the desired effector functions.

[0070] The framework regions and CDRs of the humanized antibody need not exactly correspond to the parental sequences. For example, the donor antibody CDR or consensus framework can be mutagenized by substituting, inserting, or / or deleting at least one amino acid residue so that the CDR or framework residue at that site does not correspond to the donor antibody or consensus framework. However, in a preferred embodiment, such mutations will not be extensive. Generally, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the residues of the humanized antibody will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a related family of immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, 1987)). Thus, a "consensus immunoglobulin sequence" can include a "consensus framework region" and / or a "consensus CDR". In an immunoglobulin family, each position in the consensus sequence is occupied by the amino acid that occurs most frequently in that position in the family. If two amino acids occur with equal frequency, either amino acid can be included in the consensus sequence.

[0071] As used herein, "identical" or "identity" in the context of two or more polypeptide or polynucleotide sequences can mean that the sequences have a specified percentage of identical residues over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residues occur in both sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences have different lengths or the alignment produces one or more staggered ends and the specified region to be compared includes only a single sequence, the residues of the single sequence are included in the denominator but not the numerator of the calculation.

[0072] "Injury to the head" or "head injury", as used interchangeably herein, refers to any trauma to the scalp, skull, or brain. Such injuries can range from a minor bump on the head to severe brain damage. Such injuries include primary injury to the brain and / or secondary injury to the brain. Primary brain injury occurs during the initial insult and is caused by the displacement of the physical structures of the brain. More specifically, primary brain injury is physical damage to the parenchyma (tissue, blood vessels) that occurs during a traumatic event, resulting in shear and compression of the surrounding brain tissue. Secondary brain injury occurs after the primary injury and may involve a series of cellular processes. More specifically, secondary brain injury refers to changes that develop over a period of time (from hours to days) after the primary brain injury. It includes a whole cascade of cellular, chemical, tissue, or vascular changes in the brain that lead to further destruction of the brain tissue.

[0073] A head injury can be closed or open (penetrating). A closed head injury is a trauma to the scalp, skull, or brain in which the skull is not penetrated by the impact object. An open head injury is a trauma to the scalp, skull, or brain in which the skull is penetrated by the impact object. Head injuries can be caused by the shaking of a person's body, blunt impacts produced by external mechanical or other forces that result in closed or open head trauma (e.g., traffic accidents such as in the case of cars, airplanes, trains, etc.; blows to the head such as with a baseball bat or from a firearm), cerebrovascular accidents (e.g., stroke), one or more falls (e.g., as in sports or other activities), explosions or shock waves (collectively referred to as "blast injuries"), and other types of blunt force trauma. Alternatively, a head injury may be caused by the ingestion and / or exposure to chemicals, toxins, or a combination of chemicals and toxins. Examples of such chemicals and / or toxins include fire, mold, asbestos, pesticides and insecticides, organic solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organometals (such as methylmercury, tetraethyllead, and organotin), and / or one or more abused drugs. Alternatively, a head injury may be due to a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection (e.g., SARS-CoV-2), a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof. In some cases, it is not possible to determine whether any such event or injury has occurred. For example, a patient or subject may have no medical history, the subject may be unable to speak, the subject may know the events they have experienced, etc. Such cases are described herein as the subject "may have suffered a head injury" or as a "suspected injury". In certain embodiments herein, closed head injuries do not include and specifically exclude cerebrovascular accidents such as stroke.

[0074] As used herein, "isolated polynucleotide" can mean a polynucleotide (e.g., a polynucleotide of genomic, cDNA, or synthetic origin or a combination thereof) that, as a result of its origin, is not associated with all or a portion of the polynucleotides with which the "isolated polynucleotide" is found in nature; is operably linked to a polynucleotide to which it is not linked in nature; or does not exist in nature as part of a larger sequence.

[0075] As used herein, "label" and "detectable label" refer to a moiety that is attached to an antibody or an analyte to render the reaction between the antibody and the analyte detectable, and an antibody or analyte so labeled is referred to as "detectably labeled." The label can generate a signal that is detectable by visual or instrumental means. A variety of labels include substances that generate signals, such as chromophores, fluorescent compounds, chemiluminescent compounds, radioactive compounds, and the like. Representative examples of labels include moieties that generate light, such as acridine compounds, and moieties that generate fluorescence, such as fluorescein. Other labels are described herein. In this regard, a moiety may itself be undetectable but may become detectable upon reaction with another moiety. The use of the term "detectable label" is intended to encompass such labels.

[0076] Any suitable detectable label known in the art can be used. For example, the detectable label can be a radioactive label (such as 3H, 14C, 32P, 33P, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm), an enzyme label (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, etc.), a chemiluminescent label (such as acridinium ester, thioester, or sulfonamide; luminol, isoluminol, phenanthridinium ester, etc.), a fluorescent label (such as fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, etc.)), rhodamine, phycobiliprotein, R-phycoerythrin, quantum dots (e.g., cadmium selenide capped with zinc sulfide), a thermometric label, or an immunopolymerase chain reaction label. An introduction to labels, labeling procedures, and label detection can be found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997); and Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996) (which is a combined handbook and catalog published by Molecular Probes, Inc., Eugene, Oregon). Fluorescent labels can be used in FPIA (see, for example, U.S. Patent Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are hereby incorporated by reference in their entirety). Acridinium compounds can be used as detectable labels in homogeneous chemiluminescent assays (see, for example, Adamczyk et al., Bioorg. Med Ghem. Lett. 16:1324-1328 (2006); Adamczyk et al., Bioorg. Med Chem. Lett. 4:2313-2317 (2004); Adamczyk et al., Biorg. MedChem. Lett. 14:3917-3921 (2004); and Adamczyk et al., Org. Lett. 5:3779-3782 (2003)).

[0077] In one aspect, the acridine compound is acridine-9-carboxamide. Methods for preparing acridine 9-carboxamide are described in Mattingly, J. Biolumin. Chemilumin. 6:107-114 (1991); Adamczyk et al., J. Org. Chem. 63:5636-5639 (1998); Adamczyk et al., Tetrahedron 55:10899-10914 (1999); Adamczyk et al., Org. Lett. 1:779-781 (1999); Adamczyk et al., Bioconjugate Chem. 11:714-724 (2000); Mattingly et al., In Luminescence Biotechnology: Instruments and Applications; Dyke, K.V. ed.; CRC Press: Boca Raton, pp. 77-105 (2002); Adamczyk et al., Org. Lett. 5:3779-3782 (2003); and U.S. Patent Nos. 5,468,646, 5,543,524, and 5,783,699 (the teachings of each of these references regarding this aspect are incorporated herein by reference in their entirety).

[0078] Another example of an acridine compound is an aryl acridine-9-carboxylate. An example of an aryl acridine-9-carboxylate having Formula II is 10-methyl-9-(phenoxycarbonyl)acridine fluorosulfonate (available from Cayman Chemical, Ann Arbor, MI). Methods for preparing aryl acridine-9-carboxylates are described in McCapra et al., Photochem. Photobiol. 4:1111-21 (1965); Razavi et al., Luminescence 15:245-249 (2000); Razavi et al., Luminescence 15:239-244 (2000); and U.S. Patent No. 5,241,070 (the teachings of each of these references in this regard are incorporated herein by reference in their entirety). Such aryl acridine-9-carboxylates are chemiluminescent indicators that are efficient in terms of both signal intensity and / or signal rapidity in the production of hydrogen peroxide generated by the oxidation of an analyte by at least one oxidase. The chemiluminescent emission process of aryl acridine-9-carboxylates is completed rapidly, i.e., within 1 second, while the chemiluminescent emission of acridine-9-carboxamide continues for up to 2 seconds. However, aryl acridine-9-carboxylates lose their chemiluminescent properties in the presence of proteins. Thus, their use requires the absence of proteins during signal generation and detection. Methods for separating or removing proteins from a sample are well known to those skilled in the art and include, but are not limited to, ultrafiltration, extraction, precipitation, dialysis, chromatography, and / or digestion (see, for example, Wells, High Throughput Bioanalytical Sample Preparation. Methods and Automation Strategies, Elsevier (2003)). The amount of protein removed or separated from the test sample can be about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Further details regarding aryl acridine-9-carboxylates and their use are set forth in U.S. Patent Application No. 11 / 697,835, filed April 9, 2007. Aryl acridine-9-carboxylates can be dissolved in any suitable solvent, such as degassed anhydrous N,N-dimethylformamide (DMF) or sodium cholate hydrate.

[0079] "Linker sequence" or "linker peptide sequence" refers to a natural or artificial polypeptide sequence that is linked to one or more polypeptide sequences of interest (e.g., full-length, fragment, etc.). The term "linked" refers to the conjugation of the linker sequence to the polypeptide sequence of interest. Such polypeptide sequences are preferably conjugated by one or more peptide bonds. The linker sequence can have a length of about 4 to about 50 amino acids. Preferably, the linker sequence has a length of about 6 to about 30 amino acids. Natural linker sequences can be modified by amino acid substitution, addition, or deletion to produce artificial linker sequences. Linker sequences can be used for many purposes, including in recombinant Fabs. Exemplary linker sequences include, but are not limited to: (i) a histidine (His) tag, such as a 6X His tag, having the amino acid sequence HHHHHH (SEQ ID NO:3), which can be used as a linker sequence to facilitate the isolation and purification of polypeptides and antibodies of interest; (ii) enterokinase cleavage sites, such as His tags, for the isolation and purification of proteins and antibodies of interest. Often, enterokinase cleavage sites are used in combination with His tags for the isolation and purification of proteins and antibodies of interest. Various enterokinase cleavage sites are known in the art. Examples of enterokinase cleavage sites include, but are not limited to, the amino acid sequence DDDDK (SEQ ID NO:4) and its derivatives (e.g., ADDDDK (SEQ ID NO:5), etc.); (iii) Miscellaneous sequences can be used to link or connect the light chain and / or heavy chain variable regions of single-chain variable region fragments. Examples of other linker sequences can be found in Bird et al., Science 242:423-426 (1988); Huston et al., PNAS USA 85:5879-5883 (1988); and McCafferty et al., Nature 348:552-554 (1990). Linker sequences can also be modified to achieve additional functions, such as the conjugation of drugs or attachment to solid supports. In the context of the present disclosure, monoclonal antibodies can, for example, contain linker sequences such as His tags, enterokinase cleavage sites, or both.

[0080] As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single antigen (e.g., cross-reactivity or shared reactivity may occur). In addition, unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological properties.

[0081] "Magnetic resonance imaging" or "MRI", which are used interchangeably herein, refers to a medical imaging technique used in radiology to form pictures of the anatomical structures and physiological processes of the human body in health and disease (e.g., which may be interchangeably referred to herein as "MRI", "MRI procedure", or "MRI scan"). MRI is a form of medical imaging that measures the response of atomic nuclei in body tissues to high-frequency radio waves when the body tissues are placed in a strong magnetic field and produces images of internal organs. MRI scanners, based on nuclear magnetic resonance (NMR) science, use strong magnetic fields, radio waves, and field gradients to produce images of the interior of the human body.

[0082] "Multivalent binding protein" is used herein to refer to a binding protein that contains two or more antigen-binding sites (also referred to herein as "antigen-binding domains"). Multivalent binding proteins are preferably engineered to have three or more antigen-binding sites and are generally not naturally occurring antibodies. The term "multispecific binding protein" refers to a binding protein that can bind two or more related or unrelated targets, including a binding protein capable of binding two or more different epitopes of the same target molecule.

[0083] "Negative predictive value" or "NPV", which are used interchangeably herein, refers to the probability that subjects have a negative outcome given that they have a negative test result.

[0084] "Point-of-care device" refers to a device used to provide medical diagnostic testing at or near a point of care (i.e., outside a laboratory), at the time and place of patient care (such as in a hospital, physician's office, emergency or other medical care facility, patient's home, nursing home, and / or long-term care and / or hospice facility). Examples of point-of-care devices include devices manufactured by Abbott Laboratories (Abbott Park, IL) (e.g., i-STAT and i-STAT Alinity), the Panbio biosensor (Rowville, Australia) (see US2006 / 0134713), Axis-Shield PoC AS (Oslo, Norway), and Clinical Laboratory Products (Los Angeles, USA). In some embodiments, the point-of-care device is a disposable device. The term "disposable device" or "disposable instrument" refers to a clinical diagnostic instrument that processes and performs clinical diagnostic assays on a per-unit basis (such as a disposable cartridge) for a single patient sample. A point-of-care instrument does not simultaneously assay more than one clinical sample. However, a point-of-care instrument may have the ability to measure more than one parameter (e.g., more than one analyte) in a single clinical sample on a per-unit basis.

[0085] As used interchangeably herein, "positive predictive value" or "PPV" refers to the probability that subjects have a positive outcome given that they have a positive test result.

[0086] "Quality control reagents" in the context of the immunoassays and kits described herein include, but are not limited to, calibrators, controls, and sensitivity sets. "Calibrators" or "standards" (e.g., one or more, such as multiple) are typically used to establish a calibration (standard) curve to interpolate the concentration of an analyte (such as an antibody or analyte). Alternatively, a single calibrator close to a reference level or control level (e.g., "low", "medium", or "high" level) may be used. Multiple calibrators may be used in combination (i.e., more than one calibrator or different amounts of calibrators) to constitute a "sensitivity set".

[0087] The "Receiver Operating Characteristic" curve, or "ROC" curve, is a graph that illustrates the performance of a binary classifier system as its discrimination threshold varies. For example, an ROC curve can be a plot of the true positive rate versus the false positive rate for different possible cut-off points of a diagnostic test. It is generated by plotting the true positive fraction in the positive (TPR = true positive rate) against the false positive fraction in the negative (FPR = false positive rate) at various threshold settings. The TPR is also known as sensitivity, and the FPR is one minus specificity or the true negative rate. The ROC curve demonstrates the trade-off between sensitivity and specificity (any increase in sensitivity is accompanied by a decrease in specificity); the closer the curve hugs the left boundary of the ROC space and then the top boundary, the more accurate the test; the closer the curve is to the 45-degree diagonal of the ROC space, the less accurate the test; the slope of the tangent at the cut-off point gives the likelihood ratio (LR) of that test value; and the area under the curve is a measure of the test's accuracy.

[0088] "Recombinant antibody" and "multiple recombinant antibodies" refer to antibodies prepared by one or more steps, including cloning nucleic acid sequences encoding all or part of one or more monoclonal antibodies into a suitable expression vector by recombinant techniques and subsequently expressing the antibody in a suitable host cell. The terms include, but are not limited to, recombinantly produced monoclonal antibodies, chimeric antibodies, humanized antibodies (fully or partially humanized), multi-specific or multivalent structures formed from antibody fragments, bispecific antibodies, heteroconjugate Abs, and other antibodies described herein (i). (Dual variable domain immunoglobulins and methods for their preparation are described in Wu, C et al., Nature Biotechnology, 25:1290-1297 (2007)). As used herein, the term "bispecific antibody" refers to an antibody that contains a first arm with specificity for one antigenic site and a second arm with specificity for a different antigenic site, i.e., a bispecific antibody has dual specificity.

[0089] As used herein, "reference level" refers to a determination threshold value used to evaluate diagnostic, prognostic, or therapeutic efficacy, which has been associated or correlated herein with various clinical parameters (e.g., presence of a disease, disease stage, disease severity, disease progression, non - progression or improvement, etc.). As used herein, the term "cut - off" refers to a limit (such as a number), above which there is a certain or specific clinical outcome, and below which there is a different certain or specific clinical outcome. The present disclosure provides exemplary reference levels. However, it is well known that reference levels can vary depending on the nature of the immunoassay (e.g., antibodies used, reaction conditions, sample purity, etc.), and assays can be compared and standardized. Modifying the present disclosure based on the description provided herein for other immunoassays to obtain immunoassay - specific reference levels for those other immunoassays is further fully within the capabilities of a person of ordinary skill in the art. Although the exact values of reference levels can vary between assays, the findings described herein should be generally applicable and capable of being extrapolated to other assays.

[0090] In certain aspects described herein, reference levels are described as being determined by any assay having a certain specificity and sensitivity.

[0091] As used herein, "risk assessment", "risk classification", "risk identification", or "risk stratification" of a subject (e.g., a patient) refers to the evaluation of factors including biomarkers to predict the risk of future events including disease onset or disease progression, so that treatment decisions regarding the subject can be made on a more informed basis.

[0092] As used herein, "sample", "test sample", "specimen", "sample from a subject", and "patient sample" can be used interchangeably and can be a blood sample (such as whole blood (including, for example, capillary blood, venous blood, mixed samples of venous and capillary blood, mixed samples of capillary blood and interstitial fluid, dried blood spots, etc.)), tissue, urine, serum, plasma, amniotic fluid, lower respiratory tract specimens (such as, but not limited to, sputum, endotracheal aspirate, or bronchoalveolar lavage fluid), nasal mucus, cerebrospinal fluid, placental cells or tissue, endothelial cells, white blood cells, or monocytes. The sample can be used directly as obtained from the patient or can be pre - treated, such as by filtration, dilution, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., so as to modify the characteristics of the sample in some manner discussed herein or otherwise known in the art.

[0093] A variety of cell types, tissues, or body fluids can be utilized to obtain a sample. Such cell types, tissues, and fluids can include tissue sections (such as biopsy and autopsy samples), oropharyngeal specimens, nasopharyngeal specimens, nasal mucus specimens, frozen sections obtained for histological purposes, blood (such as whole blood, capillary blood, venous blood, mixed samples of venous and capillary blood, mixed samples of capillary blood and interstitial fluid, dried blood spots, etc.), plasma, serum, red blood cells, platelets, anal samples (such as anal swab specimens), interstitial fluid, cerebrospinal fluid, etc. Cell types and tissues can also include lymph fluid, cerebrospinal fluid, or any fluid collected by aspiration. The tissue or cell type can be provided by taking a cell sample from human and non-human animals, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and / or for another purpose). Archival tissues can also be used, such as those with a history of treatment or outcome. Protein or nucleotide isolation and / or purification may not be required. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a capillary blood sample. In some embodiments, the sample is a dried blood spot. In some embodiments, the sample is a serum sample. In other embodiments, the sample is a plasma sample. In some embodiments, the sample is an oropharyngeal specimen. In other embodiments, the sample is a nasopharyngeal specimen. In other embodiments, the sample is sputum. In other embodiments, the sample is an endotracheal aspirate. In other embodiments, the sample is a bronchoalveolar lavage fluid. In still other aspects, the sample is nasal mucus.

[0094] "Sensitivity" refers to the proportion of subjects with a positive result who are correctly identified as positive (e.g., correctly identifying those subjects with the disease or medical condition for which they are being tested). For example, this may include correctly identifying a subject with ultra - mild TBI as having mild TBI from a subject with ultra - mild TBI, correctly identifying a subject with mild TBI as having ultra - mild TBI from a subject with mild TBI, correctly identifying a subject without TBI as having ultra - mild TBI, etc.

[0095] As used herein, the "specificity" of an assay refers to the proportion of subjects with a negative outcome who are correctly identified as negative (e.g., correctly identifying those subjects who do not have the disease or medical condition being tested). For example, this may include correctly identifying a subject as not having TBI, correctly identifying a subject as having ultra - mild TBI, correctly identifying a subject as not having mild TBI (i.e., correctly identifying a subject with mild TBI as having ultra - mild TBI).

[0096] "Calibration composition series" refers to a plurality of compositions comprising known concentrations of the following: (1) UCH-L1, wherein each composition differs from the other compositions in the series by the concentration of UCH-L1; and / or (2) GFAP, wherein each composition differs from the other compositions in the series by the concentration of GFAP.

[0097] As used herein, the term "single molecule detection" refers to the detection and / or measurement of single molecules of an analyte in a test sample at very low concentration levels, such as pg / mL or femtogram / mL levels. Many different single molecule analyzers or devices are known in the art and include nanopore and nanowell devices. Examples of nanopore devices are described in International Patent Publication No. WO 2016 / 161402, which is incorporated herein by reference in its entirety. Examples of nanowell devices are described in International Patent Publication No. WO 2016 / 161400, which is incorporated herein by reference in its entirety.

[0098] As used interchangeably herein, "solid phase" or "solid support" refers to any material that can be used to link and / or attract and immobilize (1) one or more capture agents or capture specific binding partners, or (2) one or more detection agents or detection specific binding partners. The solid phase can be selected for its inherent ability to attract and immobilize capture agents. Alternatively, the solid phase can have a linker attached thereto, the linker having the ability to attract and immobilize (1) a capture agent or capture specific binding partner or (2) a detection agent or detection specific binding partner. For example, the linker can include a charged substance that is oppositely charged relative to the capture agent (e.g., capture specific binding partner) or detection agent (e.g., detection specific binding partner) itself or relative to a charged substance conjugated to (1) a capture agent or capture specific binding partner or (2) a detection agent or detection specific binding partner. Generally speaking, the linker can be any binding partner (preferably heterologous) that is immobilized (linked to) on the solid phase and has the ability to immobilize (1) a capture agent or capture specific binding partner or (2) a detection agent or detection specific binding partner through a binding reaction. The linker enables the capture agent to indirectly bind to the solid phase material before or during the assay. For example, the solid phase can be plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon, including, for example, test tubes, microtiter wells, slides, beads, microparticles, chips, and other configurations known to those of ordinary skill in the art.

[0099] As used herein, "specifically binds" or "binds specifically" can refer to the interaction of an antibody, protein, or peptide with a second chemical species, where the interaction depends on the presence of a specific structure (e.g., an epitope or antigenic determinant) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than binding proteins generally. If an antibody is specific for epitope "A", the presence of molecules containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A that binds to the antibody.

[0100] A "specific binding partner" is a member of a specific binding pair. A specific binding pair consists of two different molecules that bind specifically to each other either chemically or physically. Thus, in addition to the antigen-antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin (or streptavidin); carbohydrate and lectin; complementary nucleotide sequences; effector molecule and receptor molecule; cofactor and enzyme; enzyme and enzyme inhibitor, etc. In addition, a specific binding pair can include members that are analogs of the original specific binding partners, such as analyte-analogs. Immunoreactive specific binding partners include isolated or recombinantly produced antigens, antigen fragments, and antibodies, including monoclonal and polyclonal antibodies and their complexes and fragments.

[0101] As used herein, "statistically significant" means the likelihood that the relationship between two or more variables is caused by factors other than random chance. Statistical hypothesis testing is used to determine whether the results of a data set are statistically significant. In statistical hypothesis testing, a statistically significant result is obtained whenever the p-value of the observed test statistic is less than the significance level defined by the study. The p-value is the probability of obtaining a result at least as extreme as the observed result, assuming the null hypothesis is true. Examples of statistical hypothesis analysis include the Wilcoxon signed-rank test, t-test, chi-square test, or Fisher's exact test. As used herein, "significant" refers to a change that has not been determined to be statistically significant (e.g., it may not have been subjected to statistical hypothesis testing).

[0102] As used herein, "subject" and "patient" are used interchangeably to refer to any vertebrate, including but not limited to mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice, non-human primates (e.g., monkeys such as cynomolgus monkeys or rhesus monkeys, chimpanzees, etc.), and humans). In some embodiments, the subject can be human or non-human. In some embodiments, the subject is human. The subject or patient can receive other forms of treatment. In some embodiments, the subject is a human who may be receiving other forms of treatment. In some embodiments, the subject is a human assistant subject, e.g., a horse, a dog, or another species that assists a human in performing their daily tasks (e.g., companion animals) or work (e.g., service animals). In some aspects, the subject is a human subject. In still other aspects, the subject is a pediatric subject, e.g., a human pediatric subject. In yet other aspects, the subject is an adult subject, e.g., a human adult subject.

[0103] "Treat / treating / treatment" are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progression of a disease and / or injury to which such term applies, or one or more symptoms of such disease. Depending on the condition of the subject, the term also refers to preventing a disease and includes preventing the onset of a disease or preventing symptoms associated with a disease. Treatment can be performed in an acute or chronic manner. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. Such prevention of a disease or reduction of disease severity prior to affliction means administering a pharmaceutical composition to a subject at a time when the subject is not afflicted with the disease. "Prevention" also refers to preventing the recurrence of a disease or one or more symptoms associated with such disease. "Treat" and "therapeutically" refer to the act of treating as defined above for "treatment".

[0104] "Traumatic brain injury" or "TBI", as used interchangeably herein, refers to a complex injury with a broad spectrum of symptoms and disabilities. TBI is often an acute event similar to other injuries. TBI can be classified as "ultra - mild", "mild", "moderate", "moderate - to - severe", or "severe". The causes of TBI are diverse and include, for example, the shaking of a person's body, motor vehicle accidents, firearm injuries, cerebrovascular accidents (such as strokes), falls, explosions or shock waves, and other types of blunt - force trauma. Other causes of TBI include ingestion and / or exposure to one or more chemicals or toxins (such as fire, mold, asbestos, pesticides and insecticides, organic solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organometals (such as methylmercury, tetraethyllead, and organotin), one or more abused drugs, or combinations thereof). Alternatively, TBI may occur in subjects suffering from autoimmune diseases, metabolic disorders, brain tumors, hypoxia, viral infections (such as SARS - CoV - 2, meningitis, etc.), fungal infections (such as meningitis), bacterial infections (such as meningitis), or any combination thereof. Young and old individuals are the age groups at highest risk for TBI. In some embodiments herein, traumatic brain injury or TBI does not include and specifically excludes cerebrovascular accidents, such as strokes.

