Methods for assessing brain injury in pediatric subjects

Abstract

Disclosed herein are methods and kits for use in aiding in the diagnosis and assessment of a pediatric subject for traumatic brain injury (TBI) using ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof. Also disclosed herein are methods and kits for use in aiding in the determination of whether a pediatric subject would benefit from, and therefore undergo, an imaging procedure, such as an MRI or a head computed tomography (CT) scan, based on levels of GFAP, UCH-L1, or GFAP and UCH-L1.

Description

[Technical field]

[0001] This application claims priority to U.S. Patent Application No. 63 / 189,757, filed May 18, 2021, and U.S. Patent Application No. 63 / 192,370, filed May 24, 2021, the contents of each of which are incorporated by reference in their entirety into this specification.

[0002] The computer-readable nucleotide / amino acid sequence listing submitted herewith is incorporated by reference in its entirety into this specification and is identified as follows in a single 6,541-byte ASCII (text) file named "39325-601_Sequence Listing_ST25.TXT" created on May 18, 2022.

[0003] The present disclosure relates to methods of assessing a pediatric subject for head injury. In some aspects, the present disclosure relates to methods of assessing a pediatric subject for head injury by measuring glial fibrillary acidic protein (GFAP) and / or ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in a sample obtained from the subject. [Background technology]

[0004] More than 5 million mild traumatic brain injuries (TBIs) occur annually in the United States alone. Furthermore, TBIs are the leading cause of death and disability in children. Currently, there are no simple, objective, and accurate measurements available to aid in patient evaluation. Indeed, much of TBI assessment and diagnosis is based on subjective data. Unfortunately, objective measurements such as head CT and Glasgow Coma Score (GCS) are not very comprehensive or sensitive in assessing mild TBI. Furthermore, head CT scans reveal nothing about mild TBI in most cases, are expensive, and expose patients to unnecessary radiation. In addition, a negative head CT does not mean that the patient is clearly not concussed, only that certain interventions, such as surgery, are not warranted. Physicians and patients need objective and reliable information to accurately assess this condition and facilitate appropriate triage and recovery. Currently, there is limited data available for the use of early biomarkers in acute care settings to aid in patient assessment and management. Summary of the Invention [Problem to be solved by the invention]

[0005] Mild TBI or concussion is difficult to detect objectively and presents a daily challenge in emergency rooms around the world. Concussion does not cause gross pathology, such as bleeding and abnormalities in conventional computed tomography scans of the brain, but often causes rapid onset neurological dysfunction that resolves spontaneously over days to weeks. There is an unmet need for victims of mild TBI in emergency rooms and clinics, hospitals, sports fields, and military activities (e.g., combat) to be assessed for their TBI status. [Means for solving the problem]

[0006] (Summary) In one aspect, provided herein is a method for assessing a pediatric subject for head injury. In some embodiments, provided herein is a method for assessing a pediatric subject for head injury. In some embodiments, the method comprises performing an assay on a sample taken from the subject after a real or suspected head injury to measure a level of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and / or a level of glial fibrillary acidic protein (GFAP) in the sample. In some embodiments, the method comprises determining that the subject has suffered a traumatic brain injury (TBI) if the level of GFAP in the sample is greater than a baseline level of GFAP. In some embodiments, the baseline level of GFAP is at least about 30 pg / mL. In some embodiments, the baseline level of GFAP is at least about 50 pg / mL. In some embodiments, the baseline level of GFAP is at least about 65 pg / mL. In some embodiments, the baseline level is about 1000 pg / mL.

[0007] In some embodiments, the method includes determining that the subject has suffered a TBI if the level of UCH-L1 in the sample is greater than a reference level of UCH-L1. In some embodiments, the reference level of UCH-L1 is at least about 55 pg / mL. In some embodiments, the reference level of UCH-L1 is about 300 pg / mL.

[0008] In some embodiments, the method includes determining that the subject has suffered a TBI if the level of GFAP in the sample is greater than the baseline level of GFAP and the level of UCH-L1 in the sample is greater than the baseline level of UCH-L1. In some embodiments, the baseline level of GFAP is at least about 30 pg / mL and the baseline level of UCH-L1 is about 360 pg / mL. In some embodiments, the baseline level of GFAP is about 65 pg / mL and the baseline level of UCH-L1 is about 360 pg / mL.

[0009] In some embodiments, the sample is collected within about 48 hours after the actual or suspected head injury. In other embodiments, the sample is collected within about 6 hours after the actual or suspected head injury. In other embodiments, the sample is collected within about 12 hours after the actual or suspected head injury. In other embodiments, the sample is collected within about 14 hours after the actual or suspected head injury. In other embodiments, the sample is collected within about 24 hours after the actual or suspected head injury.

[0010] In some embodiments, the subject receives a Glasgow Coma Scale (GCS) score before or after performing the assay. In some embodiments, the subject is suspected to have moderate to severe TBI based on the Glasgow Coma Scale (GCS) score. In some embodiments, the reference level correlates with the subject having moderate to severe TBI. In some embodiments, the subject is suspected to have mild TBI based on the Glasgow Coma Scale (GCS) score. In some embodiments, the reference level correlates with the subject having mild TBI.

[0011] In some embodiments, the reference level of GFAP is determined by an assay having a sensitivity of at least about 90% and a specificity of at least about 40%. In some embodiments, the reference level of GFAP is determined by an assay having a sensitivity of at least about 50% and a specificity of at least about 90%. In some embodiments, the reference level of GFAP is determined by an assay having a negative predictive value of at least about 70%. In some embodiments, the reference level of GFAP is determined by an assay having a negative predictive value of at least about 90%. In some embodiments, the reference level of GFAP is determined by an assay having a positive predictive value of at least about 50%. In some embodiments, the reference level of GFAP is determined by an assay having a positive predictive value of at least about 80%.

[0012] In some embodiments, the reference level of UCH-L1 is determined by an assay having a sensitivity of at least about 80% and a specificity of at least about 25%. In some embodiments, the reference level of UCH-L1 is determined by an assay having a sensitivity of at least about 30% and a specificity of at least about 90%. In some embodiments, the reference level of UCH-L1 is determined by an assay having a negative predictive value of at least about 65%. In some embodiments, the reference level of UCH-L1 is determined by an assay having a positive predictive value of at least about 40%. In some embodiments, the reference level of UCH-L1 is determined by an assay having a positive predictive value of at least about 80%.

[0013] In some embodiments, the reference level of GFAP and the reference level of UCH-L1 are determined by an assay having a sensitivity of at least about 70% and a specificity of at least about 10%. In some embodiments, the reference level of GFAP and the reference level of UCH-L1 are determined by an assay having a sensitivity of at least about 65% and a specificity of at least about 25%. In some embodiments, the reference level of GFAP and the reference level of UCH-L1 are determined by an assay having a positive predictive value of at least about 35%. In some embodiments, the reference level of GFAP and the reference level of UCH-L1 are determined by an assay having a negative predictive value of at least about 40%. In some embodiments, the reference level of GFAP and the reference level of UCH-L1 are determined by an assay having a negative predictive value of at least about 55%.

[0014] In some embodiments, the methods further include treating the pediatric subject determined to have TBI with a therapy for TBI, and optionally monitoring the pediatric subject after receiving said therapy.

[0015] In another aspect, provided herein are methods of assessing whether to perform a head computed tomography (CT) scan on a pediatric subject. In some embodiments, the methods include performing an assay on a sample obtained from the subject after a real or suspected head injury to measure levels of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and / or levels of glial fibrillary acidic protein (GFAP) in the sample.

[0016] In some embodiments, the method includes determining that a head CT scan should be performed in the pediatric subject if the level of GFAP in the sample is greater than the reference level of GFAP. In some embodiments, the reference level of GFAP is at least about 30 pg / mL. In some embodiments, the reference level of GFAP is at least about 50 pg / mL. In some embodiments, the reference level of GFAP is at least about 65 pg / mL. In some embodiments, the reference level is about 1000 pg / mL.

[0017] In some embodiments, the method includes determining that if the level of UCH-L1 in the sample is greater than the reference level of UCH-L1, a head CT scan should be performed on the subject. In some embodiments, the reference level of UCH-L1 is at least about 55 pg / mL. In some embodiments, the reference level of UCH-L1 is about 300 pg / mL.

[0018] In some embodiments, the method includes determining that a head CT scan should be performed on the subject if the level of GFAP in the sample is greater than the reference level of GFAP and the level of UCH-L1 in the sample is greater than the reference level of UCH-L1. In some embodiments, the reference level of GFAP is at least about 30 pg / mL and the reference level of UCH-L1 is about 360 pg / mL. In some embodiments, the reference level of GFAP is about 65 pg / mL and the reference level of UCH-L1 is about 360 pg / mL.

[0019] In some embodiments, the sample is collected within about 48 hours after the actual or suspected head injury. In other embodiments, the sample is collected within about 6 hours after the actual or suspected head injury. In other embodiments, the sample is collected within about 12 hours after the actual or suspected head injury. In other embodiments, the sample is collected within about 14 hours after the actual or suspected head injury. In other embodiments, the sample is collected within about 24 hours after the actual or suspected head injury.

[0020] Any of the methods described herein may further include performing an assay on the sample to measure or detect the level of one or more other biomarkers that are not UCH-L1 or GFAP. For example, the one or more other biomarkers may be S100β, neurospecific enolase (NSE), lipoprotein 1, Tau, C-reactive protein (CRP), free brain-derived neurotrophic factor (BDNF), p-Tau, total BDNF, troponin I (TnI), or a combination thereof. In some embodiments, measuring the level of UCH-L1 includes performing an immunoassay. In some embodiments, measuring the level of UCH-L1 comprises contacting the sample simultaneously or sequentially, in any order, with a capture antibody that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form a capture antibody-UCH-L1 antigen complex, and a detection antibody that includes a detectable label and binds to an epitope of UCH-L1 not bound by the capture antibody to form a UCH-L1 antigen-detection antibody complex, such that a capture antibody-UCH-L1 antigen-detection antibody complex is formed. In some embodiments, measuring further comprises determining 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.

[0021] In some embodiments, measuring the level of GFAP comprises performing an immunoassay. In some embodiments, measuring the level of GFAP comprises contacting the sample simultaneously or sequentially, in any order, with a capture antibody that binds to an epitope on GFAP or a GFAP fragment to form a capture antibody-GFAP antigen complex, and a detection antibody that includes a detectable label and binds to an epitope of GFAP not bound by the capture antibody to form a GFAP antigen-detection antibody complex, such that a capture antibody-GFAP antigen-detection antibody complex is formed. In some embodiments, measuring further comprises determining 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.

[0022] In some embodiments, the sample is a whole blood sample, a serum sample, a cerebrospinal fluid sample, a plasma sample, a tissue sample, a saliva sample, an oropharyngeal sample, a nasopharyngeal sample, a nasal mucus sample, or a bodily fluid. In some embodiments, the sample is obtained after the subject has suffered a head injury caused by physical shaking, an external mechanical or other force resulting in a closed or open head injury, one or more falls, a blunt impact from an explosion or blast, or other type of blunt force trauma. In some embodiments, the sample is obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin. In some embodiments, the chemical or toxin is fire, mold, asbestos, a pesticide, an insecticide, an organic solvent, a paint, a glue, a gas, an organometallic, a drug of abuse, or one or more combinations thereof. In some embodiments, the sample is obtained from a pediatric subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof.

[0023] In some embodiments, the assay is an immunoassay or a clinical chemistry assay. In some embodiments, the assay is performed using a single molecule detection or point-of-care device. [Brief description of the drawings]

[0024] [Figure 1]Figure 1 shows the ROC plot for GFAP levels in subjects ages 2 to 17. [Diagram 2] FIG. 2 shows the ROC plot for UCH-L1 levels in subjects ages 2 to 17. [Diagram 3] FIG. 3 shows a graph assessing GFAP outcome by head computed tomography (CT) status. [Figure 4] FIG. 4 shows a graph assessing UCH-L1 outcome according to CT status. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present disclosure relates to methods for aiding in or determining whether a pediatric subject who has sustained or is suspected of having sustained a head injury has sustained a traumatic brain injury (TBI), such as mild TBI (mTBI), based on levels of UCH-L1, GFAP, or a combination thereof. These methods include measuring levels of UCH-L1, GFAP, or a combination thereof in one or more samples taken from the pediatric subject. For example, a sample or samples may be taken from the subject within about 48 hours, e.g., within about 24 hours (e.g., zero to about 25 hours) of the actual or suspected head injury. Measuring levels of GFAP, UCH-L1, or GFAP and UCH-L1, fragments thereof, or combinations thereof that are equal to or greater than baseline levels of GFAP, UCH-L1, or GFAP and UCH-L1, provides for aiding or determining whether a pediatric subject has sustained a TBI, such as a mild TBI or a moderate-severe TBI, and / or is in need of further medical assessment (e.g., CT imaging and / or MRI) of a suspected damaging TBI. In some embodiments, the pediatric subject is a human subject.

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

[0027] 1.Definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of discrepancy, the present document, including definitions, will prevail. In the practice or testing of this disclosure, methods and materials similar or equivalent to those described herein may be used, but the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0028] As used herein, the terms "comprise(s)," "include(s)," "having," "having," "may be," "containing," and variations thereof are open-ended transitional phrases, transitional terms, or transitional words that do not exclude additional acts or structures. The singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise," "consist of," or "consist essentially of" the embodiments or elements presented herein, whether expressly stated or not.

[0029] In this specification, to recite numerical ranges, each intervening number with the same precision is expressly contemplated. For example, for the range of 6 to 9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated, and for the range of 6.0 to 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 expressly contemplated.

[0030] As used herein, an "affinity matured antibody" refers to an antibody that has been subjected to a process to reduce the affinity (i.e., K D , k d or k aThe term affinity matured antibody is used to refer to an antibody with one or more alterations in one or more CDRs that result in an improvement over a parent antibody that does not have the alterations. Exemplary affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Various procedures for generating affinity matured antibodies are known in the art, including screening combinatorial antibody libraries prepared using BioDisplay technology. 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 has been described by 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 positions and selective mutations at contact or hypermutation positions with activity enhancing amino acid residues are described in U.S. Patent No. 6,914,128 B1.

[0031] As used herein, "antibody" and "antibodies" include monoclonal antibodies, monospecific antibodies (e.g., which may be monoclonal or may also be produced by means other than those of producing them from common germ cells), polyspecific antibodies, human antibodies, humanized antibodies (fully humanized or partially humanized), avian (e.g., duck or goose) antibodies, shark antibodies, whale antibodies, and non-primate (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, etc.) antibodies or non-human spirit antibodies. " refers to animal antibodies, such as, but not limited to, mammalian antibodies, including long-animal (e.g., monkey, chimpanzee, etc.) antibodies, recombinant antibodies, chimeric antibodies, single-chain Fvs ("scFvs"), single-chain antibodies, single domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs ("sdFvs") 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 producing them are described in Wu, C. et al., Nature Biotechnology, 25(11):1290-1297 (2007) and PCT International Application No. 2001 / 058956, the contents of each of which are incorporated herein by reference), and functionally active epitope-binding fragments of any of the above. 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 of molecule. For simplicity, antibodies against an analyte are often referred to herein as "anti-analyte antibodies" or simply "analyte antibodies" (e.g., anti-GFAP antibodies, GFAP antibodies, anti-UCH-L1 antibodies, or UCH-L1 antibodies).

[0032] As used herein, "antibody fragment" refers to a portion of an intact antibody that contains the antigen-binding site or variable region. The portion does not contain the heavy chain domain of the Fc region of the intact antibody (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 a light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing three CDRs of a heavy chain variable region.

[0033] "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, whereas an AUC of 0.5 represents a non-significant test. A preferred AUC may 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.

[0034] As used herein, "beads" and "particles" are used interchangeably and refer to a substantially spherical solid support. One example of a bead or particle is a microparticle. Microparticles that may be used herein may be of any type known in the art. For example, the bead or particle may be a magnetic bead or particle. The magnetic beads / particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or magnetic fluid. 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 beads may be magnetic and have a solid core portion surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion may be a layer around the non-magnetic core. Microparticles can be of any size that works in the methods described herein, for example, from about 0.75 to about 5 nm, or from about 1 to about 5 nm, or from about 1 to about 3 nm.

[0035] As used herein, "binding protein" is used to refer to a monomeric or multimeric protein that binds to and forms a complex with a binding partner, such as, for example, a polypeptide, an antigen, a compound or other molecule, or a substrate of any kind. A binding protein specifically binds to a binding partner. Binding proteins include antibodies, as well as their antigen-binding fragments and various other forms and derivatives thereof known in the art and described herein below, and other molecules that contain one or more antigen-binding domains that bind to an antigen molecule or a specific site (epitope) on an antigen molecule. Thus, binding proteins include, but are not limited to, antibodies that are tetrameric immunoglobulins, IgG molecules, IgG1 molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, affinity matured antibodies, and any fragment of such antibodies that retains the ability to bind to an antigen.

[0036] As used herein, "bispecific antibodies" are used to refer to full-length antibodies generated by quadroma technology (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 knob-into-hole (KIH) method or similar techniques that introduce mutations within the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14):6444-6448 (1993)), resulting in a number of different immunoglobulin species, only one of which is a functional bispecific antibody. A bispecific antibody binds one antigen (or epitope) in one of its two binding arms (one pair of HC / LC) and binds a different antigen (or epitope) in its second arm (a different pair of HC / LC). By this definition, a bispecific antibody has two significantly different antigen-binding arms (both specificity and CDR sequences) and is monovalent for each antigen to which it binds.

[0037] As used herein, "CDR" is used to refer to the "complementarity determining region" in the variable sequence of an antibody. There are three CDRs in each of the heavy and light chain variable regions. From the N-terminus of the heavy or light chain, these regions are designated as "CDR1", "CDR2" and "CDR3" for each of the variable regions. As used herein, the term "CDR set" refers to a group of three CDRs occurring in a single variable region that bind to an antigen. Thus, an antigen-binding site may contain six CDRs, including a CDR set from each of the heavy and light chain variable regions. A polypeptide containing a single CDR (e.g., CDR1, CDR2 or CDR3) may be referred to as a "molecular recognition unit". Crystal structure analysis of antigen-antibody complexes supports that the amino acid residues of the CDRs make extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR3. Thus, the molecular recognition units may be primarily responsible for the specificity of an antigen-binding site. In general, the CDR residues are directly and most substantially involved in influencing binding to antigen.

[0038] The exact 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 an unambiguous residue numbering system that is applicable to any variable region of an antibody, but also provides the exact residue boundaries that define the three CDRs. These CDRs may 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)) have described the Kabat CDRs in a numbered system that is applicable to any variable region of an antibody. It was found that certain sub-portions within the CDRs adopt nearly identical peptide backbone conformations despite great diversity at the amino acid sequence level. These sub-portions were designated "L1", "L2" and "L3", or "H1", "H2" and "H3", with "L" and "H" designating the light and heavy chain regions, respectively. These regions are sometimes referred to as "Chothia CDRs", which have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are described in Padlan, FASEB, and others. J., 9:133-139 (1995); and MacCallum, J. Mol. Biol., 262(5):732-745 (1996). Still other CDR boundary definitions may not strictly follow the system defined herein and may be shorter or longer in light of predicted or experimental findings that a particular residue or group of residues, or even an entire CDR, does not significantly affect binding to the antigen, but still overlap with the Kabat CDRs. The methods used herein may use CDRs defined according to any of these systems, although certain embodiments use the Kabat-defined CDRs or the Chothia-defined CDRs.

[0039] "Component", "components" or "at least one component" generally refers to capture antibodies, detection reagents or detection conjugates, calibrators, controls, sensitivity panels, containers, buffers, diluents, salts, enzymes, cofactors for enzymes, detection reagents, pretreatment reagents / solutions, substrates (e.g., in solution), stop solutions, etc. that may be included in a kit for assaying a test sample, such as a patient urine sample, whole blood sample, serum sample or plasma sample, according to the methods described herein and other methods known in the art. Some components may be in solution or may be lyophilized for reconstitution for use in the assay.

[0040] As used herein, "control" generally refers to a reagent whose purpose is to assess the performance of a measurement system to ensure that it continues to provide results within acceptable boundaries (e.g., boundaries ranging from a scale appropriate for an assay for research use at one end to analytical boundaries established by quality specifications for a commercially available assay at the other end). To achieve this, the control should be indicative of patient outcome and, optionally, should somehow evaluate the effect of errors on the measurement (e.g., errors due to reagent stability, calibrator variability, instrument variability, etc.). As used herein, "control subject" refers to one or more subjects who have not suffered a traumatic brain injury (TBI). As used herein, "healthy control" or "healthy control subject" refers to a subject or subjects who are considered healthy and have not suffered an apparent TBI.

[0041] As used herein, "correlated with" refers to "compared to."

[0042] As used herein, "CT scan" refers to a 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 bones, blood vessels, and soft tissues within the body. A CT scan may use X-ray CT, positron emission tomography (PET), single photon emission computed tomography (SPECT), computer axial tomography (CAT scan), or computer-assisted tomography. A CT scan may be a conventional CT scan or a spiral / spiral CT scan. In a conventional CT scan, the scan is acquired slice by slice, stopping and moving to the next slice after each slice, e.g., from above the abdomen to the pelvis. A conventional CT scan requires the patient to hold their breath to avoid motion artifacts. A spiral / spiral CT scan is a continuous scan, acquired in a spiral, and the scanned images are continuous, making the process much quicker.

[0043] As used herein, a "derivative" of an antibody may refer to an antibody that has one or more modifications to its amino acid sequence when compared to the original or parent antibody, and may exhibit a modified domain structure. A derivative may not only adopt an amino acid sequence capable of specifically binding to a target (antigen), but may further adopt the typical domain configuration found in a natural antibody. Typical examples of antibody derivatives are antibodies coupled to other polypeptides, rearranged antibody domains or antibody fragments. A derivative may also comprise at least one further compound, e.g. a protein domain, which is linked by covalent or non-covalent bonds. Linking may be based on gene fusion according to methods known in the art. The further domain present in a fusion protein comprising an antibody may preferably be linked by a flexible linker, advantageously a peptide linker, which comprises multiple, hydrophilic, peptide-linked amino acids of sufficient length to span the distance between the C-terminus of the further protein domain and the N-terminus of the antibody or vice versa. The antibody may have a conformation suitable for biological activity or may be linked to an effector molecule that selectively binds, for example, a solid support, a biologically active substance (e.g., a cytokine or growth hormone), a chemical agent, a peptide, a protein, or a drug.

[0044] "Determined by an assay" is used herein to refer to the determination of a reference level by any suitable assay. In some embodiments, the determination of the reference level can be achieved by the same type of assay as that applied to the sample from the subject (e.g., 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, the determination of the reference level can be achieved by the same type of assay as that applied to the sample from the subject under the same assay conditions. As mentioned herein, the present disclosure presents exemplary reference levels (e.g., calculated by comparing reference levels at different time points). It is well within the skill of the art to adapt the present disclosure herein to other assays and obtain assay-specific reference levels for these other assays based on the description presented by the present disclosure. For example, assay-specific reference levels may be obtained using a set of training samples that includes samples obtained from subjects known to have sustained a head injury (e.g., samples obtained from human subjects known to have sustained (i) mild TBI and / or (ii) moderate, severe, or moderate-severe TBI) and samples obtained from subjects (e.g., human subjects) known to have not sustained a head injury. It is understood that a reference level "determined by an assay" and having a recited level of "sensitivity" and / or "specificity" is used herein to refer to a reference level that has been determined to result in the method of the recited sensitivity and / or specificity when employed in the methods of the present disclosure.It is well within the skill of one in the art to determine the sensitivity and specificity associated with a given reference level in the methods of the present disclosure, for example by repeating statistical analyses on the assay data using several different possible reference levels.

[0045] In practice, when distinguishing subjects as having or not having traumatic brain injury, or as having mild versus moderate, severe, or moderate-severe traumatic brain injury, one skilled in the art will balance the effect of raising the cutoff on sensitivity and specificity. Raising or lowering the cutoff has well-defined and predictable effects on sensitivity and specificity, as well as other standard statistical measures. It is well known that raising the cutoff will improve specificity but likely worsen sensitivity (the proportion of people who have the disease and test positive). In contrast, lowering the cutoff will improve sensitivity but worsen specificity (the proportion of people who do not have the disease and test negative). The ramifications of detecting traumatic brain injury or determining mild versus moderate, severe, or moderate-severe traumatic brain injury will be readily apparent to one skilled in the art. In discriminating whether a subject has or does not have traumatic brain injury or mild versus moderate, severe, or moderate-severe traumatic brain injury, the higher the cutoff, the better the specificity as more true negatives (i.e., subjects with no traumatic brain injury, no mild traumatic brain injury, no moderate traumatic brain injury, no severe traumatic brain injury, or no moderate-severe traumatic brain injury) are distinguished from subjects with traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury. However, at the same time, raising the cutoff necessarily reduces sensitivity, as it reduces the number of cases identified as positive overall, as well as the number of true positives. Conversely, a lower cutoff improves sensitivity as more true positives (i.e., subjects with traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury) are distinguished from subjects without traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury. At the same time, however, specificity necessarily decreases, as lowering the cutoff increases the number of cases identified as overall positives, as well as the number of false positives.

[0046] In general, high sensitivity values ​​help the skilled artisan to exclude a disease or condition (such as traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury), and high specificity values ​​help the skilled artisan to include a disease or condition. Whether the skilled artisan wants to exclude or include a disease depends on what the consequences are for the patient for each type of error. Therefore, without fully disclosing the underlying information on how the values ​​were selected, it is not possible to know or predict the exact balancing used to derive the test cutoff. Balancing specificity and sensitivity against other factors will vary depending on the individual case. For this reason, it may be preferable to provide alternative cutoff (e.g., reference) values ​​for the physician or medical practitioner to choose from.

[0047] "Drug of abuse" is used herein to refer to one or more added substances (e.g., drugs) taken for non-medical reasons (e.g., recreational and / or mood-altering effects). Excessive indulgence in, use of, or dependence on such drugs of abuse is often referred to as "substance abuse." Examples of drugs of abuse include alcohol, barbiturates, benzodiazepines, cannabis, cocaine, hallucinogens (e.g., ketamine, mescaline (peyote), PCP, psilocybin, DMT, and / or LSD), methaqualone, opioids, amphetamines (including methamphetamine), anabolic steroids, inhalants (i.e., substances containing volatile substances that contain psychoactive properties, such as, for example, nitrates, spray paints, cleaning fluids, markers, glues, etc.), and combinations thereof.

[0048] As used herein, "bispecific antibody" is used to refer to a full-length antibody (see PCT Publication No. 02 / 02773) that can bind to two different antigens (or epitopes) in each of its two binding arms (HC / LC pair). Thus, a bispecific binding protein has two identical antigen-binding arms with identical specificity and identical CDR sequences, and is bivalent for each antigen to which it binds.

[0049] As used herein, "dual variable domain" is used to refer to two or more antigen-binding sites on a binding protein, which may be a bivalent binding protein (two antigen-binding sites), a tetravalent binding protein (four antigen-binding sites), or a multivalent binding protein. A DVD may 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). A preferred DVD-binding protein comprises two heavy chain DVD polypeptides and two light chain DVD polypeptides and is referred to as a "DVD immunoglobulin" or "DVD-Ig". Such DVD-Ig binding proteins are thus tetrameric and similar to IgG molecules, but provide more antigen-binding sites than IgG molecules. Thus, each half of a tetrameric DVD-Ig molecule is similar to half of an IgG molecule and contains a heavy chain DVD polypeptide and a light chain DVD polypeptide, but unlike the heavy and light chain pair of an IgG molecule, which results in a single antigen-binding domain, the heavy and light chain pair of a DVD-Ig results in two or more antigen-binding sites.

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

[0051] The design, expression and characterization of DVD-Ig binding molecules are described in PCT Publication No. 2007 / 024715, U.S. Patent No. 7,612,181 and Wu et al., Nature Biotech., 25:1290-1297 (2007). A preferred example of such a DVD-Ig molecule comprises 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, preferably 1; and a light chain comprising the structural formula: 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, X2 does not comprise an Fc region, and n is 0 or 1, preferably 1. Such a DVD-Ig can include two such heavy chains and two such light chains, where each chain includes a variable domain linked in tandem with no intervening constant region between the variable domains, where the heavy and light chains can associate to form a functional antigen binding site in tandem, 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 include heavy and light chains including three variable domains (VD1, VD2, VD3), each linked in tandem with no intervening constant region between the variable domains, where the 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, a DVD-Ig binding protein not only binds to the same target molecule that its parent monoclonal antibodies bind, but also retains one or more desired properties of one or more of its parent monoclonal antibodies. Preferably, such additional properties are one or more antibody parameters of the parent monoclonal antibodies. Antibody parameters that can be contributed to a DVD-Ig binding protein derived from one or more of its parent 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 binding to orthologous antigens.

[0053] DVD-Ig binding proteins bind to at least one epitope of UCH-L1, GFAP, or UCH-L1 and GFAP. Non-limiting examples of DVD-Ig binding proteins include (1) DVD-Ig binding proteins that bind to one or more epitopes of UCH-L1, DVD-Ig binding proteins that bind to an epitope of human UCH-L1 and an epitope of UCH-L1 of another species (e.g., mouse), and DVD-Ig binding proteins that bind to an epitope of human UCH-L1 and an epitope of another target molecule; (2) DVD-Ig binding proteins that bind to one or more epitopes of GFAP, DVD-Ig binding proteins that bind to an epitope of human GFAP and an epitope of GFAP of another species (e.g., mouse). 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 over which the readout of an assay is proportional to the amount of target molecule or analyte in the sample being analyzed.

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

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

[0057] As used herein, "F(ab')2 fragment" refers to an antibody generated by pepsin digestion of a whole IgG antibody, removing most of the Fc region while leaving part of the hinge region intact. The F(ab')2 fragment is a bivalent fragment with two antigen-binding F(ab) portions linked together by disulfide bonds, resulting in a molecular weight of approximately 110 kDa. Bivalent antibody fragments (F(ab')2 fragments) are smaller than whole IgG molecules, allowing better tissue penetration and 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. The F(ab')2 fragment can bind and precipitate antigens.

[0058] As used herein, "framework" (FR) or "framework sequence" may refer to the remaining sequence of the variable region, excluding the CDRs. The exact definition of the CDR sequence may be determined by different systems (see, for example, above), and the meaning of the framework sequence is subject to different interpretations accordingly. 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 subregions (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. As mentioned by others, if a particular subregion is not specified as FR1, FR2, FR3, or FR4, the framework region represents the combination of FRs in the variable region of a single, naturally occurring immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FR refers to two or more of the four subregions that make up a framework region.

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

[0060] As used herein, a "functional antigen-binding site" can refer to a site on a binding protein (e.g., an antibody) that can bind to a target antigen. The antigen-binding affinity of an antigen-binding site may not be as strong as that of the parent binding protein, e.g., the parent antibody, from which the antigen-binding site is derived, but the ability to bind to an antigen must be measurable using any one of a variety of known methods for assessing proteins, e.g., antibodies, that bind to antigens. Furthermore, the antigen-binding affinity of each of the antigen-binding sites of a multivalent protein, e.g., a multivalent antibody herein, does not need to be quantitatively the same.

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

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

[0063] As used herein, "GCS (Glasgow Coma Scale)" or "GCS" refers to a 15-point scale for estimating and grading the outcome of brain injury based on overall social ability or dependency on others. The test measures motor response, language response and eye opening response with these values: I. Motor response (6: completely follows instructions; 5: locates noxious stimuli; 4: withdraws from noxious stimuli; 3: abnormal flexion, i.e., decorticate posture; 2: extension response, i.e., decorticate posture and 1: no response); II. Language response (5: clear and oriented; 4: confused but coherent speech; 3: incoherent sentences consisting of inappropriate words and words; 2: unintelligible speech and 1: no speech) and III. Eye opening (4: spontaneous eye opening; 3: eye opening in response to speech; 2: eye opening in response to pain and 1: no eye opening). The final score is determined by adding the values ​​of I+II+III. The final score may be categorized into four possible levels of survival, with lower numbers indicating more severe damage and poorer prognosis: mild (13-15); moderate disability (9-12) (loss of consciousness for more than 30 minutes; physical or cognitive impairment that may or may not be lost; would benefit from rehabilitation); severe disability (3-8) (stupor: unconscious state: no meaningful response, no spontaneous activity); and vegetative state (less than 3) (sleep-wake cycle; awakening but no interaction with environment; unlocalized response to pain). Moderate brain injury is defined as brain injury resulting in loss of consciousness for 20 minutes to 6 hours and a Glasgow Coma Scale (GCS) of 9-12. Severe brain injury is defined as brain injury resulting in loss of consciousness for more than 6 hours and a Glasgow Coma Scale (GCS) of 3-8.

[0064] As used herein, "Glasgow Outcome Scale (GOS)" refers to a global scale for functional outcome that assesses a patient's condition into one of five categories: death, vegetative state, severe disability, moderate disability, or good recovery.

[0065] The "Extended Glasgow Outcome Scale" or "GOSE", used interchangeably herein, provides a more detailed categorization into eight categories by subdividing the severely disabled, moderately disabled and well recovered categories into upper and lower categories, as shown in Table 1.

[0066] [Table 1]

[0067] As used herein, "humanized antibody" is used to describe an antibody that contains heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or VL sequences have been altered to be more "human-like", i.e., more similar to human germline variable sequences. A "humanized antibody" is an antibody, or a variant, derivative, analog, or fragment thereof, that immunospecifically binds to an antigen of interest and contains a framework (FR) region that has substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) that has substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR that has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the non-human antibody CDR. A humanized antibody comprises substantially all of at least one, but typically 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., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In certain embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically the Fc region of a human immunoglobulin. In some embodiments, the humanized antibody contains at least the variable domain of the heavy chain as well as the light chain. The antibody may also contain 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 specific embodiments, the humanized antibody contains only humanized variable domains of the light chain and / or humanized heavy chain.

[0068] The humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including, without limitation, IgG1, IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions, using techniques well known in the art.

[0069] The framework regions and CDRs of a humanized antibody need not correspond exactly to the parental sequences, for example, the donor antibody CDR or consensus framework may be mutagenized by substitution, insertion and / or deletion of at least one amino acid residue such that the CDR or framework residue at this site does not correspond to the donor antibody or consensus framework. However, in a preferred embodiment, such mutations are not extensive mutations. Typically, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues correspond to the residues of the parental FR and CDR sequences. The term "consensus framework" as used herein refers to a framework region within a consensus immunoglobulin sequence. The term "consensus immunoglobulin sequence" as used herein refers to a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, for example, Winnaker, "From Genes to Clones" (Verlagsgesellschaft, Weinheim, 1987)). Thus, a "consensus immunoglobulin sequence" may comprise a "consensus framework region" and / or a "consensus CDR." Within a family of immunoglobulins, each position within a consensus sequence is occupied by the amino acid that occurs most frequently at that position within the family. If two amino acids occur equally frequently, either may be included within the consensus sequence.

[0070] "Identical" or "identity" as used herein in the context of two or more polypeptide or polynucleotide sequences may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions in both sequences where identical residues occur to determine the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to determine the percentage of sequence identity. If the two sequences are of different length or the alignment results in one or more sticky ends and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator of the calculation, but not in the numerator.

[0071] As used herein, "imaging procedure" refers to a medical test that allows a view inside the body to diagnose, treat, and monitor a health condition. Imaging procedures can be non-invasive procedures that allow diagnosis of disease and injury without being invasive. Examples of imaging procedures include MRI, CT scans, X-rays, positron emission tomography (PET) scans, single photon emission computed tomography (SPECT), and diffusion tensor imaging (DTI) scans.

[0072] "Head injury" or "head injury", used interchangeably herein, refers to any trauma to the scalp, skull, or brain. Such injury may include only a minor bump on the head, or may be a severe brain injury. Such injury includes primary injury to the brain and / or secondary injury to the brain. Primary brain injury occurs during the initial insult and results from the displacement of the brain's physical structures. More specifically, primary brain injury is physical damage to the parenchyma (tissue, blood vessels) that occurs during the traumatic event, resulting in shearing and compression of the surrounding brain tissue. Secondary brain injury occurs subsequent to 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 (hours to days) following the primary brain injury. Secondary brain injury includes a whole cascade of cellular, chemical, tissue, or vascular changes in the brain that cause further destruction of brain tissue.

[0073] Head injuries can be closed or open (penetrating). Closed head injuries refer to trauma to the scalp, skull, or brain where the impacting object does not penetrate the skull. Open head injuries refer to trauma to the scalp, skull, or brain where the impacting object penetrates the skull. Head injuries can be caused by physical concussion of a person, blunt blows from an external machine, or other forces that result in closed or open head injuries (e.g., vehicular accidents from automobiles, airplanes, trains, etc.; blows to the head, such as blows from a baseball bat or a blow from a firearm), cerebrovascular accidents (e.g., strokes), one or more falls (e.g., during sports or other activities), explosions or blasts (collectively, "blast injuries"), and other types of blunt trauma. Alternatively, head injuries can be caused by ingestion of and / or exposure to chemicals, toxins, or combinations of chemicals and toxins. Examples of such chemicals and / or toxins include fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organometallics (such as methylmercury, tetraethyl lead, and organotins), and / or one or more drugs of abuse. Alternatively, head injury may be caused as a result of the subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, one or more viruses (e.g., SARS-CoV-2), meningitis, hydrocephalus, hypoxia, or a combination thereof. In some cases, it is not possible to ascertain whether any such event or injury has occurred or occurred. For example, a patient or subject may not have a medical history, the subject may be unable to speak, the subject may be aware of what event the subject was exposed to, etc. Such situations are described herein as the subject "may have suffered a head injury." In certain embodiments herein, closed head injury does not include and specifically excludes cerebrovascular accidents, such as stroke.

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

[0075] As used herein, "label" and "detectable label" refer to a moiety attached to an antibody or analyte such that the reaction of the antibody with the analyte is detectable, and the antibody or analyte so labeled is said to be "detectably labeled." The label may provide a signal detectable by visual or instrumental means. Various labels include signal generators such as chromogens, fluorescent compounds, chemiluminescent compounds, radioactive compounds, and the like. Representative examples of labels include moieties that provide light, e.g., acridinium compounds, and moieties that provide fluorescence, e.g., fluorescein. Other labels are also described herein. In this regard, the moiety itself may not be detectable, but may become detectable upon reaction with yet another moiety. Use of the term "detectably labeled" is intended to encompass such labels.

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

[0077] In one embodiment, the acridinium compound is acridinium-9-carboxamide. Methods for preparing acridinium 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., Luminescence Biotechnology: Instruments and Applications, edited by Dyke, KV, CRC Press: Boca Raton, 77-105 (2002); Adamczyk et al., Org. Lett., 5:3779-3782 (2003); and U.S. Pat. Nos. 5,468,646, 5,543,524, and 5,783,699, each of which is incorporated by reference in its entirety for its teachings herein.

[0078] Another example of an acridinium compound is acridinium-9-carboxylate aryl ester. An example of the acridinium-9-carboxylate aryl ester of formula II is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available from Cayman Chemical, Ann Arbor, MI). Methods for preparing acridinium-9-carboxylate aryl ester 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 (each of which is incorporated herein by reference in its entirety for its teachings therein). Such acridinium-9-carboxylate aryl esters are effective chemiluminescent indicators for hydrogen peroxide generated in the oxidation of an analyte by at least one oxidase in terms of signal intensity and / or signal rapidity. The chemiluminescence process of acridinium-9-carboxylate aryl esters is completed quickly, i.e., in less than one second, whereas the chemiluminescence of acridinium-9-carboxamides lasts for more than two seconds. However, acridinium-9-carboxylate aryl esters lose their chemiluminescent properties in the presence of proteins. Therefore, their use requires the absence of proteins at the time of signal generation and detection. Methods for separating or removing proteins in 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 acridinium-9-carboxylate aryl esters and their uses are set forth in U.S. Patent Application No. 11 / 697,835, filed April 9, 2007. Acridinium-9-carboxylate aryl esters can be dissolved in any suitable solvent, such as degassed anhydrous N,N-dimethylformamide (DMF) or aqueous sodium cholate.

[0079] A "linking sequence" or "linking peptide sequence" refers to a naturally occurring or artificial polypeptide sequence connected to one or more polypeptide sequences of interest (e.g., full length sequences, sequence fragments, etc.). The term "connected" refers to the joining of the linking sequence to the polypeptide sequence of interest. Such polypeptide sequences are preferably joined by one or more peptide bonds. The linking sequence may have a length of about 4 to about 50 amino acids. Preferably, the length of the linking sequence is about 6 to about 30 amino acids. A naturally occurring linking sequence may be modified by amino acid substitution, addition, or deletion to create an artificial linking sequence. Linking sequences may be used for many purposes, including use in recombinant Fab. Exemplary linking sequences include, but are not limited to: (i) histidine (His) tags, such as a 6×His tag, having an amino acid sequence of HHHHHH (SEQ ID NO: 3), are useful as linking sequences to facilitate isolation and purification of polypeptides and antibodies of interest; (ii) enterokinase cleavage sites, such as a His tag, are used in the isolation and purification of proteins and antibodies of interest. Enterokinase cleavage sites are often used in conjunction with His tags in the isolation and purification of proteins and antibodies of interest. A variety of enterokinase cleavage sites are known in the art. Examples of enterokinase cleavage sites include, but are not limited to, the amino acid sequence of DDDDK (SEQ ID NO: 4) and its derivatives (e.g., ADDDDK (SEQ ID NO: 5) and the like); (iii) other sequences can also be used to link or connect the light chain variable region and / or heavy chain variable region of the single chain variable region fragment. Examples of other linking 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). Linking sequences can also be modified for additional functions, such as conjugation of drugs or conjugation to solid supports. In the context of the present disclosure, the monoclonal antibody may contain linking sequences such as, for example, a His tag, an enterokinase cleavage site, or both.

[0080] As used herein, a "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical, except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigen (although, for example, cross-reactivity or shared reactivity may occur). Furthermore, in contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies herein include, inter alia, "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological properties.

[0081] "Magnetic Resonance Imaging" or "MRI," as used interchangeably herein, refers to a medical imaging technique (e.g., referred to interchangeably herein as "MRI," "MRI procedure," or "MRI exam") used in radiology to produce images of the anatomy and physiological processes of the body, both in health and disease. MRI is a form of medical imaging that measures the response of atomic nuclei in body tissues to radio frequency radio waves when placed in a strong magnetic field, producing images of internal organs. MRI scanners are based on the science of nuclear magnetic resonance (NMR) and use strong magnetic fields, radio waves, and field gradients to produce images of the inside of the body.

[0082] As used herein, "multivalent binding protein" is used 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 binding proteins that can bind to two or more related or unrelated targets, including binding proteins capable of binding to two or more different epitopes of the same target molecule.

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

[0084] As used herein, the phrase "odds ratio" refers to a number or value used to compare the relative odds of occurrence of an outcome of interest (e.g., a disease, disorder, or injury (e.g., traumatic brain injury)) given an exposure to a variable of interest (e.g., a health characteristic, an event (e.g., suffering an injury), or an aspect of a medical history). Odds ratios can also be used to determine whether a particular exposure is a risk factor for a particular outcome and to compare the magnitude of various risk factors for that outcome.

[0085] A "pediatric subject" refers to a subject who is under the age of 18 (i.e., not 18 years old or older). For example, a pediatric subject may be under about 18 years old, or about 17 years old, about 16 years old, about 15 years old, about 14 years old, about 13 years old, about 12 years old, about 11 years old, about 10 years old, about 9 years old, about 8 years old, about 7 years old, about 6 years old, about 5 years old, about 4 years old, about 3 years old, about 2 years old, about 1 year old, or less than about 1 year old. In some embodiments, a pediatric subject may be less than about 1 year old to less than about 18 years old. In some embodiments, a pediatric subject may be less than about 1 year old to about 17 years old. For example, a pediatric subject may be any of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, or about 11 months, in total, less than about 18 years old, or about 17 years old, or about 16 years old, or about 15 years old, or about 14 years old, or about 13 years old, or about 12 years old, or about 11 years old, or about 10 years old, or about 9 years old, or about 8 years old, or about 7 years old, or about 6 years old, or about 5 years old, or about 4 years old, or about 3 years old, or about 2 years old, or about 1 year old, or less than about 1 year old.

[0086] "Point-of-care device" refers to a device used to provide medical diagnostic testing at or near the point of care (i.e., outside a laboratory), at the time and place of patient care (such as a hospital, clinic, urgent care facility or other medical care facility, the patient's home, a nursing home and / or long-term care facility and / or hospice facility, etc.). Examples of point-of-care devices include point-of-care devices made by Abbott Laboratories (Abbott Park, IL) (e.g., i-STAT and i-STAT Alinity, Universal Biosensors (Rowville, Australia)) (see U.S. Patent Application Publication No. 2006 / 0134713), Axis-Shield PoC AS (Oslo, Norway) and Clinical Lab Products (Los Angeles, USA).

[0087] "Positive predictive value" or "PPV," used interchangeably herein, refers to the probability that a subject, given a positive test result, will have a positive outcome (i.e., that the proposed outcome is present) (i.e., a subject who tests positive for the proposed outcome will have the proposed outcome).

[0088] "Quality control reagents" in the context of the immunoassays and kits described herein include, but are not limited to, calibrators, controls, and sensitivity panels. "Calibrators" or "standards" (e.g., a plurality, one or more) are typically used to establish a calibration curve for interpolating the concentration of an analyte, such as an antibody or analyte. Alternatively, a single calibrator that is near a reference or control level (e.g., a "low", "medium", or "high" level) may be used. Multiple calibrators (i.e., more than one calibrator or varying amounts of calibrators) may be used to comprise a "sensitivity panel".

[0089] A "receiver operating characteristic" curve or "ROC" curve refers to a graphical plot illustrating the operation of a binary classifier system as its discrimination threshold is varied. For example, an ROC curve can be a plot of true positive rate against false positive rate for different possible cutoff points of a diagnostic test. An ROC curve is created by plotting the proportion of true positives among positives (TPR=true positive rate) against the proportion of false positives among negatives (FPR=false positive rate) at various threshold situations. TPR is also known as sensitivity, and FPR is [1-specificity or true negative rate]. ROC curves demonstrate a trade-off between sensitivity and specificity (any increase in sensitivity is accompanied by a decrease in specificity); the closer the curve is to the left and then upper boundaries of the ROC space, 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 cutoff point gives the likelihood ratio (LR) for this value of the test and the area under the curve is a measure of the accuracy of the test.

[0090] "Recombinant antibody" and "recombinant antibodies" refer to antibodies prepared by recombinant methods, by one or more steps including cloning a nucleic acid sequence encoding all or a portion of one or more monoclonal antibodies into a suitable expression vector, and then 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 antibodies), multispecific or multivalent structures formed from antibody fragments, bifunctional antibodies, heteroconjugate Abs, DVD-Ig®, and other antibodies described in (i) herein (dual variable domain immunoglobulins and methods for making them are described in Wu, C. et al., Nature Biotechnology, 25:1290-1297 (2007)). The term "bifunctional antibody" as used herein refers to an antibody that contains a first arm that has specificity for one antigenic site and a second arm that has specificity for a different antigenic site, i.e., a bifunctional antibody has dual specificity.

[0091] As used herein, a "reference level" refers to an assay cutoff value used to assess diagnostic, prognostic or therapeutic efficacy, and which is herein associated or related to various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression, non-progression or improvement of disease, etc.). As used herein, the term "cutoff" refers to a limit (e.g., a number, etc.) above which a certain or specific clinical outcome is seen and below which a different certain or specific clinical outcome is seen.