[0105] "Mild TBI", as used herein, refers to a head injury in which a subject may or may not experience loss of consciousness. For subjects who experience loss of consciousness, it is usually brief, typically lasting only a few seconds or minutes. Mild TBI is also known as concussion, minor head trauma, minor TBI, minor brain injury, and minor head injury. Although MRI and CT scans are often normal, individuals with mild TBI may have cognitive problems such as headache, difficulty thinking, memory problems, attention deficits, mood swings, and depression.

[0106] Mild TBI is the most common type of TBI and is often missed at the time of the initial injury. Typically, subjects have a Glasgow Coma Scale score between 13 - 15. Fifteen percent (15%) of patients with mild TBI have symptoms that last 3 months or longer. Mild TBI is defined as the result of a forceful movement or impact to the head that causes a brief change in mental status (confusion, disorientation, or memory loss) or loss of consciousness for less than 30 minutes. Common symptoms of mild TBI include fatigue, headache, visual disturbances, memory loss, poor attention / concentration, sleep disturbances, dizziness / loss of balance, stress - related mood disorders, depressive affect, and seizures. Other symptoms associated with mild TBI include nausea, loss of smell, sensitivity to light and sound, mood changes, confusion or disorientation, and / or slowed thinking.

[0107] "Ultra - mild TBI" as used herein refers to a subclass or subtype of mild TBI, in which the subject has a Glasgow Coma Scale score of 15. Compared with patients with mild TBI, ultra - mild TBI patients will exhibit less structural damage and less dysfunction. In some embodiments, compared with mild TBI subjects, the head CT scans of ultra - mild TBI subjects are less likely to be positive. In some embodiments, compared with mild TBI subjects, subjects with "ultra - mild" TBI may exhibit less inflammation, recover faster, exhibit less / lower symptom burden, and / or have fewer sequelae.

[0108] "Moderate TBI" as used herein refers to a brain injury in which loss of consciousness and / or confusion and disorientation are between 1 and 24 hours and the subject has a Glasgow Coma Scale score between 9 - 13 (such as 9 - 12 or 9 - 13). Moderate TBI individuals may have abnormal brain imaging findings. "Severe TBI" as used herein refers to a brain injury in which loss of consciousness exceeds 24 hours and memory loss is longer than 24 hours after injury or penetrating skull injury and the subject has a Glasgow Coma Scale score between 3 - 8. The range of deficits is from higher levels of cognitive function impairment to a comatose state. Survivors may have limited arm or leg function, speech or language abnormalities, loss of thinking ability, or emotional problems. Individuals with severe injuries may be in a non - responsive state for a long time. For many people with severe TBI, long - term rehabilitation is usually required to maximize function and independence.

[0109] As used herein, "moderate to severe" TBI refers to a spectrum of brain injuries that includes changes from moderate to severe TBI over time and thus includes, for example, (temporally) isolated moderate TBI, isolated severe TBI, and combined moderate to severe TBI. For example, in some clinical situations, a subject may initially be diagnosed with moderate TBI but over time (minutes, hours, or days) progress to having severe TBI (e.g., in the case of intracerebral hemorrhage). Alternatively, in some clinical situations, a subject may initially be diagnosed with severe TBI but over time (minutes, hours, or days) progress to having moderate TBI. Such subjects would be examples of patients classifiable as "moderate to severe." Common symptoms of moderate to severe TBI include cognitive deficits, including difficulties with attention, concentration, distractibility, memory, processing speed, confusion, perseveration, impulsivity, language processing, and / or "executive function," difficulty understanding spoken words (sensory aphasia), difficulty speaking and being understood (expressive aphasia), slurred speech, very rapid or very slow speech, reading problems, writing problems, difficulty interpreting touch, temperature, movement, limb position, and fine discrimination, integrating or patternizing sensory impressions into mentally meaningful data, partial or complete loss of vision, weak eye muscles and double vision (diplopia), blurred vision, problems judging distance, involuntary eye movements (nystagmus), intolerance to light (photophobia), hearing problems (such as reduced or lost hearing, ringing in the ears (tinnitus), increased sensitivity to sound), loss or reduction of smell (anosmia), loss or reduction of taste, seizures associated with epilepsy, which may be several types and may involve consciousness, sensory perception, or motor movement, problems with control of the bowel and bladder, insomnia, loss of stamina, changes in appetite, problems with temperature regulation, menstrual difficulties, dependent behavior, problems with emotional capacity or stability, lack of motivation, irritability, aggression, depression, disinhibition, or denial / lack of awareness. Subjects with moderate to severe TBI may have a Glasgow Coma Scale score of 3 - 12 (which includes the range of 9 - 12 for moderate TBI and the range of 3 - 8 for severe TBI).

[0110] As used interchangeably herein, "ubiquitin carboxyl-terminal hydrolase L1" or "UCH-L1" refers to the deubiquitinating enzyme encoded by the human UCH-L1 gene and UCH-L1 gene counterparts in other species. UCH-L1 (also known as ubiquitin carboxyl-terminal esterase L1 and ubiquitin thioesterase) is a member of a gene family that hydrolyzes the small C-terminal adducts of ubiquitin to produce ubiquitin monomers.

[0111] "UCH-L1 status" can refer to the UCH-L1 level or amount conveyed at a particular point in time (e.g., using a single UCH-L1 measurement), the UCH-L1 level or amount associated with monitoring (e.g., identifying an increase or decrease in the UCH-L1 amount through repeated testing of a subject), the UCH-L1 level or amount associated with the treatment of traumatic brain injury (whether primary brain injury and / or secondary brain injury), or combinations thereof.

[0112] "Variant" is used herein to describe a peptide or polypeptide that differs in amino acid sequence due to an insertion, deletion, or conservative substitution of an amino acid, but retains at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or to promote an immune response. Variant is also used herein to describe a protein whose amino acid sequence is substantially the same as a reference protein whose amino acid sequence retains at least one biological activity. Conservative substitution of an amino acid, i.e., replacement of an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), is generally recognized in the art as typically involving minor changes. As understood in the art, these minor changes can be identified in part by considering the hydrophilicity index of the amino acids. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydrophilicity index of an amino acid is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophilicity indices can be substituted and still retain protein function. In one aspect, amino acids with a hydrophilicity index of ±2 are substituted. The hydrophilicity of an amino acid can also be used to reveal substitutions that will result in a protein retaining its biological function. Considering the hydrophilicity of amino acids in the context of a peptide allows calculation of the maximum local average hydrophilicity of the peptide, which is a useful measure that has been reported to be well correlated with antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is hereby incorporated by reference in its entirety. As understood in the art, substitution of amino acids with similar hydrophilicity values can result in a peptide retaining its biological activity (e.g., immunogenicity). Substitutions can be made with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the particular side chain of the amino acid. Consistent with this observation, amino acid substitutions compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties. "Variant" can also be used to refer to an antigen-reactive fragment of an anti-UCH-L1 antibody that differs in amino acid sequence from the corresponding fragment of the anti-UCH-L1 antibody, but still has antigen reactivity and can compete with the parental fragment of the anti-UCH-L1 antibody for binding to UCH-L1. "Variant" can also be used to describe a polypeptide or fragment thereof that has been differentially processed (such as by proteolysis, phosphorylation, or other post-translational modifications), but still retains its antigen reactivity.

[0113] "Vector" is used herein to describe a nucleic acid molecule that can transport another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors can replicate autonomously in the host cells into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell after being introduced into the host cell and, thereby, replicate with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA techniques are usually in the form of plasmids. "Plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, other forms of expression vectors having equivalent functions can be used, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). In this regard, RNA forms of vectors, including RNA viral vectors, can also be used in the context of the present disclosure.

[0114] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure will have the meanings commonly understood by one of ordinary skill in the art. For example, any nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The meanings and scopes of the terms should be clear; however, if there is any implicit ambiguity, the definitions provided herein will prevail over any dictionary or extrinsic definition. Further, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular.

[0115] 2. A method of using a reference level to assist in diagnosing and evaluating whether a subject has suffered or is suspected of having suffered a head injury

[0116] Among other things, the present disclosure relates to a method for assisting in diagnosing and evaluating whether a subject (e.g., a human subject) has suffered or may have suffered a head injury. In some embodiments, the method can assist in determining the extent of traumatic brain injury in a subject (e.g., a human subject) with an actual or suspected head injury, e.g., determining whether the subject (e.g., a human subject) has a mild traumatic brain injury or a ultra-mild traumatic brain injury. For the methods described herein, control subjects (i.e., subjects who have not suffered an actual or suspected head injury) are assumed to be excluded. In other words, the methods employed herein can be used to distinguish between mild TBI and ultra-mild TBI in subjects with an actual or suspected head injury and are not intended for evaluating control subjects. In some embodiments, the method includes distinguishing between mild TBI and ultra-mild TBI. As used herein, "determining whether a subject (e.g., a human subject) has a mild traumatic brain injury or a ultra-mild traumatic brain injury" refers to the fact that the foregoing method can be used, for example, in conjunction with other information (e.g., clinical assessment data), to determine whether the subject is more likely to have a mild traumatic brain injury or a ultra-mild traumatic brain injury. The method can include assaying a sample obtained from a subject (e.g., a human subject) within about 48 hours after an actual or suspected head injury to measure or detect the level of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) and / or glial fibrillary acidic protein (GFAP) in the sample, and determining whether the subject (e.g., a human subject) has suffered a mild or ultra-mild traumatic brain injury (TBI) based on the GFAP, UCH-L1, or GFAP and UCH-L1 levels. In some aspects, the method can include assaying a sample obtained from a subject (e.g., a human subject) within about 24 hours after an actual or suspected head injury to measure or detect the level of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) and / or glial fibrillary acidic protein (GFAP) in the sample, and determining whether the subject (e.g., a human subject) has suffered a mild or ultra-mild traumatic brain injury (TBI) based on the GFAP, UCH-L1, or GFAP and UCH-L1 levels. In some embodiments, the method includes assaying a sample obtained from a subject (e.g., a human subject) within about 24 hours after an actual or suspected head injury to measure or detect the level of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) and / or glial fibrillary acidic protein (GFAP) in the sample, and distinguishing between mild TBI and ultra-mild TBI based on whether the GFAP level in the sample is equal to or below the reference level of GFAP and / or whether the UCH-L1 level in the sample is equal to or below the reference level of UCH-L1.In other aspects, the method can include assaying a sample obtained from a subject (e.g., a human subject) within about 12 hours after an actual or suspected head injury to measure or detect ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) and / or glial fibrillary acidic protein (GFAP) levels in the sample, and determining whether the subject (e.g., a human subject) has suffered a mild or ultra-mild traumatic brain injury (TBI) based on the GFAP, UCH-L1, or GFAP and UCH-L1 levels. In some embodiments, the method includes assaying a sample obtained from a subject (e.g., a human subject) within about 12 hours after an actual or suspected head injury to measure or detect ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) and / or glial fibrillary acidic protein (GFAP) levels in the sample, and differentiating mild TBI from ultra-mild TBI based on whether the GFAP level in the sample is equal to or lower than a reference level of GFAP and / or whether the UCH-L1 level in the sample is equal to or lower than a reference level of UCH-L1. In some embodiments, it is determined that the subject has mild or ultra-mild TBI based on determining whether the GFAP, UCH-L1, or GFAP and UCH-L1 levels in the sample obtained from the subject are lower than or equal to a reference level of GFAP and / or a reference level of UCH-L1.

[0117] In some embodiments, the method includes assaying a sample obtained from a subject (e.g., a human subject) to measure the glial fibrillary acidic protein (GFAP) level and / or ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) level in the sample; and determining that the subject has suffered or may have suffered mild TBI when the GFAP level in the sample is equal to a reference level of GFAP and / or the UCH-L1 level in the sample is equal to a reference level of UCH-L1; or determining that the subject has suffered or may have suffered ultra-mild TBI when the GFAP level in the sample is lower than a reference level of GFAP and / or the UCH-L1 level in the sample is lower than a reference level of UCH-L1.

[0118] In some embodiments, the method includes determining that a subject has suffered or may have suffered a mild traumatic brain injury (TBI) when the GFAP level in a sample is equal to the reference level of GFAP and / or the UCH-L1 level in the sample is equal to the reference level of UCH-L1. For example, in some embodiments, the method includes determining that a subject has suffered or may have suffered a mild TBI when the GFAP level in the sample is equal to the reference level of GFAP. As another example, in some embodiments, the method includes determining that a subject has suffered or may have suffered a mild TBI when the UCH-L1 level in the sample is equal to the reference level of UCH-L1. In some embodiments, the method includes determining that a subject has suffered or may have suffered a mild TBI when the GFAP level in the sample is equal to the reference level of GFAP and when the UCH-L1 level in the sample is equal to the reference level of UCH-L1.

[0119] In some embodiments, the method includes determining that a subject has suffered or may have suffered a ultra-mild TBI when the GFAP level in a sample is below the reference level of GFAP and / or when the UCH-L1 level in the sample is below the reference level of UCH-L1. For example, in some embodiments, the method includes determining that a subject has suffered or may have suffered a ultra-mild TBI when the GFAP level in the sample is below the reference level of GFAP. As another example, in some embodiments, the method includes determining that a subject has suffered or may have suffered a ultra-mild TBI when the UCH-L1 level in the sample is below the reference level of UCH-L1. In some embodiments, the method includes determining that a subject has suffered or may have suffered a ultra-mild TBI when the GFAP level in the sample is below the reference level of GFAP and when the UCH-L1 level in the sample is below the reference level of UCH-L1. The sample can be a biological sample.

[0120] In some embodiments, the method can include obtaining a sample within about 48 hours of an actual or suspected injury to the subject and contacting the sample with an antibody against a TBI biomarker, such as ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, to allow formation of a complex of the antibody and the biomarker. In other aspects, the method can include obtaining a sample within about 24 hours of an actual or suspected injury to the subject and contacting the sample with an antibody against a TBI biomarker, such as ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, to allow formation of a complex of the antibody and the biomarker. In yet additional aspects, the method can include obtaining a sample within about 12 hours of an actual or suspected injury to the subject and contacting the sample with an antibody against a TBI biomarker, such as ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, to allow formation of a complex of the antibody and the biomarker. The method further includes detecting the resulting antibody-biomarker complex.

[0121] In some embodiments, the sample is obtained from a subject (e.g., a human subject) within about 48 hours of an actual or suspected head injury. For example, the sample can be obtained from a subject (e.g., a human subject) within about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, within about 24 hours, within about 25 hours, within about 26 hours, within about 27 hours, within about 28 hours, within about 29 hours, within about 30 hours, within about 31 hours, within about 32 hours, within about 33 hours, within about 34 hours, within about 35 hours, within about 36 hours, within about 37 hours, within about 38 hours, within about 39 hours, within about 40 hours, within about 41 hours, within about 42 hours, within about 43 hours, within about 44 hours, within about 45 hours, within about 46 hours, within about 47 hours, or within about 48 hours after an actual or suspected head injury. In some embodiments, the sample is obtained about 2 weeks after an actual or suspected head injury.

[0122] In some embodiments, the onset of the presence of a biomarker (such as UCH-L1, GFAP, or a combination thereof) becomes apparent within about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, within about 90 minutes, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, within about 24 hours, within about 25 hours, within about 26 hours, within about 27 hours, within about 28 hours, within about 29 hours, within about 30 hours, within about 31 hours, within about 32 hours, within about 33 hours, within about 34 hours, within about 35 hours, within about 36 hours, within about 37 hours, within about 38 hours, within about 39 hours, within about 40 hours, within about 41 hours, within about 42 hours, within about 43 hours, within about 44 hours, within about 45 hours, within about 46 hours, within about 47 hours, or within about 48 hours after an actual or suspected head injury.

[0123] In some embodiments, the subject has received a Glasgow Coma Scale (GCS) score before or after the assay. In some embodiments, based on the Glasgow Coma Scale score, the subject (e.g., a human subject) is suspected of having mild or ultra-mild TBI. In some embodiments, based on the GCS score, the subject (e.g., a human subject) is suspected of having mild TBI. In some embodiments, based on the GCS score, the subject (e.g., a human subject) is suspected of having ultra-mild TBI.

[0124] In some embodiments, a reference level of a biomarker (such as UCH-L1, GFAP, or a combination thereof) is associated with a subject having a mild traumatic brain injury. In some embodiments, a reference level of a biomarker (such as UCH-L1, GFAP, or a combination thereof) is associated with a Glasgow Coma Scale score of 13 - 14 (mild TBI). In some embodiments, a reference level of a biomarker (such as UCH-L1, GFAP, or a combination thereof) is associated with a subject having an ultra-mild traumatic brain injury. In some embodiments, a reference level of a biomarker (such as UCH-L1, GFAP, or a combination thereof) is associated with a Glasgow Coma Scale score of 15 (severe TBI).

[0125] Generally speaking, the reference level of a biomarker (such as UCH-L1, GFAP, or a combination thereof) can also be used as a benchmark to evaluate the results obtained when measuring a biomarker (such as UCH-L1, GFAP, or a combination thereof) in a test sample. Generally speaking, when making such a comparison, the reference level of a biomarker (such as UCH-L1, GFAP, or a combination thereof) is obtained by running or performing a specific assay a sufficient number of times and under appropriate conditions such that the presence, amount, or concentration of the analyte can be related or associated with a specific stage or endpoint of TBI or with a specific marker. Typically, the reference level of a biomarker (such as UCH-L1, GFAP, or a combination thereof) is obtained by assaying reference subjects (or a group of subjects). The measured biomarker (such as UCH-L1, GFAP, or a combination thereof) can include fragments thereof, degradation products thereof, and / or enzymatically cleaved products thereof.

[0126] In some embodiments, the reference level can be associated with control subjects (e.g., human subjects) who have not suffered a head injury.

[0127] In some embodiments, the reference level of GFAP is from about 55 pg / mL to about 1521 pg / mL. In some embodiments, the method includes determining that a subject has suffered a mild traumatic brain injury (TBI) when the level of GFAP in a sample is equal to the reference level of GFAP, wherein the reference level of GFAP is from about 55 pg / mL to about 1521 pg / mL. In some embodiments, the method includes determining that a subject has suffered a mild TBI when the level of GFAP in a sample is equal to a reference level of GFAP from about 55 pg / mL to about 550 pg / mL, from about 500 pg / mL to about 1100 pg / mL, from about 1000 pg / mL to about 1500 pg / mL, from about 55 to about 90 g / mL, from about 75 pg / mL to about 140 pg / mL, from about 125 pg / mL to about 246 pg / mL, from about 220 pg / mL to about 502 pg / mL, from about 246 pg / mL to about 547 pg / mL, from about 526 pg / mL to about 780 pg / mL, from about 750 pg / mL to about 925 pg / mL, from about 890 pg / mL to about 1120 pg / mL, or from about 1100 pg / mL to about 1521 pg / mL. For example, in some embodiments, the method includes determining that a subject has suffered a mild TBI when the level of GFAP in a sample is equal to a reference level of GFAP of about 55 pg / mL, about 60 pg / mL, about 65 pg / mL, about 70 pg / mL, about 75 pg / mL, about 80 pg / mL, about 85 pg / mL, about 90 pg / mL, about 95 pg / mL, about 100 pg / mL, about 110 pg / mL, about 120 pg / mL, about 130 pg / mL, about 140 pg / mL, about 150 pg / mL, about 160 pg / mL, about 170 pg / mL, about 180 pg / mL, about 190 pg / mL, about 200 pg / mL, about 210 pg / mL, about 220 pg / mL, about 230 pg / mL, about 240 pg / mL, about 250 pg / mL, about 260 pg / mL, about 270 pg / mL, about 280 pg / mL, about 290 pg / mL, about 300 pg / mL, about 310 pg / mL, about 320 pg / mL, about 330 pg / mL, about 340 pg / mL, or about 350 pg / mL.As another example, in some embodiments, the method includes determining that a subject has suffered a mild TBI when the GFAP level in a sample is equal to a reference level of GFAP of about 360 pg / mL, about 370 pg / mL, about 380 pg / mL, about 390 pg / mL, about 400 pg / mL, about 410 pg / mL, about 420 pg / mL, about 430 pg / mL, about 440 pg / mL, about 450 pg / mL, about 460 pg / mL, about 470 pg / mL, about 480 pg / mL, about 490 pg / mL, about 500 pg / mL, about 510 pg / mL, about 520 pg / mL, about 530 pg / mL, about 540 pg / mL, or about 550 pg / mL. As another example, in some embodiments, the method includes determining that a subject has suffered a mild TBI when the GFAP level in a sample is equal to a reference level of GFAP of about 560 pg / mL, about 570 pg / mL, about 580 pg / mL, about 590 pg / mL, about 600 pg / mL, about 610 pg / mL, about 620 pg / mL, about 630 pg / mL, about 640 pg / mL, about 650 pg / mL, about 660 pg / mL, about 670 pg / mL, about 680 pg / mL, about 690 pg / mL, about 700 pg / mL, about 710 pg / mL, about 720 pg / mL, about 730 pg / mL, about 740 pg / mL, about 750 pg / mL, about 760 pg / mL, about 770 pg / mL, about 780 pg / mL, about 790 pg / mL, about 800 pg / mL, about 810 pg / mL, about 820 pg / mL, about 830 pg / mL, about 840 pg / mL, about 850 pg / mL, about 860 pg / mL, about 870 pg / mL, about 880 pg / mL, about 890 pg / mL, about 900 pg / mL, about 910 pg / mL, about 920 pg / mL, about 930 pg / mL, about 940 pg / mL, about 950 pg / mL, about 960 pg / mL, about 970 pg / mL, about 980 pg / mL, about 990 pg / mL, or about 1000 pg / mL. In some embodiments, the method includes determining that a subject has suffered a mild TBI when the GFAP level in a sample is equal to a reference level of GFAP of about 1100 pg / mL, about 1150 pg / mL, about 1200 pg / mL, about 1250 pg / mL, about 1300 pg / mL, about 1350 pg / mL, about 1400 pg / mL, about 1450 pg / mL, about 1500 pg / mL, or about 1521 pg / mL.

[0128] In some embodiments, the method includes determining that a subject has suffered a very mild traumatic brain injury when the GFAP level in a sample is below a reference level of GFAP, where the reference level of GFAP is about 55 pg / mL. For example, in some embodiments, the method includes determining that a subject has suffered a very mild traumatic brain injury when the GFAP level in a sample is below about 55 pg / mL, below about 50 pg / mL, below about 45 pg / mL, below about 40 pg / mL, below about 35 pg / mL, below about 30 pg / mL, below about 25 pg / mL, or below about 20 pg / mL.

[0129] In some embodiments, the sample is obtained within about 12 hours after an actual or suspected brain injury, and the reference level of GFAP is between about 40 pg / mL and about 1021 pg / mL. In some embodiments, the sample is obtained within about 12 hours after an actual or suspected head injury, and the method includes determining that the subject has suffered a mild TBI when the level of GFAP in the sample is equal to the reference level of GFAP of about 40 pg / mL to about 90 pg / mL, about 85 pg / mL to about 139 pg / mL, about 136 pg / mL to about 390 pg / mL, about 375 pg / mL to about 502 pg / mL, about 498 pg / mL to about 710 pg / mL, about 705 pg / mL to about 950 pg / mL, or about 931 pg / mL to about 1021 pg / mL.For example, in some embodiments, the method includes determining that a subject has suffered a mild TBI when the level of GFAP in a sample is equal to a reference level of GFAP of about 40 pg / mL, about 45 pg / mL, about 50 pg / mL, about 55 pg / mL, about 60 pg / mL, about 65 pg / mL, about 70 pg / mL, about 75 pg / mL, about 80 pg / mL, about 85 pg / mL, about 90 pg / mL, about 95 pg / mL, about 100 pg / mL, about 110 pg / mL, about 120 pg / mL, about 130 pg / mL, about 140 pg / mL, about 150 pg / mL, about 160 pg / mL, about 170 pg / mL, about 180 pg / mL, about 190 pg / mL, about 200 pg / mL, about 210 pg / mL, about 220 pg / mL, about 230 pg / mL, about 240 pg / mL, about 250 pg / mL, about 260 pg / mL, about 270 pg / mL, about 280 pg / mL, about 290 pg / mL, about 300 pg / mL, about 310 pg / mL, about 320 pg / mL, about 330 pg / mL, about 340 pg / mL, about 350 pg / mL, about 360 pg / mL, about 370 pg / mL, about 380 pg / mL, about 390 pg / mL, about 400 pg / mL, about 410 pg / mL, about 420 pg / mL, about 430 pg / mL, about 440 pg / mL, about 450 pg / mL, about 460 pg / mL, about 470 pg / mL, about 480 pg / mL, about 490 pg / mL, about 500 pg / mL, about 520 pg / mL, about 540 pg / mL, about 560 pg / mL, about 580 pg / mL, about 600 pg / mL, about 620 pg / mL, about 640 pg / mL, about 660 pg / mL, about 680 pg / mL, about 700 pg / mL, about 720 pg / mL, about 740 pg / mL, about 760 pg / mL, about 780 pg / mL, about 800 pg / mL, about 820 pg / mL, about 840 pg / mL, about 860 pg / mL, about 880 pg / mL, about 900 pg / mL, about 920 pg / mL, about 940 pg / mL, about 960 pg / mL, about 980 pg / mL, about 1000 pg / mL or about 1020 pg / mL.