[0092] This disclosure presents exemplary reference levels. However, it is well known that the reference levels may vary depending on the nature of the immunoassay (e.g., the antibody used, the reaction conditions, the purity of the sample, etc.), and the assays may be compared and standardized. Moreover, it is well within the skill of the artisan to adapt the disclosure herein to other immunoassays and obtain immunoassay-specific reference levels for these other immunoassays based on the descriptions presented by this disclosure. Although the exact values ​​of the reference levels may vary between assays, the findings described herein are generally applicable and may be extrapolated to other assays.

[0093] In certain aspects described herein, the reference level is said to be determined by any assay having a certain specificity and sensitivity.

[0094] As used herein, "risk assessment," "risk classification," "risk identification," or "risk stratification" of a subject (e.g., patient) refers to the assessment of factors, including biomarkers, to predict the risk of developing a future event, including disease onset or disease progression, so that treatment decisions for the subject can be made on a more informed, condition-based basis.

[0095] As used herein, "sample," "test sample," "specimen," "sample from a subject," and "patient sample" may be used interchangeably and may be a sample of blood, such as whole blood (including, e.g., capillary blood, venous blood, dried blood spots, etc.), tissue, urine, serum, plasma, amniotic fluid, sputum, lower respiratory tract specimens, such as, but not limited to, endotracheal aspirate or bronchoalveolar lavage fluid, nasal mucus, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample may be used directly as obtained from the patient or may be pretreated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., as discussed herein or otherwise known in the art to alter the characteristics of the sample.

[0096] A variety of cell types, tissues, or bodily fluids may be utilized to obtain samples. Such cell types, tissues, and bodily fluids may include biopsy and autopsy samples, oropharyngeal specimens, nasopharyngeal specimens, nasal mucus specimens, sections of tissue such as frozen sections taken for histological purposes, blood (such as whole blood, dried blood spots, etc.), plasma, serum, red blood cells, platelets, anal samples (such as anal swabs), interstitial fluid, cerebrospinal fluid, etc. Cell types and tissues may include lymphatic fluid, cerebrospinal fluid, or any fluid collected by aspiration. Tissues or cell types may be provided by removing cell samples from humans and non-human animals, but may also be arrived at by using previously isolated cells (e.g., isolated by another person, at another time, and / or for another purpose). Archival tissues, such as tissues with treatment history or outcome history, may also be used. Protein or nucleotide isolation and / or purification may not be necessary. 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 still 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 still other embodiments, the sample is bronchoalveolar lavage fluid. In still other embodiments, the sample is nasal mucus.

[0097] "Sensitivity" refers to the proportion of subjects whose outcome is positive who are correctly identified as positive (e.g., the proportion of subjects who correctly identify those with the disease or medical condition for which they are being tested). For example, this can include correctly identifying subjects with TBI from subjects without TBI, correctly identifying subjects with moderate, severe, or moderate-severe TBI from subjects with mild TBI, correctly identifying subjects with mild TBI from subjects with moderate, severe, or moderate-severe TBI, correctly identifying subjects with moderate, severe, or moderate-severe TBI from subjects without TBI, or correctly identifying subjects with mild TBI from subjects without TBI, etc.).

[0098] As used herein, the "specificity" of an assay refers to the proportion of subjects whose outcome is negative who are correctly identified as negative (e.g., the proportion of subjects who correctly identify that they do not have the disease or medical condition for which they are being tested). For example, this can include correctly identifying subjects with TBI from subjects without TBI, correctly identifying subjects without moderate, severe, or moderate-severe TBI from subjects with mild TBI, correctly identifying subjects without mild TBI from subjects with moderate, severe, or moderate-severe TBI, or correctly identifying subjects without any TBI, or correctly identifying subjects with mild TBI from subjects without TBI, etc.

[0099] A "series of calibration compositions" refers to a plurality of compositions comprising a known concentration of (1) UCH-L1, each of which differs from the other compositions in the series in its concentration of UCH-L1; and / or a known concentration of (2) GFAP, each of which differs from the other compositions in the series in its concentration of GFAP.

[0100] The term "single molecule detection" as used herein refers to the detection and / or measurement of a single molecule of an analyte in a test sample at very low levels of concentration (such as pg / mL or femtogram / mL levels). Many different single molecule analyzers or devices are known in the art, including 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.

[0101] As used interchangeably herein, "solid phase" or "solid support" refers to any material that can be attached to and / or attract and immobilize (1) one or more capture agents or specific binding partners for capture, or (2) one or more detection agents or specific binding partners for detection. The solid phase can be selected for its intrinsic ability to attract and immobilize the capture agent. Alternatively, the solid phase has a linking agent attached thereto that has the ability to attract and immobilize (1) the capture agent or specific binding partner for capture, or (2) the detection agent or specific binding partner for detection. For example, the linking agent can include a charged substance that is oppositely charged to the capture agent (e.g., specific binding partner for capture) or detection agent (e.g., specific binding partner for detection) itself, or a charged substance conjugated to (1) the capture agent or specific binding partner for capture, or (2) the detection agent or specific binding partner for detection. In general, the linking agent can be any binding partner (preferably a specific binding partner) that is capable of being immobilized (attached to) a solid phase and immobilizing (1) a capture agent or a specific binding partner for capture, or (2) a detection agent or a specific binding partner for detection, via a binding reaction. The linking agent allows the indirect binding of the capture agent to the solid phase material before the performance of the assay or during the performance of 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, sheets, beads, microparticles, chips, and other configurations known to those skilled in the art.

[0102] As used herein, "specific binding" or "specifically binding to" can refer to the interaction of an antibody, protein, or peptide with a second chemical species, where the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure, rather than proteins in general. If an antibody is specific for epitope "A", then the presence of a molecule containing epitope A (or free A, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[0103] A "specific binding partner" is a member of a specific binding pair. A specific binding pair includes two different molecules that specifically bind to each other through chemical or physical means. Thus, in addition to the typical immunoassay specific binding pair of antigen and antibody, other specific binding pairs can include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzymes and enzyme inhibitors, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding member, e.g., analyte analogs. Immunoreactive specific binding members include antigens, antigen fragments, and monoclonal and polyclonal antibodies, as well as antibodies, including complexes and fragments thereof, whether isolated or recombinantly produced.

[0104] "Statistically significant" as used herein refers to the likelihood that a relationship between two or more variables is caused by something 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 reached whenever the observed p-value for the test statistic is less than the significance level defined for the study. The p-value is the probability of obtaining a result at least as extreme as the result observed if the null hypothesis is true. Examples of statistical hypothesis analysis include Wilcoxon's matched rank test, t-test, chi-square test, or Fisher's exact test. "Significant" as used herein refers to a change that has not been determined to be statistically significant (e.g., may not have been subjected to statistical hypothesis testing).

[0105] As used interchangeably herein, "subject" and "patient" 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 or rhesus monkeys, chimpanzees) and humans). In some embodiments, the subject can be human or non-human. In some embodiments, the subject is human. The subject or patient may be undergoing other forms of treatment. In some embodiments, the subject is a human who may be undergoing other forms of treatment. In some embodiments, the subject is a human helper subject, e.g., a horse, dog, or other species that helps a human perform their daily tasks (e.g., companion animals) or occupations (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 further aspects, the subject is an adult subject, e.g., a human adult subject.

[0106] As used herein, "treating", "treating" or "treatment" are each used interchangeably to describe preventing, alleviating or inhibiting the progression of a disease and / or injury or one or more symptoms of such disease to which such term applies. Depending on the subject's condition, the term also refers to preventing disease, including preventing the onset of a disease or preventing symptoms associated with a disease. Treatment can be performed acutely or chronically. The term also refers to reducing the severity of a disease or symptoms associated with such disease before contraction of the disease. Such prevention or reduction of the severity of a disease before contraction refers to administration of a pharmaceutical composition to a subject who is not affected by the disease at the time of administration. "Preventing" also refers to preventing the recurrence of a disease or one or more symptoms associated with such disease. "Treatment" and "therapeutically" refer to the act of treating, where "treating" is as defined above.

[0107] "Traumatic brain injury" or "TBI", used interchangeably herein, refers to a complex injury with a wide range of symptoms and disability. TBI is most often an acute event, like other injuries. TBI can be classified as "mild", "moderate", "moderate-severe", or "severe". The causes of TBI are diverse. For example, in some aspects, the cause of TBI can be physical, such as from a person, shaking from a vehicle accident, injury from a firearm, cerebrovascular accident (e.g., stroke), fall, explosion or blast, and other types of blunt force trauma. Other causes of TBI include ingestion of and / or exposure to one or more chemicals or toxins (fire, mold, asbestos, pesticides and insecticides, organic solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide, and cyanides), organometallics (such as methylmercury, tetraethyl lead, and organotins), one or more drugs of abuse, or combinations thereof). Alternatively, in other aspects, TBI may occur in subjects suffering from autoimmune disease, metabolic disorder, brain tumor, hypoxia, one or more viruses, meningitis, hydrocephalus, or a combination thereof.Young adults and the elderly are the age groups most at risk for TBI.In certain embodiments herein, traumatic brain injury or TBI does not include and expressly excludes cerebrovascular accidents such as stroke.

[0108] As used herein, "mild TBI" refers to brain injury in which loss of consciousness is brief, usually for a few seconds or minutes, and / or confusion and disorientation lasts for less than an hour. Mild TBI is also referred to as concussion, mild head trauma, mild TBI, mild brain injury, and mild head injury. MRI and CT scans may be normal, but individuals with mild TBI may have cognitive problems such as headaches, difficulty thinking, memory problems, attention deficits, mood swings, and frustration.

[0109] Mild TBI is the most common type of TBI and is often overlooked at the time of initial injury. Typically, subjects have a Glasgow Coma Scale (GCS) score between 13-15 (such as 13-15 or 14-15). Of people with mild TBI, fifteen percent (15%) have symptoms that last for more than three months. Mild TBI is defined as the result of a forceful movement of the head or an impact 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 impairment, memory loss, decreased attention / concentration, sleep disorders, vertigo / loss of balance, irritability (emotional disturbance), feelings of depression, and seizures. Other symptoms associated with mild TBI include nausea, loss of smell, sensitivity to light and sound, mood changes, confusion or confusion, and / or slowed thinking.

[0110] As used herein, "moderate TBI" refers to a brain injury in which loss of consciousness and / or confusion and disorientation occurs for between 1 and 24 hours and the subject has a Glasgow Coma Scale (GCS) score between 9 and 12. Individuals with moderate TBI have abnormalities in brain imaging results.

[0111] As used herein, "severe TBI" refers to brain injury in which loss of consciousness exceeds 24 hours, memory loss after injury or penetrating skull injury exceeds 24 hours, and the subject has a Glasgow Coma Scale score between 3 and 8. Deficits range from high level cognitive impairment to coma. Survivors may have limited arm or leg function, speech or language abnormalities, loss of thinking ability, or emotional problems. Individuals with severe injuries may remain in a prolonged refractory state. For many people with severe TBI, prolonged rehabilitation is often required to maximize function and independence.

[0112] Common symptoms of moderate to severe TBI include problems with attention, concentration, distractibility, memory, processing speed, confusion, perseveration, impulsivity, language processing and / or "executive function", not understanding spoken words (receptive aphasia), difficulty speaking and being understood (expressive aphasia), slurred speech, very fast or very slow speech, problems reading, problems writing, difficulties interpreting touch, temperature, movement, position and fine discrimination of the lower extremities, difficulties integrating or patterning sensory impressions into psychologically meaningful data, partial or total loss of vision, eye muscle weakness and double vision (diplopia), blurred vision, problems judging distances, involuntary eye movements (nystagmus), light intolerance (photophobia), hearing problems, reduced or lost hearing, ringing in the ears (tinnitus), hypersensitivity to sound, loss or reduced sense of smell (anosmia), loss or reduced sense of taste, convulsions associated with epilepsy which can be of several types and which may involve disruption of consciousness, sensory awareness or movement, bowel and bladder control, sleep disorders, loss of energy, changes in appetite, regulation of internal temperature, menstrual difficulties, addictive behaviour, emotional capacity, lack of motivation, irritability, aggression, depression, disinhibition or cognitive deficits including denial / lack of consciousness.

[0113] "Ubiquitin carboxy-terminal hydrolase L1" or "UCH-L1," as used interchangeably herein, refers to the deubiquitinating enzyme encoded by the UCH-L1 gene in humans and by UCH-L1 gene counterparts in other species. UCH-L1, also known as ubiquitin carboxyl-terminal esterase L1 and ubiquitin thiolesterase, is a member of a gene family whose products hydrolyze small C-terminal appendages of ubiquitin to generate ubiquitin monomers.

[0114] "UCH-L1 status" can mean the level or amount of UCH-L1 at a point in time (such as a point in time involving a single measurement of UCH-L1), the level or amount of UCH-L1 associated with monitoring (such as monitoring involving repeated testing to identify increases or decreases in the amount of UCH-L1 in a subject), the level or amount of UCH-L1 associated with treatment for traumatic brain injury (whether primary brain injury and / or secondary brain injury), or a combination thereof.

[0115] As used herein, "variant" is used to describe a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or the ability to stimulate an immune response. As used herein, variant is also used to describe a protein with an amino acid sequence that is substantially identical to a reference protein with an amino acid sequence that retains at least one biological activity. It is recognized in the art that conservative substitutions of amino acids, i.e., replacing an amino acid with a different amino acid that has similar properties (e.g., hydrophilicity, degree of charge, and distribution of charged regions), typically involve small changes. As understood in the art, these small changes can be identified, in part, by considering the hydrophobicity index of an amino acid (Kyte et al., J. Mol. Biol. 157:105-132 (1982)). The hydrophobicity index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophilicity indexes may be substituted and still retain protein function. In one embodiment, amino acids with hydrophobicity indexes of ±2 are substituted. The hydrophilicity of amino acids may also be used to identify substitutions that will result in proteins that retain biological function. Consideration of the hydrophilicity of amino acids in the context of a peptide allows for the calculation of the maximum local average hydrophilicity of the peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity (U.S. Pat. No. 4,554,101, incorporated herein by reference). Substitution of amino acids with similar hydrophilicity values ​​may result in peptides that retain biological activity, such as immunogenicity as understood in the art. Substitutions may be made with amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the particular side chain of the amino acid.Consistent with this observation is the understanding that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size and other properties, and in particular on the side chains of these amino acids. "Variants" may also be used to refer to antigenically reactive fragments of anti-analyte (such as GFAP, UCH-L1 or GFAP and UCH-L1) antibodies that differ in amino acid sequence from the corresponding fragments of anti-analyte (such as GFAP, UCH-L1 or GFAP and UCH-L1) antibodies, but are still antigenically reactive and can compete with the corresponding fragments of anti-analyte (such as GFAP, UCH-L1 or GFAP and UCH-L1) antibodies for binding to the analyte (such as GFAP, UCH-L1 or GFAP and UCH-L1). "Variants" may also be used to describe polypeptides or fragments thereof that are differentially processed, such as by proteolysis, phosphorylation or other post-translational modifications, but that retain their antigenic reactivity.

[0116] As used herein, "vector" is used to describe a nucleic acid molecule that can carry another nucleic acid to which it has been 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 are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., mammalian non-episomal vectors) can be integrated into the genome of the host cell upon introduction into the host cell, and thereby replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Since plasmids are the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions, may also be used. In this regard, RNA forms of vectors, including viral RNA vectors, may also be used in the context of the present disclosure.

[0117] As used herein, "Y" refers to Youden's J statistic (also called Youden's index), which is a single statistic that captures the performance of a dichotomous diagnostic test. Y is expressed by the following formula:

[0118] J=sensitivity+specificity-1 The two right hand side quantities are sensitivity and specificity. The expanded formula is shown below.

[0119]

number

[0120] Unless otherwise specified, scientific and technical terms used herein shall have the same meaning as commonly understood by those skilled in the art. For example, the terminology and techniques used in connection with cell and tissue culture methods, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well-known and commonly used in the art. The meaning and scope of the terms shall be clear, but in the unlikely event of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0121] 2. Methods for Assessing Pediatric Subjects The present disclosure relates, inter alia, to a method of assessing a pediatric subject for head injury. The method can help determine whether a pediatric subject with a real or suspected head injury has sustained a traumatic brain injury (TBI), such as a mild TBI. As used herein, "determining whether a subject has sustained a TBI" refers to the fact that the aforementioned method can be used, for example, together with other information (e.g., clinical assessment data), to determine whether a subject is more likely than not to have sustained a TBI, such as a mild TBI, and / or to show a positive or negative finding for a head imaging procedure, such as a positive or negative MRI head result or a positive or negative CT scan result.Specifically, such methods may be used within about 48 hours (about 0 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, 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, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about 81 hours, In some embodiments, the assay is performed on a sample obtained from a pediatric subject, such as a sample obtained within 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 to detect GFAP, U in the sample. The method can include measuring or detecting levels of UCH-L1 or GFAP and UCH-L1, and (a) determining that the pediatric subject has suffered a traumatic brain injury (TBI), such as a mild TBI, if (i) the level of GFAP in the sample is equal to or greater than a reference level of GFAP, (ii) the level of UCH-L1 in the sample is equal to or greater than a reference level of UCH-L1; or (iii) the level of GFAP in the sample is equal to or greater than a reference level of GFAP and the level of UCH-L1 in the sample is equal to or greater than a reference level of UCH-L1, and (b) determining that the pediatric subject has not suffered a TBI if the level of GFAP in the sample is lower than the reference level of GFAP and / or the level of UCH-L1 in the sample is lower than the reference level of UCH-L1. The sample can be a biological sample, such as a human (e.g., pediatric) sample. In some embodiments, baseline levels of GFAP and / or UCH-L1 correlate with and distinguish subjects with TBI (e.g., pediatric subjects) from subjects without TBI, while in other embodiments, baseline levels of GFAP and / or UCH-L1 correlate with and distinguish subjects with mild TBI (e.g., pediatric subjects) from subjects without mild TBI.

[0122] In some embodiments, the pediatric subject is determined to have suffered a traumatic brain injury if the level of GFAP in the sample is equal to or greater than the reference level of GFAP. In some embodiments, the reference level of GFAP is at least about 30 pg / mL. In some embodiments, the reference level of GFAP is between about 30 pg / mL and about 1000 pg / mL. In some embodiments, the reference level of GFAP is 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 90 pg / mL, about 95 pg / mL, or about 100 pg / mL. In some embodiments, the reference level of GFAP is between about 100 pg / mL and about 1000 pg / mL. For example, the baseline level of GFAP can be at least about 100 pg / mL, at least about 150 pg / mL, at least about 200 pg / mL, at least about 250 pg / mL, at least about 300 pg / mL, at least about 350 pg / mL, at least about 400 pg / mL, at least about 450 pg / mL, at least about 500 pg / mL, at least about 550 pg / mL, at least about 600 pg / mL, at least about 650 pg / mL, at least about 700 pg / mL, at least about 750 pg / mL, at least about 800 pg / mL, at least about 850 pg / mL, at least about 900 pg / mL, at least about 950 pg / mL, or about 1000 pg / mL.

[0123] In some embodiments, a pediatric subject is determined to have suffered a traumatic brain injury if the level of GFAP in the sample is equal to or greater than a reference level of GFAP of at least about 30 pg / mL. For example, a pediatric subject may be determined to have suffered a traumatic brain injury if the level of GFAP in the sample is equal to or greater than 30 pg / mL. As another example, a pediatric subject may be determined to have suffered a traumatic brain injury if the level of GFAP in the sample is equal to or greater than about 50 pg / mL. In some embodiments, a pediatric subject may be determined to have suffered a traumatic brain injury if the level of GFAP in the sample is equal to or greater than about 65 pg / mL. In some embodiments, a pediatric subject may be determined to have suffered a traumatic brain injury if the level of GFAP in the sample is equal to or greater than about 1000 pg / mL.

[0124] In some embodiments, the pediatric subject is determined to have suffered a traumatic brain injury if the level of UCH-L1 in the sample is equal to or greater than the reference level of UCH-L1. In some embodiments, the reference level is at least about 55 pg / mL. In some embodiments, the reference level of UCH-L1 is between about 55 pg / mL and about 1000 pg / mL. In some embodiments, the reference level of UCH-L1 is between 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. In some embodiments, the reference level of UCH-L1 is between about 100 pg / mL and about 1000 pg / mL. In some embodiments, the reference level of UCH-L1 is about 100 pg / mL, about 120 pg / mL, about 140 pg / mL, about 160 pg / mL, about 180 pg / mL, about 200 pg / mL, about 220 pg / mL, about 240 pg / mL, about 260 pg / mL, about 280 pg / mL, about 300 pg / mL, about 320 pg / mL, about 340 pg / mL, about 360pg / mL, about 380pg / mL, about 400pg / mL, about 450pg / mL, about 500pg / mL, about 550pg / mL, about 600pg / mL, about 650pg / mL, about 700pg / mL, about 750pg / mL, about 800pg / mL, about 850pg / mL, about 900pg / mL, about 950pg / mL, or about 1000pg / mL.

[0125] In some embodiments, the pediatric subject is determined to have a traumatic brain injury if the level of UCH-L1 in the sample is greater than the reference level of UCH-L1 of at least about 55 pg / mL. For example, the pediatric subject may be determined to have a traumatic brain injury if the level of UCH-L1 in the sample is equal to or greater than 55 pg / mL. As another example, the pediatric subject may be determined to have a traumatic brain injury if the level of UCH-L1 in the sample is equal to or greater than 300 pg / mL. As another example, the pediatric subject may be determined to have a traumatic brain injury if the level of UCH-L1 in the sample is equal to or greater than 360 pg / mL.

[0126] In some embodiments, the pediatric subject is determined to have a traumatic brain injury if the level of GFAP in the sample is greater than the reference level of GFAP and the level of UCH-L1 in the sample is greater than the reference level of UCH-L1. In some embodiments, the reference level of UCH-L1 is at least about 30 pg / mL and the reference level of UCH-L1 is about 360 pg / mL. For example, the pediatric subject may be determined to have a traumatic brain injury if the level of GFAP in the sample is equal to or greater than 30 pg / mL and the level of UCH-L1 in the sample is equal to or greater than 360 pg / mL. In some embodiments, the reference level of GFAP is about 65 pg / mL and the reference level of UCH-L1 is about 360 pg / mL. For example, a pediatric subject may be determined to have suffered a traumatic brain injury if the level of GFAP in the sample is equal to or greater than 65 pg / mL and the level of UCH-L1 in the sample is equal to or greater than 360 pg / mL.

[0127] The present disclosure also relates, inter alia, to a method of assessing a pediatric subject to determine whether the subject would benefit from and therefore undergo an imaging procedure, such as an MRI or a head computed tomography (CT) scan. As used herein, "determining whether a pediatric subject would benefit from and therefore undergo an imaging procedure" refers to the fact that the aforementioned method can be used, for example, together with other information (e.g., clinical evaluation data), to determine whether a pediatric subject is more likely than not to have suffered a TBI, such as a mild TBI, and more likely than not to show a positive finding for a head imaging procedure, such as a positive MRI head result or a positive CT scan result. In some embodiments, a method of assessing whether a pediatric subject should undergo a head CT scan is provided herein. Specifically, such methods can include performing an assay on a sample obtained from the pediatric subject, such as within about 48 hours (e.g., zero to about 48 hours); and (a) determining that a head CT scan should be performed on the pediatric subject if (i) the level of GFAP in the sample is equal to or greater than a reference level of GFAP, (ii) the level of UCH-L1 in the sample is equal to or greater than a reference level of UCH-L1; or (iii) the level of GFAP in the sample is equal to or greater than a reference level of GFAP and the level of UCH-L1 in the sample is equal to or greater than a reference level of UCH-L1. In some embodiments, the method further includes providing a head imaging procedure (e.g., a head CT scan). In some embodiments, the method includes determining that the subject has not suffered a TBI if the level of GFAP in the sample is lower than the reference level of GFAP and / or the level of UCH-L1 in the sample is lower than the reference level of UCH-L1. The sample can be a biological sample, such as a human sample. Conversely, as described herein, low levels of one or more of the GFAP and UCH-L1 biomarkers can predict whether a scan is likely to be negative.

[0128] In some embodiments, if the level of GFAP in the sample is equal to or greater than the reference level of GFAP, it is determined that a head CT scan should be performed. In some embodiments, the reference level of GFAP is at least about 30 pg / mL. In some embodiments, the reference level of GFAP is between about 30 pg / mL and about 1000 pg / mL. In some embodiments, the reference level of GFAP is 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 90 pg / mL, about 95 pg / mL, or about 100 pg / mL. In some embodiments, the reference level of GFAP is between about 100 pg / mL and about 1000 pg / mL. For example, the baseline level of GFAP can be at least about 100 pg / mL, at least about 150 pg / mL, at least about 200 pg / mL, at least about 250 pg / mL, at least about 300 pg / mL, at least about 350 pg / mL, at least about 400 pg / mL, at least about 450 pg / mL, at least about 500 pg / mL, at least about 550 pg / mL, at least about 600 pg / mL, at least about 650 pg / mL, at least about 700 pg / mL, at least about 750 pg / mL, at least about 800 pg / mL, at least about 850 pg / mL, at least about 900 pg / mL, at least about 950 pg / mL, or about 1000 pg / mL.

[0129] In some embodiments, it is determined that a head CT scan should be performed if the level of GFAP in the sample is equal to or greater than a reference level of GFAP of at least about 30 pg / mL. For example, it may be determined that a head CT scan should be performed if the level of GFAP in the sample is equal to or greater than 30 pg / mL. As another example, it may be determined that a head CT scan should be performed if the level of GFAP in the sample is equal to or greater than about 50 pg / mL. In some embodiments, it is determined that a head CT scan should be performed if the level of GFAP in the sample is equal to or greater than about 65 pg / mL. In some embodiments, it is determined that a head CT scan should be performed if the level of GFAP in the sample is equal to or greater than about 1000 pg / mL.

[0130] In some embodiments, if the level of UCH-L1 in the sample is equal to or greater than the reference level of UCH-L1, it is determined that a head CT scan should be performed. In some embodiments, the reference level is at least about 55 pg / mL. In some embodiments, the reference level of UCH-L1 is between about 55 pg / mL and about 1000 pg / mL. In some embodiments, the reference level of UCH-L1 is 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. In some embodiments, the reference level of UCH-L1 is between about 100 pg / mL and about 1000 pg / mL. In some embodiments, the reference level of UCH-L1 is about 100 pg / mL, about 120 pg / mL, about 140 pg / mL, about 160 pg / mL, about 180 pg / mL, about 200 pg / mL, about 220 pg / mL, about 240 pg / mL, about 260 pg / mL, about 280 pg / mL, about 300 pg / mL, about 320 pg / mL, about 340 pg / mL, about 360pg / mL, about 380pg / mL, about 400pg / mL, about 450pg / mL, about 500pg / mL, about 550pg / mL, about 600pg / mL, about 650pg / mL, about 700pg / mL, about 750pg / mL, about 800pg / mL, about 850pg / mL, about 900pg / mL, about 950pg / mL, or about 1000pg / mL.

[0131] In some embodiments, if the level of UCH-L1 in the sample is greater than the reference level of UCH-L1 of at least about 55 pg / mL, it is determined that a head CT scan should be performed. For example, if the level of UCH-L1 in the sample is equal to or greater than 55 pg / mL, it may be determined that a head CT scan should be performed. As another example, if the level of UCH-L1 in the sample is equal to or greater than 300 pg / mL, it may be determined that a head CT scan should be performed. As another example, if the level of UCH-L1 in the sample is equal to or greater than 360 pg / mL, it may be determined that a head CT scan should be performed.

[0132] In some embodiments, if the level of GFAP in the sample is greater than the reference level of GFAP and the level of UCH-L1 in the sample is greater than the reference level of UCH-L1, it is determined that a head CT scan should be performed. In some embodiments, the reference level of UCH-L1 is at least about 30 pg / mL, and the reference level of UCH-L1 is about 360 pg / mL. For example, if the level of GFAP in the sample is equal to or greater than 30 pg / mL and the level of UCH-L1 in the sample is equal to or greater than 360 pg / mL, it may be determined that a head CT scan should be performed. In some embodiments, the reference level of GFAP is about 65 pg / mL and the reference level of UCH-L1 is about 360 pg / mL. For example, if the level of GFAP in the sample is equal to or greater than 65 pg / mL and the level of UCH-L1 in the sample is equal to or greater than 360 pg / mL, it may be determined that a head CT scan should be performed.

[0133] In any of the methods described herein, the method can include obtaining a sample from a pediatric subject within about 48 hours, such as within about 24 hours (e.g., zero to about 25 hours) of an actual or suspected head injury, and contacting the sample with an antibody to GFAP or an antibody to UCH-L1 to allow for formation of an antibody-GFAP or antibody-UCH-L1 complex. The method can also include detecting the antibody-GFAP complex or antibody-UCH-L1 complex thus obtained.

[0134] In some embodiments, the sample is collected from the human (e.g., pediatric) subject within 48 hours of actual or suspected head injury, e.g., about 0 to about 4 hours, about 0 to about 8 hours, about 0 to about 12 hours, about 0 to about 16 hours, about 0 to about 20 hours, about 0 to about 24 hours, and about 0 to about 48 hours. In some embodiments, the sample is collected from the human (e.g., pediatric) subject within about 4 hours to about 8 hours, about 8 hours to about 12 hours, about 12 hours to about 16 hours, about 16 hours to about 20 hours, about 20 hours to about 24 hours, and about 24 hours to about 48 hours. In other embodiments, the samples are incubated for about 0 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, 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, or about 28 hours after the actual or suspected injury. In some embodiments, the antibody can be harvested from a human (e.g., pediatric) subject within 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. In some embodiments, the onset of presence of GFAP, UCH-L1, or GFAP and UCH-L1 occurs at about 0 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, 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 after head injury. , 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.

[0135] In some embodiments, the subject may have a Glasgow Coma Scale (GCS) score at one or more time points before or after the levels of GFAP, UCH-L1, or GFAP and UCH-L1 are determined. In certain embodiments, the subject may be suspected of having mild traumatic brain injury based on the Glasgow Coma Scale (GCS) score. In certain embodiments, the pediatric subject may be suspected of having mild traumatic brain injury based on an abnormal head CT. In some embodiments, the pediatric subject has a CT scan before or after the assay is performed. In some embodiments, the subject has a normal head CT.

[0136] In some embodiments, the reference levels of GFAP, UCH-L1 or GFAP and UCH-L1 correlate with pediatric subjects having moderate to severe traumatic brain injury. In some embodiments, the reference levels of GFAP, UCH-L1 or GFAP and UCH-L1 correlate with a Glasgow Coma Scale (GCS) score of 3-12. In some embodiments, the pediatric subject is suspected to have mild traumatic brain injury based on a Glasgow Coma Scale (GCS) score. In some embodiments, the reference levels of GFAP, UCH-L1 or GFAP and UCH-L1 correlate with subjects having mild traumatic brain injury. In some embodiments, the reference levels of GFAP, UCH-L1 or GFAP and UCH-L1 correlate with a Glasgow Coma Scale (GCS) score of 13-15.

[0137] Generally, the reference level of GFAP, UCH-L1 or GFAP and UCH-L1 can also be used as a benchmark for evaluating the results obtained when a test sample is assayed for GFAP, UCH-L1 or GFAP and UCH-L1. Generally, when making such a comparison, the reference level of GFAP, UCH-L1 or GFAP and UCH-L1 is obtained by performing a particular assay a sufficient number of times and under appropriate conditions to establish a link or association of the presence, amount or concentration of the analyte with a particular stage or endpoint or a particular symptom of TBI. Typically, the reference level of GFAP, UCH-L1 or GFAP and UCH-L1 is obtained by assaying a reference subject (or population of subjects). The measured GFAP, UCH-L1 or GFAP and UCH-L1 can include fragments thereof, degradation products thereof and / or enzymatic cleavage products thereof.

[0138] In certain embodiments, the baseline level may be correlated to control subjects who have not suffered a head injury.

[0139] In some embodiments, the reference levels of GFAP, UCH-L1, or GFAP and UCH-L1 are determined by an assay having a sensitivity (for GFAP, UCH-L1, or GFAP and UCH-L1) of at least about 30% to about 100% and a specificity of at least about 30% to about 100%. In some embodiments, the sensitivity is between at least about 30% to about 100%, between at least about 30% to about 99%, between at least about 30% to about 97%, between at least about 30% to about 95%, between at least about 30% to about 90%, between at least about 30% to about 85%, between at least about 30% to about 80%, between at least about 30% to about 75%, between at least about 35% to about 100%, between at least about 35% to about 99%, between at least about 35% to about 97%, between at least about 35% to about 95%, between at least about 35% to about 90%, between at least about 35% to about 85%, between at least about 35% to about 80%, between at least about 35% to about 75%, between at least about 70% to about 100%, at least about 70% to about 99%, at least about 70% to about 97%, at least about 70% to about 95%, at least about 70% to about 90%, at least about 70% to about 85%, at least about 70% to about 80%, at least about 70% to about 75%, at least about 75% to about 100%, at least about 75% to about 99%, at least about 75% to about 95%, at least about 75% to about 90%, at least about 75% to about 85%, at least about 75% to about 80%, at least about 85% to about 100%, at least about 85% to about 99%, at least about 85% to about 95%, at least about 85% to about 90%, at least about 95% to about 100%, or at least about 95% to about 99%.In some embodiments, the sensitivity is at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 81.0%, at least about 85.0%, at least about 87.5%, at least about 90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%.

[0140] In some embodiments, the specificity (of GFAP, UCH-L1, or GFAP and UCH-L1) is between at least about 10% and about 100%, between at least about 15% and about 99%, between at least about 20% and about 95%, between at least about 25% and about 90%, between at least about 30% and about 85%, between at least about 30% and about 80%, between at least about 30% and about 75%, between at least about 30% and about 70%, between at least about 30% and about 60%, between at least about 30% and about 50%, between at least about 30% and about 40%, at least about 30% between about 35%, at least about 40% to about 100%, at least about 40% to about 99%, at least about 40% to about 95%, at least about 40% to about 90%, at least about 40% to about 85%, at least about 40% to about 80%, at least about 40% to about 75%, at least about 40% to about 70%, at least about 40% to about 60%, at least about 40% to about 50%, at least about 50% to about 100%, at least about 50% to about 99%, at least about 50% to about 95%, at least about 50% to about 9 between 0%, between at least about 50% and about 85%, between at least about 50% and about 80%, between at least about 50% and about 75%, between at least about 50% and about 70%, between at least about 50% and about 60%, between at least about 60% and about 100%, between at least about 60% and about 99%, between at least about 60% and about 95%, between at least about 60% and about 90%, between at least about 60% and about 85%, between at least about 60% and about 80%, between at least about 60% and about 75%, between at least about 60% and about 70%, at least about 70% to about 100% between about 70% and about 99%, between about 70% and about 95%, between about 70% and about 90%, between about 70% and about 85%, between about 70% and about 80%, between about 70% and about 75%, between about 80% and about 100%, between about 80% and about 99%, between about 80% and about 95%, between about 80% and about 90%, between about 80% and about 85%, between about 90% and about 100%, between about 90% and about 99%, between about 90% and about 95%,Specificity is at least about 30.0%, at least about 31.0%, at least about 32.0%, at least about 33.0%, at least about 34.0%, at least about 35.0%, at least about 36.0%, at least about 37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%, at least about 45.0%, at least about 50.0%, at least about 55.0%, at least about 60.0%, at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 81.0%, at least about 82.0%, at least about 83.0%, at least about 84.0%, at least about 85.0%, at least about 90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%, at least about 94.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%. For example, the sensitivity is at least about 99% and the specificity is at least about 75%, the sensitivity is at least about 99% and the specificity is at least about 99%, or the sensitivity is at least about 100% and the specificity is at least about 100%.

[0141] In some embodiments, the reference level of GFAP is determined by an assay having a sensitivity of at least about 90% and a specificity of at least about 40%. For example, the reference level of GFAP can be determined by an assay having a sensitivity of about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% and a sensitivity of at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0142] In some embodiments, the reference level of GFAP is determined by an assay having a sensitivity of at least about 50% and a specificity of at least about 90%. For example, the reference level of GFAP can be determined by an assay having a sensitivity of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% and a specificity of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0143] In some embodiments, the reference level of GFAP is determined by an assay having a negative predictive value of at least about 70%. For example, the reference level of GFAP can be determined by an assay having a negative predictive value of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%. In some embodiments, the reference level of GFAP is determined by an assay having a negative predictive value of at least about 90%.

[0144] In some embodiments, the reference level of GFAP is determined by an assay having a positive predictive value of at least about 50%. For example, the reference level of GFAP can be determined by an assay having a positive predictive value of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%. In some embodiments, the reference level of GFAP is determined by an assay having a positive predictive value of at least about 80%.

[0145] In some embodiments, the reference level of UCH-L1 is determined by an assay having a sensitivity of at least about 80% and a specificity of at least about 25%. In some embodiments, the reference level of UCH-L1 is determined by an assay having a sensitivity of at least about 85%, at least about 90%, at least about 95%, or about 100% and a specificity of at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0146] In some embodiments, the reference level of UCH-L1 is determined by an assay having a sensitivity of at least about 30% and a specificity of at least about 90%. In some embodiments, the reference level of UCH-L1 is determined by an assay having a sensitivity of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% and a specificity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%.

[0147] In some embodiments, the reference level of UCH-L1 is determined by an assay having a negative predictive value of at least about 65%. In some embodiments, the reference level of UCH-L1 is determined by an assay having a negative predictive value of at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%.

[0148] In some embodiments, the reference level of UCH-L1 is determined by an assay having a positive predictive value of at least about 40%. For example, the reference level of UCH-L1 can be determined by an assay having a positive predictive value of at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%. In some embodiments, the reference level of UCH-L1 is determined by an assay having a positive predictive value of at least about 80%.

[0149] In some embodiments, the reference level of GFAP and the reference level of UCH-L1 are determined by an assay having a sensitivity of at least about 70% and a specificity of at least about 10%. For example, the reference level of GFAP and the reference level of UCH-L1 can be determined by an assay having a sensitivity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% and a specificity of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0150] In some embodiments, the reference level of GFAP and the reference level of UCH-L1 are determined by an assay having a sensitivity of at least about 65% and a specificity of at least about 25%. For example, the reference level of GFAP and the reference level of UCH-L1 can be determined by an assay having a sensitivity of at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% and a specificity of at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0151] In some embodiments, the baseline level of GFAP and the baseline level of UCH-L1 are determined by an assay having a positive predictive value of at least about 35%. For example, the baseline level of GFAP and the baseline level of UCH-L1 can be determined by an assay having a positive predictive value of at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0152] In some embodiments, the reference level of GFAP and the reference level of UCH-L1 are determined by an assay having a negative predictive value of at least about 40%. For example, the reference level of GFAP and the reference level of UCH-L1 can be determined by an assay having a negative predictive value of at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%. In some embodiments, the reference level of GFAP and the reference level of UCH-L1 are determined by an assay having a negative predictive value of at least about 55%.

[0153] In some embodiments, the sample is selected from the group consisting of a whole blood sample, a serum sample, a cerebrospinal fluid sample, a tissue sample, a body fluid sample, and a plasma sample. In some embodiments, the sample is a whole blood sample obtained from a human, such as a pediatric human. In other embodiments, the sample is a serum sample obtained from a human, such as a pediatric human. In yet other embodiments, the sample is a cerebrospinal fluid sample obtained from a human, such as a pediatric human. In yet other embodiments, the sample is a plasma sample obtained from a human, such as a pediatric human. More specifically, the pediatric human from whom the sample is collected may be less than about 18 years of age.

[0154] In some embodiments, the methods may be performed on any pediatric subject, regardless of factors selected from the group consisting of the pediatric subject's clinical condition, the subject's laboratory values, and the subject's classification as suffering from mild, moderate, or severe traumatic brain injury.

[0155] In some embodiments, the method may further include treating the pediatric subject determined to have suffered a mild TBI with a traumatic brain injury treatment, as described below. In some embodiments, the method may further include monitoring the pediatric subject determined to have suffered a mild TBI, as described below.

[0156] The nature of the assay used in the methods described herein is not critical, and the test can be any assay known in the art, such as, for example, 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). The assay can also be used in clinical chemistry formats known to those skilled in the art. Such assays are described in more detail herein. It is known in the art that values ​​(e.g., reference levels, cut-offs, thresholds, specificity, sensitivity, calibrator and / or control concentrations, etc.) used in assays that utilize specific sample types (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 in which assay standardization can be performed is by applying a factor to the calibrators used in the assay to make the sample concentration readings higher or lower to obtain a slope commensurate with the comparative method. Other methods of standardizing the results obtained in one assay to another are also well known and described in the literature (see, for example, David Wild, "Immunoassay Handbook", 4th Edition, Chapter 3.5, pages 315-322, the contents of which are incorporated herein by reference).

[0157] 3. Treatment and monitoring of pediatric subjects with traumatic brain injury In the methods described above, subjects identified or assessed as having traumatic brain injury, such as mild traumatic brain injury or moderate to severe traumatic brain injury, may be treated or monitored. In some embodiments, the method further comprises treating the human pediatric subject assessed as having traumatic brain injury with a traumatic brain injury treatment, such as any treatment known in the art. For example, traumatic brain injury treatment may take a variety of forms depending on the severity of the injury to the head. For example, for a pediatric subject suffering from mild TBI, treatment may include symptom management, such as resting, refraining from events (such as sports) that exacerbate symptoms, avoiding light or wearing sunglasses when going out in the sun, administering medications to relieve headaches or migraines, anti-nausea medications, etc. Treatment for patients suffering from severe TBI may include one or more appropriate drugs (e.g., diuretics, anticonvulsant drugs, drugs to sedate the individual and put them into a drug-induced coma, etc.) or other pharmaceutical or biopharmaceutical drugs (known or future developed drugs for the treatment of TBI), one or more surgical procedures (e.g., removal of hematomas, repair of skull fractures, decompressive craniectomy, etc.), and one or more therapies (e.g., one or more of rehabilitation, cognitive behavioral therapy, anger management, psychological counseling, etc.). In some embodiments, the method further includes monitoring the human pediatric subject assessed as having a traumatic brain injury (e.g., mild or moderate-severe traumatic brain injury). In some embodiments, pediatric subjects identified as having a traumatic brain injury, such as mild traumatic brain injury or severe traumatic brain injury, may be monitored by CT scan or MRI.

[0158] 4. Methods for Measuring UCH-L1 Levels In the methods described above, UCH-L1 levels can be measured by any means, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, antibody-dependent methods such as SDS-PAGE and Western blot analysis or protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, or chromatographic methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS) or spectroscopic methods. Also, clinical chemistry format assays known to those skilled in the art can be used.

[0159] In some embodiments, measuring the level of UCH-L1 comprises contacting the 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 level of UCH-L1 comprises contacting the sample with, simultaneously or sequentially, in any order, (1) at least one capture antibody (e.g., a UCH-L1 capture antibody) that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form at least one capture antibody-UCH-L1 antigen complex (e.g., a UCH-L1 capture antibody-UCH-L1 antigen complex), and (2) a UCH-L1 antigen-at least one capture antibody comprising a detectable label that binds to an epitope on UCH-L1 not bound by the capture antibody. The method includes contacting the sample with at least one detection antibody (e.g., a UCH-L1 detection antibody) to form a detection antibody complex (e.g., a UCH-L1 antigen-UCH-L1 detection antibody complex) to form at least one capture antibody-UCH-L1 antigen-at least one detection antibody complex (e.g., a UCH-L1 capture antibody-UCH-L1 antigen-UCH-L1 detection antibody complex), and measuring the amount or concentration of UCH-L1 in the sample based on a signal generated by a detectable label in the capture antibody-UCH-L1 antigen-detection antibody complex.

[0160] In some embodiments, the method further includes a third specific binding member, such as a second detection antibody, that comprises a detectable label and binds to an epitope on UCH-L1 that is not bound by the capture antibody and the first detection antibody.

[0161] In some embodiments, the first specific binding member is immobilized on a solid support. In some embodiments, the second specific binding member is immobilized on a solid support. In some embodiments, the first specific binding member is a UCH-L1 antibody described below.

[0162] In some embodiments, the sample is diluted or undiluted. The sample may be about 1 to about 25 microliters, about 1 to about 24 microliters, about 1 to about 23 microliters, about 1 to about 22 microliters, about 1 to about 21 microliters, about 1 to about 20 microliters, about 1 to about 18 microliters, about 1 to about 17 microliters, about 1 to about 16 microliters, about 15 microliters, or 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, or about 25 microliters. In some embodiments, the sample is from about 1 to about 150 microliters or less, or from about 1 to about 25 microliters or less.

[0163] Some meters other than point-of-care devices (e.g., the Abbott Laboratories ARCHITECT® meter and other core testing meters) may be capable of measuring UCH-L1 levels in samples of 25,000 pg / mL or greater.

[0164] Other detection methods may include or be adapted for use in nanopore or nanowell devices, for example for single molecule detection. Examples of nanopore devices are described in International Patent Publication No. 2016 / 161402, which is incorporated herein by reference in its entirety. Examples of nanowell devices are described in International Patent Publication No. 2016 / 161400, which is incorporated herein by reference in its entirety. Other devices and methods suitable for single molecule detection may also be utilized.

[0165] 5. Methods for Measuring GFAP Levels In the methods described above, GFAP levels may be measured by any means, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, antibody-dependent methods such as SDS-PAGE and Western blot analysis or protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, or chromatographic methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS), or spectroscopic methods. Clinical chemistry formats of assays known to those skilled in the art may also be utilized.

[0166] In some embodiments, measuring the level of GFAP comprises contacting the sample with a first specific binding member and a second specific binding member, hi some embodiments, the first specific binding member is a capture antibody and the second specific binding member is a detection antibody. In some embodiments, the step of measuring the level of GFAP includes contacting the sample simultaneously or sequentially, in any order, with (1) at least one capture antibody (e.g., a GFAP capture antibody) that binds to an epitope on GFAP or a GFAP fragment to form at least one capture antibody-GFAP antigen complex (e.g., a GFAP capture antibody-GFAP antigen complex), and (2) at least one detection antibody (e.g., a GFAP detection antibody) that comprises a detectable label and binds to an epitope on GFAP to which the capture antibody is not bound to form at least one GFAP antigen-at least one detection antibody complex (e.g., a GFAP antigen-GFAP detection antibody complex), to form at least one capture antibody-GFAP antigen-at least one detection antibody complex (e.g., a GFAP capture antibody-GFAP antigen-GFAP detection antibody complex), and measuring the amount or concentration of GFAP in the sample based on a signal generated by the detectable label in the at least one capture antibody-GFAP antigen-at least one detection antibody complex.

[0167] In some embodiments, the first specific binding member is immobilized on a solid support. In some embodiments, the second specific binding member is immobilized on a solid support. In some embodiments, the first specific binding member is a GFAP antibody as described below.

[0168] In some embodiments, the sample is diluted or undiluted. The sample may be about 1 to about 25 microliters, about 1 to about 24 microliters, about 1 to about 23 microliters, about 1 to about 22 microliters, about 1 to about 21 microliters, about 1 to about 20 microliters, about 1 to about 18 microliters, about 1 to about 17 microliters, about 1 to about 16 microliters, about 15 microliters, or 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, or about 25 microliters. In some embodiments, the sample is from about 1 to about 150 microliters or less, or from about 1 to about 25 microliters or less.

[0169] Some meters other than point-of-care devices (e.g., the Abbott Laboratories ARCHITECT® meter and other core testing meters) may be capable of measuring GFAP levels in samples of 50,000 pg / mL or greater.

[0170] Other detection methods may include or be adapted for use in nanopore or nanowell devices, for example for single molecule detection. Examples of nanopore devices are described in International Patent Publication No. 2016 / 161402, which is incorporated herein by reference in its entirety. Examples of nanowell devices are described in International Patent Publication No. 2016 / 161400, which is incorporated herein by reference in its entirety. Other devices and methods suitable for single molecule detection may also be utilized.