[0130] In some embodiments, the sample is obtained within about 12 hours after an actual or suspected head injury, and the method includes determining that the subject has suffered a super - mild TBI when the GFAP level in the sample is less than a reference level of GFAP of about 40 pg / mL. For example, in some embodiments, the sample is obtained within about 12 hours after an actual or suspected head injury, and the method includes determining that the subject has suffered a super - mild traumatic brain injury when the GFAP level in the sample is below about 40 pg / mL, below about 38 pg / mL, below about 36 pg / mL, below about 34 pg / mL, below about 32 pg / mL, below about 30 pg / mL, below about 28 pg / mL, or below about 26 pg / mL, below about 24 pg / mL, or below about 22 pg / mL, or below about 20 pg / mL.

[0131] In some embodiments, the sample is obtained within about 2 weeks after an actual or suspected head injury, and the reference level of GFAP is between about 15 pg / mL and about 169 pg / mL. In some embodiments, the sample is obtained about 2 weeks after an actual or suspected head injury, and the method includes determining that the subject has suffered a mild TBI when the GFAP level in the sample is equal to a reference level of GFAP of about 15 pg / mL to about 23 pg / mL, about 15 pg / mL to about 55 pg / mL, about 23 pg / mL to about 88 pg / mL, about 75 pg / mL to about 143 pg / mL, or about 141 pg / mL to about 169 pg / mL. In some embodiments, the sample is obtained about 2 weeks after an actual or suspected head injury, and the method includes determining that the subject may have suffered a mild TBI when the GFAP level in the sample is equal to a reference level of GFAP of about 15 pg / mL, about 20 pg / mL, about 25 pg / mL, about 30 pg / mL, about 35 pg / mL, about 40 pg / mL, about 45 pg / mL, about 50 pg / mL, about 55 pg / mL, about 60 pg / mL, about 65 pg / mL, about 70 pg / mL, about 75 pg / mL, about 80 pg / mL, about 85 pg / mL, about 90 pg / mL, about 95 pg / mL, about 100 pg / mL, about 105 pg / mL, about 110 pg / mL, about 115 pg / mL, about 120 pg / mL, about 125 pg / mL, about 130 pg / mL, about 135 pg / mL, about 140 pg / mL, about 145 pg / mL, about 150 pg / mL, about 155 pg / mL, about 160 pg / mL, about 165 pg / mL, or about 169 pg / mL.

[0132] In some embodiments, the sample is obtained about 2 weeks after an actual or suspected head injury, and the method includes determining that a subject may have suffered a very mild TBI when the GFAP level in the sample is below a reference level of GFAP of about 15 pg / mL.

[0133] In some embodiments, the reference level of UCH-L1 is from about 160 pg / mL to about 533 pg / mL. In some embodiments, the method includes determining that a subject has suffered a mild traumatic brain injury (TBI) when the UCH-L1 level in the sample is equal to the reference level of UCH-L1, wherein the reference level of UCH-L1 is from about 160 pg / mL to about 533 pg / mL. In some embodiments, the method includes determining that a subject has suffered a mild traumatic brain injury (TBI) when the UCH-L1 level in the sample is equal to a reference level of UCH-L1 of from about 160 pg / mL to about 300 pg / mL, from about 250 pg / mL to about 450 pg / mL, from about 300 pg / mL to about 500 pg / mL, from about 350 pg / mL to about 533 pg / mL, from about 160 to about 200 pg / mL, from about 191 pg / mL to about 240 pg / mL, from about 235 pg / mL to about 300 pg / mL, from about 290 pg / mL to about 350 pg / mL, from about 325 pg / mL to about 475 pg / mL or from about 450 pg / mL to about 533 pg / mL. For example, in some embodiments, the method includes determining that a subject may have suffered a mild traumatic brain injury (TBI) when the UCH-L1 level in the sample is equal to a reference level of UCH-L1 of about 160 pg / mL, about 170 pg / mL, about 180 pg / mL, about 190 pg / mL, about 200 pg / mL, about 210 pg / mL, about 220 pg / mL, about 230 pg / mL, about 240 pg / mL, about 250 pg / mL, about 260 pg / mL, about 270 pg / mL, about 280 pg / mL, about 290 pg / mL, about 300 pg / mL, about 310 pg / mL, about 320 pg / mL, about 330 pg / mL, about 340 pg / mL, about 350 pg / mL, about 360 pg / mL, about 370 pg / mL, about 380 pg / mL, about 390 pg / mL, about 400 pg / mL, about 410 pg / mL, about 420 pg / mL, about 430 pg / mL, about 440 pg / mL, about 450 pg / mL, about 460 pg / mL, about 470 pg / mL, about 480 pg / mL, about 490 pg / mL, about 500 pg / mL, about 520 pg / mL, about 530 pg / mL or about 533 pg / mL.

[0134] In some embodiments, the method includes determining that a subject has suffered a very mild traumatic brain injury when the level of UCH-L1 in a sample is below a reference level of UCH-L1, wherein the reference level of UCH-L1 is about 160 pg / mL. For example, in some embodiments, the method includes determining that a subject has suffered a very mild TBI when the level of UCH-L1 in a sample is below about 160 pg / mL, below about 150 pg / mL, below about 140 pg / mL, below about 130 pg / mL, below about 120 pg / mL, below about 110 pg / mL, below about 100 pg / mL, below about 95 pg / mL, below about 90 pg / mL, below about 85 pg / mL, below about 80 pg / mL, below about 75 pg / mL, below about 70 pg / mL, below about 65 pg / mL, below about 60 pg / mL, below about 55 pg / mL, below about 50 pg / mL, below about 45 pg / mL, below about 40 pg / mL, below about 35 pg / mL, below about 30 pg / mL, below about 25 pg / mL, or below about 20 pg / mL. In some embodiments, the sample is obtained within about 12 hours after an actual or suspected head injury, and the reference level of UCH-L1 is between about 144 pg / mL and about 533 pg / mL. In some embodiments, the sample is obtained within about 12 hours after an actual or suspected head injury, and the method includes determining that a subject has suffered a mild TBI when the level of UCH-L1 in the sample is equal to a reference level of UCH-L1 of about 144 pg / mL to about 184 pg / mL, about 175 pg / mL to about 363 pg / mL, about 359 pg / mL to about 433 pg / mL, or about 425 pg / mL to about 533 pg / mL.For example, in some embodiments, the sample is obtained within about 12 hours after an actual or suspected head injury, and the method includes determining that the subject has suffered a mild TBI when the UCH-L1 level in the sample equals a reference level of UCH-L1 of about 144 pg / mL, about 150 pg / mL, about 160 pg / mL, about 170 pg / mL, about 180 pg / mL, about 190 pg / mL, about 200 pg / mL, about 210 pg / mL, about 220 pg / mL, about 230 pg / mL, about 240 pg / mL, about 250 pg / mL, about 260 pg / mL, about 270 pg / mL, about 280 pg / mL, about 290 pg / mL, about 300 pg / mL, about 310 pg / mL, about 320 pg / mL, about 330 pg / mL, about 340 pg / mL, about 350 pg / mL, about 360 pg / mL, about 370 pg / mL, about 380 pg / mL, about 390 pg / mL, about 400 pg / mL, about 410 pg / mL, about 420 pg / mL, about 430 pg / mL, about 440 pg / mL, about 450 pg / mL, about 460 pg / mL, about 470 pg / mL, about 480 pg / mL, about 490 pg / mL, about 500 pg / mL, about 520 pg / mL, about 530 pg / mL or about 533 pg / mL.

[0135] In some embodiments, the sample is obtained within about 12 hours after an actual or suspected head injury, and the method includes determining that the subject has suffered a very mild TBI when the UCH-L1 level in the sample is below a reference level of UCH-L1 of about 144 pg / mL. For example, in some embodiments, the sample is obtained within about 12 hours after an actual or suspected head injury, and the method includes determining that the subject has suffered a very mild TBI when the UCH-L1 level in the sample is below about 144 pg / mL, below about 140 pg / mL, below about 130 pg / mL, below about 120 pg / mL, below about 110 pg / mL, below about 100 pg / mL, below about 95 pg / mL, below about 90 pg / mL, below about 85 pg / mL, below about 80 pg / mL, below about 75 pg / mL, below about 70 pg / mL, below about 65 pg / mL, below about 60 pg / mL, below about 55 pg / mL, below about 50 pg / mL, below about 45 pg / mL, below about 40 pg / mL, below about 35 pg / mL, below about 30 pg / mL, below about 25 pg / mL, or below about 20 pg / mL. In some embodiments, the sample is obtained about 2 weeks after an actual or suspected head injury, and the reference level of UCH-L1 is between about 64 pg / mL and about 154 pg / mL. In some embodiments, the sample is obtained about 2 weeks after an actual or suspected head injury, and the method includes determining that the subject has suffered a mild TBI when the UCH-L1 level in the sample is equal to a reference level of UCH-L1 of about 64 pg / mL to about 69 pg / mL, about 68 pg / mL to about 131 pg / mL, or about 128 pg / mL to about 154 pg / mL. In some embodiments, the sample is obtained about 2 weeks after an actual or suspected head injury, and the method includes determining that the subject has suffered a mild TBI when the UCH-L1 level in the sample is equal to a reference level of UCH-L1 of about 64 pg / mL, about 70 pg / mL, about 75 pg / mL, about 80 pg / mL, about 85 pg / mL, about 90 pg / mL, about 95 pg / mL, about 100 pg / mL, about 105 pg / mL, about 110 pg / mL, about 115 pg / mL, about 120 pg / mL, about 125 pg / mL, about 130 pg / mL, about 135 pg / mL, about 140 pg / mL, about 145 pg / mL, about 150 pg / mL, or about 154 pg / mL.

[0136] In some embodiments, the sample is obtained about 2 weeks after an actual or suspected head injury, and the method includes determining that the subject has suffered a very mild TBI when the UCH-L1 level in the sample is below a reference level of UCH-L1 of about 64 pg / mL. For example, in some embodiments, the sample is obtained about 2 weeks after an actual or suspected head injury, and the method includes determining that the subject has suffered a very mild TBI when the UCH-L1 level in the sample is below about 64 pg / mL, below about 60 pg / mL, below about 55 pg / mL, below about 50 pg / mL, below about 45 pg / mL, below about 40 pg / mL, below about 35 pg / mL, below about 30 pg / mL, below about 25 pg / mL, or below about 20 pg / mL.

[0137] In some embodiments, the method further includes treating a subject (e.g., a human subject) evaluated as having a mild traumatic brain injury with a traumatic brain injury treatment, as described below. In some embodiments, the method further includes monitoring a subject (e.g., a human subject) evaluated as having a mild traumatic brain injury, as described below. In other embodiments, the method further includes monitoring a subject (e.g., a human subject) evaluated as having a very mild traumatic brain injury, as described below.

[0138] The nature of the assays employed in the methods described herein is not critical, and the assays can be any assay known in the art, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, or protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays or chromatographic or spectroscopic methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). However, assays or determinations that are capable of performing the claimed methods will be employed, such as assays having various sensitivities and sensitivities as described herein. In addition, the assays used in the methods described herein can be employed in a clinical chemistry format, such as will be known to those of ordinary skill in the art. Such assays are described in more detail herein. It is known in the art that values (e.g., reference levels, cut-off values, thresholds, specificities, sensitivities, concentrations of calibrators and / or controls) used in assays that employ a particular sample type (e.g., immunoassays using serum or point-of-care devices using whole blood) can be extrapolated to other assay formats using techniques known in the art (such as assay standardization). For example, one way to perform assay standardization is by applying a factor to the calibrator used in the assay to make the sample concentration readings higher or lower to obtain a slope that aligns with a comparative method. Other methods of standardizing the results obtained on one assay to another assay are well known and have been described in the literature (see, e.g., David Wild, Immunoassay Handbook, 4th Edition, Chapter 3.5, pages 315-322, the contents of which are incorporated herein by reference).

[0139] At least one assay for GFAP and at least one assay for UCH-L1 can be performed simultaneously. Alternatively, the assay for GFAP and the assay for UCH-L1 can be performed sequentially. The assays can be performed sequentially in any order. For example, the assay for GFAP can be performed first, followed by the assay for UCH-L1. As another example, the assay for UCH-L1 can be performed first, followed by the assay for GFAP.

[0140] In some embodiments, at least one determination for GFAP and / or at least one determination for UCH-L1 is each performed within about 10 to about 20 minutes. In some embodiments, at least one determination for GFAP and / or at least one determination for UCH-L1 is each performed within about 10 minutes. In some embodiments, at least one determination for GFAP and / or at least one determination for UCH-L1 is each performed within about 11 minutes. In some embodiments, at least one determination for GFAP and / or at least one determination for UCH-L1 is each performed within about 12 minutes. In some embodiments, at least one determination for GFAP and / or at least one determination for UCH-L1 is each performed within about 13 minutes. In some embodiments, at least one determination for GFAP and / or at least one determination for UCH-L1 is each performed within about 14 minutes. In some embodiments, at least one determination for GFAP and / or at least one determination for UCH-L1 is each performed within about 15 minutes. In some embodiments, at least one determination for GFAP and / or at least one determination for UCH-L1 is each performed within about 16 minutes. In some embodiments, at least one determination for GFAP and / or at least one determination for UCH-L1 is each performed within about 17 minutes. In some embodiments, at least one determination for GFAP and / or at least one determination for UCH-L1 is each performed within about 18 minutes. In some embodiments, at least one determination for GFAP and / or at least one determination for UCH-L1 is each performed within about 19 minutes. In some embodiments, at least one determination for GFAP and / or at least one determination for UCH-L1 is each performed within about 20 minutes. The nature of the determination employed in the methods described herein is not critical, and the test can be any determination known in the art, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, or protein immunostaining, electrophoretic analysis, protein determination, competitive binding assays, functional protein assays or chromatography or spectroscopy, such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). However, a test or determination capable of performing the claimed method will be employed, such as a determination having the various sensitivities and sensitivities as described herein. Additionally, the determinations used in the methods described herein can be employed in a clinical chemistry format, such as will be known to those of ordinary skill in the art. Such determinations are further described in detail in Sections 5-9 herein.It is known in the art that values used in assays employing a particular sample type (e.g., immunoassays using serum or point-of-care devices using whole blood), such as reference levels, cut-off values, thresholds, specificities, sensitivities, concentrations of calibrators and / or controls, can be extrapolated to other assay formats using techniques known in the art, such as assay standardization. For example, one way to perform assay standardization is to apply a factor to the calibrator used in the assay to make the sample concentration reading higher or lower to obtain a slope that aligns with a comparative method. Other methods of standardizing the results obtained on one assay to another assay are well known and have been described in the literature (see, e.g., David Wild, Immunoassay Handbook, 4th Edition, Chapter 3.5, pages 315 - 322, the contents of which are incorporated herein by reference).

[0141] 3. Treating and Monitoring Subjects Suffering from Traumatic Brain Injury

[0142] It is possible to treat or monitor a subject (e.g., a human subject) identified or evaluated as having a traumatic brain injury (e.g., mild TBI or ultra-mild TBI) in the methods described above. In some embodiments, the method further comprises treating a subject (e.g., a human subject (e.g., a human adult or a human pediatric subject)) determined to have TBI with a treatment for traumatic brain injury, such as any treatment known in the art. For example, the treatment of traumatic brain injury can take various forms depending on the severity of the head injury. For example, for a subject suffering from mild TBI, the treatment can include one or more of the following: (1) rest (e.g., physical and / or mental rest); (2) avoiding physical activity, such as exercise, work, school, play, or any combination thereof; (3) avoiding light or wearing sunglasses in the light;(4) Administer one or more types of medications, such as medications for relieving headaches or migraines (e.g., steroid anti-inflammatory drugs (e.g., corticosteroids (prednisone or dexamethasone)) or non-steroidal anti-inflammatory drugs (NSAIDs, such as aspirin, ibuprofen, naproxen sodium, etc.), medications for treating dizziness, anti-nausea medications (e.g., antiemetics), motion sickness medications (e.g., scopolamine, promethazine, dimenhydrinate, etc.), antidepressant medications (e.g., SSRIs, such as fluoxetine (Prozac), paroxetine (Paxil), fluvoxamine (Luvox), citalopram (Celexa), escitalopram (Cipralex), sertraline (Zoloft) and / or hallucinogenic drugs, such as psilocybin and MDMA, etc.), anti-anxiety medications (e.g., SSRIs, such as fluoxetine (Prozac), paroxetine (Paxil), fluvoxamine (Luvox), citalopram (Celexa), escitalopram (Cipralex), sertraline (Zoloft) and / or hallucinogenic drugs, such as psilocybin and MDMA, etc.), sleep aid medications (e.g., melatonin, estazolam, flurazepam, quazepam, temazepam, triazolam, etc.), muscle relaxants (e.g., in cases where there is injury or involvement of the muscles in or around the neck, reducing inflammation that may exacerbate TBI), anti-inflammatory drugs (e.g., steroid anti-inflammatory drugs or NSAIDs), medications for enhancing attention and / or concentration (e.g., psychostimulants, such as methylphenidate, etc.) and / or one or more natural therapy medications or treatments (e.g., non-hallucinogenic mushrooms, herbal teas, acupuncture, medical marijuana, etc.); (5) Provide one or more devices for treating nausea, such as acupressure wristbands (e.g., Psi Band); (6) Administer one or more nutraceuticals and / or nutritional compositions containing one or more omega-3 fatty acids, one or more vitamins (e.g., vitamin B and / or vitamin D), one or more fatty acids and / or one or more antioxidants; (7) Provide hyperbaric oxygen therapy; (8) Light therapy (i.e., helping to relieve mood swings, irritability and / or depression caused by TBI); (9) Physical therapy for TBI (e.g., treating dizziness, improving motor skills, etc.);(10) Occupational therapy for TBI (e.g., helping to improve memory, attention, concentration, etc.); (11) Individual, group counseling, and / or psychotherapy to help treat depression and / or anxiety caused by TBI; (12) Sleep therapy to help treat sleep disorders (e.g., insomnia or hypersomnia) caused by TBI; or (13) Any combination of (1)-(12).;

[0143] For subjects suffering from ultra - mild TBI, treatment may include providing the same or similar treatments as those for mild TBI, but the treatment may require a smaller dose or a shorter duration. For example, for ultra - mild TBI, treatment can include one or more of the following: (1) rest (e.g., physical and / or mental rest); (2) avoiding physical activities such as exercise, work, school, play, or any combination thereof; (3) avoiding light or wearing sunglasses in the light; (4) administering one or more types of drugs, which can be provided in a smaller dose or at a lower dosing frequency than recommended for mild TBI subjects. Such drugs can include drugs for relieving headache or migraine (e.g., steroid anti - inflammatory drugs (e.g., corticosteroids (prednisone or dexamethasone)) or non - steroid anti - inflammatory drugs (NSAIDs, such as aspirin, ibuprofen, naproxen sodium, etc.)), drugs for treating dizziness, anti - nausea drugs (e.g., antiemetics), motion sickness drugs (e.g., scopolamine, promethazine, dimenhydrinate, etc.), antidepressant drugs (e.g., SSRIs, such as fluoxetine (Prozac), paroxetine (Paxil), fluvoxamine (Luvox), citalopram (Celexa), escitalopram (Cipralex), sertraline (Zoloft) and / or hallucinogenic drugs, such as psilocybin and MDMA, etc.), anti - anxiety drugs (e.g., SSRIs, such as fluoxetine (Prozac), paroxetine (Paxil), fluvoxamine (Luvox), citalopram (Celexa), escitalopram (Cipralex), sertraline (Zoloft) and / or hallucinogenic drugs, such as psilocybin, MDMA, etc.), sleep - aid drugs (e.g., melatonin, estazolam, flurazepam, quazepam, temazepam, triazolam, etc.), muscle relaxants (e.g., in cases where there is injury or involvement of the muscles in or around the neck, reducing inflammation that may exacerbate TBI), anti - inflammatory drugs (e.g., steroid anti - inflammatory drugs or NSAIDs), drugs for improving attention and / or concentration (e.g., psychostimulants, such as methylphenidate, etc.) and / or one or more natural therapy drugs or treatments (e.g., non - hallucinogenic mushrooms, herbal teas, acupuncture, medical marijuana, etc.); (5) providing one or more devices for treating nausea, such as acupressure wristbands (e.g., Psi Band); (6) administering one or more nutraceuticals and / or nutritional compositions containing one or more omega - 3 fatty acids, one or more vitamins (e.g., vitamin B and / or vitamin D), one or more fatty acids and / or one or more antioxidants; (7) providing hyperbaric oxygen therapy; (8) light therapy (i.e., helping to relieve mood swings, irritability, and / or depression caused by TBI); (9) physical therapy for TBI (e.g., treating dizziness, improving motor skills, etc.); (10) occupational therapy for TBI (e.g., helping to improve memory, attention, concentration, etc.);(11)Individual, group counseling, and / or psychotherapy to assist in treating depression and / or anxiety caused by TBI; (12)Sleep therapy to assist in treating sleep disorders (e.g., insomnia or hypersomnia) caused by TBI; or (13)Any combination of (1)-(12). In some embodiments, the method further comprises monitoring a subject (e.g., a human subject) assessed as having a traumatic brain injury, such as a mild TBI or ultra-mild TBI).;

[0144] 4. Method for measuring UCH-L1 level

[0145] In the methods described above, the UCH-L1 level can be measured by any means, such as antibody-dependent methods, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, or chromatographic or spectroscopic methods, such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS), for example, those described in WO 2018 / 067468, WO2018 / 191531, WO2018 / 218169, and WO 2019 / 112860, the content of each patent being incorporated herein by reference. Additionally, the assay can be employed in a clinical chemistry format, such as would be known to those of skill in the art.

[0146] In some embodiments, measuring the UCH-L1 level comprises contacting a sample with a first specific binding member and a second specific binding member. In some embodiments, the first specific binding member is a capture antibody and the second specific binding member is a detection antibody. In some embodiments, measuring the UCH-L1 level comprises contacting the sample simultaneously or sequentially in any order with: (1) a capture antibody (e.g., a UCH-L1 capture antibody) that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form a capture antibody-UCH-L1 antigen complex (e.g., a UCH-L1 capture antibody-UCH-L1 antigen complex), and (2) a detection antibody (e.g., a UCH-L1 detection antibody) that comprises a detectable label and binds to an epitope on UCH-L1 that is not bound by the capture antibody to form a UCH-L1 antigen-detection antibody complex (e.g., a UCH-L1 antigen-UCH-L1 detection antibody complex), such that a capture antibody-UCH-L1 antigen-detection antibody complex (e.g., a UCH-L1 capture antibody-UCH-L1 antigen-UCH-L1 detection antibody complex) is formed; and measuring the amount or concentration of UCH-L1 in the sample based on a signal generated by the detectable label in the capture antibody-UCH-L1 antigen-detection antibody complex.

[0147] In some embodiments, the first specific binding member is immobilized on a solid support. In some embodiments, the second specific binding partner is immobilized on a solid support. In some embodiments, the first specific binding partner is a UCH-L1 antibody as described below.

[0148] In some embodiments, the sample is diluted or undiluted. In some embodiments, the sample is from about 1 to about 100 microliters. In some embodiments, the sample is from about 10 to about 90 microliters. In some embodiments, the sample is about 1 microliter, about 5 microliters, about 10 microliters, about 20 microliters, about 30 microliters, about 40 microliters, about 50 microliters, about 60 microliters, about 70 microliters, about 80 microliters, or about 90 microliters. In some embodiments, the sample is from about 1 to about 85 microliters, from about 1 to about 80 microliters, from about 1 to about 75 microliters, from about 1 to about 65 microliters, from about 1 to about 50 microliters, from about 1 to about 40 microliters, from about 1 to about 30 microliters, from about 1 to about 20 microliters, from about 1 to about 10 microliters, or from about 1 to about 5 microliters. In some embodiments, the sample is about 1 microliter, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, about 10 microliters, about 11 microliters, about 12 microliters, about 13 microliters, about 14 microliters, about 15 microliters, about 16 microliters, about 17 microliters, about 18 microliters, about 19 microliters, about 20 microliters, about 21 microliters, about 22 microliters, about 23 microliters, about 24 microliters, about 25 microliters, about 26 microliters, about 27 microliters, about 28 microliters, about 29 microliters, about 30 microliters, about 40 microliters, about 50 microliters, about 60 microliters, about 70 microliters, about 80 microliters, about 90 microliters, or about 100 microliters. In some embodiments, the sample is from about 1 to about 150 microliters or less or from about 1 to about 80 microliters or less.

[0149] Some instruments of point-of-care devices (such as Abbott Laboratories instruments Alinity and other core laboratory instruments) may be able to measure UCH-L1 levels in samples that are higher than or greater than 25,000 pg / mL.

[0150] Other detection methods include using nanopore devices or nanowell devices or may be adapted to be used on nanopore devices or nanowell devices. Examples of nanopore devices are described in International Patent Publication No. WO 2016 / 161402, which is incorporated herein by reference in its entirety. Examples of nanowell devices are described in International Patent Publication No. WO 2016 / 161400, which is incorporated herein by reference in its entirety.