[0171] 6. Antibodies The methods described herein may use isolated antibodies that specifically bind to GFAP, UCH-L1, or GFAP and UCH-L1.

[0172] a.UCH-L1 antibody The methods described herein may use isolated antibodies that specifically bind to ubiquitin carboxy-terminal hydrolase L1 ("UCH-L1") (or fragments thereof), referred to as "UCH-L1 antibodies." UCH-L1 antibodies may be used to assess UCH-L1 status as a measure of traumatic brain injury, or to detect the presence of UCH-L1 in a sample, to quantitate the amount of UCH-L1 present in a sample, or to detect the presence of UCH-L1 in a sample and quantitate the amount of UCH-L1 in a sample.

[0173] (1) Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), also known as "ubiquitin C-terminal hydrolase" ("UCH-L1"), is a deubiquitinating enzyme. UCH-L1 is a member of a gene family whose products hydrolyze small C-terminal adducts of ubiquitin to generate ubiquitin monomers. Expression of UCH-L1 is highly specific to cells of the neurons and diffuse neuroendocrine system and their tumors. UCH-L1 is abundant in all neurons (accounting for 1-2% of total brain protein) and is specifically expressed in neurons and testis / ovary. The catalytic triad of UCH-L1 contains a cysteine ​​at position 90, an aspartic acid at position 176, and a histidine at position 161, which contribute to its hydrolase activity.

[0174] Human UCH-L1 has the following amino acid sequence:

[0175] [ka] may have.

[0176] Human UCH-L1 can be a fragment or variant of SEQ ID NO: 1. Fragments 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 in length. Fragments can include consecutively numbered amino acids derived from SEQ ID NO: 1.

[0177] (2)UCH-L1 recognition antibody The antibody is an antibody that binds to UCH-L1, a fragment thereof, an epitope of UCH-L1 or a variant thereof. The antibody may be a fragment of an anti-UCH-L1 antibody or a variant or derivative thereof. The antibody may be a polyclonal or monoclonal antibody. The antibody may 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. The antibody fragment or derivative may include a F(ab')2 fragment, an Fv fragment or a scFv fragment. The antibody derivative may be produced by peptidomimetics. Furthermore, techniques described for producing single chain antibodies may be adapted to produce single chain antibodies.

[0178] The anti-UCH-L1 antibody may be a chimeric or humanized anti-UCH-L1 antibody. In one embodiment, both the humanized and chimeric antibodies are monovalent. In one embodiment, both the humanized and chimeric antibodies comprise a single Fab region linked to an Fc region.

[0179] Human antibodies can be derived by phage display technology or transgenic mice expressing human immunoglobulin genes. Human antibodies can be generated and isolated as a result of a human immune response in vivo. See, for example, Funaro et al., BMC Biotechnology, 2008(8):85. Thus, the antibody can be a product of a human and may not be an animal repertoire. Because the antibody is of human origin, the risk of reactivity against self-antigens can be minimized. Alternatively, standard yeast display libraries and yeast display technology can be used to select and isolate human anti-UCH-L1 antibodies. For example, a library of naive human single chain variable fragments (scFv) can be used to select human anti-UCH-L1 antibodies. Transgenic animals can be used to express human antibodies.

[0180] A humanized antibody can be an antibody molecule derived from a non-human species antibody that binds to a desired antigen and has one or more complementarity determining regions (CDRs) from the non-human species and a framework region derived from a human immunoglobulin molecule.

[0181] The antibody is distinguishable from known antibodies in that it possesses a biological function distinct from antibodies known in the art.

[0182] i. Epitope The antibody may immunospecifically bind to UCH-L1 (SEQ ID NO: 1), a fragment thereof, or a variant thereof. The antibody may immunospecifically recognize and bind to at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, or at least 10 amino acids within the epitope region. The antibody may immunospecifically recognize and bind to an epitope having at least 3 consecutive amino acids, at least 4 consecutive amino acids, at least 5 consecutive amino acids, at least 6 consecutive amino acids, at least 7 consecutive amino acids, at least 8 consecutive amino acids, at least 9 consecutive amino acids, or at least 10 consecutive amino acids within the epitope region.

[0183] (3) Exemplary Anti-UCH-L1 Antibodies Anti-UCH-L1 antibodies can be made using the techniques described herein, as well as using routine techniques known in the art. In some embodiments, the anti-UCH-L1 antibody is available from United State Biological (product number: 031320), Cell Signaling Technology (product number: 3524), Sigma-Aldrich (product number: HPA005993), Santa Cruz Biotechnology, Inc. (product number: sc-58593 or sc-58594), R&D Systems (product number: MAB6007), Novus Biologicals (product number: NB600-1160), Biobyt (product number: orb33715), Enzo Life Sciences, Inc. (product number: ADI-905-520-1), Bio-Rad (product number: VMA00004), BioVision (product number: 6130-50), Abcam (product number: ab75275 or ab104938), Invitrogen The anti-UCH-L1 antibody may be an unconjugated UCH-L1 antibody, such as the UCH-L1 antibody commercially available from ThermoFisher Scientific (Model No.: MA1-46079, MA5-17235, MA1-90008, or MA1-83428), EMD Millipore (Model No.: MABN48) or Sino Biological Inc. (Model No.: 50690-R011). The anti-UCH-L1 antibody may be conjugated to a fluorophore, such as the conjugated UCH-L1 antibody commercially available from BioVision (Model No.: 6960-25) or Aviva Systems Biology (Model No.: OAAF01904-FITC). Other UCH-L1 antibodies that may be used in the methods described herein include the UCH-L1 antibodies described in WO2018 / 081649, the contents of which are incorporated herein by reference.

[0184] b.GFAP antibody The methods described herein may use isolated antibodies that specifically bind to glial fibrillary acidic protein ("GFAP") (or fragments thereof), referred to as "GFAP antibodies." GFAP antibodies may be used to assess GFAP status as a measure of traumatic brain injury, to detect the presence of GFAP in a sample, to quantitate the amount of GFAP present in a sample, or to detect the presence of GFAP in a sample and quantitate the amount of GFAP in a sample.

[0185] (1) Glial fibrillary acidic protein (GFAP) Glial fibrillary acidic protein (GFAP) is a 50 kDa cytoplasmic fibrillary protein that constitutes part of the cytoskeleton in astrocytes and has been demonstrated to be the most specific marker for cells of astrocyte origin. GFAP protein is encoded by the GFAP gene in humans. GFAP is the major intermediate fiber of mature astrocytes. In the central rod domain of the molecule, GFAP shares a large structural homology with other intermediate fibers. GFAP is involved in astrocyte motility and shape by providing structural stability to astrocyte processes. Glial fibrillary acidic protein and its degradation products (GFAP-BDP) are brain-specific proteins released into the blood as part of the pathophysiological response after traumatic brain injury (TBI). Following injury to the human CNS caused by trauma, genetic injury or chemicals, astrocytes proliferate and show extensive hypertrophy of cell bodies and cell processes, and GFAP is significantly upregulated. In contrast, there is a progressive loss of GFAP production in growing astrocytic malignancies.GFAP can also be detected in Schwann cells, gastrointestinal glial cells, salivary gland neoplasms, metastatic renal carcinomas, epiglottic cartilage, pituitary cells, immature oligodendrocytes, papillary meningiomas, and myoepithelial cells of the mammary gland.

[0186] Human GFAP has the following amino acid sequence:

[0187] [ka] may have.

[0188] The human GFAP may be a fragment or variant of SEQ ID NO: 2. The fragment of GFAP may 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 in length. The fragment may comprise consecutive amino acids from SEQ ID NO: 2. The fragment or variant of human GFAP of SEQ ID NO: 2 may be a GFAP degradation product (BDP). The GFAP BDP may be 38 kDa, 42 kDa (faint band: 41 kDa), 47 kDa (faint band: 45 kDa); 25 kDa (faint band: 23 kDa); 19 kDa or 20 kDa.

[0189] It has been found that the use of at least two antibodies that bind to non-overlapping epitopes within a GFAP degradation 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 embodiment, the at least two antibodies bind to non-overlapping epitopes near the N-terminus of the 38 kDa BDP. In another embodiment, the at least two antibodies bind to non-overlapping epitopes between amino acids 60-383 of SEQ ID NO:2. In another embodiment, at least one first antibody (e.g., a capture antibody) binds to an epitope near the N-terminus of the 38 kDa BDP and at least one second antibody (e.g., a detection antibody) binds to an epitope near the middle of the 38 kDa BDP that does not overlap with the first antibody. In another embodiment, 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 that does not overlap with the first antibody. The epitope bound by the first antibody can be 10, 11, 12, 13, 14, or 15 amino acids in length. The epitope bound by the second antibody can be 10, 11, 12, 13, 14, or 15 amino acids in length. One of skill 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 defined techniques known in the art.

[0190] Similarly, other antibodies may be selected that may help maintain the dynamic range and low end sensitivity of the immunoassay. For example, it may be useful to select at least one first antibody (such as a capture antibody) that binds to an epitope near the N-terminus of the 38 kDa BDP and at least one second antibody (such as a detection antibody) that binds to an epitope near the center of the 38 kDa BDP, for example, an epitope that is in the center of the 38 kDa BDP and does not overlap with the first antibody. Other variations are possible and can be easily examined by those skilled in the art, such as by examining binding to short peptides to confirm that the antibodies bind to different epitopes, and then screening antibody pairs using low calibrator concentrations. Furthermore, the selection of antibodies with different affinities for GFAP may also help maintain or increase the dynamic range of the assay. GFAP antibodies have been described in the literature and are commercially available.

[0191] (2)GFAP recognition antibody The antibody is an antibody that binds to GFAP, a fragment thereof, an epitope of GFAP or a variant thereof. The antibody may be a fragment of an anti-GFAP antibody or a variant or derivative thereof. The antibody may be a polyclonal or monoclonal antibody. The antibody may 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. The antibody fragment or derivative may include a F(ab')2 fragment, an Fv fragment or a scFv fragment. The antibody derivative may be produced by peptidomimetics. Furthermore, techniques described for producing single chain antibodies may be adapted to produce single chain antibodies.

[0192] The anti-GFAP antibody may be a chimeric or humanized anti-GFAP antibody. In one embodiment, both the humanized and chimeric antibodies are monovalent. In one embodiment, both the humanized and chimeric antibodies comprise a single Fab region linked to an Fc region.

[0193] Human antibodies can be derived from phage display technology or transgenic mice expressing human immunoglobulin genes. Human antibodies can be generated and isolated as a result of a human in vivo immune response. See, for example, Funaro et al., BMC Biotechnology, 2008(8):85. Thus, the antibody can be a product of a human and may not be an animal repertoire. Because the antibody is of human origin, the risk of reactivity against self-antigens can be minimized. Alternatively, standard yeast display libraries and yeast display technology can be used to select and isolate human anti-GFAP antibodies. For example, a library of naive human single chain variable fragments (scFv) can be used to select human anti-GFAP antibodies. Transgenic animals can be used to express human antibodies.

[0194] A humanized antibody can be an antibody molecule derived from a non-human species antibody that binds to a desired antigen and has one or more complementarity determining regions (CDRs) from the non-human species and a framework region derived from a human immunoglobulin molecule.

[0195] The antibody is distinguishable from known antibodies in that it possesses a biological function distinct from antibodies known in the art.

[0196] i. Epitope The antibody may immunospecifically bind to GFAP (SEQ ID NO: 2), a fragment or variant thereof. The antibody may immunospecifically recognize and bind to at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acids within the epitope region. The antibody may immunospecifically recognize and bind to an epitope having at least 3 consecutive amino acids, at least 4 consecutive amino acids, at least 5 consecutive amino acids, at least 6 consecutive amino acids, at least 7 consecutive amino acids, at least 8 consecutive amino acids, at least 9 consecutive amino acids or at least 10 consecutive amino acids within the epitope region.

[0197] (3) Exemplary Anti-GFAP Antibodies Anti-GFAP antibodies can be made using the techniques described herein, as well as using routine techniques known in the art. In some embodiments, the anti-GFAP antibody is from Dako (product no. M0761), ThermoFisher Scientific (product no. MA5-12023, A-21282, 13-0300, MA1-19170, MA1-19395, MA5-15086, MA5-16367, MA1-35377, MA1-06701, or MA1-20035), AbCam (product no. ab10062, ab4648, ab68428, ab33922, ab207165, ab190288, ab115898, or ab21837), EMD Millipore (product no. FCMAB257P, MAB360, MAB3402, 04-1031, 04-1062, MAB5628), Santa The antibody may be an unconjugated GFAP antibody, such as a GFAP antibody commercially available from Cruz (Model No.: sc-166481, sc-166458, sc-58766, sc-56395, sc-51908, sc-135921, sc-71143, sc-65343 or sc-33673), Sigma-Aldrich (Model No.: G3893 or G6171) or Sino Biological Inc. (Model No.: 100140-R012-50). The anti-GFAP antibody may be conjugated to a fluorophore, such as the conjugated GFAP antibodies commercially available from ThermoFisher Scientific (Cat. No.: A-21295 or A-21294), EMD Millipore (Cat. No.: MAB3402X, MAB3402B, MAB3402B or MAB3402C3) or AbCam (Cat. No.: ab49874 or ab194325). Other GFAP antibodies that may be used in the methods described herein include the GFAP antibodies described in WO2018 / 081649, the contents of which are incorporated herein by reference.

[0198] c. Preparation / production of antibodies Antibodies may be prepared by any of a variety of techniques, including techniques well known to those of skill in the art. In general, antibodies may be produced by cell culture methods, including the production of monoclonal antibodies, via conventional techniques or by transfection of the antibody genes, heavy and / or light chains into a suitable bacterial or mammalian cell host to allow for the production of antibodies, which may be recombinant. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. 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, since such eukaryotic cells (and particularly mammalian cells) are more likely than prokaryotic cells to assemble, properly fold, and secrete immunologically active antibodies.

[0199] 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)) used with a DHFR selectable marker as described, for example, 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 sufficient time to allow for expression of the antibody within the host cell or, more preferably, secretion of the antibody into the culture medium in which the host cell is grown. The antibody may be recovered from the culture medium using standard protein purification methods.

[0200] The host cells may also be used to generate functional antibody fragments, such as Fab fragments or scFv molecules. It is understood that variations on the above procedures may be implemented. For example, it may be desirable to transfect the host cells with DNA encoding functional fragments of the light and / or heavy chains of the antibody. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also encompassed by antibody. In addition, bifunctional antibodies, in which one heavy and one light chain is an antibody (i.e., binds to an analyte, e.g., human troponin I, human UCH-L1, or human GFAP), and the other heavy and light chains are specific for an antigen other than the analyte, may also be generated by crosslinking the antibody to a second antibody via standard chemical crosslinking methods.

[0201] In a preferred system for recombinantly expressing an antibody or antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, each of the antibody heavy chain gene and the antibody light chain gene is operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene that allows for selection of CHO cells transfected with the vector using methotrexate selection / amplification. The host cells of the selected transformants are cultured to allow expression of the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology methods are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. Still further, a method for synthesizing a recombinant antibody is by culturing the host cells in an appropriate culture medium until the recombinant antibody is synthesized. The method may further comprise the step of isolating the recombinant antibody from the culture medium.

[0202] The method of preparing monoclonal antibodies involves the preparation of immortal cell lines 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 the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) or fragments and / or variants thereof. The peptide used to immunize the animals can include amino acids encoding human Fc, e.g., the Fc (fragment crystallizable) region or tail region of a human antibody. The spleen cells are then immunized, e.g., by fusion with a myeloma cell fusion partner. Various fusion methods can be utilized. For example, spleen cells and myeloma cells are combined with a non-ionic detergent for several 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 involves electrofusion. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants are tested for binding activity to the polypeptide. Hybridomas with high reactivity and specificity can be used.

[0203] Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. In addition, various techniques can be utilized to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies can then be harvested from the ascites fluid or blood. Contaminants can be removed from the antibody by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. Affinity chromatography is an example of a method that can be used in the process of purifying the antibody.

[0204] The proteolytic enzyme papain preferentially cleaves IgG molecules into several fragments, two of which (F(ab) fragments) contain a covalent heterodimer, each of which contains an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules into several fragments, including the F(ab')2 fragment, which contains both antigen-binding sites.

[0205] Fv fragments can be generated by preferential proteolytic cleavage of IgM, and in rare cases, IgG or IgA immunoglobulin molecules. Fv fragments can be derived using recombinant methods. Fv fragments contain a non-covalent VH::VL heterodimer that contains an antigen-binding site that retains much of the antigen recognition and binding capabilities of a native antibody molecule.

[0206] Each antibody, antibody fragment or derivative may comprise a set of heavy and light chain complementarity determining regions ("CDRs") interposed between a set of heavy and light chain frameworks ("FRs") that provide support for the CDRs and define the spatial relationship of the CDRs to each other. The set of CDRs may contain three hypervariable regions, consisting of heavy chain V regions or light chain V regions.

[0207] Other suitable methods of making or isolating antibodies with the requisite specificity may be used, including, but not limited to, selecting recombinant antibodies from peptide or protein libraries (e.g., but not limited to, display libraries such as bacteriophage libraries, ribosomal libraries, oligonucleotide libraries, RNA libraries, cDNA libraries, yeast libraries, etc.) commercially available from a variety of commercial sources, 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; and 5,976,862. An alternative method relies on immunization of transgenic animals capable of generating a repertoire of human antibodies (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 described herein.Such techniques include 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 generation techniques (e.g., the selected lymphocyte antibody assay ("SLAM") (U.S. Patent No. 5,627,052; Wen et al. (1987) J. Immunol., 17:887-892; Babcook 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)).

[0208] Affinity matured antibodies can be produced by any one of a number of procedures known in the art, for example, Marks et al., BioTechnology, 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues has been described by 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 positions and selective mutations at contact or hypermutation positions with activity enhancing amino acid residues have been described in U.S. Patent No. 6,914,128 B1.

[0209] Antibody variants can also be prepared using delivery of a polynucleotide encoding the antibody into a suitable host to produce a transgenic animal or mammal, such as a goat, cow, horse, sheep, etc., which produces such antibodies in their milk. These methods are known in the art and 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.

[0210] Antibody variants may also be prepared by delivering polynucleotides to produce transgenic plants and cultured plant cells (such as, but not limited to, tobacco, corn, and duckweed) that produce such antibodies, specified portions, or variants in plant parts or cells cultured therefrom. For example, Cramer et al. (1999), Curr. Top. Microbiol. Immunol., 240:95-118 and references cited therein, describe the production of transgenic tobacco leaves that express large amounts of recombinant proteins, for example, using inducible promoters. Transgenic corn has been used to express mammalian proteins at commercial production levels with biological activity equivalent to mammalian proteins 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 references cited therein. Antibody variants have also been produced in large quantities from transgenic plant seeds that contain antibody fragments, such as single chain antibodies (scFv), including tobacco seeds and potato tubers. See, e.g., Conrad et al. (1998), Plant Mol. Biol., 38:101-109 and references cited therein. Thus, antibodies can also be produced using transgenic plants according to known methods.

[0211] Derivatives of antibodies can be made, for example, by adding exogenous sequences to modify immunogenicity or to reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life or any other suitable characteristic. Generally, the non-human sequences of the variable and constant regions are replaced with human or other amino acids while maintaining some or all of the non-human or human CDR sequences.

[0212] A small antibody fragment may be a diabody having two antigen-binding sites, where the fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) (VH-VL) on the same polypeptide chain. See, e.g., EP 404,097; WO 93 / 1161 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 on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. See also U.S. Pat. No. 6,632,926 by Chen et al., which is incorporated by reference in its entirety and which also discloses antibody mutants in which one or more amino acids are inserted into a hypervariable region of a parent antibody and which have a binding affinity for a target antigen that is at least about two-fold stronger than the binding affinity of the parent antibody for that antigen.

[0213] The antibody may be a linear antibody. Procedures for making linear antibodies are known in the art and are 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) that form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific.

[0214] Antibodies can be recovered and purified from recombinant cell culture 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.

[0215] It may be useful to detectably label the antibody. Methods for conjugating the antibody to these agents are known in the art. By way of example only, the antibody may be labeled with a detectable moiety, such as a radioactive atom, a chromophore, a fluorophore, etc. Such labeled antibodies may be used for diagnostic methods in vivo or in an isolated test sample. Such labeled antibodies may be linked to a cytokine, a ligand, another antibody. Drugs suitable for coupling to antibodies to achieve an antitumor effect include cytokines such as interleukin 2 (IL-2) and tumor necrosis factor (TNF); photosensitizers for use in photodynamic therapy, including aluminum (III) phthalocyanine tetrasulfonic acid, 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; diphtheria toxin, Pseudomonas aeruginosa exotoxin A, Staphylococcus aureus enterotoxin A, and the like. bacterial, plant and other toxins such as toxin A, abrin A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-alpha toxin, cytotoxin derived from Taiwan cobra (Naja atra) and gelonin (a plant toxin); ribosome-inactivating proteins derived from plants, bacteria and fungi such as restrictocin (a ribosome-inactivating protein produced by Aspergillus restrictus), saporin (a ribosome-inactivating protein derived from Saponaria officinalis) and RNase; tyrosine kinase inhibitors; ly207702 (a difluorinated purine nucleoside); liposomes containing anticystants (e.g., antisense oligonucleotides, plasmids encoding toxins, methotrexate, etc.) and other antibodies or antibody fragments such as F(ab).

[0216] The production of antibodies through hybridoma technology, selected lymphocyte antibody technique (SLAM), transgenic animals and the use of recombinant antibody libraries are described in more detail below.

[0217] (1) Anti-analyte monoclonal antibodies using hybridoma technology Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma methods known in the art, including those taught in, for example, Harlow et al., Antibodies: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, NY, 1981). It is also noted that the term "monoclonal antibody" as used herein is not limited to antibodies produced via hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0218] The method of producing monoclonal antibodies and the antibodies produced by the method may include a step of culturing hybridoma cells secreting the antibodies, where the hybridomas are preferably produced by fusing spleen cells isolated from an animal, e.g., rat or mouse, immunized with the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) with myeloma cells, and then screening the hybridomas resulting from the fusion for hybridoma clones secreting antibodies capable of binding to the polypeptide. Briefly, rats may be immunized with the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) antigen. In a preferred embodiment, the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) antigen is administered with an adjuvant to stimulate the immune response. Such adjuvants include Freund's complete or incomplete adjuvants, RIBI (muramyl dipeptides) or ISCOMs (immunostimulating complexes). Such adjuvants may protect the polypeptide from rapid dispersion by encapsulating it in a local deposit, or they may contain substances which stimulate the host to secrete factors which are chemotactic for macrophages and other components of the immune system. If a polypeptide is administered, the immunization schedule preferably involves two or more administrations of the polypeptide, spread out over a period of weeks, although a single administration of the polypeptide may also be used.

[0219] After immunization of an animal with an analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1) antigen, antibodies and / or antibody-producing cells can be obtained from the animal. Anti-analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1) antibody-containing serum can be obtained from the animal by bleeding the animal or sacrificing the animal. The serum can be used as obtained from the animal, an immunoglobulin fraction can be obtained from the serum, or anti-analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1) antibodies can be purified from the serum. The serum or immunoglobulins thus obtained are polyclonal and therefore have a heterogeneous set of characteristics.

[0220] Once an immune response is detected, for example, antibodies specific to the antigen analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) are detected in the rat serum, the rat spleen is removed and spleen cells are isolated. The spleen cells are then fused to any suitable myeloma cells, for example cells derived from SP20, a cell line available from American Type Culture Collection (ATCC, Manassas, Va., US), by well-known techniques. Hybridomas are selected and cloned by limiting dilution. Hybridoma clones are then assayed for cells secreting antibodies capable of binding to the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) by methods known in the art. Rats can be immunized with positive hybridoma clones to produce ascites fluid, which generally contains high levels of antibodies.

[0221] In another embodiment, immunized hybridomas producing antibodies can be prepared from the immunized animal. After immunization, the animal is sacrificed and splenic B cells are fused to immunized 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-secretory cell lines). After fusion and antibiotic selection, the hybridomas are screened using the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) or a portion thereof or cells expressing the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1). In a preferred embodiment, initial screening is performed using an enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), preferably an ELISA. An example of ELISA screening is provided in PCT Publication No. 00 / 37504.

[0222] Hybridomas producing anti-analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1) antibodies are selected, cloned and further screened for desired characteristics, including robust hybridoma growth, high antibody production and desired antibody characteristics. Hybridomas can be cultured and expanded in vivo in syngeneic animals, animals lacking an immune system such as nude mice, or in in vitro cell culture. Methods for selecting, cloning and expanding hybridomas are well known to those of skill in the art.

[0223] In a preferred embodiment, the hybridomas are rat hybridomas. In another embodiment, the hybridomas are made in a non-human, non-rat species, such as mouse, sheep, pig, goat, cow, or horse. In yet another preferred embodiment, the hybridomas are human hybridomas in which a human non-secretory myeloma has been fused with a human cell expressing an anti-analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) antibody.

[0224] Antibody fragments that recognize specific epitopes can be produced by known techniques. For example, Fab and F(ab')2 fragments can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (yielding two identical Fab fragments) or pepsin (yielding 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, including both light chains (containing the light chain variable and constant regions), the CH1 domain of the heavy chain, and the disulfide-forming hinge region of the parent IgG molecule. Thus, the F(ab')2 fragment is capable of cross-linking antigen molecules in the same way as the parent IgG molecule.

[0225] (2) Anti-analyte monoclonal antibody using SLAM In another embodiment, recombinant antibodies are made from single isolated lymphocytes using a procedure referred to in the art as the selected lymphocyte antibody method (SLAM), as described in U.S. Pat. No. 5,627,052; PCT Publication No. 92 / 02551, and Babcook et al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996). In this method, the antigen analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1), a subunit of the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1), or a fragment thereof is coupled to sheep red blood cells using a linker such as biotin, and a single cell, e.g., a lymphocyte from any one of the immunized animals, secreting the antibody of interest is screened using an antigen-specific hemolytic plaque assay used to identify a single cell that secretes an antibody with specificity for the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1). After identifying the antibody-secreting cell of interest, cDNAs for the heavy and light chain variable regions are rescued from the cells by reverse transcriptase PCR (RT-PCR), and these variable regions are then expressed in the context of an appropriate immunoglobulin constant region (e.g., human constant region) in a mammalian host cell, such as COS or CHO cells. Host cells transfected with amplified immunoglobulin sequences derived from in vivo selected lymphocytes may then undergo further analysis and selection in vitro, for example, by panning the transfected cells to isolate cells expressing antibodies to the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1). The amplified immunoglobulin sequences may be further manipulated in vitro, such as by in vitro affinity maturation methods. See, e.g., PCT Publication Nos. 97 / 29131 and 00 / 56772.

[0226] (3) Anti-analyte monoclonal antibodies using transgenic animals In another embodiment, the antibodies are produced by immunizing a non-human animal that contains some or all of the human immunoglobulin loci with the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) antigen. In one embodiment, the non-human animal is a XENOMOUSE® transgenic mouse, an engineered mouse strain that contains large fragments of the human immunoglobulin loci and is deficient in the production of mouse antibodies. 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. 91 / 10741; PCT Publication No. 94 / 02602; PCT Publication No. 96 / 34096; PCT Publication No. 96 / 33735; PCT Publication No. 98 / 16654; PCT Publication No. 98 / 24893; PCT Publication No. 98 / 50433; PCT Publication No. 99 / 45031; PCT Publication No. 99 / 53049; PCT Publication No. 00 / 09560, and PCT Publication No. 00 / 37504. XENOMOUSE® transgenic mice produce an adult-like human repertoire of fully human antibodies and develop antigen-specific human monoclonal antibodies. The XENOMOUSE® transgenic mice contain approximately 80% of the human antibody repertoire by introducing megabase-sized, germline-configured YAC fragments of human heavy chain and x light chain loci. 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 by reference.

[0227] (4) Anti-analyte monoclonal antibodies using recombinant antibody libraries In vitro methods may also be used to generate antibodies, where antibody libraries are screened to identify antibodies with the desired analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1) binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art, and are described, for example, in U.S. Pat. No. 5,223,409 (Ladner et al.); PCT Publication No. 92 / 18619 (Kang et al.); PCT Publication No. 91 / 17271 (Dower et al.); PCT Publication No. 92 / 20791 (Winter et al.); PCT Publication No. 92 / 15679 (Markland et al.); PCT Publication No. 93 / 01288 (Breitling et al.); PCT Publication No. 92 / 01047 (McCafferty et al.); PCT Publication No. 92 / 09690 (Garrard et al.); Fuchs et al., Bio / Technology, 9:1369-1372 (1991); Hay et al., Bio / Technology, 9:1369-1372 (1992 ... et al., Hum. Antibod. Hybrodomas, 3:81-85 (1992); Huse et al., Science, 246:1275-1281 (1989); McCafferty et al., Nature, 348:552-554 (1990); Griffiths et al., EMBOJ., 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. 97 / 29131.

[0228] The recombinant antibody library may be derived from a subject immunized with the analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1) or a portion of the analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1). Alternatively, the recombinant antibody library may be derived from a naive subject, i.e., a subject not immunized with the analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1), such as a human antibody library derived from a human subject not immunized with the human analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1). Antibodies are selected by screening the recombinant antibody library with a peptide comprising the human analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1), thereby selecting antibodies that recognize the analyte (e.g., GFAP, UCH-L1 or GFAP and 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. An antibody having a specific binding affinity for an analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1), and capable of binding to a specific K off To select antibodies that dissociate with a desired K off Surface plasmon resonance techniques known in the art can be used to select antibodies with rate constants. Antibodies with specific neutralizing activity against an analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) and with specific IC 50 To select antibodies, such as antibodies with, standard methods known in the art for assessing inhibition of analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) activity can be used.

[0229] In one aspect, the disclosure relates to an isolated antibody, or antigen-binding portion thereof, that binds to a human analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1). Preferably, the antibody is a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody.

[0230] For example, antibodies can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. Such phage can be used to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen-binding domain that binds to the antigen of interest can be selected or identified with the antigen, for example, using labeled antigen or antigen bound or captured to a solid surface or bead. The phage used in these methods are typically filamentous phage that contain phage-expressed fd and M13 binding domains, together with Fab, Fv or disulfide-stabilized Fv antibody domains recombinantly fused to phage gene III or phage gene VIII proteins.Examples of phage display methods which can be used to make antibodies include those described in 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. 92 / 01047; PCT Publication No. 90 / 02809; PCT Publication No. 91 / 10737; PCT Publication No. 92 / 01047; PCT Publication No. 92 / 18619; PCT Publication No. 93 / 11236; PCT Publication No. 95 / 15982; PCT Publication No. 95 / 20401 and U.S. Pat. Nos. 5,698,426 and 5,223,409. Nos. 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.

[0231] After phage selection, as described in the above references, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, which can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described in detail below. Techniques can also be utilized to recombinantly generate, for example, Fab fragments, Fab' fragments, and F(ab')2 fragments, using methods known in the art, such as those disclosed in PCT Publication No. 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 make single chain Fvs and antibodies include those described in U.S. Pat. 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).

[0232] Other methods known in the art for screening large combinatorial libraries, which are alternatives to screening recombinant antibody libraries by phage display, can also be applied to identify antibodies. One type of alternative expression system is the expression system in which recombinant antibody libraries are expressed as RNA-protein fusions, as described in PCT Publication No. 98 / 31700 (Szostak and Roberts) and Roberts and Szostak, Proc. Natl. Acad. Sci. USA, 94:12297-12302 (1997). In this system, covalent fusions are created between the mRNA and the peptide or protein it encodes by in vitro translation of synthetic mRNA carrying the peptidyl acceptor antibiotic puromycin at its 3' end. Thus, specific mRNAs can be enriched from a complex mixture of mRNAs (e.g., combinatorial libraries) based on the properties of the encoded peptides or proteins, e.g., antibodies or portions thereof, such as the binding of the antibodies or portions thereof to bispecific antigens. Nucleic acid sequences encoding antibodies or portions thereof recovered from screening of such libraries can be expressed by recombinant means (e.g., in mammalian host cells) as described above and subjected to further affinity maturation by further rounds of screening of mRNA-peptide fusions in which mutations are introduced into the originally selected sequences, or by other methods for affinity maturation of recombinant antibodies in vitro as described above. A preferred example of this methodology is the PROfusion display technology.

[0233] In another approach, antibodies can also be made using yeast display methods known in the art. In yeast display methods, genetic methods are used to tether antibody domains to the yeast cell wall and display them on the surface of the yeast. In particular, 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 make antibodies include the yeast display method disclosed in U.S. Patent No. 6,699,658 (Wittrup et al.), which is incorporated herein by reference.

[0234] d. Generation of recombinant analyte antibodies Antibodies may be produced by any of a number of techniques known in the art, such as expression from a host cell in which expression vectors encoding the heavy and light chains have been transfected into the host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although antibodies can be expressed in prokaryotic or eukaryotic host cells, expression of the antibody in eukaryotic cells is preferred, and most preferred in mammalian host cells, since such eukaryotic cells (and particularly mammalian cells) are more likely than prokaryotic cells to assemble, properly fold, and secrete immunologically active antibodies.

[0235] 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)), used with a DHFR selectable marker as described, for example, 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 sufficient time to allow for expression of the antibody within the host cell or, more preferably, secretion of the antibody into the culture medium in which the host cell is grown. The antibody may be recovered from the culture medium using standard protein purification methods.

[0236] The host cells may also be used to generate functional antibody fragments, such as Fab fragments or scFv molecules. It is understood that variations on the above procedures may be performed. For example, it may be desirable to transfect the host cells with DNA encoding functional fragments of the antibody light and / or heavy chains. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also encompassed by antibody. In addition, bifunctional antibodies, in which one heavy chain and one light chain is an antibody (i.e., an antibody that binds to a human analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1)) and the other heavy and light chains are specific for an antigen other than the human analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1), may also be generated by crosslinking the antibody to a second antibody via standard chemical crosslinking methods.

[0237] In a preferred system for recombinantly expressing an antibody or antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy and light chains is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, each of the antibody heavy and light chain genes is operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, allowing for selection of CHO cells transfected with the vector using methotrexate selection / amplification. The host cells of the selected transformants are cultured to allow expression of the antibody heavy and light chains and for intact antibody to be recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. Still further, the present disclosure provides a method of synthesizing a recombinant antibody by culturing the host cells in an appropriate culture medium until the recombinant antibody is synthesized. The method may further comprise the step of isolating the recombinant antibody from the culture medium.

[0238] (1) Humanized antibodies A humanized antibody may be an antibody, or a variant, derivative, analog 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 a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody. A humanized antibody may be derived from a non-human species antibody that binds to a desired antigen and has one or more complementarity determining regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule.

[0239] As used herein, the term "substantially" in the context of a CDR refers to a CDR that has 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, but typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. According to one aspect, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically the Fc region of a human immunoglobulin. In some embodiments, the humanized antibody contains both a light chain and at least the variable domains of a heavy chain. The antibody may also comprise 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 specific embodiments, a humanized antibody contains only humanized variable domains of the light and / or heavy chain.

[0240] The humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including, without limitation, IgG1, IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions, using techniques well known in the art.

[0241] The framework regions and CDRs of a humanized antibody need not correspond exactly to the parental sequences, for example, the donor antibody CDR or consensus framework may be mutagenized by substitution, insertion and / or deletion of at least one amino acid residue such that the CDR or framework residue at this site does not correspond to the donor antibody or consensus framework. However, in one embodiment, such mutations are not extensive mutations. Typically, at least 90%, at least 95%, at least 98% or at least 99% of the humanized antibody residues correspond to the residues of the parental FR and CDR sequences. The term "consensus framework" as used herein refers to a framework region in a consensus immunoglobulin sequence. The term "consensus immunoglobulin sequence" as used herein refers to a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, for example, Winnaker, "From Genes to Clones" (Verlagsgesellschaft, Weinheim, Germany, 1987)). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

[0242] Humanized antibodies can be designed to minimize unwanted immune responses against anti-human rodent antibodies that limit the duration and effectiveness of therapeutic applications of these moieties in human recipients. A humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human residues are often referred to as "import" residues, typically taken from the variable domain. Humanization can be performed by substituting hypervariable region sequences with the corresponding sequences of a human antibody. Such "humanized" antibodies are thus chimeric antibodies in which substantially less than intact human variable domains are substituted by the corresponding sequences from a non-human species. See, for example, U.S. Pat. No. 4,816,567, the contents of which are incorporated herein by reference. Humanized antibodies can be human antibodies in which some hypervariable region residues, and possibly some FR residues, are substituted by residues from analogous sites in rodent antibodies. Humanization or engineering of the antibodies of the present disclosure may be carried out using any known method, 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.

[0243] Humanized antibodies may retain high affinity for the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) and other favorable biological properties. Humanized antibodies may be prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models for the parental and humanized sequences. Three-dimensional immunoglobulin models are publicly available. Computer programs are available which illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these representations allows analysis of the residues for their key role in the functioning of the candidate immunoglobulin sequence, i.e., for residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues may be selected and combined from the recipient and import sequences such that the desired antibody characteristic, such as increased affinity for the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1), is achieved. In general, the hypervariable region residues may be directly and most substantially involved in influencing antigen binding.

[0244] As an alternative to humanization, human antibodies (also referred to herein as "fully human antibodies") can be produced. For example, human antibodies can be isolated from libraries via PROfusion and / or yeast-related technologies. It is also possible to produce transgenic animals (e.g., mice that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production). For example, the expression of the antibody heavy chain joining region (JC) in chimeric and germline mutant mice can be expressed in a variety of ways. HHomozygous deletion of the .) gene results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. Humanized or fully human antibodies may be prepared according to the methods described in U.S. Pat. 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.

[0245] 7. Method Variations The disclosed methods for determining the presence or amount of an analyte of interest (GFAP, UCH-L1 or GFAP and UCH-L1) present in a sample can be as described above. The methods can also be adapted with other methods for analyzing the analyte in mind. Examples of well-known variations include, but are not limited to, sandwich immunoassays (e.g., monoclonal-polyclonal sandwich immunoassays), competitive inhibition immunoassays (e.g., forward competitive inhibition immunoassays and reverse competitive inhibition immunoassays), enzyme multiplexed immunoassay method (EMIT), competitive binding assays, bioluminescence energy transfer (BRET), one-step antibody detection assays, homogeneous assays, heterogeneous assays, capture-on-the-fly assays, single molecule detection assays, and the like, including enzyme detection (enzyme immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISA)).

[0246] Immunoassay Analytes of interest and / or peptides or fragments thereof (e.g., GFAP, UCH-L1 or GFAP and UCH-L1 and / or peptides or fragments thereof, i.e., GFAP, UCH-L1 or GFAP and UCH-L1 fragments) can be analyzed using GFAP, UCH-L1 or GFAP and UCH-L1 antibodies in an immunoassay. The presence or amount of an analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1) can be determined using an antibody to detect specific binding to the analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1). For example, an antibody or an antibody fragment thereof can specifically bind to the analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1). If desired, one or more of the antibodies can be used in combination with one or more commercially available monoclonal / polyclonal antibodies. Such antibodies are commercially available from companies such as R&D Systems, Inc. (Minneapolis, MN) and Enzo Life Sciences International, Inc. (Plymouth Meeting, PA).

[0247] The presence or amount of an analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) present in a body sample can be readily determined using immunoassays such as sandwich immunoassays (e.g., monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonal sandwich immunoassays, including radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme immunometric assay (ELISA) (e.g., Quantikine ELISA Assay, R&D Systems, Minneapolis, MN)). An example of a point-of-care device that can be used is i-STAT® (Abbott Laboratories, Abbott Park, IL). Another method that can be used is chemiluminescent microparticle immunoassays, specifically, by way of example, the ARCHITECT® automated analyzer (Abbott Laboratories, Abbott Other methods include, for example, chemiluminescent microparticle immunoassays utilizing immunofluorescence assays (e.g., chemiluminescent microparticle immunoassays utilizing immunofluorescence assays utilizing immunofluorescence microscopy (e.g., chemiluminescent microparticle immunoassay ... This includes the detection methods 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, which are incorporated herein by reference in their entireties.Specific immunological binding of an antibody to an analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) can be detected via a direct label, such as a fluorescent or luminescent tag, metals and radionuclides, or via an indirect label, such as alkaline phosphatase or horseradish peroxidase, conjugated to the antibody.

[0248] The use of immobilized antibodies or fragments thereof can be incorporated into immunoassays. Antibodies can be immobilized to a variety of supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (such as a microtiter plate), or a small piece of solid substrate material. An assay strip can be prepared by coating an antibody or antibodies in an array on a solid support. The strip can then be dipped into the test sample and rapidly processed through washing and detection steps to generate a measurable signal, such as a colored spot.

[0249] A homogeneous format may be used. For example, a test sample may be obtained from a subject, and then a mixture may be prepared. The mixture contains a test sample to be evaluated for an analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1), 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 with the first specific binding partner and the second specific binding partner simultaneously. In some embodiments, the first specific binding partner and any GFAP, UCH-L1, or GFAP and UCH-L1 contained in the test sample can form a first specific binding partner-analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) antigen complex, and the second specific binding partner can form a first specific binding partner-analyte of interest (e.g., GFAP, UCH-L1, or GFAP and UCH-L1)-second specific binding partner complex. In some embodiments, the second specific binding partner and any GFAP, UCH-L1, or GFAP and UCH-L1 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., GFAP, UCH-L1, or GFAP and UCH-L1)-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 an amino acid sequence comprising at least 3 consecutive amino acids of SEQ ID NO:1 or an anti-GFAP antibody that binds to an epitope having an amino acid sequence comprising at least 3 consecutive 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 an amino acid sequence comprising at least 3 consecutive amino acids of SEQ ID NO:1 or an anti-GFAP antibody that binds to an epitope having an amino acid sequence comprising at least 3 consecutive amino acids of SEQ ID NO:2).Additionally, the second specific binding partner is labeled with or contains a detectable label, as described above.

[0250] Heterogeneous formats may be used. For example, a first mixture may be prepared after a test sample is obtained from a subject. The mixture contains the test sample to be evaluated for an analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1P) and a first specific binding partner, where the first specific binding partner and any GFAP, UCH-L1, or GFAP and UCH-L1 contained in the test sample form a first specific binding partner-analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) antigen complex. The first specific binding partner may be an anti-analyte antibody (e.g., an anti-UCH-L1 antibody that binds to an epitope having an amino acid sequence that includes at least 3 consecutive amino acids of SEQ ID NO: 1, or an anti-GFAP antibody that binds to an epitope having an amino acid sequence that includes at least 3 consecutive amino acids of SEQ ID NO: 2). The order in which the test sample and the first specific binding partner are added to form the mixture is not critical.

[0251] The first specific binding partner may be immobilized on a solid phase. The solid phase used in the immunoassay (for the first specific binding partner and, optionally, the second specific binding partner) may be any solid phase known in the art, including, but not limited to, magnetic particles, beads, test tubes, microtiter plates, cuvettes, membranes, scaffolding molecules, thin films, filter paper, disks and chips. In embodiments where the solid phase is a bead, the bead may be a magnetic bead or a magnetic particle. The magnetic bead / particle may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic or magnetic fluid. 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 beads may be magnetic and have a solid core portion surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion may be a layer around a non-magnetic core. The solid support on which the first specific binding member is immobilized may be stored in dry form or in liquid. The magnetic beads may be placed under a magnetic field before or after contacting the sample with the magnetic beads on which the first specific binding member is immobilized.

[0252] After the mixture containing the first specific binding partner-analyte (e.g., UCH-L1 and / or GFAP) antigen complex is formed, any unbound analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) is removed from the complex using any technique known in the art. For example, unbound analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) can be removed by washing. However, it is desirable that the first specific binding partner is present in excess of any analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) present in the test sample, so that the first specific binding partner binds to all analytes (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) present in the test sample.

[0253] After any unbound analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) is removed, a second specific binding partner is added to the mixture to form a first specific binding partner-analyte of interest (e.g., GFAP, UCH-L1, or GFAP and UCH-L1)-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 an amino acid sequence that includes at least 3 consecutive amino acids of SEQ ID NO: 1 or an anti-GFAP antibody that binds to an epitope having an amino acid sequence that includes at least 3 consecutive amino acids of SEQ ID NO: 2). Additionally, the second specific binding partner is labeled with or contains a detectable label, as described above.

[0254] The use of immobilized antibodies or fragments thereof can be incorporated into immunoassays. Antibodies can be immobilized to a variety of supports, such as magnetic particles 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 an assay plate (such as a microtiter plate), or small pieces of solid substrate material. An assay strip can be prepared by coating an antibody or multiple antibodies in an array on a solid support. The strip can then be dipped into the test sample and rapidly processed through washing and detection steps to generate a measurable signal, such as a colored spot.

[0255] (1) Sandwich immunoassay Sandwich immunoassays measure the amount of antigen between two layers of antibodies (i.e., at least one capture antibody) and detection antibodies (i.e., at least one detection antibody). The capture antibody and detection antibody bind to different epitopes on the analyte of interest, such as an antigen, e.g., GFAP, UCH-L1, or GFAP and UCH-L1. It is desirable that the binding of the capture antibody to the epitope does not interfere with the binding of the detection antibody to the epitope. Either monoclonal or polyclonal antibodies can be used as the capture antibody and detection antibody in sandwich immunoassays.

[0256] Generally, at least two antibodies are utilized to separate and quantify an analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) in a test sample. More specifically, at least two antibodies bind to a specific epitope of the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) to form an immune complex called a "sandwich." One or more antibodies may be used to capture the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) in the test sample (these antibodies are often referred to as "capture" antibodies or "capture" antibodies), and one or more antibodies are used to bind a detectable (i.e., quantifiable) label to the sandwich (these antibodies are often referred to as "detection" antibodies or "detection" antibodies). In a sandwich assay, the binding of an antibody to its epitope is not diminished by the binding of any other antibody in the assay to its respective epitope. The antibodies are selected such that one or more first antibodies contacted with a test sample suspected of containing an analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) do not bind to all or a portion of the epitope recognized by a second antibody or subsequent antibody, thereby interfering with the ability of one or more second detection antibodies to bind to the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1).

[0257] The antibody may be used as a first antibody in the immunoassay. The antibody immunospecifically binds to an epitope on the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1). In addition to the antibody of the present disclosure, the immunoassay may include a second antibody that immunospecifically binds to an epitope not recognized or bound by the first antibody.

[0258] A test sample suspected of containing an analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) can be contacted simultaneously or sequentially with at least one first capture antibody (or antibodies) and at least one second detection antibody. In a sandwich assay format, a test sample suspected of containing an analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) is first contacted with at least one first capture antibody that specifically binds to a particular epitope under conditions that allow the formation of a first antibody-analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) antigen complex. When more than one capture antibody is used, a first plurality of capture antibodies-UCH-L1, GFAP, or UCH-L1 and GFAP antigen complexes are formed. In a sandwich assay, an antibody, preferably at least one capture antibody, is used in molar excess relative to the maximum amount of analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) expected in the test sample. For example, about 5 μg / ml to about 1 mg / ml of antibody per ml of microparticle coating buffer can be used.

[0259] i. Anti-GFAP, UCH-L1 or GFAP and UCH-L1 capture antibodies Optionally, prior to contacting the test sample with the at least one first capture antibody, the at least one first capture antibody may be bound to a solid support that facilitates separation of the first antibody-analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) complex from the test sample. Any solid support known in the art may be used, including, but not limited to, solid supports made of polymeric materials in the form of wells, tubes, or beads (such as microparticles). The antibodies (or antibodies) may be bound to the solid support by adsorption, by covalent binding using chemical coupling agents, or by other means known in the art, provided that such binding does not interfere with the ability of the antibody to bind to the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1). Furthermore, if necessary, the solid support may be derivatized to allow reactivity 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.