[0151] 5. UCH-L1 Antibody

[0152] The methods described herein can use isolated antibodies that specifically bind to ubiquitin carboxyl-terminal hydrolase L1 (“UCH-L1”) (or fragments thereof), referred to as “UCH-L1 antibodies”. UCH-L1 antibodies can be used to assess UCH-L1 status as a measure of traumatic brain injury, detect the presence of UCH-L1 in a sample, quantify the amount of UCH-L1 present in a sample, or detect the presence of UCH-L1 in a sample and quantify its amount.

[0153] a. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1)

[0154] Ubiquitin carboxyl-terminal esterase L1 (“UCH-L1”), also known as “ubiquitin C-terminal hydrolase”, is a deubiquitinating enzyme. UCH-L1 is a member of a gene family that hydrolyzes the small C-terminal adducts of ubiquitin to produce ubiquitin monomers as products. The expression of UCH-L1 is highly specific for neurons and for cells of the diffuse neuroendocrine system and their tumors. It is present in large amounts in all neurons (1-2% of total brain protein), and is expressed particularly in neurons and testis / ovary. The catalytic triad of UCH-L1 contains cysteine at position 90, aspartic acid at position 176, and histidine at position 161, which are responsible for its hydrolase activity.

[0155] Human UCH-L1 can have the following amino acid sequence:

[0156] MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFPLTAQHENFRKKQIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGSVLKQFLSETEKMSPEDRAKCFEKNEAIQAAHDAVAQEGQCRVDDKVNFHFILFNNVDGHLYELDGRMPFPVNHGASSEDTLLKDAAKVCREFTEREQGEVRFSAVALCKAA (SEQ ID NO:1).

[0157] Human UCH-L1 can be a fragment or variant of SEQ ID NO:1. The length of a fragment of UCH-L1 can be between 5 and 225 amino acids, between 10 and 225 amino acids, between 50 and 225 amino acids, between 60 and 225 amino acids, between 65 and 225 amino acids, between 100 and 225 amino acids, between 150 and 225 amino acids, between 100 and 175 amino acids, or between 175 and 225 amino acids. The fragment can contain a number of contiguous amino acids from SEQ ID NO:1.

[0158] b. UCH-L1 recognition antibody

[0159] The antibody is an antibody that binds to UCH-L1, its fragments, epitopes of UCH-L1 or variants thereof. The antibody can be a fragment or a variant or derivative of an anti-UCH-L1 antibody. The antibody can be a polyclonal or monoclonal antibody. The antibody can be a chimeric antibody, a single-chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, a fully human antibody or an antibody fragment (such as a Fab fragment) or a mixture thereof. Antibody fragments or derivatives can include F(ab')2, Fv or scFv fragments. Antibody derivatives can be produced from peptidomimetics. In addition, the techniques described for generating single-chain antibodies can be adapted to produce single-chain antibodies.

[0160] The anti-UCH-L1 antibody can be a chimeric anti-UCH-L1 antibody or a humanized anti-UCH-L1 antibody. In one embodiment, both the humanized antibody and the chimeric antibody are monovalent. In one embodiment, both the humanized antibody and the chimeric antibody comprise a single Fab region linked to an Fc region.

[0161] Human antibodies can be derived from phage display technology or transgenic mice expressing human immunoglobulin genes. Human antibodies can be produced as a result of an immune response in a human body and isolated. See, for example, Funaro et al., BMC Biotechnology, 2008(8):85. Thus, the antibody can be a product of a human rather than an animal repertoire. Since it is derived from a human, the risk of a reaction against self-antigens can be minimized. Alternatively, standard yeast display libraries and display techniques can be used to select and isolate human anti-UCH-L1 antibodies. For example, an initial human single-chain variable fragment (scFv) library can be used to select human anti-UCH-L1 antibodies. Transgenic animals can be used to express human antibodies.

[0162] A humanized antibody can be an antibody molecule from an antibody of a non-human species that binds a desired antigen having one or more complementarity determining regions (CDRs) from a non-human species and framework regions from a human immunoglobulin molecule.

[0163] The antibody is distinguished from known antibodies in that it has a biological function different from those known in the art.

[0164] (1) Epitope

[0165] The antibody can immunospecifically bind to UCH-L1 (SEQ ID NO:1), its fragment or its variant. The antibody can immunospecifically recognize and bind to at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids or at least ten amino acids within the epitope region. The antibody can immunospecifically recognize and bind to an epitope having at least three consecutive amino acids, at least four consecutive amino acids, at least five consecutive amino acids, at least six consecutive amino acids, at least seven consecutive amino acids, at least eight consecutive amino acids, at least nine consecutive amino acids or at least ten consecutive amino acids within the epitope region.

[0166] c. Antibody preparation / production

[0167] The antibody can be prepared by any of a variety of techniques, including those well known to those skilled in the art. Generally, antibodies can be produced by cell culture techniques, which include monoclonal antibody production via conventional techniques or via transfection of antibody genes, heavy and / or light chains into suitable bacterial or mammalian cell hosts to permit antibody production (where the antibody can be recombinant). The various forms of the term "transfection" are intended to encompass the various techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although antibodies can be expressed in prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells is preferred and most preferred in mammalian host cells because such eukaryotic cells (especially mammalian cells) are more likely to express assembled and secreted properly folded and immunologically active antibodies than prokaryotic cells.

[0168] Exemplary mammalian host cells for expressing recombinant antibodies include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), which are used with a DHFR selection marker, such as described in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982); NS0 myeloma cells, COS cells and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a time sufficient to permit expression of the antibody in the host cell or more preferably to secrete the antibody into the medium in which the host cell is grown. The antibody can be recovered from the medium using standard protein purification methods.

[0169] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It should be understood that variations of the above procedures can be made. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of the light and / or heavy chains of an antibody. Recombinant DNA techniques can also be used to remove some or all of the DNA encoding one or both of the light or heavy chains that are not necessary for binding to the antigen of interest. The antibodies also encompass molecules expressed from such truncated DNA molecules. In addition, bispecific antibodies can be generated by cross-linking an antibody with a second antibody using standard chemical cross-linking methods, where one heavy chain and one light chain are the antibody (i.e., bind to human UCH-L1) and the other heavy chain and the other light chain are specific for an antigen other than human UCH-L1.

[0170] In a preferred system for recombinant expression of an antibody or an antigen-binding portion thereof, recombinant expression vectors encoding the antibody heavy chain and the antibody light chain are introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. In the recombinant expression vector, the antibody heavy chain and light chain genes are each operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high-level transcription of the genes. The recombinant expression vector also carries the DHFR gene, which allows selection / amplification using methotrexate to select CHO cells that have been transfected with the vector. The selected transformed host cells are cultured to express the antibody heavy chain and light chain, and the intact antibody is recovered from the culture medium. Recombinant expression vectors are prepared using standard molecular biology techniques, host cells are transfected, transformants are selected, host cells are cultured, and the antibody is recovered from the culture medium. Further, a method for synthesizing a recombinant antibody can be by culturing host cells in a suitable medium until the recombinant antibody is synthesized. The method can also include isolating the recombinant antibody from the culture medium.

[0171] The method for preparing monoclonal antibodies involves preparing an immortal cell line capable of producing antibodies with the desired specificity. Such cell lines can be generated from spleen cells obtained from immunized animals. The animals can be immunized with UCH-L1 or a fragment and / or variant thereof. The peptide used for immunizing the animals can contain amino acids encoding human Fc (e.g., the crystallizable fragment region or the tail region of a human antibody). The spleen cells can then be immortalized, for example, by fusion with a myeloma cell fusion partner. A variety of fusion techniques can be employed. For example, spleen cells and myeloma cells can be mixed with a non-ionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells but not myeloma cells. One such technique uses hypoxanthine, aminopterin, thymidine (HAT) selection. Another technique includes electrofusion. After a sufficient time (usually about 1 to 2 weeks), colonies of hybrids are observed. Individual colonies are selected and their culture supernatants are tested for binding activity to the polypeptide. Hybridomas with high reactivity and specificity can be used.

[0172] Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. In addition, various techniques can be employed to increase the yield, such as injecting the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host (such as a mouse). The monoclonal antibodies can then be harvested from the ascites or blood. Contaminants can be removed from the antibodies by conventional techniques such as chromatography, gel filtration, precipitation, and extraction. Affinity chromatography is an example of a method that can be used to purify antibodies.

[0173] The proteolytic enzyme papain preferentially cleaves the IgG molecule to produce several fragments, two of which (F(ab) fragments) each contain a covalently linked heterodimer with a complete antigen-binding site. Pepsin is capable of cleaving the IgG molecule to provide several fragments, including the F(ab’)2 fragment that contains two antigen-binding sites.

[0174] Fv fragments can be generated by proteolytic cleavage, preferably of IgM, and in rare cases can be IgG or IgA immunoglobulin molecules. Fv fragments can be derived using recombinant techniques. Fv fragments comprise a non-covalently linked VH::VL heterodimer that contains an antigen-binding site that retains many of the antigen recognition and binding capabilities of the native antibody molecule.

[0175] An antibody, antibody fragment, or derivative can comprise a set of heavy-chain and light-chain complementarity-determining regions (“CDRs”) respectively inserted between sets of heavy-chain and light-chain framework (“FR”) regions that provide support for the CDRs and define the spatial relationship between the CDRs. The set of CDRs can comprise the three hypervariable regions of the heavy-chain or light-chain V region.

[0176] Other suitable methods for generating or isolating antibodies with the required specificity can be used, including but not limited to methods for selecting recombinant antibodies from peptide or protein libraries (such as but not limited to phage, ribosome, oligonucleotide, RNA, cDNA, yeast, etc. display libraries); for example, recombinant antibodies can be obtained from various commercial suppliers such as Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid / Planegg, Del.), Biovation (Aberdeen, Scotland, UK), BioInvent (Lund, Sweden) using methods known in the art. See U.S. Patent Nos. 4,704,692, 5,723,323, 5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862. Alternative methods rely on immunizing transgenic animals capable of generating a human antibody repertoire (e.g., SCID mice, Nguyen et al. (1997) Microbiol. Immunol. 41:901-907; Sandhu et al. (1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol. 93:154-161), as known in the art and / or as described herein. Such techniques include but are not limited to ribosome display (Hanes et al. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998) Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibody production techniques (e.g., selected lymphocyte antibody method ("SLAM") (U.S. Patent No. 5,627,052; Wen et al. (1987) J. Immunol. 17:887-892; Babcock et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848); gel microdroplets and flow cytometry (Powell et al. (1990) Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass).; Gray et al. (1995) J. Imm. Meth. 182:155-163; Kenny et al. (1995) Bio / Technol. 13:787-790); B cell selection (Steenbakkers et al. (1994) Molec. Biol. Reports 19:125-134 (1994)).

[0177] Affinity matured antibodies can be generated by any of a variety of procedures known in the art. For example, see Marks et al., BioTechnology, 10:779-783 (1992) which describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described in Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-3319 (1995); Hawkins et al., J. Mol. Biol., 226:889-896 (1992). Selective mutagenesis at selected mutagenic positions and with activity enhancing amino acid residues at contact or hypermutable positions is described in U.S. Patent No. 6,914,128 B1.

[0178] Antibody variants can also be prepared by delivering a polynucleotide encoding the antibody to a suitable host to provide a transgenic animal or mammal, such as goats, cows, horses, and sheep, etc., which produce such antibodies in their milk. These methods are known in the art and are described, for example, in U.S. Patent Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362 and 5,304,489.

[0179] Antibody variants can also be prepared by delivering polynucleotides to provide transgenic plants and cultured plant cells (such as, but not limited to, tobacco, corn, and duckweed), which produce such antibodies, specific portions, or variants in plant parts or cells cultured therefrom. For example, Cramer et al. (1999) Curr. Top. Microbiol. Immunol. 240:95-118 and the references cited therein describe, for example, the use of inducible promoters to produce transgenic tobacco leaves expressing large amounts of recombinant proteins. Transgenic corn has been used to express mammalian proteins at commercial production levels, and their biological activities are the same as those produced in other recombinant systems or purified from natural sources. See, for example, Hood et al., Adv. Exp. Med. Biol. (1999) 464:127-147 and the references cited therein. Antibody variants have also been produced in large quantities by transgenic plant seeds (including tobacco seeds and potato tubers) including antibody fragments such as single-chain antibodies (scFv). See, for example, Conrad et al. (1998) Plant Mol. Biol. 38:101-109 and the references cited therein. Thus, transgenic plants can also be used to produce antibodies according to known methods.

[0180] Antibody derivatives can be produced, for example, by adding foreign sequences to modify immunogenicity or to reduce, enhance, or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life, or any other suitable characteristic. Generally, some or all of the non-human or human CDR sequences are maintained, while the non-human sequences of the variable and constant regions are replaced with human or other amino acids.

[0181] Small antibody fragments can be diabodies having two antigen-binding sites, where the fragment contains a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH VL). See, for example, EP 404,097; WO93 / 11161; and Hollinger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. By using a linker that is too short to allow pairing between the two domains of the same chain, the domains are forced to pair with the complementary domains of the other chain and produce two antigen-binding sites. See also U.S. Patent No. 6,632,926 to Chen, which is hereby incorporated by reference in its entirety and discloses antibody variants having one or more amino acids inserted into the hypervariable regions of the parental antibody and having a binding affinity for the target antigen that is at least about twice as strong as the binding affinity of the parental antibody for the antigen.

[0182] The antibody can be a linear antibody. Procedures for preparing linear antibodies are known in the art and described in Zapata et al., (1995) Protein Eng. 8(10):1057-1062. Briefly, these antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1), which form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0183] Antibodies can be recovered and purified from recombinant cell cultures by known methods, including but not limited to protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification.

[0184] Detectably labeled antibodies may be useful. Methods for conjugating antibodies to such reagents are known in the art. For illustrative purposes only, antibodies can be labeled with a detectable moiety, such as a radioactive atom, a chromophore, or a fluorophore, etc. Such labeled antibodies can be used in diagnostic techniques in vivo or in isolated test samples. They can be linked to cytokines, ligands, and another antibody. Suitable agents for conjugation to an antibody for anti-tumor effects include cytokines, such as interleukin 2 (IL-2) and tumor necrosis factor (TNF); photosensitizers for photodynamic therapy, including aluminum(III) phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine; radionuclides, such as iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), technetium-99m (99mTc), rhenium-186 (186Re), and rhenium-188 (188Re); antibiotics, such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacteria, plant, and other toxins, such as diphtheria toxin, Pseudomonas exotoxin A, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-α toxin, cytotoxins from Naja naja atra (cobra), and gelonin (a plant toxin); ribosome-inactivating proteins from plants, bacteria, and fungi, such as restrictocin (a ribosome-inactivating protein produced by Aspergillus restrictus), saporin (a ribosome-inactivating protein from Saponaria officinalis), and ribonuclease; tyrosine kinase inhibitors; ly207702 (difluoropurine nucleoside); liposomes containing antisense agents (e.g., antisense oligonucleotides, plasmids encoding toxins, methotrexate, etc.); and other antibodies or antibody fragments, such as F(ab).

[0185] Antibody production via the use of hybridoma technology, selected lymphocyte antibody method (SLAM), transgenic animals, and recombinant antibody libraries is described in more detail below.

[0186] (1) Anti-UCH-L1 monoclonal antibody using hybridoma technology

[0187] A variety of techniques known in the art can be used to prepare monoclonal antibodies, including the use of hybridoma, recombinant, and phage display techniques or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma technology, which includes those known in the art and taught, for example, in the following: Harlow et al., Antibodies: A Laboratory Manual, 2nd edition, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988); Hammerling et al., In Monoclonal Antibodies and T-Cell Hybridomas, (Elsevier, N.Y., 1981). It should also be noted that the term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not to the method by which the antibody is produced.

[0188] Methods for producing monoclonal antibodies and the antibodies produced by such methods can include culturing hybridoma cells that secrete the antibodies of the present disclosure, wherein the hybridomas are preferably produced by fusing spleen cells isolated from an animal immunized with UCH-L1, such as a rat or a mouse, with myeloma cells, and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete antibodies capable of binding the polypeptides of the present disclosure. Briefly, a rat can be immunized with the UCH-L1 antigen. In a preferred embodiment, the UCH-L1 antigen is administered together with an adjuvant to stimulate the immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immunostimulating complex). Such adjuvants can protect the polypeptide from rapid diffusion by sequestering the polypeptide in local deposits, or they can contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. Preferably, if the polypeptide is administered, the immunization protocol will involve two or more administrations of the polypeptide, carried out over a period of several weeks; however, a single administration of the polypeptide can also be used.

[0189] After immunizing an animal with the UCH-L1 antigen, antibodies and / or antibody-producing cells can be obtained from the animal. Serum containing anti-UCH-L1 antibodies is obtained from the animal by bleeding or sacrificing the animal. The serum obtained from the animal can be used, the immunoglobulin fraction can be obtained from the serum, or the anti-UCH-L1 antibody can be purified from the serum. The serum or immunoglobulin obtained in this way is polyclonal and thus has a range of heterogeneity.

[0190] Once an immune response is detected, e.g., antibodies specific for the antigen UCH-L1 are detected in rat serum, the rat spleen is harvested and the spleen cells are isolated. The spleen cells are then fused with any suitable myeloma cells (e.g., cells from the cell line SP20 available from the American Type Culture Collection (ATCC, Manassas, Va., US)) by well-known techniques. Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed for cells that secrete antibodies capable of binding UCH-L1 by methods known in the art. Ascites containing generally high levels of antibodies can be produced by immunizing rats with positive hybridoma clones.

[0191] In another embodiment, immortalized hybridomas that produce antibodies can be prepared from immunized animals. After immunization, the animals are sacrificed and the splenic B cells are fused with immortalized myeloma cells, as is well known in the art. See, e.g., Harlow and Lane, supra. In a preferred embodiment, the myeloma cells do not secrete immunoglobulin polypeptides (non-secreting cell lines). After fusion and antibiotic selection, the hybridomas are screened using UCH-L1, or a portion thereof, or cells expressing UCH-L1. In a preferred embodiment, an enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), preferably ELISA, is used for the initial screening. Examples of ELISA screening are provided in PCT Publication No. WO 00 / 37504.

[0192] Hybridomas that produce anti-UCH-L1 antibodies are selected, cloned, and further screened for desired characteristics, including robust hybridoma growth, high antibody production, and desired antibody characteristics. The hybridomas can be cultured and amplified in vivo in syngeneic animals, in animals lacking an immune system (e.g., nude mice), or in vitro in cell culture. Methods for selecting, cloning, and amplifying hybridomas are well known to those of ordinary skill in the art.

[0193] In a preferred embodiment, the hybridoma is a rat hybridoma. In another embodiment, the hybridoma is produced in a non-human, non-rat species, such as a mouse, sheep, pig, goat, cow, or horse. In another preferred embodiment, the hybridoma is a human hybridoma, wherein a human non-secretory myeloma is fused with a human cell expressing an anti-UCH-L1 antibody.

[0194] Antibody fragments that recognize specific epitopes can be produced by known techniques. For example, Fab and F(ab')2 fragments of the present disclosure can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce two identical Fab fragments) or pepsin (to produce F(ab')2 fragments). The F(ab')2 fragment of an IgG molecule retains the two antigen-binding sites of the larger (“parent”) IgG molecule, which includes two light chains (containing variable and constant light chain regions), the CH1 domain of the heavy chain, and the disulfide-bond-forming hinge region of the parent IgG molecule. Thus, the F(ab')2 fragment is still able to cross-link antigen molecules like the parent IgG molecule.

[0195] (2) Anti-UCH-L1 monoclonal antibody using SLAM

[0196] In another aspect of the present disclosure, recombinant antibodies are generated from single, isolated lymphocytes using a method known in the art as the selected lymphocyte antibody method (SLAM), as described in U.S. Patent No. 5,627,052; PCT Publication No. WO 92 / 02551; and Babcock et al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996). In this method, single cells secreting an antibody of interest are screened using an antigen-specific hemolytic plaque assay, e.g., lymphocytes derived from any immunized animal, wherein an antigen such as UCH-L1, a subunit of UCH-L1, or a fragment thereof is conjugated to sheep red blood cells using a linker (such as biotin), and used to identify single cells secreting antibodies specific for UCH-L1. After identifying the antibody-secreting cells of interest, the heavy and light chain variable region cDNAs are rescued from the cells by reverse transcriptase-PCR (RT-PCR), and these variable regions can then be expressed in the context of an appropriate immunoglobulin constant region (e.g., a human constant region) in a mammalian host cell (such as a COS or CHO cell). Host cells transfected with the amplified immunoglobulin sequences (derived from the in vivo selected lymphocytes) can then be further analyzed and selected in vitro, e.g., by panning the transfected cells to isolate cells expressing an antibody against UCH-L1. The amplified immunoglobulin sequences can be further manipulated in vitro, such as by in vitro affinity maturation methods. See, e.g., PCT Publication No. WO 97 / 29131 and PCT Publication No. WO 00 / 56772.

[0197] (3) Anti-UCH-L1 monoclonal antibodies using transgenic animals

[0198] In another embodiment of the present disclosure, antibodies are generated by immunizing a non-human animal comprising some or all of the human immunoglobulin loci with the UCH-L1 antigen. In one embodiment, the non-human animal is Transgenic mice, an engineered mouse strain that contains a large fragment of the human immunoglobulin locus and lacks murine antibody production. See, e.g., Green et al., Nature Genetics, 7:13-21 (1994) and U.S. Patent Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364. See also PCT Publication Nos. WO 91 / 10741, WO 94 / 02602, WO 96 / 34096, WO 96 / 33735, WO 98 / 16654, WO 98 / 24893, WO 98 / 50433, WO 99 / 45031, WO 99 / 53049, WO 00 / 09560 and WO 00 / 37504. Transgenic mice generate a human-like repertoire of fully human antibodies and produce antigen-specific human monoclonal antibodies. Transgenic mice contain approximately 80% of the human antibody repertoire by introduction of megabase-sized, germline configuration YAC fragments of the human heavy chain locus and the λ light chain locus. See Mendez et al., Nature Genetics, 15:146-156 (1997); Green and Jakobovits, J. Exp. Med., 188:483-495 (1998), the disclosures of which are incorporated herein by reference.

[0199] (4) Anti-UCH-L1 monoclonal antibodies using recombinant antibody libraries

[0200] Antibodies of the present disclosure can also be prepared using in vitro methods, where antibody libraries are screened to identify antibodies having the desired UCH-L1 binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include those described in the following documents: for example, U.S. Patent No. 5,223,409 (Ladner et al.); PCT Publication No. WO 92 / 18619 (Kang et al.); PCT Publication No. WO 91 / 17271 (Dower et al.); PCT Publication No. WO 92 / 20791 (Winter et al.); PCT Publication No. WO 92 / 15679 (Markland et al.); PCT Publication No. WO 93 / 01288 (Breitling et al.); PCT Publication No. WO 92 / 01047 (McCafferty et al.); PCT Publication No. WO 92 / 09690 (Garrard et al.); Fuchs et al., Bio / Technology, 9:1369-1372 (1991); Hay et al., Hum. Antibod. Hybridomas, 3:81-85 (1992); Huse et al., Science, 246:1275-1281 (1989); McCafferty et al., Nature, 348:552-554 (1990); Griffiths et al., EMBO J., 12:725-734 (1993); Hawkins et al., J. Mol. Biol., 226:889-896 (1992); Clackson et al., Nature, 352:624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992); Garrard et al., Bio / Technology, 9:1373-1377 (1991); Hoogenboom et al., Nucl. Acids Res., 19:4133-4137 (1991); Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991); U.S. Patent Application Publication No. 2003 / 0186374; and PCT Publication No. WO 97 / 29131, the content of each of which is incorporated herein by reference.

[0201] The recombinant antibody library can be from a subject immunized with UCH-L1 or a portion of UCH-L1. Alternatively, the recombinant antibody library can be from a naïve subject, i.e., a human not immunized with UCH-L1, such as a human antibody library from a human subject not immunized with human UCH-L1. The antibodies of the present disclosure are selected by screening a recombinant antibody library with a peptide comprising human UCH-L1, thereby selecting those antibodies that recognize UCH-L1. Methods for performing such screening and selection are well known in the art, such as those described in the references in the preceding paragraph. To select the antibodies of the present disclosure that have a specific binding affinity for UCH-L1, such as those that dissociate from human UCH-L1 at a specific K off rate constant, surface plasmon resonance methods known in the art can be used to select antibodies having the desired K off rate constant. To select the antibodies of the present disclosure that have a specific neutralizing activity against hUCH-L1, such as those having a specific IC 50 , standard methods known in the art for assessing inhibition of UCH-L1 activity can be used.

[0202] In one aspect, the present disclosure relates to an isolated antibody or antigen-binding portion thereof that binds human UCH-L1. Preferably, the antibody is a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody.