[0260] The test sample suspected of containing the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) is then incubated to allow for the formation of a first capture antibody (or antibodies)-analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) complex. The incubation may be carried out at a pH of about 4.5 to about 10.0, at a temperature of about 2° C. to about 45° C., for a period of at least about 1 minute to about 18 minutes, about 2 to 6 minutes, about 7 to 12 minutes, about 5 to 15 minutes, or about 3 to 4 minutes.

[0261] ii. Detection antibody After formation of the first / multiple capture antibody-analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1) complex, the complex is then contacted with at least one second detection antibody (under conditions that allow for the formation of a first / multiple antibody-analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1) antigen-second antibody complex). In some embodiments, the test sample is contacted with the detection antibody simultaneously with the capture antibody. When the first antibody-analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1) complex is contacted with more than one detection antibody, a first / multiple capture antibody-analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1)-multiple antibody detection complex is formed. When at least the second (and subsequent) antibody is contacted with the first antibody-analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1) complex, an incubation time under conditions similar to those described above as for the first antibody is required for the formation of the first / multiple antibody-analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1)-second / multiple antibody complex. Preferably, at least one second antibody contains a detectable label. The detectable label can be attached to at least one second antibody before, simultaneously with, or after the formation of the first / multiple antibody-analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1)-second / multiple antibody complex. Any detectable label known in the art can be used.

[0262] Chemiluminescence assays 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 USA, LLC, Oak Ridge, TN) allows for rapid assay of multiple samples in small volumes. The chemiluminometer can be equipped with multiple reagent injectors using 96-well black polystyrene microplates (Costar, model no. 3792). Each sample can be added to an individual well, followed by simultaneous / sequential addition of other reagents as determined by the type of assay used. It is desirable to avoid the formation of pseudobases in neutral or basic solutions that utilize acridinium aryl esters, such as by acidification. The chemiluminescence response is then recorded for each well. In this regard, the time to record a chemiluminescent response will depend, in part, on the delay between the addition of the reagent utilized and the addition of the particular acridinium.

[0263] The order in which the test sample and the specific binding partner are added to form a mixture for chemiluminescence assay is not critical. When the first specific binding partner is detectably labeled with an acridinium compound, a detectably labeled first specific binding partner-antigen (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) complex is formed. Alternatively, when 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., GFAP, UCH-L1, or GFAP and UCH-L1)-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.

[0264] Hydrogen peroxide can be generated in situ in the mixture or can be administered or provided to the mixture prior to, simultaneously with, or after the addition of the acridinium compound described above. Hydrogen peroxide can be generated in situ in a number of ways, such as those apparent to those skilled in the art.

[0265] Alternatively, a source of hydrogen peroxide may simply be added to the mixture. For example, the source of hydrogen peroxide may be one or more buffers or other solutions known to contain hydrogen peroxide. In this regard, a solution of hydrogen peroxide may simply be added.

[0266] Upon addition of at least one basic solution to the sample, either simultaneously or subsequently, a detectable signal, i.e., a chemiluminescent signal, is generated that indicates the presence of the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1). The basic solution contains at least one base and has a pH of 10 or more, preferably 12 or more. 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, one skilled in the art can easily determine the amount of basic solution added to the sample. Labels other than chemiluminescent labels can also be used. For example, enzyme labels (including but not limited to alkaline phosphatase) can be used.

[0267] The generated chemiluminescent or other signal can be detected using routine techniques known to those skilled in the art. Based on the intensity of the generated signal, the amount of the analyte of interest (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) in the sample can be quantified. Specifically, the amount of analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) in the sample is proportional to the intensity of the generated signal. The amount of analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) present can be quantified by comparing the amount of light generated to a calibration curve for the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) or by comparison to a reference standard. Calibration curves can be generated by mass spectrometry, gravimetry, and other techniques known in the art using serial dilutions or solutions of known concentrations of the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1). Quantitation methods for panel and multiplex assays are also described in the scientific literature and are known to those of skill in the art.

[0268] (2) Forward competitive inhibition assay In a forward competition format, an aliquot of a known concentration of labeled analyte of interest (e.g., an analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) having a fluorescent label, a tag conjugated with a cleavable linker, etc.) is used to compete with the analyte of interest (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) in a test sample for binding to an analyte of interest antibody (e.g., GFAP, UCH-L1, or GFAP and UCH-L1 antibody).

[0269] In a forward competitive assay, an immobilized specific binding partner (such as an antibody) can be contacted sequentially or simultaneously with the test sample and the labeled analyte of interest, analyte of interest fragment, or analyte of interest variant. The analyte of interest peptide, analyte of interest fragment, or analyte of interest variant can be labeled with any detectable label, including detectable labels composed of a tag conjugated with a cleavable linker. In this assay, the antibody can be immobilized to a solid support. Alternatively, the antibody can be coupled to an antibody, such as an anti-species antibody, immobilized on a solid support, such as a microparticle or planar substrate.

[0270] The labeled analyte of interest, the test sample and the antibody are incubated under conditions similar to those described above in relation to the sandwich assay format. Two or more different molecular 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 (such as a fluorescent label), while the other antibody-analyte of interest complexes does not contain a detectable label. The antibody-analyte of interest complexes can be, but do not have to be, separated from the rest of the test sample before quantifying the detectable label. The amount of detectable label in the antibody-analyte of interest complex is then quantified, regardless of whether the antibody-analyte of interest complexes are separated from the rest of the test sample. The concentration of the analyte of interest (such as a membrane-associated analyte of interest, a soluble analyte of interest, a fragment of a soluble analyte of interest, a mutant of the analyte of interest (either a membrane-associated analyte of interest or a soluble analyte of interest) or any combination thereof) in the test sample can then be determined, for example, as described above.

[0271] (3) Reverse competitive inhibition assay In a reverse competition assay, an immobilized analyte of interest (e.g., GFAP, UCH-L1, or GFAP and UCH-L1) can be contacted sequentially or simultaneously with the test sample and at least one labeled antibody.

[0272] The analyte of interest is bound to a solid support, such as those solid supports discussed above in connection with the sandwich assay format.

[0273] The immobilized analyte of interest, the test sample and at least one labeled antibody are incubated under conditions similar to those described above in connection with the sandwich assay format. Two different molecular species of analyte of interest-antibody complexes can then be generated. Specifically, one of the generated analyte of interest-antibody complexes is immobilized and contains a detectable label (such as a fluorescent label), while the other analyte of interest-antibody complex is not immobilized and does not contain a detectable label. The non-immobilized analyte of interest-antibody complex and the remaining test sample are removed from the presence of the immobilized analyte of interest-antibody complex through techniques known in the art, such as washing. Then, once the non-immobilized analyte of interest-antibody complex is removed, the amount of detectable label in the immobilized analyte of interest-antibody complex after cleavage of the tag is quantified. The concentration of the analyte of interest in the test sample can then be determined by comparing the quantity of detectable label as described above.

[0274] (4) One-step immunoassay or "capture-on-the-fly" assay In a capture-on-the-fly assay, the solid substrate is pre-coated with an immobilizing agent. The capture agent, analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1) and detection agent are added together to the solid substrate, followed by a washing step before detection. The capture agent is capable of binding to the analyte (e.g., GFAP, UCH-L1 or GFAP and UCH-L1) and includes a ligand for the immobilizing agent. The capture agent and detection agent can be an antibody or any other moiety capable of capture or detection, as described herein or known in the art. The ligand can include a peptide tag, and the immobilizing agent can include an anti-peptide tag antibody. Alternatively, the ligand and immobilizing agent can be any pair of agents capable of binding together (e.g., specific binding pairs and other pairs known in the art) as utilized for the capture-on-the-fly assay. More than one analyte can be measured. In some embodiments, the solid substrate may be coated with an antigen and the analyte being analyzed is an antibody.

[0275] In certain other embodiments of one-step immunoassays or "capture on the fly", a solid support (such as a microparticle) pre-coated with an immobilization agent (such as biotin, streptavidin, etc.) and at least a first specific binding member and a second specific binding member (functioning as a capture reagent and a detection reagent, respectively) are used. The first specific binding member comprises a ligand for the immobilization agent (e.g., if the immobilization agent on the solid support is streptavidin, the ligand on the first specific binding member can be biotin) and also binds to the analyte of interest (e.g., GFAP, UCH-L1, or GFAP and UCH-L1). The second specific binding member comprises a detectable label and binds to the analyte of interest (e.g., GFAP, UCH-L1, or GFAP and UCH-L1). The solid support and the first specific binding member and the second specific binding member can be added (sequentially or simultaneously) to the test sample. The ligand on the first specific binding member binds to the immobilizing agent on the solid support to form 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 wash step may be utilized prior to detection. In certain embodiments in a one-step assay, more than one analyte may be measured. In certain other embodiments, more than two specific binding members may be utilized. In certain other embodiments, multiple detectable labels may be added. In certain other embodiments, multiple analytes of interest may be detected or their amounts, levels or concentrations may be measured, determined or assessed.

[0276] The use of capture on the fly assays can be in a variety of formats, as described herein and known in the art, for example, the format can be a sandwich assay, such as those described above, but can alternatively be a competitive assay, can utilize monospecific binding members, or can use other variations, such as those known in the art.

[0277] (5) Single molecule detection assay Single molecule detection assays and methods may also be used, such as using nanopore or nanowell devices. Examples of nanopore devices are described in International Patent Publication No. 2016 / 161402, which is incorporated herein by reference in its entirety. Examples of nanowell devices are described in International Patent Publication No. 2016 / 161400, which is incorporated herein by reference in its entirety. Other devices and methods suitable for single molecule detection may also be utilized.

[0278] 8. Other Factors The diagnostic, prognostic and / or evaluation methods described above may further include the use of other factors for diagnosis, prognosis and evaluation. In some embodiments, the other factors may include measuring and / or determining blood urea nitrogen (BUN) levels and / or sodium / creatinine ratios. In some embodiments, the methods may further include measuring and / or determining other biomarkers, such as myelin basic protein (MBP), neurofilament light protein (NFL) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR).

[0279] In some embodiments, traumatic brain injury can be diagnosed using the Glasgow Coma Scale (GCS), or the outcome of traumatic brain injury can be predicted using the Extended Glasgow Outcome Scale (GOSE). Other tests, scales, or indices can also be used alone or in combination with the Glasgow Coma Scale (GCS). An example is the Ranchos Los Amigos Scale (RLAS). The Ranchos Los Amigos Scale (RLAS) measures the level of consciousness, cognition, behavior, and interaction with the environment. The Ranchos Los Amigos Scale (RLAS) includes the following scales: Level I: no response; Level II: general response; Level III: local response; Level IV: confusion (agitation); Level V: confusion (inappropriate); Level VI: confusion (appropriate); Level VII: automatic (appropriate), and Level VIII: purposeful (appropriate).

[0280] Other classification systems based on CT scan results, such as any classification system known in the art, may also be used to predict outcomes in patients. An example is the Marshall Classification of Traumatic Brain Injury, which classifies patients into one of six types (I-VI) of increasing severity based on findings on non-contrast CT scans of the brain. The more severe types have poorer prognosis and lower survival rates. The Marshall Classification is primarily concerned with two features: 1) the degree of swelling, determined by midline shift and / or compression of the basal cisterns, and 2) the presence and size of contusion / hemorrhage, referred to as "dense or mixed density lesions." Another example is the Rotterdam Score, which incorporates additional variables (e.g., subarachnoid hemorrhage) and attempts to address some of the recognized limitations of the Marshall System, such as efforts to classify patients with multiple types of injury. The Rotterdam Classification includes four independently assessed elements. Like the Marshall system, the Rotterdam classification includes 1) the degree of compression of the basal cisterns and 2) the degree of midline shift. However, it does not include contusion and limits large lesions to 3) epidural hematoma and adds 4) intraventricular hemorrhage and / or subarachnoid hemorrhage. Each of these is given a score, which is then added together to a grand total of 1. The higher the score, the worse the prognosis and the lower the survival rate.

[0281] 9. Sample In some embodiments, the sample is obtained after the human pediatric subject has suffered a head injury or other type of blunt trauma caused by physical shaking, external mechanical or other forces resulting in closed or open head trauma, one or more falls, blunt impact from an explosion or blast. In some embodiments, the sample is obtained after the human pediatric subject has ingested or been exposed to a chemical, a toxin, or a combination of chemicals and toxins. Examples of such chemicals and / or toxins include fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide, and cyanides), organometallics (such as methylmercury, tetraethyl lead, and organotins), and / or one or more drugs of abuse. In some embodiments, the sample is obtained from a human pediatric subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, one or more viruses, meningitis, hydrocephalus, or a combination thereof.

[0282] In yet another embodiment, the methods described herein use samples that can also be used to determine whether a subject has or is at risk for developing mild traumatic brain injury by determining the levels of GFAP, UCH-L1, or GFAP and UCH-L1 in the subject using the anti-GFAP and anti-UCH-L1 antibodies or antibody fragments thereof described above.Thus, in certain embodiments, the present disclosure also presents methods for determining whether a pediatric subject has or is at risk for traumatic brain injury is a candidate for therapy or treatment, as discussed herein and known in the art. Generally, pediatric subjects are at least (i) subjects who have suffered a head injury; (ii) subjects who have ingested and / or been exposed to one or more chemicals and / or toxins; (iii) subjects suffering from an autoimmune disease, a metabolic disorder, brain tumor, hypoxia, one or more viruses, meningitis, hydrocephalus, or any combination thereof, or (iv) subjects who have been diagnosed as having or at risk for a TBI (such as, for example, a subject suffering from an autoimmune disease, a metabolic disorder, brain tumor, hypoxia, one or more viruses, meningitis, hydrocephalus, or a combination thereof) and / or subjects who exhibit undesirable (i.e., clinically undesirable) concentrations or amounts of GFAP, UCH-L1, GFAP fragments and / or UCH-L1 fragments as described herein.

[0283] A test subject or biological sample As used herein, the terms "sample", "test sample", and "biological sample" refer to a bodily fluid sample that contains or is suspected to contain GFAP, UCH-L1, or GFAP and UCH-L1. The sample may be from any suitable source. In some cases, the sample may include a liquid, a flowable particulate solid, or a fluid with a suspension of solid particles. In some cases, the sample may be processed prior to the analysis described herein. For example, the sample may be separated or purified from its source prior to analysis, although in certain embodiments, an unprocessed sample containing GFAP, UCH-L1, or GFAP and UCH-L1 may be assayed directly. In certain examples, the source containing GFAP, UCH-L1 or GFAP and UCH-L1 is a human (e.g., pediatric) body substance (e.g., body fluids, blood such as whole blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, lung lavage fluid, cerebrospinal fluid, feces, tissue, organ, etc.). Tissues can include, but are 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 a tissue, such as an organ or a biopsy sample, which can be solubilized by tissue digestion / cell lysis.

[0284] A wide range of volumes of bodily fluid samples can be analyzed. In a few 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 bodily 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.

[0285] In some cases, the body fluid sample may be diluted before use in the assay. For example, in an embodiment in which the source containing GFAP, UCH-L1 or GFAP and UCH-L1 is a human body fluid (e.g., blood, serum), the body fluid may be diluted with a suitable solvent (e.g., a buffer such as PBS buffer). The body fluid sample may 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 body fluid sample is not diluted before use in the assay.

[0286] In some cases, the sample may undergo pre-analysis treatment. Pre-analysis treatment may provide additional functionality, such as removal of non-specific proteins and / or effective but inexpensively implementable mixed functionality. Common methods of pre-analysis treatment may include the use of electrokinetic trapping, AC electrokinetics, surface acoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, or other pre-concentration methods known in the art. In some cases, the body fluid sample may be concentrated before use in the assay. For example, in embodiments where the source containing GFAP, UCH-L1, or GFAP and UCH-L1 is a human (e.g., pediatric) body fluid (e.g., blood, serum), the body fluid may be concentrated by precipitation, evaporation, filtration, centrifugation, or a combination thereof. The body fluid sample may 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.

[0287] b. Control It may be desirable to include controls (such as positive and / or negative controls as known in the art). The controls may be analyzed contemporaneously with the samples from the subject, as described above. Results obtained from the subject may be compared to results or information obtained from the control samples. A calibration curve may be prepared to which the assay results for the samples may be compared. Such a calibration curve presents the level of the marker as a function of assay units, i.e., fluorescent signal intensity if a fluorescent label is used. Samples taken from multiple donors may be used to provide calibration curves for baseline levels of GFAP, UCH-L1, or GFAP and UCH-L1 in normal healthy subjects, as well as for "risk" levels of GFAP, UCH-L1, or GFAP and UCH-L1 in tissue taken from donors that may have one or more of the characteristics described above. In some cases, the controls may be with respect to (e.g., based on) samples or information taken from healthy subjects who are considered healthy and have not suffered an apparent TBI ("healthy controls").

[0288] Therefore, with the above in mind, there is provided a method for determining the presence, amount or concentration of GFAP, UCH-L1, or GFAP and UCH-L1 in a test sample. The method includes, for example, assaying the test sample for GFAP, UCH-L1 or GFAP and UCH-L1 by an immunoassay utilizing at least one capture antibody that binds to an epitope on GFAP, UCH-L1 or GFAP and UCH-L1 and at least one detection antibody that binds to an epitope on GFAP, UCH-L1 or GFAP and UCH-L1 different from the epitope for the capture antibody and optionally comprises a detectable label, and comparing a signal generated by the detectable label as a direct or indirect indication of the presence, amount or concentration of GFAP, UCH-L1 or GFAP and UCH-L1 in the test sample to a signal generated in a calibrator as a direct or indirect indication of the presence, amount or concentration of GFAP, UCH-L1 or GFAP and UCH-L1. The calibrator is optionally, and preferably is, part of a series of calibrators, each of which differs in concentration of GFAP, UCH-L1, or GFAP and UCH-L1 from the other calibrators in the series.

[0289] 10. Kit Provided herein are kits that can be used in the methods described herein to assay or evaluate a test sample for GFAP, UCH-L1, or GFAP and UCH-L1 fragments. The kits include at least one component for assaying a test sample for GFAP, UCH-L1, or GFAP and UCH-L1, and instructions for assaying the test sample for. For example, the kits can include instructions for assaying a test sample for GFAP, UCH-L1, or GFAP and UCH-L1 by immunoassay, e.g., chemiluminescent microparticle immunoassay. Instructions included within the kits can be affixed to the packaging material, or included as a package insert, or viewed or downloaded from a particular website listed as part of the kit packaging material or insert. Instructions can typically be written or printed, but are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), etc. "Instructions" as used herein may include an address of an internet site that provides the instructions.

[0290] At least one component may include at least one composition comprising one or more isolated antibodies or antibody fragments thereof that specifically bind to GFAP, UCH-L1 or GFAP and UCH-L1. The antibodies may be GFAP, UCH-L1 or GFAP and UCH-L1 capture antibodies and / or GFAP, UCH-L1 or GFAP and UCH-L1 detection antibodies.

[0291] Alternatively or in addition, the kit may include calibrators or controls as described above, such as purified, optionally lyophilized GFAP, UCH-L1 or GFAP and UCH-L1, and / or at least one container for performing the assay (e.g., a tube, microtiter plate or strip, which may be pre-coated with anti-GFAP, UCH-L1 or GFAP and UCH-L1 monoclonal antibody) and / or a buffer, such as an assay buffer or a wash buffer, one of which may serve as a concentration solution, a substrate solution for a detectable label (e.g., an enzyme label) or a stop solution. Preferably, the kit includes all components, i.e., reagents, standards, buffers, diluents, etc., necessary to perform the assay. The instructions may also include instructions for generating a calibration curve.

[0292] The kit may further comprise a reference standard for quantifying GFAP, UCH-L1, or GFAP and UCH-L1, which may be utilized to establish a calibration curve for interpolating and / or extrapolating GFAP, UCH-L1, or GFAP and UCH-L1 concentrations. The reference standard may be a high concentration level of GFAP, UCH-L1, or GFAP and UCH-L1, for example, 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; a medium concentration level of GFAP, UCH-L1, or GFAP and UCH-L1, for example, 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 concentration levels of GFAP, UCH-L1, or GFAP and UCH-L1, for example, 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.

[0293] Any antibody provided in the kit, such as recombinant antibodies specific for GFAP, UCH-L1, or GFAP and UCH-L1, can incorporate a detectable label, such as a fluorophore, a radioactive moiety, an enzyme, a biotin / avidin label, a chromophore, a chemiluminescent label, 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., GFAP, UCH-L1, or GFAP and UCH-L1) or for detecting the analyte (e.g., GFAP, UCH-L1, or GFAP and UCH-L1). Antibodies, calibrators and / or controls can be provided in separate containers or can be pre-dispensed into an appropriate assay format, e.g., a microtiter plate.

[0294] Optionally, the kit includes components for quality control (e.g., sensitivity panels, calibrators, and positive controls). Preparation of quality control reagents is well known in the art and is described on the insert sheets for various immunodiagnostic products. Sensitivity panel members are optionally used to establish assay performance characteristics and, further, are optionally useful indicators of immunoassay kit reagent integrity and assay standardization.

[0295] The kit may also optionally include other reagents required to perform the diagnostic assay or to facilitate quality control assessment, such as buffers, salts, enzymes, enzyme cofactors, substrates, detection reagents, etc. Other components may also be included in the kit, such as buffers and solutions for isolating and / or treating the test sample (e.g., pretreatment reagents). In addition, the kit may also include one or more other controls. One or more of the components of the kit may be lyophilized, in which case the kit may further include reagents suitable for reconstituting the lyophilized components.

[0296] The various components of the kit are optionally provided in suitable containers, such as microtiter plates, as needed. The kit may further include containers for holding or storing samples (e.g., containers or cartridges for urine samples, whole blood samples, plasma samples, or serum samples). Where appropriate, the kit may also optionally contain reaction vessels, mixing vessels, and reagents or other components that facilitate preparation of test samples. The kit may also include one or more instruments to aid in obtaining test samples, such as syringes, pipettes, forceps, measuring spoons, etc.

[0297] When the detectable label is at least one acridinium compound, the kit can include at least one acridinium-9-carboxamide, at least one acridinium-9-carboxylate aryl ester, or any combination thereof.When the detectable label is at least one acridinium compound, the kit can also include a source of hydrogen peroxide, such as a buffer, solution, and / or at least one basic solution.If desired, the kit can include a solid phase, such as magnetic particles, beads, test tubes, microtiter plates, cuvettes, membranes, scaffolding molecules, thin films, filter paper, disks, or chips.

[0298] If desired, the kit may further comprise one or more components, either alone or further combined with instructions for assaying the test sample for another analyte, which may be a biomarker, such as a biomarker for traumatic brain injury or disorder.

[0299] a. Kit and method compatibility Kits (or components thereof) and methods for assessing or determining the concentration of GFAP, UCH-L1, or GFAP and UCH-L1 in a test sample by the immunoassays described herein are described, e.g., in U.S. Pat. No. 5,063,081, U.S. Patent Application Publication Nos. 2003 / 0170881, 2004 / 0018577, 2005 / 0054078, and 2006 / 0160164, and include a variety of automated and semi-automated systems (including systems in which the solid phase comprises microparticles) commercially available, e.g., by Abbott Laboratories (Abbott Park, Ill.) as Abbott Point of Care (i-STAT® or i-STAT Alinity, Abbott Laboratories), as well as U.S. Pat. Nos. 5,089,424 and 5,006,309, and may be used in conjunction with any of the various systems commercially available, e.g., by Abbott Laboratories (Abbott Park, Ill.). The devices may be adapted for use in systems commercially available from Abbott Medical, Inc. (Park, Ill.) as ARCHITECT® or the Abbott Alinity series of devices.

[0300] Some differences of automated or semi-automated systems compared to non-automated systems (e.g., ELISA) include the length and timing of the substrate to which the first specific binding partner (e.g., analyte antibody or capture antibody) is conjugated (which may affect sandwich formation and analyte reactivity) as well as the capture, detection and / or optional wash steps. Non-automated formats such as ELISA may require relatively long incubation times with the sample and capture reagent (e.g., about 2 hours), whereas automated or semi-automated formats (e.g., ARCHITECT® and any successor platforms, Abbott Laboratories) may have relatively short incubation times (e.g., about 18 minutes for ARCHITECT®). Similarly, whereas non-automated formats such as ELISAs incubate a detection antibody such as a conjugate reagent for a relatively long incubation time (e.g., about 2 hours), an automated or semi-automated format (e.g., ARCHITECT® and any successor platforms) may have a relatively short incubation time (e.g., about 4 minutes for ARCHITECT® and any successor platforms).