[0203] For example, various phage display methods known in the art can also be used to generate antibodies. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. Such phages can be used to display antigen-binding domains expressed from combinatorial libraries or combinatorial antibody libraries (e.g., human or murine). Phages expressing antigen-binding domains that bind to an antigen of interest can be identified using, for example, a labeled antigen or an antigen bound or captured to a solid surface or bead. The phages used in these methods are typically filamentous phages, including fd and M13 binding domains expressed from phage, and Fab, Fv, or disulfide-stabilized Fv antibody domains are recombinantly fused to phage gene III or gene VIII proteins. Examples of phage display methods that can be used to prepare antibodies include those disclosed in the following references: Brinkmann et al., J. Immunol. Methods, 182:41-50 (1995); Ames et al., J. Immunol. Methods, 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic et al., Gene, 187:9-18 (1997); Burton et al., Advances in Immunology, 57:191-280 (1994); PCT Publication No. WO 92 / 01047; PCT Publication No. WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0204] As described in the above references, after phage selection, the antibody-encoding regions can be isolated from the phage and used to generate full antibodies, including human antibodies or any other desired antigen-binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, as detailed below. For example, techniques known in the art for recombinant production of Fab, Fab', and F(ab')2 fragments can also be used, such as those disclosed in the following documents: PCT Publication No. WO 92 / 22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); Sawai et al., Am. J. Reprod. Immunol., 34:26-34 (1995); and Better et al., Science, 240:1041-1043 (1988). Examples of techniques that can be used to generate single-chain Fv and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88 (1991); Shu et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999 (1993); and Skerra et al., Science, 240:1038-1041 (1988).

[0205] As an alternative to screening a recombinant antibody library by phage display, other methods known in the art for screening large combinatorial libraries can be applied to identify the antibodies of the present disclosure. One type of alternative expression system is a system in which a recombinant antibody library is expressed as an RNA-protein fusion, as described in PCT Publication No. WO 98 / 31700 (Szostak and Roberts) and Roberts and Szostak, Proc. Natl. Acad. Sci. USA, 94:12297-12302 (1997). In this system, a synthetic mRNA carrying puromycin (a peptidyl acceptor antibiotic) at its 3' end is produced by in vitro translation, resulting in a covalent fusion between the mRNA and the peptide or protein it encodes. Thus, specific mRNAs can be enriched from a complex mixture of mRNAs (e.g., a combinatorial library) based on the properties of the encoded peptide or protein (e.g., an antibody) or a portion thereof, such as the binding of the antibody or a portion thereof to a bispecific antigen. The nucleic acid sequences encoding the antibody or a portion thereof recovered from screening such libraries can be expressed by recombinant means (e.g., in mammalian host cells) as described above, and can additionally be subjected to further affinity maturation by additional rounds of screening of the mRNA-peptide fusions in which mutations have been introduced into the initially selected sequences or by other methods for in vitro affinity maturation of recombinant antibodies as described above. A preferred example of such a method is the PROfusion display technology.

[0206] In another method, yeast display methods known in the art can also be used to generate antibodies. In yeast display methods, antibody domains are tethered to the yeast cell wall using genetic methods and displayed on the yeast surface. Specifically, such yeast can be used to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Examples of yeast display methods that can be used to prepare antibodies include the methods disclosed in U.S. Patent No. 6,699,658 (Wittrup et al.), which is incorporated herein by reference.

[0207] d. Generation of recombinant UCH-L1 antibodies

[0208] Antibodies can be produced by any of a number of techniques known in the art. For example, from host cell expression, where expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to cover the various techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although it is possible to express the antibodies of the present disclosure in prokaryotic or eukaryotic host cells, it is preferred to express the antibodies in eukaryotic cells and most preferably in mammalian host cells, because such eukaryotic cells (and especially mammalian cells) are more likely to assemble and secrete correctly folded and immunologically active antibodies compared to prokaryotic cells.

[0209] Exemplary mammalian host cells for expressing the recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), which are used with a DHFR selection marker, such as described in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982); NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a time sufficient to allow the antibody to be expressed in the host cell or more preferably secreted into the medium in which the host cell is grown. The antibody can be recovered from the medium using standard protein purification methods.

[0210] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It should be understood that variations of the above procedures can be made. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of the light and / or heavy chains of the antibodies of the present disclosure. Recombinant DNA techniques can also be used to remove some or all of the DNA encoding portions of one or both of the light or heavy chains that are not necessary for binding to the antigen of interest. The antibodies of the present disclosure also cover molecules expressed from such truncated DNA molecules. In addition, bispecific antibodies can be produced by cross-linking the antibodies of the present disclosure with a second antibody using standard chemical cross-linking methods, where one heavy chain and one light chain are the antibodies of the present disclosure (i.e., bind to human UCH-L1) and the other heavy chain and the other light chain are specific for an antigen other than human UCH-L1.

[0211] In a preferred system for recombinantly expressing an antibody of the present disclosure, or an antigen-binding portion thereof, recombinant expression vectors encoding the antibody heavy chain and the antibody light chain are introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. In the recombinant expression vector, the antibody heavy chain and light chain genes are each operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high-level transcription of the genes. The recombinant expression vector also carries the DHFR gene, which allows selection / amplification using methotrexate to select CHO cells that have been transfected with the vector. The selected transformed host cells are cultured to express the antibody heavy chain and light chain, and the intact antibody is recovered from the culture medium. The recombinant expression vector is prepared using standard molecular biology techniques, the host cells are transfected, the transformants are selected, the host cells are cultured, and the antibody is recovered from the culture medium. Further, the present disclosure provides a method for synthesizing a recombinant antibody of the present disclosure, the method comprising culturing the host cells of the present disclosure in a suitable medium until the recombinant antibody of the present disclosure is synthesized. The method may further comprise isolating the recombinant antibody from the culture medium.

[0212] (1) Humanized antibody

[0213] A humanized antibody can be an antibody or a variant, derivative, analogue or portion thereof that immunospecifically binds to an antigen of interest and comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and complementarity-determining regions (CDRs) having substantially the amino acid sequence of a non-human antibody. A humanized antibody can be derived from a non-human species antibody that binds to a desired antigen having one or more complementarity-determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule.

[0214] As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one and usually two variable domains (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., the donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. According to one aspect, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc) (usually the constant region of a human immunoglobulin). In some embodiments, a humanized antibody contains the variable domains of a light chain as well as at least the heavy chain. An antibody may also include the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, a humanized antibody contains only a humanized light chain. In some embodiments, a humanized antibody contains only a humanized heavy chain. In certain embodiments, a humanized antibody contains only the humanized variable domains of the light chain and / or the heavy chain.

[0215] A humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including but not limited to IgG1, IgG2, IgG3 and IgG4. A humanized antibody can contain sequences from more than one class or isotype, and specific constant domains can be selected using techniques well known in the art to optimize the desired effector functions.

[0216] The framework and CDR regions of a humanized antibody need not precisely correspond to the parental sequences, e.g., the donor antibody CDR or consensus framework can be mutagenized by substituting, inserting or / or deleting at least one amino acid residue such that the CDR or framework residue at that site does not correspond to the donor antibody or consensus framework. However, in one embodiment, such mutations will not be extensive. Generally, at least 90%, at least 95%, at least 98% or at least 99% of the residues of a humanized antibody will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, 1987)). In an immunoglobulin family, each position in the consensus sequence is occupied by the amino acid that occurs most frequently in that position in the family. If two amino acids occur with equal frequency, either amino acid can be included in the consensus sequence.

[0217] Humanized antibodies can be designed to minimize unwanted immune responses to rodent anti - human antibodies, which limits the duration and effectiveness of the therapeutic applications of those moieties in human recipients. Humanized antibodies can have one or more amino acid residues introduced therein from non - human sources. These non - human residues are often referred to as "import" residues and are typically taken from variable domains. Humanization can be performed by replacing the corresponding sequences of a human antibody with hypervariable region sequences. Thus, such "humanized" antibodies are chimeric antibodies in which substantially less than the complete human variable domain has been replaced by the corresponding sequences from non - human species. See, for example, U.S. Patent No. 4,816,567, the content of which is incorporated herein by reference. A humanized antibody can be a human antibody in which some hypervariable region residues and possibly some FR residues are replaced with residues from similar sites in a rodent antibody. Any known method can be used for humanizing or engineering the antibodies of the present disclosure, such as, but not limited to, those described in U.S. Patent Nos. 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539, and 4,816,567.

[0218] Humanized antibodies can retain high affinity for UCH - L1 and other favorable biological properties. Humanized antibodies can be prepared by a process of analyzing the parental sequence and various conceptual humanized products using three - dimensional models of the parental and humanized sequences. Three - dimensional immunoglobulin models are generally available. Computer programs that illustrate and display the possible three - dimensional conformational structures of selected candidate immunoglobulin sequences are available. Examining these displays allows analysis of the possible role of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from acceptor and import sequences such that the desired antibody characteristics, such as increased affinity for UCH - L1, are achieved. Generally speaking, hypervariable region residues can be directly and most substantially involved in affecting antigen binding.

[0219] As an alternative to humanization, human antibodies (also referred to herein as "fully human antibodies") can be produced. For example, it is possible to isolate human antibodies from libraries via PROfusion and / or yeast - related technologies. Transgenic animals (e.g., mice) can also be produced that are capable of generating a complete human antibody repertoire in the absence of endogenous immunoglobulin production after immunization. For example, in chimeric and germline mutant mice, the antibody heavy - chain joining region (J H)Homozygous deletion of the gene results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies upon antigen challenge. Humanized or fully human antibodies can be prepared according to the methods described in U.S. Patent Nos. 5,770,429, 5,833,985, 5,837,243, 5,922,845, 6,017,517, 6,096,311, 6,111,166, 6,270,765, 6,303,755, 6,365,116, 6,410,690, 6,682,928, and 6,984,720, the contents of each of which are incorporated herein by reference.

[0220] e. anti-UCH-L1 antibody

[0221] Anti-UCH-L1 antibodies can be generated using the techniques described above and using conventional techniques known in the art. In some embodiments, the anti-UCH-L1 antibody can be an unconjugated UCH-L1 antibody, such as UCH-L1 available from: United State Biological (Catalog No.: 031320); Cell Signaling Technology (Catalog No.: 3524); Sigma-Aldrich (Catalog No.: HPA005993); Santa Cruz Biotechnology, Inc. (Catalog No.: sc-58593 or sc-58594); R&D Systems (Catalog No.: MAB6007); Novus Biologicals (Catalog No.: NB600-1160); Biorbyt (Catalog No.: orb33715); Enzo Life Sciences, Inc. (Catalog No.: ADI-905-520-1); Bio-Rad (Catalog No.: VMA00004); BioVision (Catalog No.: 6130-50); Abcam (Catalog No.: ab75275 or ab104938); Invitrogen Antibodies (Catalog No.: 480012); ThermoFisher Scientific (Catalog No.: MA1-46079, MA5-17235, MA1-90008 or MA1-83428); EMD Millipore (Catalog No.: MABN48); or Sino Biological Inc. (Catalog No.: 50690-R011). The anti-UCH-L1 antibody can be conjugated to a fluorophore, such as a conjugated UCH-L1 antibody available from BioVision (Catalog No.: 6960-25) or Aviva Systems Biology (Catalog No. OAAF01904-FITC).

[0222] Alternatively, the antibodies described in WO 2018 / 067474, WO2018 / 081649, U.S. Patent No. 11,078,298, U.S. Publication No. 2019 / 0502127, and / or Bazarian et al., “Accuracy of a rapid GFAP / UCH-L1 test for the prediction of intracranial injuries on head CT after mild traumatic brain injury”, Acad. Emerg. Med., (August 6, 2021) can also be used, the contents of which are incorporated herein by reference.

[0223] 6. Method for measuring GFAP level

[0224] In the methods described above, the GFAP level can be measured by any means, such as antibody-dependent methods, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, or protein immunostaining, electrophoretic analysis, protein determination, competitive binding assays, functional protein assays, or chromatographic or spectroscopic methods, such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS), for example, those described in WO 2018 / 067474, WO2018 / 191531, WO2018 / 218169 and WO 2019 / 112860, the content of each patent being incorporated herein by reference. Additionally, the determination can be in the form of clinical chemistry, such as would be known to those skilled in the art.

[0225] In some embodiments, measuring the GFAP level comprises contacting a sample with a first specific binding member and a second specific binding member. In some embodiments, the first specific binding member is a capture antibody and the second specific binding member is a detection antibody. In some embodiments, measuring the GFAP level comprises contacting the sample simultaneously or sequentially in any order with: (1) a capture antibody (e.g., a GFAP capture antibody) that binds to an epitope on GFAP or a GFAP fragment to form a capture antibody-GFAP antigen complex (e.g., a GFAP capture antibody-GFAP antigen complex), and (2) a detection antibody (e.g., a GFAP detection antibody) that comprises a detectable label and binds to an epitope on GFAP that is not bound by the capture antibody to form a GFAP antigen-detection antibody complex (e.g., a GFAP antigen-GFAP detection antibody complex), such that a capture antibody-GFAP antigen-detection antibody complex (e.g., a GFAP capture antibody-GFAP antigen-GFAP detection antibody complex) is formed; and measuring the amount or concentration of GFAP in the sample based on a signal generated by the detectable label in the capture antibody-GFAP antigen-detection antibody complex.

[0226] In some embodiments, the first specific binding member is immobilized on a solid support. In some embodiments, the second specific binding partner is immobilized on a solid support. In some embodiments, the first specific binding partner is a GFAP antibody as described below.

[0227] In some embodiments, the sample is diluted or undiluted. In some embodiments, the sample is from about 1 to about 30 microliters. In some embodiments, the sample is from about 10 to about 30 microliters. In some embodiments, the sample is about 20 microliters. In some embodiments, the sample is from about 1 to about 25 microliters, from about 1 to about 24 microliters, from about 1 to about 23 microliters, from about 1 to about 22 microliters, from about 1 to about 21 microliters, from about 1 to about 20 microliters, from about 1 to about 18 microliters, from about 1 to about 17 microliters, from about 1 to about 16 microliters, or about 15 microliters. In some embodiments, the sample is about 1 microliter, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, about 10 microliters, about 11 microliters, about 12 microliters, about 13 microliters, about 14 microliters, about 15 microliters, about 16 microliters, about 17 microliters, about 18 microliters, about 19 microliters, about 20 microliters, about 21 microliters, about 22 microliters, about 23 microliters, about 24 microliters, about 25 microliters, about 26 microliters, about 27 microliters, about 28 microliters, about 29 microliters, or about 30 microliters. In some embodiments, the sample is from about 1 to about 150 microliters or less or from about 1 to about 30 microliters or less.

[0228] Some instruments of point-of-care devices, such as Abbott Laboratories instruments Alinity and other core laboratory instruments, may be able to measure GFAP levels in a sample that are higher than or greater than 25,000 pg / mL.

[0229] Other detection methods include using nanopore devices or nano-well devices or may be adapted to be used on nanopore devices or nano-well devices. Examples of nanopore devices are described in International Patent Publication No. WO 2016 / 161402, which is incorporated herein by reference in its entirety. Examples of nano-well devices are described in International Patent Publication No. WO 2016 / 161400, which is incorporated herein by reference in its entirety.

[0230] 7. GFAP Antibody

[0231] The methods described herein may use an isolated antibody that specifically binds to glial fibrillary acidic protein (“GFAP”) (or a fragment thereof), referred to as a “GFAP antibody”. The GFAP antibody can be used to assess the GFAP status as a measure of traumatic brain injury, detect the presence of GFAP in a sample, quantify the amount of GFAP present in a sample, or detect the presence of GFAP in a sample and quantify its amount.

[0232] a. Glial Fibrillary Acidic Protein (GFAP)

[0233] Glial fibrillary acidic protein (GFAP) is a 50 kDa cytoplasmic filamentous protein that forms part of the cytoskeleton in astrocytes and has been shown to be the most specific marker for astrocyte-derived cells. The GFAP protein is encoded by the human GFAP gene. GFAP is the major intermediate filament in mature astrocytes. In the central rod domain of the molecule, GFAP has considerable structural homology with other intermediate filaments. GFAP participates in the movement and shape of astrocytes by providing structural stability to astrocytic processes. Glial fibrillary acidic protein and its breakdown products (GFAP-BDP) are brain-specific proteins released into the blood as part of the pathophysiological response following traumatic brain injury (TBI). After injury to the human CNS by trauma, genetic disorders, or chemicals, astrocytes proliferate and exhibit extensive hypertrophy of the cell body and processes, and GFAP is significantly upregulated. In contrast, GFAP production gradually decreases as astrocytic malignancies increase. GFAP can also be detected in Schwann cells, enteric glial cells, salivary gland tumors, metastatic renal cell carcinomas, epiglottic cartilage, pituitary cells, immature oligodendrocytes, papillary meningiomas, and mammary myoepithelial cells.

[0234] Human GFAP can have the following amino acid sequence:

[0235] MERRRITSAARRSYVSSGEMMVGGLAPGRRLGPGTRLSLARMPPPLPTRVDFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTKLADVYQAELRELRLRLDQLTANSARLEVERDNLAQDLATVRQKLQDETNLRLEAENNLAAYRQEADEATLARLDLERKIESLEEEIRFLRKIHEEEVRELQEQLARQQVHVELDVAKPDLTAALKEIRTQYEAMASSNMHEAEEWYRSKFADLTDAAARNAELLRQAKHEANDYRRQLQSLTCDLESLRGTNESLERQMREQEERHVREAASYQEALARLEEEGQSLKDEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKESKQEHKDVM (SEQ ID NO:2).

[0236] Human GFAP can be a fragment or variant of SEQ ID NO:2. The length of the fragment of GFAP can be between 5 and 400 amino acids, between 10 and 400 amino acids, between 50 and 400 amino acids, between 60 and 400 amino acids, between 65 and 400 amino acids, between 100 and 400 amino acids, between 150 and 400 amino acids, between 100 and 300 amino acids, or between 200 and 300 amino acids. The fragment can contain a number of contiguous amino acids from SEQ ID NO:2. The human GFAP fragment or variant of SEQ ID NO:2 can be a GFAP breakdown product (BDP). The GFAP BDP can be 38 kDa, 42 kDa (weaker 41 kDa), 47 kDa (weaker 45 kDa), 25 kDa (weaker 23 kDa), 19 kDa, or 20 kDa. In some embodiments, the human GFAP fragment or variant can be a GFAP BDP containing between 5 and 25 amino acids, between 5 and 50 amino acids, between 5 and 100 amino acids, or between 5 and 200 amino acids.

[0237] It has been found that using at least two antibodies that bind to non-overlapping epitopes within a GFAP breakdown product (BDP), such as the 38 kDa BDP defined by amino acids 60 - 383 of the GFAP protein sequence (SEQ ID NO:2), can help maintain the dynamic range and low-end sensitivity of an immunoassay. In one aspect, at least two antibodies bind to non-overlapping epitopes near the N-terminus of the 38 kDa BDP. In another aspect, at least two antibodies bind to non-overlapping epitopes between amino acids 60 - 383 of SEQ ID NO:2. In another aspect, at least one first antibody, such as a capture antibody, binds to an epitope near the N-terminus of the 38 kDa BDP, and at least one second antibody, such as a detection antibody, binds to an epitope near the middle of the 38 kDa BDP, with the second antibody not overlapping with the first antibody. In another aspect, at least one first antibody, such as a capture antibody, binds to an epitope between amino acids 60 - 383 of SEQ ID NO:2, and at least one second antibody binds to an epitope between amino acids 60 - 383 of SEQ ID NO:2, with the second antibody not overlapping with the first antibody. The length of the epitope bound by the first antibody can be 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, or 15 amino acids. The length of the epitope bound by the second antibody can be 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, or 15 amino acids. A person skilled in the art can readily determine antibodies that bind to non-overlapping epitopes within the 38 kDa BDP defined by amino acids 60 - 383 of SEQ ID NO:2 using conventional techniques known in the art.

[0238] b. GFAP - recognizing antibody

[0239] The antibody is an antibody that binds to GFAP, its fragments, epitopes of GFAP, or variants thereof. The antibody can be a fragment or a variant or derivative of an anti - GFAP antibody. The antibody can be a polyclonal or monoclonal antibody. The antibody can be a chimeric antibody, a single - chain antibody, an affinity - matured antibody, a human antibody, a humanized antibody, a fully human antibody, or an antibody fragment (such as a Fab fragment) or a mixture thereof. Antibody fragments or derivatives can include F(ab')2, Fv, or scFv fragments. Antibody derivatives can be produced from peptidomimetics. In addition, the techniques described for generating single - chain antibodies can be adapted to generate single - chain antibodies.

[0240] The anti - GFAP antibody can be a chimeric anti - GFAP or a humanized anti - GFAP antibody. In one embodiment, both the humanized antibody and the chimeric antibody are monovalent. In one embodiment, both the humanized antibody and the chimeric antibody comprise a single Fab region linked to an Fc region.

[0241] Human antibodies can be derived from phage - display technology or transgenic mice expressing human immunoglobulin genes. Human antibodies can be produced as a result of an immune response in a human body and isolated. See, for example, Funaro et al., BMC Biotechnology, 2008(8):85. Thus, the antibody can be a product of a human rather than an animal repertoire. Since it is derived from a human, the risk of a reaction against self - antigens can be minimized. Alternatively, standard yeast display libraries and display techniques can be used to select and isolate human anti - GFAP antibodies. For example, an initial human single - chain variable fragment (scFv) library can be used to select human anti - GFAP antibodies. Transgenic animals can be used to express human antibodies.

[0242] A humanized antibody can be an antibody molecule from an antibody of a non - human species that binds a desired antigen having one or more complementarity - determining regions (CDRs) from a non - human species and framework regions from a human immunoglobulin molecule.

[0243] The antibody is distinguished from known antibodies in that it has a biological function different from those known in the art.

[0244] (1) Epitope

[0245] The antibody can immunospecifically bind to GFAP (SEQ ID NO: 2), its fragment or its variant. The antibody can immunospecifically recognize and bind to at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids or at least ten amino acids within an epitope region. The antibody can immunospecifically recognize and bind to an epitope having at least three consecutive amino acids, at least four consecutive amino acids, at least five consecutive amino acids, at least six consecutive amino acids, at least seven consecutive amino acids, at least eight consecutive amino acids, at least nine consecutive amino acids or at least ten consecutive amino acids within an epitope region.

[0246] c. Antibody preparation / production

[0247] The antibody can be prepared by any of a variety of techniques, including those well known to those skilled in the art. Generally, antibodies can be produced by cell culture techniques, which include monoclonal antibody production via conventional techniques or via transfection of antibody genes, heavy and / or light chains into suitable bacterial or mammalian cell hosts to permit antibody production (wherein the antibody can be recombinant). The various forms of the term "transfection" are intended to encompass the various techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although antibodies can be expressed in prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells is preferred, and most preferably in mammalian host cells, because such eukaryotic cells (especially mammalian cells) are more likely than prokaryotic cells to express assembled and secreted properly folded and immunologically active antibodies.

[0248] Exemplary mammalian host cells for expressing recombinant antibodies include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), which are used with a DHFR selectable marker, such as described in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982); NS0 myeloma cells, COS cells and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a time sufficient to permit antibody expression in the host cell or, more preferably, secretion of the antibody into the medium in which the host cell is grown. The antibody can be recovered from the medium using standard protein purification methods.

[0249] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It should be understood that variations of the above procedures can be made. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of the light and / or heavy chains of an antibody. Recombinant DNA techniques can also be used to remove some or all of the DNA encoding portions of one or both of the light or heavy chains that are not necessary for binding to the antigen of interest. The antibodies also encompass molecules expressed from such truncated DNA molecules. In addition, bispecific antibodies can be generated by cross-linking an antibody with a second antibody using standard chemical cross-linking methods, where one heavy chain and one light chain are the antibody (i.e., bind to human GFAP) and the other heavy chain and the other light chain are specific for an antigen other than human GFAP.

[0250] In a preferred system for recombinant expression of an antibody or an antigen-binding portion thereof, recombinant expression vectors encoding the antibody heavy chain and the antibody light chain are introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. In the recombinant expression vector, the antibody heavy chain and light chain genes are each operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high-level transcription of the genes. The recombinant expression vector also carries the DHFR gene, which allows selection / amplification using methotrexate to select CHO cells that have been transfected with the vector. The selected transformed host cells are cultured to express the antibody heavy chain and light chain, and the intact antibody is recovered from the culture medium. Recombinant expression vectors are prepared using standard molecular biology techniques, host cells are transfected, transformants are selected, host cells are cultured, and the antibody is recovered from the culture medium. Further, a method for synthesizing a recombinant antibody can be by culturing host cells in a suitable medium until the recombinant antibody is synthesized. The method can also include isolating the recombinant antibody from the culture medium.

[0251] The method for preparing monoclonal antibodies involves preparing an immortal cell line capable of producing antibodies with the desired specificity. Such cell lines can be generated from spleen cells obtained from immunized animals. The animals can be immunized with GFAP or a fragment and / or variant thereof. The peptide used to immunize the animals can contain amino acids encoding human Fc (e.g., the crystallizable fragment region or the tail region of a human antibody). The spleen cells can then be immortalized by fusing, for example, with a myeloma cell fusion partner. A variety of fusion techniques can be employed. For example, spleen cells and myeloma cells can be mixed with a non-ionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells but not myeloma cells. One such technique uses hypoxanthine, aminopterin, thymidine (HAT) selection. Another technique includes electrofusion. After a sufficient time (usually about 1 to 2 weeks), colonies of hybrids are observed. Individual colonies are selected and their culture supernatants are tested for binding activity to the polypeptide. Hybridomas with high reactivity and specificity can be used.

[0252] Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. In addition, various techniques can be employed to increase the yield, such as injecting the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host (such as a mouse). The monoclonal antibodies can then be harvested from the ascites or blood. Contaminants can be removed from the antibodies by conventional techniques such as chromatography, gel filtration, precipitation, and extraction. Affinity chromatography is an example of a method that can be used to purify antibodies.

[0253] The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each contain a covalently linked heterodimer with a complete antigen-binding site. Pepsin is capable of cleaving IgG molecules to provide several fragments, including the F(ab’)2 fragment that contains two antigen-binding sites.

[0254] Fv fragments can be generated by proteolytic cleavage, preferably of IgM, and in rare cases can be IgG or IgA immunoglobulin molecules. Fv fragments can be derived using recombinant techniques. Fv fragments comprise a non-covalently linked VH::VL heterodimer that contains an antigen-binding site that retains many of the antigen recognition and binding capabilities of the native antibody molecule.

[0255] An antibody, antibody fragment, or derivative can comprise a set of heavy-chain and light-chain complementarity-determining regions (“CDRs”) respectively inserted between sets of heavy-chain and light-chain framework (“FR”) regions that provide support for the CDRs and define the spatial relationship between the CDRs. The set of CDRs can comprise the three hypervariable regions of the heavy-chain or light-chain V region.

[0256] Other suitable methods for generating or isolating antibodies with the required specificity can be used, including but not limited to methods for selecting recombinant antibodies from peptide or protein libraries (such as but not limited to phage, ribosome, oligonucleotide, RNA, cDNA, yeast, etc. display libraries); for example, as can be obtained from various commercial suppliers such as Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid / Planegg, Del.), Biovation (Aberdeen, Scotland, UK), BioInvent (Lund, Sweden) using methods known in the art. See U.S. Patent Nos. 4,704,692, 5,723,323, 5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862. Alternative methods rely on immunizing transgenic animals capable of generating a human antibody repertoire (e.g., SCID mice, Nguyen et al. (1997) Microbiol. Immunol. 41:901-907; Sandhu et al. (1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol. 93:154-161), as known in the art and / or as described herein. Such techniques include but are not limited to ribosome display (Hanes et al. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998) Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibody production techniques (e.g., selected lymphocyte antibody method ("SLAM") (U.S. Patent No. 5,627,052; Wen et al. (1987) J. Immunol. 17:887-892; Babcock et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848); gel microdroplets and flow cytometry (Powell et al. (1990) Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass).; Gray et al. (1995) J. Imm. Meth. 182:155-163; Kenny et al. (1995) Bio / Technol. 13:787-790); B cell selection (Steenbakkers et al. (1994) Molec. Biol. Reports 19:125-134 (1994)).

[0257] Affinity matured antibodies can be generated by any of a variety of procedures known in the art. For example, see Marks et al., BioTechnology, 10:779-783 (1992) which describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described in Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-3319 (1995); Hawkins et al., J. Mol. Biol., 226:889-896 (1992). Selective mutagenesis at selected mutagenic positions and with activity enhancing amino acid residues at contact or hypermutable positions is described in U.S. Patent No. 6,914,128 B1.

[0258] Antibody variants can also be prepared by delivering a polynucleotide encoding the antibody to a suitable host to provide a transgenic animal or mammal, such as goats, cows, horses, and sheep, etc., which produce such antibodies in their milk. These methods are known in the art and are described, for example, in U.S. Patent Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362 and 5,304,489.

[0259] Antibody variants can also be prepared by delivering polynucleotides to provide transgenic plants and cultured plant cells (such as, but not limited to, tobacco, corn, and duckweed), which produce such antibodies, specific portions, or variants in plant parts or cells cultured therefrom. For example, Cramer et al. (1999) Curr. Top. Microbiol. Immunol. 240:95-118 and the references cited therein describe, for example, the use of inducible promoters to produce transgenic tobacco leaves expressing large amounts of recombinant proteins. Transgenic corn has been used to express mammalian proteins at commercial production levels, and their biological activities are the same as those produced in other recombinant systems or purified from natural sources. See, for example, Hood et al., Adv. Exp. Med. Biol. (1999) 464:127-147 and the references cited therein. Antibody variants have also been produced in large quantities by transgenic plant seeds (including tobacco seeds and potato tubers) including antibody fragments such as single-chain antibodies (scFv). See, for example, Conrad et al. (1998) Plant Mol. Biol. 38:101-109 and the references cited therein. Thus, transgenic plants can also be used to produce antibodies according to known methods.

[0260] Antibody derivatives can be produced, for example, by adding exogenous sequences to modify immunogenicity or to reduce, enhance, or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life, or any other suitable characteristic. Generally, some or all of the non-human or human CDR sequences are maintained, while the non-human sequences of the variable and constant regions are replaced with human or other amino acids.

[0261] Small antibody fragments can be diabodies having two antigen-binding sites, where the fragment contains a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH VL). See, for example, EP 404,097; WO93 / 11161; and Hollinger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. By using a linker that is too short to allow pairing between the two domains of the same chain, the domains are forced to pair with the complementary domains of the other chain and produce two antigen-binding sites. See also U.S. Patent No. 6,632,926 to Chen et al., which is hereby incorporated by reference in its entirety and discloses antibody variants having one or more amino acids inserted into the hypervariable regions of the parental antibody and having a binding affinity for the target antigen that is at least about two-fold stronger than the binding affinity of the parental antibody for the antigen.

[0262] The antibody can be a linear antibody. Procedures for preparing linear antibodies are known in the art and described in Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, these antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1), which form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0263] Antibodies can be recovered and purified from recombinant cell cultures by known methods, including but not limited to protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification.

[0264] Detectably labeled antibodies may be useful. Methods for conjugating antibodies to these reagents are known in the art. For illustrative purposes only, antibodies can be labeled with a detectable moiety, such as a radioactive atom, a chromophore, or a fluorophore, etc. Such labeled antibodies can be used in diagnostic techniques in vivo or in isolated test samples. They can be linked to cytokines, ligands, and another antibody. Suitable agents for conjugation to antibodies to achieve anti-tumor effects include cytokines, such as interleukin 2 (IL-2) and tumor necrosis factor (TNF); photosensitizers for photodynamic therapy, including aluminum(III) phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine; radionuclides, such as iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), technetium-99m (99mTc), rhenium-186 (186Re), and rhenium-188 (188Re); antibiotics, such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacteria, plant, and other toxins, such as diphtheria toxin, Pseudomonas exotoxin A, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-α toxin, cytotoxins from Naja naja atra (cobra), and gelsemium elegans Benth. toxin (a plant toxin); ribosome-inactivating proteins from plants, bacteria, and fungi, such as restrictocin (a ribosome-inactivating protein produced by Aspergillus restrictus), saporin (a ribosome-inactivating protein from Saponaria officinalis), and ribonuclease; tyrosine kinase inhibitors; ly207702 (difluoropurine nucleoside); liposomes containing antisense agents (e.g., antisense oligonucleotides, plasmids encoding toxins, methotrexate, etc.); and other antibodies or antibody fragments, such as F(ab).

[0265] Antibody production via the use of hybridoma technology, selected lymphocyte antibody method (SLAM), transgenic animals, and recombinant antibody libraries is described in more detail below.

[0266] (1) Anti-GFAP monoclonal antibodies using hybridoma technology

[0267] A variety of techniques known in the art can be used to prepare monoclonal antibodies, including the use of hybridoma, recombinant, and phage display techniques or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma technology, which includes those known in the art and taught, for example, in the following: Harlow et al., Antibodies: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988); Hammerling et al., In Monoclonal Antibodies and T-Cell Hybridomas, (Elsevier, N.Y., 1981). It should also be noted that the term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to antibodies derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not to the method by which the antibodies are produced.

[0268] Methods for producing monoclonal antibodies and the antibodies produced by such methods can include culturing hybridoma cells that secrete the antibodies of the present disclosure, wherein the hybridomas are preferably produced by fusing spleen cells isolated from an animal immunized with GFAP, such as a rat or a mouse, with myeloma cells, and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete antibodies capable of binding the polypeptides of the present disclosure. Briefly, a rat can be immunized with the GFAP antigen. In a preferred embodiment, the GFAP antigen is administered together with an adjuvant to stimulate the immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immunostimulating complex). Such adjuvants can protect the polypeptide from rapid diffusion by sequestering the polypeptide in local deposits, or they can contain substances that stimulate the host to secrete factors chemotactic for macrophages and other components of the immune system. Preferably, if the polypeptide is administered, the immunization protocol will involve two or more administrations of the polypeptide, carried out over several weeks; however, a single administration of the polypeptide can also be used.

[0269] After immunizing an animal with GFAP antigen, antibodies and / or antibody-producing cells can be obtained from the animal. Serum containing anti-GFAP antibodies is obtained from the animal by bleeding or sacrificing the animal. The serum obtained from the animal can be used, the immunoglobulin fraction can be obtained from the serum, or the anti-GFAP antibody can be purified from the serum. The serum or immunoglobulin obtained in this way is polyclonal and thus has a range of heterogeneity.

[0270] Once an immune response is detected, for example, antibodies specific for the antigen GFAP are detected in rat serum, the rat spleen is harvested and the spleen cells are isolated. The spleen cells are then fused with any suitable myeloma cells (e.g., cells from the cell line SP20 available from the American Type Culture Collection (ATCC, Manassas, Va., US)) by well-known techniques. Hybridomas are selected and cloned by limiting dilution. The ability of the hybridoma clones to secrete antibodies that can bind GFAP is then determined by methods known in the art. Ascites containing generally high levels of antibodies can be produced by immunizing rats with positive hybridoma clones.

[0271] In another embodiment, immortalized hybridomas that produce antibodies can be prepared from immunized animals. After immunization, the animals are sacrificed and the splenic B cells are fused with immortalized myeloma cells, as is well known in the art. See, e.g., Harlow and Lane, supra. In a preferred embodiment, the myeloma cells do not secrete immunoglobulin polypeptides (non-secreting cell lines). After fusion and antibiotic selection, the hybridomas are screened using GFAP, or a portion thereof, or cells expressing GFAP. In a preferred embodiment, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), preferably ELISA, is used for the initial screening. Examples of ELISA screening are provided in PCT Publication No. WO 00 / 37504.

[0272] Hybridomas that produce anti-GFAP antibodies are selected, cloned, and further screened for their desired characteristics, including robust hybridoma growth, high antibody production, and the desired antibody characteristics. Hybridomas can be cultured and amplified in vivo in syngeneic animals, in animals lacking an immune system (e.g., nude mice), or in vitro in cell culture. Methods for selecting, cloning, and amplifying hybridomas are well known to those of ordinary skill in the art.

[0273] In a preferred embodiment, the hybridomas are rat hybridomas. In another embodiment, the hybridomas are produced in non-human, non-rat species such as mice, sheep, pigs, goats, cattle, or horses. In another preferred embodiment, the hybridomas are human hybridomas, in which human non-secreting myelomas are fused with human cells expressing anti-GFAP antibodies.

[0274] Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, the Fab and F(ab')2 fragments of the present disclosure can be generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce two identical Fab fragments) or pepsin (to produce F(ab')2 fragments). The F(ab')2 fragment of an IgG molecule retains the two antigen-binding sites of the larger (“parent”) IgG molecule, which includes two light chains (containing variable and constant light chain regions), the CH1 domain of the heavy chain, and the disulfide-bond-forming hinge region of the parent IgG molecule. Thus, the F(ab')2 fragment is still able to crosslink antigen molecules like the parent IgG molecule.

[0275] (2) Anti-GFAP monoclonal antibodies using SLAM

[0276] In another aspect of the present disclosure, recombinant antibodies are generated from single, isolated lymphocytes using a method known in the art as the selected lymphocyte antibody method (SLAM), as described in U.S. Patent No. 5,627,052; PCT Publication No. WO 92 / 02551; and Babcock et al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996). In this method, single cells secreting an antibody of interest are screened using an antigen-specific hemolytic plaque assay, such as lymphocytes from any immunized animal, wherein an antigen such as GFAP, a subunit of GFAP, or a fragment thereof is conjugated to sheep red blood cells using a linker such as biotin, and used to identify single cells secreting antibodies specific for GFAP. After identifying the antibody-secreting cells of interest, the heavy and light chain variable region cDNAs are rescued from the cells by reverse transcriptase-PCR (RT-PCR), and these variable regions can then be expressed in the context of appropriate immunoglobulin constant regions (e.g., human constant regions) in mammalian host cells such as COS or CHO cells. Host cells transfected with the amplified immunoglobulin sequences (derived from in vivo selected lymphocytes) can then be further analyzed and selected in vitro, e.g., by panning the transfected cells to isolate cells expressing antibodies against GFAP. The amplified immunoglobulin sequences can be further manipulated in vitro, such as by in vitro affinity maturation methods. See, e.g., PCT Publication No. WO 97 / 29131 and PCT Publication No. WO 00 / 56772.

[0277] (3) Anti-GFAP monoclonal antibodies using transgenic animals

[0278] In another embodiment of the present disclosure, antibodies are produced by immunizing a non-human animal comprising some or all of the human immunoglobulin loci with a GFAP antigen. In one embodiment, the non-human animal is a transgenic mouse, an engineered mouse strain that contains a large fragment of the human immunoglobulin locus and lacks murine antibody production. See, e.g., Green et al., Nature Genetics, 7:13-21 (1994) and U.S. Patent Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, and 6,130,364. See also PCT Publication Nos. WO 91 / 10741, WO 94 / 02602, WO 96 / 34096, WO 96 / 33735, WO 98 / 16654, WO 98 / 24893, WO 98 / 50433, WO 99 / 45031, WO 99 / 53049, WO 00 / 09560, and WO 00 / 37504. The transgenic mouse generates a human-like fully human antibody repertoire and produces antigen-specific human monoclonal antibodies. The transgenic mouse contains approximately 80% of the human antibody repertoire by introduction of megabase-sized, germline configuration YAC fragments of the human heavy chain locus and lambda light chain locus. See Mendez et al., Nature Genetics, 15:146-156 (1997); Green and Jakobovits, J. Exp. Med., 188:483-495 (1998), the disclosures of which are incorporated herein by reference.

[0279] (4) Anti-GFAP Monoclonal Antibodies Using a Recombinant Antibody Library

[0280] Antibodies of the present disclosure can also be prepared using in vitro methods, in which an antibody library is screened to identify antibodies having the desired GFAP binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include those described in the following documents: for example, U.S. Patent No. 5,223,409 (Ladner et al.); PCT Publication No. WO 92 / 18619 (Kang et al.); PCT Publication No. WO 91 / 17271 (Dower et al.); PCT Publication No. WO 92 / 20791 (Winter et al.); PCT Publication No. WO 92 / 15679 (Markland et al.); PCT Publication No. WO 93 / 01288 (Breitling et al.); PCT Publication No. WO92 / 01047 (McCafferty et al.); PCT Publication No. WO 92 / 09690 (Garrard et al.); Fuchs et al., Bio / Technology, 9:1369-1372 (1991); Hay et al., Hum. Antibod. Hybridomas, 3:81-85 (1992); Huse et al., Science, 246:1275-1281 (1989); McCafferty et al., Nature, 348:552-554 (1990); Griffiths et al., EMBO J., 12:725-734 (1993); Hawkins et al., J. Mol. Biol., 226:889-896 (1992); Clackson et al., Nature, 352:624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992); Garrard et al., Bio / Technology, 9:1373-1377 (1991); Hoogenboom et al., Nucl. Acids Res., 19:4133-4137 (1991); Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991); U.S. Patent Application Publication No. 2003 / 0186374; and PCT Publication No. WO 97 / 29131, the content of each of which is incorporated herein by reference.

[0281] A recombinant antibody library can be from a subject immunized with GFAP or a portion of GFAP. Alternatively, a recombinant antibody library can be from a naïve subject, i.e., a human not immunized with GFAP, such as a human antibody library from a human subject not immunized with human GFAP. The antibodies of the present disclosure are selected by screening a recombinant antibody library with a peptide comprising human GFAP, thereby selecting those antibodies that recognize GFAP. Methods for performing such screening and selection are well known in the art, as described in the references in the previous paragraph. To select the antibodies of the present disclosure that have a specific binding affinity for GFAP, such as those that dissociate from human GFAP at a specific K off rate constant, surface plasmon resonance methods known in the art can be used to select antibodies having the desired K off rate constant. To select the antibodies of the present disclosure that have a specific neutralizing activity against hGFAP, such as those having a specific IC 50 , standard methods known in the art for assessing inhibition of GFAP activity can be used.

[0282] In one aspect, the present disclosure relates to an isolated antibody or antigen-binding portion thereof that binds human GFAP. Preferably, the antibody is a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody.

[0283] For example, various phage display methods known in the art can also be used to generate antibodies. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. Such phages can be used to display antigen-binding domains expressed from combinatorial libraries or combinatorial antibody libraries (e.g., human or murine). Phages expressing antigen-binding domains that bind to an antigen of interest can be identified using, for example, a labeled antigen or an antigen bound or captured to a solid surface or bead. The phages used in these methods are typically filamentous phages, including fd and M13 binding domains expressed from phage, and Fab, Fv, or disulfide-stabilized Fv antibody domains are recombinantly fused to the phage gene III or gene VIII protein. Examples of phage display methods that can be used to prepare antibodies include those disclosed in the following references: Brinkmann et al., J. Immunol. Methods, 182:41-50 (1995); Ames et al., J. Immunol. Methods, 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic et al., Gene, 187:9-18 (1997); Burton et al., Advances in Immunology, 57:191-280 (1994); PCT Publication No. WO 92 / 01047; PCT Publication No. WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0284] As described in the above references, after phage selection, the antibody-encoding regions can be isolated from the phage and used to generate full antibodies, including human antibodies or any other desired antigen-binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, as detailed below. For example, techniques known in the art for recombinantly generating Fab, Fab', and F(ab')2 fragments can also be used, such as those disclosed in the following documents: PCT Publication No. WO 92 / 22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); Sawai et al., Am. J. Reprod. Immunol., 34:26-34 (1995); and Better et al., Science, 240:1041-1043 (1988). Examples of techniques that can be used to generate single-chain Fv and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88 (1991); Shu et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999 (1993); and Skerra et al., Science, 240:1038-1041 (1988).

[0285] As an alternative to screening a recombinant antibody library by phage display, other methods known in the art for screening large combinatorial libraries can be applied to identify the antibodies of the present disclosure. One type of alternative expression system is a system in which a recombinant antibody library is expressed as an RNA-protein fusion, as described in PCT Publication No. WO 98 / 31700 (Szostak and Roberts) and Roberts and Szostak, Proc. Natl. Acad. Sci. USA, 94:12297-12302 (1997). In this system, a synthetic mRNA that carries puromycin (a peptidyl acceptor antibiotic) at its 3' end by in vitro translation generates a covalent fusion between the mRNA and the peptide or protein it encodes. Thus, specific mRNAs can be enriched from a complex mixture of mRNAs (e.g., a combinatorial library) based on the properties of the encoded peptide or protein (e.g., an antibody) or a portion thereof, such as the binding of the antibody or a portion thereof to a bispecific antigen. The nucleic acid sequences encoding the antibody or a portion thereof recovered from screening such libraries can be expressed by recombinant means (e.g., in mammalian host cells) as described above, and can additionally be subjected to further affinity maturation by additional rounds of screening of mRNA-peptide fusions in which mutations have been introduced into the initially selected sequences or by other methods for in vitro affinity maturation of recombinant antibodies as described above. A preferred example of such a method is the PROfusion display technology.

[0286] In another method, yeast display methods known in the art can also be used to generate antibodies. In yeast display methods, antibody domains are tethered to the yeast cell wall using genetic methods and displayed on the yeast surface. Specifically, such yeast can be used to display antigen-binding domains expressed from phage libraries or combinatorial antibody libraries (e.g., human or murine). Examples of yeast display methods that can be used to prepare antibodies include the methods disclosed in U.S. Patent No. 6,699,658 (Wittrup et al.), which is incorporated herein by reference.

[0287] d. Generation of recombinant GFAP antibodies

[0288] Antibodies can be produced by any of a number of techniques known in the art. For example, from host cell expression, where expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass the various techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although it is possible to express the antibodies of the present disclosure in prokaryotic or eukaryotic host cells, it is preferred to express the antibodies in eukaryotic cells and most preferably in mammalian host cells, because such eukaryotic cells (and especially mammalian cells) are more likely to assemble and secrete correctly folded and immunologically active antibodies compared to prokaryotic cells.

[0289] Exemplary mammalian host cells for expressing the recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), which are used with a DHFR selection marker, such as described in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982); NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a time sufficient to allow expression of the antibody in the host cell or more preferably to secrete the antibody into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0290] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It should be understood that variations of the above procedures can be made. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of the light and / or heavy chains of the antibodies of the present disclosure. Recombinant DNA techniques can also be used to remove some or all of the DNA encoding portions of one or both of the light or heavy chains that are not necessary for binding to the antigen of interest. The antibodies of the present disclosure also encompass molecules expressed from such truncated DNA molecules. In addition, bispecific antibodies can be produced by cross-linking the antibodies of the present disclosure with a second antibody using standard chemical cross-linking methods, where one heavy chain and one light chain are the antibodies of the present disclosure (i.e., bind to human GFAP) and the other heavy chain and the other light chain are specific for an antigen other than human GFAP.

[0291] In one preferred system for recombinantly expressing an antibody of the present disclosure, or an antigen-binding portion thereof, recombinant expression vectors encoding the antibody heavy chain and the antibody light chain are introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. In the recombinant expression vectors, the antibody heavy chain and light chain genes are each operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high-level transcription of the genes. The recombinant expression vectors also carry the DHFR gene, which allows selection / amplification using methotrexate to select CHO cells that have been transfected with the vectors. The selected transformant host cells are cultured to express the antibody heavy chain and light chain, and the intact antibody is recovered from the culture medium. The recombinant expression vectors are prepared using standard molecular biology techniques, the host cells are transfected, the transformants are selected, the host cells are cultured, and the antibody is recovered from the culture medium. Further, the present disclosure provides a method for synthesizing a recombinant antibody of the present disclosure, the method being carried out by culturing the host cells of the present disclosure in a suitable medium until the recombinant antibody of the present disclosure is synthesized. The method may further include isolating the recombinant antibody from the culture medium.

[0292] (1) Humanized antibody

[0293] A humanized antibody can be an antibody or a variant, derivative, analogue or portion thereof that immunospecifically binds an antigen of interest and comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and complementarity-determining regions (CDRs) having substantially the amino acid sequence of a non-human antibody. A humanized antibody can be derived from a non-human species antibody that binds a desired antigen having one or more complementarity-determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule.

[0294] As used herein, in the context of a CDR, the term "substantially" refers to a CDR having at least 90%, at least 95%, at least 98% or at least 99% identity to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one and usually two variable domains (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., the donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. According to one aspect, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc) (usually the constant region of a human immunoglobulin). In some embodiments, a humanized antibody contains a light chain and at least the variable domain of the heavy chain. The antibody may also include the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, a humanized antibody contains only a humanized light chain. In some embodiments, a humanized antibody contains only a humanized heavy chain. In certain embodiments, a humanized antibody contains only the humanized variable domains of the light chain and / or heavy chain.

[0295] Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including but not limited to IgG1, IgG2, IgG3 and IgG4. Humanized antibodies can contain sequences from more than one class or isotype, and specific constant domains can be selected using techniques well known in the art to optimize the desired effector functions.

[0296] The framework and CDR regions of a humanized antibody need not precisely correspond to the parental sequences, for example, the donor antibody CDR or consensus framework can be mutagenized by substituting, inserting or / or deleting at least one amino acid residue such that the CDR or framework residue at that site does not correspond to the donor antibody or consensus framework. However, in one embodiment, such mutations will not be extensive. Generally, at least 90%, at least 95%, at least 98% or at least 99% of the residues of a humanized antibody will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed by the amino acids (or nucleotides) that occur most frequently in a related family of immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, 1987)). In an immunoglobulin family, each position in the consensus sequence is occupied by the amino acid that occurs most frequently in that position in the family. If two amino acids occur with equal frequency, either amino acid can be included in the consensus sequence.

[0297] Humanized antibodies can be designed to minimize unwanted immune responses to rodent anti - human antibodies, which limits the duration and effectiveness of the therapeutic applications of those moieties in human recipients. A humanized antibody can have one or more amino acid residues introduced therein from a non - human source. These non - human residues are often referred to as "import" residues and are typically taken from the variable domain. Humanization can be performed by replacing the corresponding sequences of a human antibody with hypervariable region sequences. Thus, such "humanized" antibodies are chimeric antibodies in which substantially less than the complete human variable domain has been replaced by the corresponding sequences from a non - human species. See, for example, U.S. Patent No. 4,816,567, the contents of which are incorporated herein by reference. A humanized antibody can be a human antibody in which some hypervariable region residues and possibly some FR residues are replaced with residues from similar sites in a rodent antibody. Any known method can be used for humanizing or engineering the antibodies of the present disclosure, such as, but not limited to, those described in U.S. Patent Nos. 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539, and 4,816,567.

[0298] Humanized antibodies can retain high affinity for GFAP and other favorable biological properties. Humanized antibodies can be prepared by a process of analyzing the parental sequence and various conceptual humanized products using three - dimensional models of the parental and humanized sequences. Three - dimensional immunoglobulin models are generally available. Computer programs that illustrate and display the possible three - dimensional conformational structures of selected candidate immunoglobulin sequences are available. Examining these displays allows analysis of the possible role of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the acceptor and import sequences such that the desired antibody characteristics, such as increased affinity for GFAP, are achieved. Generally speaking, hypervariable region residues can be directly and most substantially involved in affecting antigen binding.

[0299] As an alternative to humanization, human antibodies (also referred to herein as "fully human antibodies") can be produced. For example, it is possible to isolate human antibodies from libraries via PROfusion and / or yeast - related technologies. Transgenic animals (e.g., mice) can also be produced that are capable of generating a fully human antibody repertoire upon immunization in the absence of endogenous immunoglobulin production. For example, in chimeric and germline mutant mice, the antibody heavy - chain joining region (J H)Homozygous deletion of the gene results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies upon antigen challenge. Humanized or fully human antibodies can be prepared according to the methods described in U.S. Patent Nos. 5,770,429, 5,833,985, 5,837,243, 5,922,845, 6,017,517, 6,096,311, 6,111,166, 6,270,765, 6,303,755, 6,365,116, 6,410,690, 6,682,928, and 6,984,720, the content of each of which is incorporated herein by reference.

[0300] e. anti-GFAP antibody

[0301] Anti-GFAP antibodies can be generated using the techniques described above and using conventional techniques known in the art. In some embodiments, the anti-GFAP antibody can be an unconjugated GFAP antibody, such as a GFAP antibody available from: Dako (Catalog No.: M0761); ThermoFisher Scientific (Catalog No.: MA5-12023, A-21282, 13-0300, MA1-19170, MA1-19395, MA5-15086, MA5-16367, MA1-35377, MA1-06701 or MA1-20035); AbCam (Catalog No.: ab10062, ab4648, ab68428, ab33922, ab207165, ab190288, ab115898 or ab21837); EMD Millipore (Catalog No.: FCMAB257P, MAB360, MAB3402, 04-1031, 04-1062, MAB5628); Santa Cruz (Catalog No.: sc-166481, sc-166458, sc-58766, sc-56395, sc-51908, sc-135921, sc-71143, sc-65343 or sc-33673); Sigma-Aldrich (Catalog No.: G3893 or G6171); Sino Biological Inc. (Catalog No.: 100140-R012-50). The anti-GFAP antibody can be conjugated to a fluorophore, such as a conjugated GFAP antibody available from: ThermoFisher Scientific (Catalog No.: A-21295 or A-21294); EMD Millipore (Catalog No.: MAB3402X, MAB3402B, MAB3402B or MAB3402C3); or AbCam (Catalog No.: ab49874 or ab194325).

[0302] Alternatively, the antibodies described in WO 2018 / 067474, WO2018 / 081649, U.S. Patent No. 11,078,298, U.S. Publication No. 2019 / 0502127 and / or Bazarian et al., “Accuracy of a rapid GFAP / UCH-L1testfor the prediction of intracranial injuries on head CT after mild traumaticbrain injury”, Acad. Emerg. Med., (August 6, 2021) can also be used, the contents of which are incorporated herein by reference.

[0303] 8. Variations of the method

[0304] The disclosed method for determining the presence or amount of an analyte of interest (UCH-L1 and / or GFAP) present in a sample can be as described herein. The method can also be adjusted according to other methods for analyzing analytes. Examples of well-known variations include, but are not limited to, immunoassays such as sandwich immunoassays (e.g., monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonal sandwich immunoassays, including enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA)), competitive inhibition immunoassays (e.g., forward and reverse), enzyme multiplied immunoassay technique (EMIT), competitive binding assays, bioluminescence resonance energy transfer (BRET), one-step antibody detection assays, homogeneous assays, heterogeneous assays, capture on the fly assays, etc.).

[0305] a. Immunoassay

[0306] UCH-L1 and / or GFAP antibodies can be used in an immunoassay to analyze the analyte of interest and / or its peptide or fragment (e.g., UCH-L1 and / or GFAP, and / or its peptide or fragment, i.e., UCH-L1 and / or GFAP fragment). Antibodies can be used and the specific binding to the analyte (e.g., UCH-L1 and / or GFAP) can be detected to determine the presence or amount of the analyte (e.g., UCH-L1 and / or GFAP). For example, an antibody or its antibody fragment can specifically bind to the analyte (e.g., UCH-L1 and / or GFAP). If desired, one or more antibodies can be used in combination with one or more commercially available monoclonal / polyclonal antibodies. Such antibodies can be purchased from companies such as R&D Systems, Inc. (Minneapolis, MN) and Enzo Life Sciences International, Inc. (Plymouth Meeting, PA).

[0307] The presence or amount of an analyte (e.g., UCH-L1 and / or GFAP) present in a body sample can be readily determined using an immunoassay such as a sandwich immunoassay (e.g., monoclonal-monoclonal sandwich immunoassay, monoclonal-polyclonal sandwich immunoassay, including radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay ELISA) (e.g., Quantikine ELISA assays, R&D Systems, Minneapolis, MN)). Examples of point-of-care devices that can be used are (Abbott Laboratories, Abbott Park, IL). Other methods that can be used include, for example, chemiluminescent microparticle immunoassays, particularly methods employing an analyzer or methods of the Alinity automated series (Abbott Laboratories, Abbott Park, IL). Other methods include, for example, mass spectrometry, and immunohistochemistry (e.g., using sections from a tissue biopsy), which use anti-analyte (e.g., anti-UCH-L1 and / or anti-GFAP) antibodies (monoclonal, polyclonal, chimeric, humanized, human, etc.) or antibody fragments thereof to the analyte (e.g., UCH-L1 and / or GFAP). Other detection methods include those described in U.S. Patent Nos. 6,143,576, 6,113,855, 6,019,944, 5,985,579, 5,947,124, 5,939,272, 5,922,615, 5,885,527, 5,851,776, 5,824,799, 5,679,526, 5,525,524, and 5,480,792, each of which is hereby incorporated by reference. Specific immunobinding of the antibody to the analyte (e.g., UCH-L1 and / or GFAP) can be detected via a direct label attached to the antibody, such as a fluorescent or luminescent tag, metal, and radionuclide, or via an indirect label, such as alkaline phosphatase or horseradish peroxidase.

[0308] The use of immobilized antibodies or antibody fragments thereof can be incorporated into immunoassays. The antibody can be immobilized on a variety of supports, such as magnetic or chromatographic matrix particles, the surface of assay plates (such as microtiter wells), sheets of solid substrate material, etc. A test strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. The strip can then be immersed in a test sample and rapidly processed through washing and detection steps to produce a measurable signal, such as a colored spot.

[0309] A homogeneous format can be used. For example, a mixture is prepared after obtaining a test sample from a subject. The mixture contains a test sample to be evaluated for an analyte (e.g., UCH-L1 and / or GFAP), a first specific binding partner, and a second specific binding partner. The order in which the test sample, the first specific binding partner, and the second specific binding partner are added to form the mixture is not critical. The test sample is contacted simultaneously with the first specific binding partner and the second specific binding partner. In some embodiments, the first specific binding partner and any UCH-L1 and / or GFAP contained in the test sample can form a first specific binding partner-analyte (e.g., UCH-L1 and / or GFAP)-antigen complex, and the second specific binding partner can form a first specific binding partner-analyte of interest (e.g., UCH-L1 and / or GFAP)-second specific binding partner complex. In some embodiments, the second specific binding partner and any UCH-L1 and / or GFAP contained in the test sample can form a second specific binding partner-analyte (e.g., UCH-L1)-antigen complex, and the first specific binding partner can form a first specific binding partner-analyte of interest (e.g., UCH-L1 and / or GFAP)-second specific binding partner complex. The first specific binding partner can be an anti-analyte antibody (e.g., an anti-UCH-L1 antibody that binds to an epitope having at least three consecutive (3) amino acids of SEQ ID NO:1; or an anti-GFAP antibody that binds to an epitope having at least three consecutive (3) amino acids of SEQ ID NO:2). The second specific binding partner can be an anti-analyte antibody (e.g., an anti-UCH-L1 antibody that binds to an epitope having at least three consecutive (3) amino acids of SEQ ID NO:1; or an anti-GFAP antibody that binds to an epitope having at least three consecutive (3) amino acids of SEQ ID NO:2). Additionally, the second specific binding partner is labeled with a detectable label as described above or contains a detectable label.

[0310] Heterogeneous formats can be used. For example, a first mixture is prepared after obtaining a test sample from a subject. The mixture contains the test sample to be evaluated for an analyte (e.g., UCH-L1 and / or GFAP) and a first specific binding partner, wherein the first specific binding partner and any UCH-L1 and / or GFAP contained in the test sample form a first specific binding partner-analyte (e.g., UCH-L1 and / or GFAP)-antigen complex. The first specific binding partner can be an anti-analyte antibody (e.g., an anti-UCH-L1 antibody that binds to an epitope having at least three consecutive (3) amino acids of SEQ ID NO:1; or an anti-GFAP antibody that binds to an epitope having at least three consecutive (3) amino acids of SEQ ID NO:2). The order of adding the test sample and the first specific binding partner to form the mixture is not critical.

[0311] The first specific binding partner can be immobilized on a solid phase. The solid phase used in an immunoassay (for the first specific binding partner and optionally a second specific binding partner) can be any solid phase known in the art, such as but not limited to magnetic particles, beads, test tubes, microtiter plates, colorimetric tubes, membranes, scaffold molecules, films, filter papers, discs, and chips. In those embodiments where the solid phase is a bead, the bead can be a magnetic bead or a magnetic particle. The magnetic bead / particle can be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or ferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO2, MnAs, MnBi, EuO, and NiO / Fe. Examples of ferrimagnetic materials include NiFe2O4, CoFe2O4, Fe3O4 (or FeO . Fe2O3). The bead can have a magnetic solid core portion and be surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion can be a layer surrounding a non-magnetic core. The solid support to which the first specific binding member is immobilized can be in a dry form or stored in a liquid. The magnetic bead can be subjected to a magnetic field before or after contact with a sample having a magnetic bead with the first specific binding member immobilized thereon.

[0312] After forming a mixture containing a first specific binding partner-analyte (e.g., UCH-L1 or GFAP) antigen complex, any unbound analyte (e.g., UCH-L1 and / or GFAP) is removed from the complex using any technique known in the art. For example, the unbound analyte (e.g., UCH-L1 and / or GFAP) can be removed by washing. However, it is desirable for the first specific binding partner to be present in an amount in excess of any analyte (e.g., UCH-L1 and / or GFAP) present in the test sample such that all of the analyte (e.g., UCH-L1 and / or GFAP) present in the test sample is bound by the first specific binding partner.

[0313] After removing any unbound analyte (e.g., UCH-L1 and / or GFAP), a second specific binding partner is added to the mixture to form a first specific binding partner-analyte of interest (e.g., UCH-L1 and / or GFAP)-second specific binding partner complex. The second specific binding partner can be an anti-analyte antibody (e.g., an anti-UCH-L1 antibody that binds to an epitope having at least three consecutive (3) amino acids of SEQ ID NO:1; or an anti-GFAP antibody that binds to an epitope having at least three consecutive (3) amino acids of SEQ ID NO:2). In addition, the second specific binding partner is labeled with a detectable label as described above or contains a detectable label.

[0314] The use of immobilized antibodies or antibody fragments can be incorporated into immunoassays. The antibody can be immobilized on a variety of supports such as magnetic or chromatographic matrix particles (such as magnetic beads), latex particles or surface-modified latex particles, polymers or polymer films, plastics or plastic films, planar substrates, the surface of assay plates (such as microtiter wells), pieces of solid substrate material, etc. An assay strip can be prepared by coating the antibody or antibodies in an array manner on a solid support. The strip can then be dipped into a test sample and rapidly processed through washing and detection steps to produce a measurable signal such as a colored spot.

[0315] (1) Sandwich immunoassay

[0316] A sandwich immunoassay measures the amount of antigen between two layers of antibodies (i.e., at least one capture antibody) and a detection antibody (i.e., at least one detection antibody). The capture antibody and the detection antibody bind to different epitopes on the antigen such as an analyte of interest (such as UCH-L1 and / or GFAP). Desirably, the binding of the capture antibody to the epitope does not interfere with the binding of the detection antibody to the epitope. Either monoclonal antibodies or polyclonal antibodies can be used as the capture antibody and the detection antibody in a sandwich immunoassay.

[0317] Generally, at least two antibodies are used to isolate and quantitatively test for an analyte (e.g., UCH-L1 and / or GFAP) in a test sample. More specifically, at least two antibodies bind to certain epitopes of the analyte (e.g., UCH-L1 and / or GFAP), thereby forming an immune complex, which is referred to as a "sandwich". One or more antibodies can be used to capture the analyte (e.g., UCH-L1 and / or GFAP) in the test sample (these antibodies are often referred to as "capture" antibodies) and one or more antibodies can be used to bind a detectable (i.e., quantifiable) label to the sandwich (these antibodies are often referred to as "detection" antibodies). In a sandwich assay, the binding of an antibody to its epitope is ideally not diminished by the binding of any other antibody in the assay to its corresponding epitope. Antibodies are selected such that one or more first antibodies that come into contact with a test sample suspected of containing an analyte (e.g., UCH-L1 and / or GFAP) do not bind all or a portion of the epitopes recognized by a second or subsequent antibody, thereby interfering with the ability of one or more second detection antibodies to bind the analyte (e.g., UCH-L1 and / or GFAP).

[0318] An antibody can be used as a first antibody in the immunoassay. The antibody immunospecifically binds to an epitope on the analyte (e.g., UCH-L1 and / or GFAP). In addition to the antibodies of the present disclosure, the immunoassay can include a second antibody that immunospecifically binds to an epitope not recognized or bound by the first antibody.

[0319] A test sample suspected of containing an analyte (e.g., UCH-L1 and / or GFAP) can be contacted with at least one first capture antibody (or multiple first capture antibodies) and at least one second detection antibody simultaneously or sequentially. In a sandwich assay format, first, the test sample suspected of containing an analyte (e.g., UCH-L1 and / or GFAP) is contacted with at least one first capture antibody that specifically binds to a particular epitope under conditions that permit the formation of a first antibody-analyte (e.g., UCH-L1 and / or GFAP) antigen complex. If more than one capture antibody is used, a first multiple capture antibody-UCH-L1 and / or GFAP antigen complex is formed. In a sandwich assay, the antibody, preferably at least one capture antibody, is used in a molar excess relative to the maximum amount of analyte (e.g., UCH-L1 and / or GFAP) expected in the test sample. For example, about 5 μg / mL to about 1 mg / mL of antibody can be used per milliliter of particle coating buffer.

[0320] i. Anti-UCH-L1 capture antibody

[0321] Optionally, prior to contacting the test sample with at least one first capture antibody, the at least one first capture antibody can be bound to a solid support that facilitates separation of the first antibody - analyte (e.g., UCH - L1 and / or GFAP) complex from the test sample. Any solid support known in the art can be used, including but not limited to solid supports in the form of pores, tubes, or beads (such as microparticles) made of polymeric materials. The antibody(ies) can be bound to the solid support by adsorption, by covalent bonding using a chemical coupling agent, or by other means known in the art, provided that such binding does not interfere with the ability of the antibody to bind the analyte (e.g., UCH - L1 and / or GFAP). Additionally, if desired, the solid support can be derivatized to allow reaction with various functional groups on the antibody. Such derivatization requires the use of certain coupling agents, such as but not limited to maleic anhydride, N - hydroxysuccinimide, and 1 - ethyl - 3 - (3 - dimethylaminopropyl)carbodiimide.

[0322] Thereafter, a test sample suspected of containing an analyte (e.g., UCH - L1 and / or GFAP) is incubated to allow formation of the first capture antibody (or multiplex capture antibody) - analyte (e.g., UCH - L1 and / or GFAP) complex. The incubation can be carried out at a pH of from about 4.5 to about 10.0, at a temperature of from about 2°C to about 45°C, and for a period of at least about one (1) minute to about eighteen (18) hours, about 2 - 6 minutes, about 7 - 12 minutes, about 5 - 15 minutes, or about 3 - 4 minutes.

[0323] ii. Detection antibody

[0324] After forming the first / multiple capture antibody-analyte (e.g., UCH-L1 and / or GFAP) complex, the complex is then contacted with at least one second detection antibody (under conditions that permit formation of the first / multiple antibody-analyte (e.g., UCH-L1 and / or GFAP) antigen-second antibody complex). In some embodiments, the test sample is contacted with the capture antibody simultaneously with the detection antibody. If the first antibody-analyte (e.g., UCH-L1 and / or GFAP) complex is contacted with more than one detection antibody, a first / multiple capture antibody-analyte (e.g., UCH-L1 and / or GFAP)-multiple antibody detection complex is formed. As with the first antibody, when the at least second (and subsequent) antibody is contacted with the first antibody-analyte (e.g., UCH-L1 and / or GFAP) complex, incubation for a period of time under conditions similar to those described above is required to form the first / multiple antibody-analyte (e.g., UCH-L1 and / or GFAP)-second / multiple antibody complex. Preferably, at least one second antibody contains a detectable label. The detectable label may be bound to the at least one second antibody before, simultaneously with, or after formation of the first / multiple antibody-analyte (e.g., UCH-L1 and / or GFAP)-second / multiple antibody complex. Any detectable label known in the art may be used.

[0325] The chemiluminescent assay can be performed according to the method described in Adamczyk et al., Anal. Chim. Acta 579(1):61-67 (2006). While any suitable assay format can be used, a microplate chemiluminometer (Mithras LB-940, Berthold Technologies U.S.A., LLC, Oak Ridge, TN) enables rapid assay of multiple small volume samples. When using a 96-well black polystyrene microplate (Costar #3792), the chemiluminometer can be equipped with multiple reagent syringes. Each sample can be added to a separate well, followed by simultaneous / sequential addition of other reagents as determined by the type of assay employed. Desirably, false base formation in neutral or basic solutions of acridinium aryl esters is avoided, such as by acidification. The chemiluminescent response is then recorded well by well. In this regard, the time at which the chemiluminescent response is recorded depends in part on the delay between addition of the reagent and the particular acridinium employed.

[0326] The order of adding the test sample and the specific binding partner to form a mixture for chemiluminescent assay is not critical. If the first specific binding partner is detectably labeled with an acridinium compound, a detectably labeled first specific binding partner - antigen (e.g., UCH-L1 and / or GFAP) complex is formed. Alternatively, if a second specific binding partner is used and the second specific binding partner is detectably labeled with an acridinium compound, a detectably labeled first specific binding partner - analyte (e.g., UCH-L1 and / or GFAP) - second specific binding partner complex is formed. Any unbound specific binding partner (whether labeled or unlabeled) can be removed from the mixture using any technique known in the art, such as washing.

[0327] Hydrogen peroxide can be generated in situ in the mixture, or provided or supplied to the mixture before, simultaneously with, or after adding the above acridinium compound. Hydrogen peroxide can be generated in situ in a variety of ways that will be apparent to those skilled in the art.

[0328] Alternatively, a source of hydrogen peroxide can simply be added to the mixture. For example, the source of hydrogen peroxide can be one or more buffers or other solutions known to contain hydrogen peroxide. In this regard, a hydrogen peroxide solution can simply be added.

[0329] After adding at least one basic solution to the sample simultaneously or sequentially, a detectable signal indicating the presence of the analyte (e.g., UCH-L1 and / or GFAP), i.e., a chemiluminescent signal, is generated. The basic solution contains at least one base and has a pH greater than or equal to 10, preferably greater than or equal to 12. Examples of basic solutions include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide, calcium carbonate, and calcium bicarbonate. The amount of basic solution added to the sample depends on the concentration of the basic solution. Based on the concentration of the basic solution used, those skilled in the art can readily determine the amount of basic solution to add to the sample. Other labels other than chemiluminescent labels can be employed. For example, enzyme labels (including but not limited to alkaline phosphatase) can be employed.

[0330] Chemiluminescent signals or other signals generated can be detected using conventional techniques known to those skilled in the art. Based on the intensity of the generated signals, the amount of the analyte of interest (e.g., UCH-L1 and / or GFAP) in the sample can be quantified. Specifically, the amount of the analyte (e.g., UCH-L1 and / or GFAP) in the sample is proportional to the intensity of the generated signals. The amount of the analyte present (e.g., UCH-L1 and / or GFAP) can be quantified by comparing the amount of light generated with a standard curve of the analyte (e.g., UCH-L1 and / or GFAP) or by comparison with a reference standard. A standard curve can be generated using serial dilutions or solutions of known concentrations of the analyte (e.g., UCH-L1 and / or GFAP) by mass spectrometry, gravimetric methods, and other techniques known in the art.

[0331] (2) Forward competitive inhibition assay

[0332] In the forward competitive format, aliquots of a labeled analyte of interest at a known concentration (e.g., an analyte (e.g., UCH-L1 and / or GFAP) with a fluorescent label (a tag linked to a cleavable linker), etc.) are used to compete with the analyte of interest (e.g., UCH-L1 and / or GFAP) in the test sample for binding to an analyte of interest antibody (e.g., UCH-L1 and / or GFAP antibody).

[0333] In a forward competitive assay, a fixed specific binding partner (such as an antibody) can be contacted with the test sample and the labeled analyte of interest, a fragment of the analyte of interest, or a variant of the analyte of interest either sequentially or simultaneously. The analyte peptide, fragment of the analyte of interest, or variant of the analyte of interest can be labeled with any detectable label, including a detectable label consisting of a tag linked to a cleavable linker. In this assay, the antibody can be immobilized on a solid support. Alternatively, the antibody can be conjugated to an antibody, such as an anti-species antibody, immobilized on a solid support (such as a microparticle or planar substrate).

[0334] Incubate the labeled analyte of interest, the test sample, and the antibody under conditions similar to those described above for the binding sandwich assay format. Two different species of antibody-analyte of interest complexes can then be generated. Specifically, one of the generated antibody-analyte of interest complexes contains a detectable label (e.g., a fluorescent label, etc.), while the other antibody-analyte of interest does not contain a detectable label. The antibody-analyte of interest complexes can, but need not, be separated from the remainder of the test sample prior to quantifying the detectable label. Whether or not the antibody-analyte of interest complexes are separated from the remainder of the test sample, the amount of detectable marker in the antibody-analyte of interest complexes is then quantified. The concentration of the analyte of interest in the test sample (such as a membrane-associated analyte of interest, a soluble analyte of interest, a fragment of a soluble analyte of interest, a variant of the analyte of interest (membrane-associated or soluble analyte of interest), or any combination thereof) can then be determined, e.g., as described above.

[0335] (3) Reverse competitive inhibition assay

[0336] In the reverse competitive assay, the immobilized analyte of interest (e.g., UCH-L1 and / or GFAP) can be contacted with the test sample and at least one labeled antibody sequentially or simultaneously.

[0337] The analyte of interest can be bound to a solid support, such as the solid support discussed above in connection with the binding sandwich assay format.

[0338] Incubate the immobilized analyte of interest, the test sample, and at least one labeled antibody under conditions similar to those described above for the binding sandwich assay format. Two different species of analyte of interest-antibody complexes are then generated. Specifically, one of the generated analyte of interest-antibody complexes is immobilized and contains a detectable label (e.g., a fluorescent label, etc.), while the other analyte of interest-antibody is not immobilized and does not contain a detectable label. By techniques known in the art, such as washing, the unimmobilized analyte of interest-antibody complexes and the remainder of the test sample are removed from the presence of the immobilized analyte of interest-antibody complexes. Once the unimmobilized analyte of interest antibody complexes are removed, the amount of detectable label in the analyte of the immobilized analyte of interest-antibody complexes is quantified after cleaving the tag. The concentration of each analyte of interest in the test sample can then be determined by comparing the amounts of detectable label as described above.

[0339] (4) One-step immunoassay or "instant capture" assay

[0340] In an immediate capture immunoassay, a solid substrate is pre-coated with a fixative. A capture agent and a detection agent for an analyte (e.g., UCH-L1 and / or GFAP) are added together to the solid substrate, followed by a washing step and then detection. The capture agent can bind the analyte (e.g., UCH-L1 and / or GFAP) and comprises a ligand for the fixative. The capture agent and the detection agent can be antibodies or any other moiety capable of capturing or detecting as described herein or known in the art. The ligand can comprise a peptide tag and the fixative can comprise an anti-peptide tag antibody. Alternatively, the ligand and the fixative can be any pair of reagents capable of binding together for use in an immediate capture assay (e.g., a specific binding pair, and other reagent pairs known in the art). More than one analyte can be measured. In some embodiments, the solid substrate can be coated with an antigen and the analyte to be assayed is an antibody.

[0341] In certain other embodiments, in a one-step immunoassay or “immediate capture,” a solid support (such as a microparticle) pre-coated with a fixative (such as biotin, streptavidin, etc.) and at least a first specific binding member and a second specific binding member (used as a capture reagent and a detection reagent, respectively) are used. The first specific binding member comprises a ligand for the fixative (e.g., if the fixative on the solid support is streptavidin, the ligand on the first specific binding member can be biotin) and also binds the analyte of interest (e.g., UCH-L1 and / or GFAP). The second specific binding member comprises a detectable label and binds the analyte of interest (e.g., UCH-L1 and / or GFAP). The solid support and the first and second specific binding members can be added (sequentially or simultaneously) to a test sample. The ligand on the first specific binding member binds to the fixative on the solid support, forming a solid support / first specific binding member complex. Any analyte of interest present in the sample binds to the solid support / first specific binding member complex to form a solid support / first specific binding member / analyte complex. The second specific binding member binds to the solid support / first specific binding member / analyte complex, and the detectable label is detected. An optional washing step can be employed before detection. In certain embodiments, in a one-step assay, more than one analyte can be measured. In certain other embodiments, more than two specific binding members can be employed. In certain other embodiments, multiple detectable labels can be added. In certain other embodiments, multiple analytes of interest can be detected, or their amounts, levels, or concentrations can be measured, determined, or evaluated.

[0342] The use of an immediate capture assay can be performed in a variety of forms as described herein and known in the art. For example, the form can be a sandwich assay as described above, but alternatively can be a competitive assay, can employ a single specific binding member, or use other variants such as known.

[0343] 9. Other Factors

[0344] The methods of diagnosis, prognosis, and / or assessment as described above can also include using other factors for diagnosis, prognosis, and assessment. In some embodiments, the Glasgow Coma Scale or the Extended Glasgow Outcome Scale (GOSE) can be used to diagnose traumatic brain injury. Other tests, scales, or indices can also be used alone or in combination with the Glasgow Coma Scale. One example is the Ranchos Los Amigos Scale. The Ranchos Los Amigos Scale measures the levels of consciousness, cognition, behavior, and interaction with the environment. The Ranchos Los Amigos Scale includes: Level I: No Response; Level II: Generalized Response; Level III: Localized Response; Level IV: Confused-Agitated; Level V: Confused-Inappropriate; Level VI: Confused-Appropriate; Level VII: Automatic-Appropriate; and Level VIII: Purposeful-Appropriate. Another example is the Rivermead Post-Concussion Symptoms Questionnaire, which is a self-report scale for measuring the severity of post-concussion symptoms after TBI. The patient is asked to rate the severity of each of 16 symptoms (e.g., headache, dizziness, nausea, vomiting) experienced in the past 24 hours. In each case, the symptoms are compared to the severity before the injury (pre-injury). The symptoms are reported on a scale of 0 to 4: not experienced, no longer a problem, mild problem, moderate problem, and severe problem.

[0345] 10. Samples

[0346] In some embodiments, a sample is obtained from a subject (e.g., a human subject) who has suffered or is suspected of having suffered a head injury, which may have been caused by or has been caused by any one factor or combination of factors. In some embodiments, the sample is obtained after the subject has suffered a head injury caused by a blunt impact, one or more falls, an explosion or shock wave, or other types of blunt force trauma resulting from body shaking, an external mechanical force or other force that causes closed or open head trauma. In some embodiments, the sample is obtained after the subject has ingested or been exposed to a chemical, toxin, or combination of chemicals and toxins. In some embodiments, the chemical or toxin is fire, mold, asbestos, pesticides, insecticides, organic solvents, paint, glue, gas, organometals, abused drugs, or one or more combinations thereof. In some embodiments, the sample is obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection (e.g., such as SARS-CoV-2), a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof.

[0347] In yet another embodiment, the sample used in the methods described herein can also be used to determine whether a subject has or is at risk of having a mild traumatic brain injury by measuring UCH-L1 and / or GFAP levels in the subject using the anti-UCH-L1 and / or anti-GFAP antibodies or antibody fragments described below. Thus, in certain embodiments, the present disclosure also provides a method for determining whether a subject having or at risk of having a traumatic brain injury as described herein and known in the art is a candidate for therapy or treatment.

[0348] b. Test or biological sample

[0349] As used herein, "sample", "test sample", "biological sample" refers to a fluid sample that contains or is suspected of containing GFAP and / or UCH-L1. The sample can be derived from any suitable source. In some cases, the sample can comprise a liquid, a flowing particulate solid, or a fluid suspension of solid particles. In some cases, the sample can be processed prior to the assays described herein. For example, the sample can be separated or purified from its source prior to the assay; however, in certain embodiments, an unprocessed sample containing GFAP and / or UCH-L1 can be assayed directly. In a specific instance, the source containing GFAP and / or UCH-L1 is human (e.g., pediatric or adult human) material or material from another species. As used herein, "pediatric" or "pediatric subject" refers to a subject less than 18 years of age (i.e., not 18 years of age or older). For example, a pediatric subject can be less than about 18 years of age, or about 17 years, about 16 years, about 15 years, about 14 years, about 13 years, about 12 years, about 11 years, about 10 years, about 9 years, about 8 years, about 7 years, about 6 years, about 5 years, about 4 years, about 3 years, about 2 years, about 1 year, or less than about 1 year. In some aspects, a pediatric subject can be less than about 1 year to about less than 18 years. In some aspects, a pediatric subject can be less than about 1 year to about 17 years. For example, a pediatric subject can be about one day, about two days, about three days, about four days, about five days, about six days, about one week, about two weeks, about three weeks, about one month, about two months, about three months, about four months, about five months, about six months, about seven months, about eight months, about nine months, about ten months, or about eleven months, with a total less than about 18 years, or about 17 years, or about 16 years, or about 15 years, or about 14 years, or about 13 years, or about 12 years, or about 11 years, or about 10 years, or about 9 years, or about 8 years, or about 7 years, or about 6 years, or about 5 years, or about 4 years, or about 3 years, or about 2 years, or about 1 year, or less than about 1 year. "Adult" or "adult subject" refers to a subject 18 years of age or older.

[0350] The material is optionally human material (e.g., body fluids, blood (such as whole blood, serum, plasma), urine, saliva, sweat, sputum, semen, mucus, tears, lymph fluid, amniotic fluid, interstitial fluid, bronchoalveolar lavage fluid, cerebrospinal fluid, feces, tissue, organs, etc.). The tissue can include but is not limited to skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, myocardial tissue, brain tissue, bone marrow, cervical tissue, skin, etc. The sample can be a liquid sample or a liquid extract of a solid sample. In certain cases, the source of the sample can be an organ or tissue, such as a biopsy sample, which can be solubilized by tissue dissociation / cell lysis. In some embodiments, the sample is a whole blood sample, a serum sample, a cerebrospinal fluid sample, a mixed sample of venous blood and capillary blood, a mixed sample of capillary blood and interstitial fluid, a tissue sample, a body fluid, or a plasma sample.

[0351] A series of fluid samples of various volumes can be analyzed. In some exemplary embodiments, the sample volume can be about 0.5 nL, about 1 nL, about 3 nL, about 0.01 μL, about 0.1 μL, about 1 μL, about 5 μL, about 10 μL, about 100 μL, about 1 mL, about 5 mL, about 10 mL, etc. In some cases, the volume of the fluid sample is between about 0.01 μL and about 10 mL, between about 0.01 μL and about 1 mL, between about 0.01 μL and about 100 μL, or between about 0.1 μL and about 10 μL.

[0352] In some cases, the fluid sample can be diluted before being used in the assay. For example, in embodiments where the source of GFAP and / or UCH-L1 is a human body fluid (e.g., blood, serum), the fluid can be diluted with an appropriate solvent (e.g., a buffer such as PBS buffer). The fluid sample can be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold or more before use. In other cases, the fluid sample is not diluted before being used in the assay.

[0353] In some cases, the sample can undergo pretreatment before analysis. The pretreatment can provide additional functionality, such as non-specific protein removal and / or effective but inexpensive mixing functionality. General methods of pretreatment can include using electrokinetic capture, AC electrokinetics, surface acoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, or other preconcentration techniques known in the art. In some cases, the fluid sample can be concentrated before being used in the assay. For example, in embodiments where the source of GFAP and / or UCH-L1 is a body fluid (e.g., blood, serum) from a subject (e.g., human or other substance), the fluid can be concentrated by precipitation, evaporation, filtration, centrifugation, or a combination thereof. The fluid sample can be concentrated about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold or more before use.

[0354] c. Control

[0355] It may be desirable to include control samples. The control samples can be analyzed simultaneously with the samples from the subject as described above. The results obtained from the subject samples can be compared with the results obtained from the control samples. A standard curve can be provided, and the assay results of the samples can be compared therewith. If fluorescence labeling is used, such a standard curve presents the marker level as a function of the assay unit, i.e., the fluorescence signal intensity. Using samples taken from multiple donors, standard curves can be provided for the reference levels of UCH-L1 and / or GFAP in normal healthy tissues, as well as for the "at-risk" levels of UCH-L1 and / or GFAP in tissues taken from donors who may have one or more of the characteristics described above.

[0356] Accordingly, in view of the foregoing, there is provided a method for determining the presence, amount or concentration of UCH-L1 and / or GFAP in a test sample. The method includes assaying the UCH-L1 and / or GFAP of the test sample by immunoassay, such as using at least one capture antibody that binds to an epitope on the UCH-L1 and / or GFAP and at least one detection antibody that binds to an epitope on the UCH-L1 and / or GFAP that is different from the epitope bound by the capture antibody and optionally includes a detectable label, and includes comparing a signal generated by the detectable label, which is a direct or indirect indication of the presence, amount or concentration of UCH-L1 and / or GFAP in the test sample, with a signal generated as a direct or indirect indication of the presence, amount or concentration of UCH-L1 and / or GFAP in a calibrator. The calibrator is optionally and preferably part of a series of calibrators, wherein each calibrator differs from the other calibrators in the series in terms of the UCH-L1 and / or GFAP concentration.

[0357] 11. Kit

[0358] Provided herein is a kit that can be used to assay or evaluate UCH-L1 and / or GFAP or UCH-L1 and / or GFAP fragments of a test sample. The kit includes at least one component for assaying the UCH-L1 and / or GFAP of the test sample and instructions for assaying the UCH-L1 and / or GFAP of the test sample. For example, the kit can include instructions for assaying the UCH-L1 and / or GFAP of the test sample by immunoassay (such as chemiluminescent microparticle immunoassay). The instructions included in the kit can be affixed to the packaging material or can be included as a package insert. While the instructions are typically written or printed materials, they are not limited thereto. The present disclosure encompasses any medium capable of storing such instructions and communicating them to the end user. Such media include, but are not limited to, electronic storage media (e.g., disk, tape, cartridge, chip), optical media (e.g., CD ROM), etc. As used herein, the term "instructions" can include the address of an Internet website that provides the instructions.

[0359] The at least one component can include at least one composition that includes one or more isolated antibodies or antibody fragments that specifically bind to UCH-L1 and / or GFAP. The antibodies can be UCH-L1 and / or GFAP capture antibodies and / or UCH-L1 and / or GFAP detection antibodies.

[0360] Alternatively or additionally, the kit may include a calibrator or control (e.g., purified and optionally lyophilized UCH-L1 and / or GFAP) and / or at least one container for performing the assay (e.g., a tube, microtiter plate or strip, which may have been coated with anti-UCH-L1 and / or GFAP monoclonal antibodies) and / or a buffer, such as an assay buffer or wash buffer, either of which may be provided as a concentrated solution, a substrate solution for a detectable label (e.g., an enzyme label), or a stop solution. Preferably, the kit includes all components necessary for performing the assay, i.e., reagents, standards, buffers, diluents, etc. The instructions may also include instructions for generating a standard curve.

[0361] The kit may further include reference standards for quantifying UCH-L1 and / or GFAP. The reference standards can be used to establish a standard curve for interpolating and / or extrapolating the concentration of UCH-L1 and / or GFAP. The reference standards may contain high UCH-L1 and / or GFAP concentration levels, such as about 100,000 pg / mL, about 125,000 pg / mL, about 150,000 pg / mL, about 175,000 pg / mL, about 200,000 pg / mL, about 225,000 pg / mL, about 250,000 pg / mL, about 275,000 pg / mL, or about 300,000 pg / mL; medium UCH-L1 and / or GFAP concentration levels, such as about 25,000 pg / mL, about 40,000 pg / mL, about 45,000 pg / mL, about 50,000 pg / mL, about 55,000 pg / mL, about 60,000 pg / mL, about 75,000 pg / mL or about 100,000 pg / mL; and / or low UCH-L1 and / or GFAP concentration levels, such as about 1 pg / mL, about 5 pg / mL, about 10 pg / mL, about 12.5 pg / mL, about 15 pg / mL, about 20 pg / mL, about 25 pg / mL, about 30 pg / mL, about 35 pg / mL, about 40 pg / mL, about 45 pg / mL, about 50 pg / mL, about 55 pg / mL, about 60 pg / mL, about 65 pg / mL, about 70 pg / mL, about 75 pg / mL, about 80 pg / mL, about 85 pg / mL, about 90 pg / mL, about 95 pg / mL or about 100 pg / mL.

[0362] Any antibodies provided in the kit, such as recombinant antibodies specific for UCH-L1 and / or GFAP, can be incorporated with a detectable label, such as a fluorophore, a radioactive moiety, an enzyme, a biotin / avidin label, a chromophore, a chemiluminescent label, etc., or the kit can include reagents for labeling the antibody or for detecting the antibody (e.g., a detection antibody) and / or reagents for labeling the analyte (e.g., UCH-L1 and / or GFAP) or for detecting the analyte (e.g., UCH-L1 and / or GFAP). The antibodies, calibrators, and / or controls can be provided in separate containers or pre-allocated into a suitable assay format, such as pre-allocated into a microtiter plate.

[0363] Optionally, the kit includes quality control components (e.g., sensitivity sets, calibrators, and positive controls). The preparation of quality control reagents is well known in the art and described i...

Claims

1. A method for assisting in diagnosing or determining whether a human subject who has suffered an actual or suspected head injury has ultra - mild traumatic brain injury (TBI), the method comprising: a) Perform at least one assay on a sample obtained from the subject within about 24 hours after the actual or suspected head injury to measure the level of glial fibrillary acidic protein (GFAP) and / or the level of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) in the sample; and b) Determine that the subject has not suffered a very mild TBI when the level of GFAP in the sample is below the reference level of GFAP and / or the level of UCH-L1 in the sample is below the reference level of UCH-L1.

2. The method according to claim 1, wherein the reference level of GFAP is about 55 pg / mL and the reference level of UCH - L1 is about 160 pg / mL.

3. The method according to claim 1, wherein the sample is obtained from the subject within about 12 hours after the actual or suspected head injury, and wherein the reference level of GFAP is about 40 pg / mL and the reference level of UCH - L1 is about 144 pg / mL.

4. A method for assisting in diagnosing or determining whether a human subject who has suffered an actual or suspected head injury has mild traumatic brain injury (TBI) or ultra - mild traumatic brain injury (TBI), the method comprising: a) Perform at least one assay on a sample obtained from the subject within about 24 hours after the actual or suspected head injury to measure the level of glial fibrillary acidic protein (GFAP) and / or the level of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) in the sample; and b) Distinguish mild TBI from very mild TBI based on whether the level of GFAP in the sample is equal to or below the reference level of GFAP and / or whether the level of UCH-L1 in the sample is equal to or below the reference level of UCH-L1.

5. The method according to claim 4, wherein the subject has been scored using the Glasgow Coma Scale (GCS) before or after the at least one determination, and wherein based on the GCS score, the subject is suspected of having mild TBI.

6. The method according to claim 5, wherein based on a GCS score of 13 - 14, the reference level of GFAP and / or the reference level of UCH - L1 are associated with mild TBI subjects.

7. The method according to claim 4, wherein the subject has been scored using the Glasgow Coma Scale (GCS) before or after the at least one determination, and wherein based on the GCS score, the subject is suspected of having ultra - mild TBI.

8. The method according to claim 7, wherein based on a GCS score of 15, the reference level of GFAP and the reference level of UCH - L1 are associated with ultra - mild TBI subjects.

9. The method according to any one of claims 4 - 8, wherein differentiating mild TBI from ultra - mild TBI comprises: a) Determine that the subject has suffered a mild TBI when the level of GFAP in the sample is equal to the reference level of GFAP from about 55 pg / mL to about 1521 pg / mL and / or the level of UCH-L1 in the sample is equal to the reference level of UCH-L1 from about 160 pg / mL to about 533 pg / mL; or b) Determine that the subject has suffered a very mild TBI when the level of GFAP in the sample is below the reference level of GFAP of about 55 pg / mL and / or the level of UCH-L1 in the sample is below the reference level of UCH-L1 of about 160 pg / mL.

10. The method according to claim 9, wherein differentiating mild TBI from ultra - mild TBI comprises: a) When the GFAP level in the sample is equal to the reference level of GFAP of about 55 pg / mL to about 550 pg / mL, about 500 pg / mL to about 1100 pg / mL, about 1000 pg / mL to about 1500 pg / mL, about 55 pg / mL to about 90 pg / mL, about 75 pg / mL to about 140 pg / mL, about 125 pg / mL to about 246 pg / mL, about 220 pg / mL to about 502 pg / mL, about 246 pg / mL to about 547 pg / mL, about 526 pg / mL to about 780 pg / mL, about 750 pg / mL to about 925 pg / mL, about 890 pg / mL to about 1120 pg / mL or about 1100 pg / mL to about 1521 pg / mL and / or when the UCH-L1 level in the sample is equal to the reference level of UCH-L1 of about 160 pg / mL to about 300 pg / mL, about 250 pg / mL to about 450 pg / mL, about 300 pg / mL to about 500 pg / mL, about 350 pg / mL to about 533 pg / mL, about 160 to about 200 pg / mL, about 191 pg / mL to about 240 pg / mL, about 235 pg / mL to about 300 pg / mL, about 290 pg / mL to about 350 pg / mL, about 325 pg / mL to about 475 pg / mL or about 450 pg / mL to about 533 pg / mL, it is determined that the subject has suffered a mild TBI; or b) When the GFAP level in the sample is lower than the reference level of GFAP of about 55 pg / mL and / or when the UCH-L1 level in the sample is lower than the reference level of UCH-L1 of about 160 pg / mL, it is determined that the subject has suffered a ultra - mild TBI.

11. A method according to any one of claims 4 - 8, wherein the sample is obtained from the subject within about 12 hours after the actual or suspected injury, and wherein differentiating mild TBI from ultra - mild TBI comprises: a) When the GFAP level in the sample is equal to the reference level of GFAP of about 40 pg / mL to about 1021 pg / mL and / or when the UCH-L1 level in the sample is equal to the reference level of UCH-L1 of about 144 pg / mL to about 533 pg / mL, it is determined that the subject may have suffered a mild TBI; or b) When the GFAP level in the sample is lower than the reference level of GFAP of about 40 pg / mL and / or when the UCH-L1 level in the sample is lower than the reference level of UCH-L1 of about 144 pg / mL, it is determined that the subject may have suffered a ultra - mild TBI.

12. A method according to claim 11, wherein differentiating mild TBI from ultra - mild TBI comprises: a) When the GFAP level in the sample is equal to the reference level of GFAP of about 40 pg / mL to about 90 pg / mL, about 85 pg / mL to about 139 pg / mL, about 136 pg / mL to about 390 pg / mL, about 375 pg / mL to about 502 pg / mL, about 498 pg / mL to about 710 pg / mL, about 705 pg / mL to about 950 pg / mL, or about 931 pg / mL to about 1021 pg / mL and / or the UCH-L1 level in the sample is equal to the reference level of UCH-L1 of about 144 pg / mL to about 184 pg / mL, about 175 pg / mL to about 363 pg / mL, about 359 pg / mL to about 433 pg / mL, or about 425 pg / mL to about 533 pg / mL, it is determined that the subject may have suffered a mild TBI; or b) When the GFAP level in the sample is lower than the reference level of GFAP of about 40 pg / mL and / or the UCH-L1 level in the sample is lower than the reference level of UCH-L1 of about 144 pg / mL, it is determined that the subject may have suffered a ultra-mild TBI.

13. A method of assisting in diagnosing or determining whether a human subject who has suffered an actual or suspected head injury has mild traumatic brain injury (TBI) or ultra - mild traumatic brain injury (TBI), the method comprising: Determine the level of glial fibrillary acidic protein (GFAP) and / or ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) in a sample obtained from the subject at about 2 weeks after the actual or suspected head injury; and Distinguish mild TBI from ultra-mild TBI based on whether the GFAP level in the sample is equal to or lower than the reference level of GFAP and / or whether the UCH-L1 level in the sample is equal to or lower than the reference level of UCH-L1.

14. A method according to claim 13, wherein the subject has been scored using the Glasgow Coma Scale (GCS) before or after the determination, and wherein based on the GCS score, the subject is suspected of having mild TBI.

15. A method according to claim 14, wherein based on a GCS score of 13 - 14, the reference level of GFAP and / or the reference level of UCH - L1 are associated with mild TBI subjects.

16. A method according to claim 13, wherein the subject has been scored using the Glasgow Coma Scale (GCS) before or after the determination, and wherein based on the GCS score, the subject is suspected of having ultra - mild TBI.

17. A method according to claim 16, wherein based on a GCS score of 15, the reference level of GFAP and the reference level of UCH - L1 are associated with ultra - mild TBI subjects.

18. A method according to any one of claims 13 - 17, wherein differentiating mild TBI from ultra - mild TBI comprises: a) When the GFAP level in the sample is equal to the reference level of GFAP of about 15 pg / mL to about 169 pg / mL and / or the UCH-L1 level in the sample is equal to the reference level of UCH-L1 of about 64 pg / mL to about 154 pg / mL, it is determined that the subject may have suffered a mild TBI; or b) When the GFAP level in the sample is lower than the reference level of GFAP of about 15 pg / mL and / or the UCH-L1 level in the sample is lower than the reference level of UCH-L1 of about 64 pg / mL, it is determined that the subject may have suffered a ultra-mild TBI.

19. A method according to any one of claims 1 - 18, wherein measuring the GFAP level comprises: (a) Contact the sample simultaneously or sequentially in any order with the following: (1) At least one GFAP capture antibody that binds to an epitope on GFAP or a GFAP fragment to form at least one GFAP capture antibody-GFAP antigen complex, and (2) At least one GFAP detection antibody that contains a detectable label and binds to an epitope on GFAP that is not bound by the GFAP capture antibody to form a GFAP antigen-at least one GFAP detection antibody complex, such that at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex is formed; And (b) Measure the amount or concentration of GFAP in the sample based on the signal generated by the detectable label in the at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex.

20. The method according to any one of claims 1-19, wherein measuring the UCH-L1 level comprises: (a) Contact the sample simultaneously or sequentially in any order with the following: (1) At least one UCH-L1 capture antibody that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form at least one UCH-L1 capture antibody-UCH-L1 antigen complex, and (2) At least one UCH-L1 detection antibody that comprises a detectable label and binds to an epitope on UCH-L1 that is not bound by the at least one UCH-L1 capture antibody to form a UCH-L1 antigen-at least one UCH-L1 detection antibody complex, such that at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex is formed; And (b) Measure the amount or concentration of UCH-L1 in the sample based on the signal generated by the detectable label in the at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex.

21. The method according to any one of claims 1-20, wherein the sample is a whole blood sample, a serum sample, a cerebrospinal fluid sample, a plasma sample, a tissue sample, or a body fluid.

22. The method according to any one of claims 1-21, wherein the sample: (a) is obtained after the subject has suffered a head injury caused by physical shaking, an external mechanical force or other force that causes closed or open head trauma, blunt impact, one or more falls, an explosion or shock wave, or other types of blunt force trauma; (b) is obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin; or (c) is obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof.

23. The method according to any one of claims 1-22, wherein the method can be performed on any subject, regardless of factors selected from the group consisting of: the clinical condition of the subject; the laboratory values of the subject; the subject being classified as suffering from mild, moderate, or severe traumatic brain injury; the subject exhibiting a low or high UCH-L1 level; and the timing of any event in which the human subject may have suffered a head injury.

24. The method according to any one of claims 1-23, further comprising treating the subject determined to have mild traumatic brain injury with a mild traumatic brain injury treatment.

25. The method according to any one of claims 1-23, further comprising monitoring the subject.

26. The method according to any one of claims 1-25, wherein the sample is: (a) a whole blood sample; (b) a serum sample; or (c) a plasma sample.

27. The method according to any one of claims 1-26, wherein the assay is an immunoassay or a clinical chemistry assay.

28. The method according to any one of claims 1-26, wherein the assay is a single molecule detection assay or a point-of-care assay.

29. The method according to any one of claims 1-28, wherein the amount of the at least one sample is from about 10 μL to about 30 μL.

30. The method according to claim 29, wherein the amount of the at least one sample is about 20 μL.

31. The method according to any one of claims 1-30, wherein at least one determination for UCH-L1, at least one determination for GFAP, or at least one determination for UCH-L1 and at least one determination for GFAP are carried out in about 10 to about 20 minutes.

32. The method according to claim 31, wherein at least one determination for UCH-L1, at least one determination for GFAP, or at least one determination for UCH-L1 and at least one determination for GFAP are carried out in about 15 minutes.

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