[0301] Other platforms available commercially from Abbott Laboratories include, but are not limited to, AxSYM®, IMx® (see, e.g., U.S. Pat. No. 5,294,404, incorporated herein by reference in its entirety), PRISM®, EIA(beads), and Quantum™ II, as well as other platforms. Additionally, the assays, kits, and kit components may be utilized in other formats, for example, on electrochemical assay systems or other portable or point-of-care assay systems. As previously mentioned, the present disclosure is applicable to, for example, the commercially available Abbott Point of Care (i-STAT®, Abbott Laboratories) electrochemical immunoassay system, which performs sandwich immunoassays. Immunosensors and their manufacture and operation in single-use test devices are described, for example, in U.S. Pat. No. 5,063,081, U.S. Patent Application Publication Nos. 2003 / 0170881, 2004 / 0018577, 2005 / 0054078, and 2006 / 0160164, which are incorporated by reference herein for their teachings in this regard.

[0302] In particular, with regard to adapting the assay to the i-STAT® system, the following configuration is preferred: A microfabricated silicon chip is fabricated with a pair of gold amperometric working electrodes and a silver-silver chloride reference electrode. At one of the working electrodes, polystyrene beads (0.2 mm in diameter) with immobilized capture antibodies are attached to a patterned polyvinyl alcohol polymer coating on the electrode. This chip is assembled into an i-STAT® cartridge with a fluidic format suitable for immunoassays. On the silicon chip portion are present specific binding partners for GFAP, UCH-L1 or GFAP and UCH-L1, such as one or more GFAP, UCH-L1 or GFAP and UCH-L1 antibodies (one or more monoclonal / polyclonal antibodies or fragments thereof, variants thereof or fragments of variants thereof capable of binding to GFAP, UCH-L1 or GFAP and UCH-L1) or one or more anti-GFAP, UCH-L1 or GFAP and UCH-L1 DVD-Ig (or fragments thereof, variants thereof or fragments of variants thereof capable of binding to GFAP, UCH-L1 or GFAP and UCH-L1), any of which may be detectably labeled. Within the fluid pouch of the cartridge is an aqueous reagent comprising p-aminophenol phosphate.

[0303] In operation, a sample from a subject suspected of having TBI is added to the holding chamber of the test cartridge and the cartridge is inserted into the i-STAT® reader. A pump element in the cartridge pushes the sample into a conduit containing a chip. The sample is contacted with the sensor, dissolving the enzyme conjugate into the sample. The sample is oscillated across the sensor to promote the formation of a sandwich for approximately 2-12 minutes. In the penultimate step of the assay, the sample is pushed into a waste chamber and a wash solution containing a substrate for the alkaline phosphatase enzyme is used to wash excess enzyme conjugate and sample off the sensor chip. In the final step of the assay, the alkaline phosphatase label reacts with p-aminophenol phosphate to cleave the phosphate group and electrochemically oxidize free p-aminophenol at the working electrode. Based on the measured current, the reader is able to calculate the amount of GFAP, UCH-L1, or GFAP and UCH-L1 in the sample using built-in algorithms and factory determined calibration curves. Adaptation of cartridges such as those used in the i-Stat for multiplexed use is described in patent literature, such as, for example, U.S. Patent No. 6,438,498, the contents of which are incorporated herein by reference.

[0304] The methods and kits described herein necessarily include other reagents and methods for performing immunoassays. For example, various buffers are included that are known in the art and / or can be easily prepared or optimized and used as conjugate diluents and / or calibrator diluents, for example for washing. An exemplary conjugate diluent is ARCHITECT® conjugate diluent, which is used in certain kits (Abbott Laboratories, Abbott Park, Ill.), and contains 2-(N-morpholino)ethanesulfonic acid (MES), salts, protein blocking agents, antimicrobial agents, and surfactants. An exemplary calibrator diluent is ARCHITECT® human calibrator diluent, which is used in certain kits (Abbott Laboratories, Abbott Park, Ill.), and contains a buffer containing MES, other salts, protein blocking agents, and antimicrobial agents. Additionally, as described in U.S. Patent Application No. 61 / 142,048, filed December 31, 2008, improved signal generation can be obtained in the i-STAT® cartridge format using, for example, a nucleic acid sequence linked to a signal antibody as a signal amplifier.

[0305] While certain embodiments herein are advantageously used to assess diseases such as traumatic brain injury, the assays and kits may also be used, as appropriate and optionally, to assess GFAP, UCH-L1, or GFAP and UCH-L1 in other diseases, disorders and conditions.

[0306] The assay method can also be used to identify compounds that improve diseases such as traumatic brain injury. For example, cells expressing GFAP, UCH-L1, or GFAP and UCH-L1 can be contacted with a candidate compound. Using the assay method described herein, the expression level of GFAP, UCH-L1, or GFAP and UCH-L1 in the cells contacted with the compound can be compared with the expression level in control cells. EXAMPLES

[0307] The present disclosure has multiple aspects, illustrated by the following non-limiting examples.

[0308] 11. Working Example It will be readily apparent to one skilled in the art that other suitable modifications and adaptations of the disclosed methods described herein are readily applicable and recognizable, and may be made using appropriate equivalents without departing from the scope of the disclosure or the aspects and embodiments disclosed herein. Now, the disclosure has been described in detail, which will be more clearly understood by reference to the following examples, which are intended to illustrate only certain aspects and embodiments of the disclosure, and should not be considered as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents and publications mentioned herein are incorporated herein by reference in their entirety.

[0309] The present disclosure has multiple aspects, illustrated by the following non-limiting examples.

[0310] [Example 1] Assays used in the examples i-STAT® UCH-L1 assay: Monoclonal antibody pairs were investigated, including antibody A as the capture monoclonal antibody and antibody B and antibody C as the detection monoclonal antibodies. Antibody A is an exemplary anti-UCH-L1 antibody developed in-house at Abbott Laboratories (Abbott Park, IL). Antibody B and antibody C, developed by Banyan Biomarkers (Alachua, Florida), recognize different epitopes of UCH-L1 and enhance the detection of the antigen in the sample. The antibody combinations, when used together, produce synergistic effects, resulting in increased signal compared to the use of uncombined antibodies. Other antibodies developed in-house at Abbott Laboratories (Abbott Park, IL) also show, or are expected to show, similar signal enhancement when used together in various combinations as capture or detection antibodies. The UCH-L1 assay design was assessed for key performance attributes. Cartridge configuration was: Antibody Configuration: Antibody A (capture antibody) / Antibody B+C (detection antibody); Reagent Conditions: 0.8% solids, 125 μg / mL Fab alkaline phosphatase cluster conjugate and sample inlet imprint: UCH-L1 standard. Assay time was 10-15 minutes (with sample capture time of 7-12 minutes). The i-STAT® UCH-L1 assay was used in the TBI patient population study.

[0311] i-STAT® GFAP Assay: The i-STAT® GFAP assay was used in the TBI patient population study. A monoclonal antibody pair was used, with antibody A as the capture monoclonal antibody and antibody B as the detection monoclonal antibody. Antibody A and antibody B are exemplary anti-GFAP antibodies developed in-house at Abbott Laboratories (Abbott Park, IL). Both antibody A and antibody B bind to epitopes within the same GFAP degradation product (BDP). The antibody combination, when used together, produced a synergistic effect, resulting in increased signal compared to the use of uncombined antibodies. The GFAP assay design was assessed against key performance attributes. The cartridge configuration was antibody configuration: antibody A (capture antibody) / antibody B+C (detection antibody); reagent conditions: 0.8% solids, 250 μg / mL Fab alkaline phosphatase cluster conjugate and sample inlet imprinting: GFAP specific. The assay time was 10-15 minutes (with sample capture time of 7-12 minutes).

[0312] [Example 2] Pediatric assessment Pediatric patients were assessed in the Transforming Research and Clinical Knowledge in Traumatic Brain Injury (TRACK-TBI) study, a study involving trauma patients from Hennepin County Medical Center (Hennepin) in Minnesota. The TRACK-TBI study is a collaborative, public-private partnership involving over 11 clinical sites, seven cores, and nearly 50 collaborating institutions, companies, and advocacy groups. For trauma patients recruited at HCMC, participants included trauma patients who presented to the HCMC emergency department (ED), trauma surgery suite, or as a direct transfer to neurosurgery.

[0313] Subject group - TRACK-TBI: Pediatric TBI patients were assessed. Subjects consisted of 50 pediatric subjects with biomarker results, 40 pediatric subjects with valid CT scans, 23 pediatric subjects with valid MRI scans, and 0 controls.

[0314] Subject group - Hennepin: Pediatric TBI patients were assessed. Subjects consisted of one pediatric subject with biomarker results and valid CT scans and three controls (four subjects total).

[0315] Subject eligibility: Pediatric patients aged 17 years or younger presenting to the emergency department (ED) with a history of acute TBI according to the American Congress of Rehabilitation Medicine (ACRM) Criteria and who suffered trauma-induced physiological disruption of brain function as manifested by ≥1 of the following: loss of consciousness (LOC) for any period of time; any memory loss event (e.g., amnesia) immediately prior to or immediately after the incident; any alteration in mental status (sensation of dazedness, disorientation, and / or confusion) at the time of the incident; and / or focal neurological deficits, which may or may not be permanent. Trauma-induced included blows to the head, hitting the head against an object, or brain undergoing acceleration / deceleration motion without direct external trauma to the head (e.g., whiplash conditions). GFAP and UCH-L1 levels in samples obtained from pediatric subjects aged 2-17 were measured using the prototype iSTAT GFAP and UCH-L1 assays (Abbott Laboratories). A total of 50 subjects were evaluated, of which 40 had valid CT scans. Sixteen of the 40 subjects had positive CT scans. Nine subjects had mild GCS scores (13-15). It should be noted that GCS scores were not available for the majority of subjects.

[0316] Figure 1 shows the receiver operating characteristic (ROC) analysis of GFAP levels for these 40 TBI samples compared to GFAP levels in all control samples (AUC=0.77, 95% CI .62, .93). Table 2 shows the sensitivity, specificity, negative predictive value, and positive predictive value for cutoff values ​​(i.e., reference values) of 50 pg / mL and 1000 pg / mL, respectively.

[0317] [Table 2]

[0318] Figure 2 shows the receiver operating characteristic (ROC) analysis of UCH-L1 levels for these 40 TBI samples compared to UCH-L1 levels in all control samples (AUC=0.67, 95% CI .48, .86). Table 3 shows the sensitivity, specificity, negative predictive value, and positive predictive value for cutoff values ​​(i.e., reference values) of 55 pg / mL and 300 pg / mL, respectively.

[0319] [Table 3]

[0320] The cutoff values ​​were further assessed. Cutoffs of at least 30 pg / mL for GFAP and at least 360 pg / mL for UCH-L1 were tested. The results are shown in Tables 4 and 5 below.

[0321] [Table 4]

[0322] [Table 5]

[0323] One potential outlier was identified and the results are shown in Table 6. Excluding this outlier, a sensitivity and negative predictive value of 100% was obtained.

[0324] [Table 6]

[0325] Next, cutoffs of at least 65 pg / mL for GFAP and at least 360 pg / mL for UCH-L1 were assessed, and the results are shown in Tables 7 and 8 below.

[0326] [Table 7]

[0327] [Table 8]

[0328] A comparison of GFAP results against CT status was performed for subjects for whom CT status was available. The results are shown in Table 9 and Figure 3.

[0329] [Table 9]

[0330] A comparison of GFAP results against CT status was performed for subjects for whom CT status was available. The results are shown in Table 10 and Figure 4.

[0331] [Table 10]

[0332] [Example 3] Assessment of pediatric subjects against adult controls Further comparisons were performed of the pediatric data described in Example 2 to data in adult subjects (e.g., subjects ages 18-79). The resulting cohorts, median ages, GFAP levels, and UCH-L1 levels are shown in Table 11.

[0333] [Table 11]

[0334] It is understood that the foregoing detailed description and the accompanying examples are exemplary only and are not to be taken as limitations on the scope of the present disclosure, which is defined solely by the appended claims and their equivalents.

[0335] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including, without limitation, changes and modifications to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the present disclosure, may be made without departing from the spirit and scope thereof.

[0336] Article 1: A method of assessing a pediatric subject for head injury, comprising: a) performing an assay on a sample obtained from a subject following actual or suspected head injury to measure the level of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and / or the level of glial fibrillary acidic protein (GFAP) in the sample; and b) i. the level of GFAP in the sample is greater than a baseline level of GFAP, the baseline level of GFAP being at least about 30 pg / mL; ii. the level of UCH-L1 in the sample is greater than a reference level of UCH-L1, the reference level of UCH-L1 being at least about 55 pg / mL; or iii. the level of GFAP in the sample is greater than the baseline level of GFAP, the level of UCH-L1 in the sample is greater than the baseline level of UCH-L1, the baseline level of GFAP is at least about 30 pg / mL, and the baseline level of UCH-L1 is about 360 pg / mL; determining that the subject has suffered a traumatic brain injury (TBI) if

[0337] Clause 2: The method of clause 1, wherein the subject is determined to have suffered a TBI if the level of GFAP in the sample is greater than a baseline level of GFAP, the baseline level of GFAP being at least about 50 pg / mL.

[0338] Clause 3: The method of clause 2, wherein the baseline level of GFAP is at least about 65 pg / mL.

[0339] Clause 4: The method of clause 3, wherein the reference level of GFAP is about 1000 pg / mL.

[0340] Clause 5: The method of clause 1, wherein the subject is determined to have suffered a TBI if the level of UCH-L1 in the sample is greater than the reference level of UCH-L1, the reference level of UCH-L1 being about 300 pg / mL.

[0341] Clause 6: The method of clause 1, wherein the subject is determined to have suffered a TBI if the level of GFAP in the sample is greater than the baseline level of GFAP, the level of UCH-L1 in the sample is greater than the baseline level of UCH-L1, the baseline level of GFAP is about 65 pg / mL, and the baseline level of UCH-L1 is about 360 pg / mL.

[0342] Clause 7: The method of any one of clauses 1 to 6, wherein the sample is collected within about 48 hours after actual or suspected head injury.

[0343] Clause 8: The method of any one of clauses 1 to 7, wherein the subject receives a GCS (Glasgow Coma Scale) score before or after the assay is performed.

[0344] Clause 9: The method of clause 8, wherein the subject is suspected to have moderate to severe TBI based on a Glasgow Coma Scale (GCS) score or a reference level correlates with a subject having moderate to severe TBI.

[0345] Clause 10: The method of clause 8, wherein the subject is suspected to have mild TBI based on a Glasgow Coma Scale (GCS) score or a reference level correlates with a subject having mild TBI.

[0346] Clause 11:a) the reference level of GFAP is (i) determined by an assay having a sensitivity of at least about 90% and a specificity of at least about 40%; (ii) determined by an assay having a sensitivity of at least about 50% and a specificity of at least about 90%; (iii) determined by an assay having a negative predictive value of at least about 70%; (iv) determined by an assay having a negative predictive value of at least about 90%; (v) determined by an assay having a positive predictive value of at least about 50%; or (vi) determined by an assay having a positive predictive value of at least about 80%; b) the reference level of UCH-L1 is (i) determined by an assay having a sensitivity of at least about 80% and a specificity of at least about 25%; (ii) determined by an assay having a sensitivity of at least about 30% and a specificity of at least about 90%; (iii) determined by an assay having a negative predictive value of at least about 65%; (iv) determined by an assay having a positive predictive value of at least about 40%; or (v) determined by an assay having a positive predictive value of at least about 80%; and / or c) The method of any of clauses 1-10, wherein the baseline level of GFAP and the baseline level of UCH-L1 are (i) determined by an assay having a sensitivity of at least about 70% and a specificity of at least about 10%; (ii) determined by an assay having a sensitivity of at least about 65% and a specificity of at least about 25%; (iii) determined by an assay having a positive predictive value of at least about 35%; (iv) determined by an assay having a negative predictive value of at least about 40%; or (v) determined by an assay having a negative predictive value of at least about 55%.

[0347] Clause 12: The method of any one of clauses 1-11, further comprising treating a pediatric subject determined to have TBI with a therapy for TBI, and optionally monitoring the pediatric subject after receiving said therapy.

[0348] Clause 13: The method of any one of clauses 1-12, wherein the pediatric subject is a human.

[0349] Article 14: A method of assessing whether to perform a head computed tomography (CT) scan on a pediatric subject, comprising: a) performing an assay on a sample obtained from a subject following actual or suspected head injury to measure levels of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and / or levels of glial fibrillary acidic protein (GFAP) in the sample; and b) i. the level of GFAP in the sample is greater than a baseline level of GFAP, the baseline level of GFAP being at least about 30 pg / mL; ii. the level of UCH-L1 in the sample is greater than a reference level of UCH-L1, the reference level of UCH-L1 being at least about 55 pg / mL; or iii. the level of GFAP in the sample is greater than the baseline level of GFAP and the level of UCH-L1 in the sample is greater than the baseline level of UCH-L1, the baseline level of GFAP being at least about 30 pg / mL and the baseline level of UCH-L1 being at least about 360 pg / mL; To determine whether a head CT scan should be performed in children if The method includes:

[0350] Clause 15: The method of clause 14, wherein if the level of GFAP in the sample is greater than the reference level of GFAP, and the reference level of GFAP is at least about 50 pg / mL, it is determined that a head CT scan should be performed.

[0351] Clause 16: The method of clause 15, wherein the baseline level of GFAP is at least about 65 pg / mL.

[0352] Clause 17: The method of clause 16, wherein the reference level of GFAP is about 1000 pg / mL.

[0353] Clause 18: The method of clause 14, wherein if the level of UCH-L1 in the sample is greater than the reference level of UCH-L1, and the reference level of UCH-L1 is about 300 pg / mL, it is determined that a head CT scan should be performed.

[0354] Clause 19: The method of clause 14, wherein if the level of GFAP in the sample is greater than the reference level of GFAP and the level of UCH-L1 in the sample is greater than the reference level of UCH-L1, the reference level of GFAP being about 65 pg / mL and the reference level of UCH-L1 being about 360 pg / mL, it is determined that a head CT scan should be performed.

[0355] Clause 20:a) the reference level of GFAP is (i) determined by an assay having a sensitivity of at least about 90% and a specificity of at least about 40%; (ii) determined by an assay having a sensitivity of at least about 50% and a specificity of at least about 90%; (iii) determined by an assay having a negative predictive value of at least about 70%; (iv) determined by an assay having a negative predictive value of at least about 90%; (v) determined by an assay having a positive predictive value of at least about 50%; or (vi) determined by an assay having a positive predictive value of at least about 80%; b) the reference level of UCH-L1 is (i) determined by an assay having a sensitivity of at least about 80% and a specificity of at least about 25%; (ii) determined by an assay having a sensitivity of at least about 30% and a specificity of at least about 90%; (iii) determined by an assay having a negative predictive value of at least about 65%; (iv) determined by an assay having a positive predictive value of at least about 40%; or (v) determined by an assay having a positive predictive value of at least about 80%; and / or c) The method of any one of clauses 14-19, wherein the baseline level of GFAP and the baseline level of UCH-L1 are (i) determined by an assay having a sensitivity of at least about 70% and a specificity of at least about 10%; (ii) determined by an assay having a sensitivity of at least about 65% and a specificity of at least about 25%; (iii) determined by an assay having a positive predictive value of at least about 35%; (iv) determined by an assay having a negative predictive value of at least about 40%; or (v) determined by an assay having a negative predictive value of at least about 55%.

[0356] Clause 21: The method of any one of clauses 14 to 20, wherein the sample is collected within about 48 hours after actual or suspected head injury.

[0357] Clause 22: The method of any one of clauses 1 to 21, further comprising performing an assay on the sample to measure or detect the level of one or more other biomarkers that are not UCH-L1 or GFAP.

[0358] Clause 23: The method of clause 22, wherein the one or more other biomarkers are selected from the group consisting of S100β, neuron-specific enolase (NSE), lipoprotein 1, Tau, C-reactive protein (CRP), free brain-derived neurotrophic factor (BDNF), p-Tau, total BDNF, troponin I (TnI), and combinations thereof.

[0359] Clause 24: The method of any one of clauses 1 to 23, wherein measuring the level of UCH-L1 comprises performing an immunoassay.

[0360] Clause 25: Measuring the level of UCH-L1 (a) To form a capture antibody-UCH-L1 antigen-detection antibody complex, (1) a capture antibody that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form a capture antibody-UCH-L1 antigen complex; and (2) a detection antibody that contains a detectable label and binds to an epitope of UCH-L1 to which the capture antibody is not bound, thereby forming a UCH-L1 antigen-detection antibody complex; contacting the samples simultaneously or sequentially, in any order; and (b) 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; 25. The method according to claim 24, comprising:

[0361] Clause 26: The method of any one of clauses 1-25, wherein measuring the level of GFAP comprises performing an immunoassay.

[0362] Article 27: Measuring the level of GFAP (a) To form a capture antibody-GFAP antigen-detection antibody complex, (1) a capture antibody that binds to an epitope on GFAP or a GFAP fragment to form a capture antibody-GFAP antigen complex; and (2) a detection antibody that contains a detectable label and that binds to an epitope of GFAP to which the capture antibody is not bound, forming a GFAP antigen-detection antibody complex; contacting the samples simultaneously or sequentially, in any order; and (b) 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; 27. The method according to claim 26, comprising:

[0363] Clause 28: The method of any one of clauses 1 to 28, wherein the sample is a whole blood sample, a serum sample, a cerebrospinal fluid sample, a plasma sample, a tissue sample, a saliva sample, an oropharyngeal sample, a nasopharyngeal sample, a nasal mucus sample, or a body fluid.

[0364] Clause 29: The method of any one of clauses 1 to 29, wherein the sample is obtained after the subject has suffered a head injury or other type of blunt trauma caused by physical shaking, an external mechanical or other force resulting in a closed or open head injury, one or more falls, blunt impact from an explosion or blast.

[0365] Clause 30: The method of any one of clauses 1 to 28, wherein the sample is obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin.

[0366] Clause 31: The method of clause 30, wherein the chemical or toxin is fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organometallics, drugs of abuse, or a combination of one or more thereof.

[0367] Clause 32: The method of any one of clauses 1 to 31, wherein the sample is obtained from a pediatric subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus or a combination thereof.

[0368] Clause 33: The method of any one of clauses 1 to 32, wherein the assay is an immunoassay or a clinical chemistry assay.

[0369] Clause 34: The method of any one of clauses 1 to 33, wherein the assay is performed using a single molecule detection or point-of-care device.

[0370] Clause 35: The method of any one of clauses 1 to 6, wherein the sample is collected within about 6 hours after actual or suspected head injury.

[0371] Clause 36: The method of any one of clauses 1 to 6, wherein the sample is collected within about 12 hours after actual or suspected head injury.

[0372] Clause 37: The method of any one of clauses 1 to 6, wherein the sample is collected within about 14 hours after an actual or suspected head injury.

[0373] Clause 38: The method of any one of clauses 1 to 6, wherein the sample is collected within about 24 hours after an actual or suspected head injury.

[0374] Article 39: A method of assessing a pediatric subject for head injury, comprising: c) performing an assay on a sample obtained from the subject following actual or suspected head injury to measure the level of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and / or the level of glial fibrillary acidic protein (GFAP) in the sample; and d) i) the level of GFAP in the sample is greater than the baseline level of GFAP, the baseline level of GFAP being at least about 30 pg / mL; ii) the level of UCH-L1 in the sample is greater than a reference level of UCH-L1, the reference level of UCH-L1 being at least about 55 pg / mL; or iii) determining that the subject is more likely to have suffered a traumatic brain injury (TBI) than not having suffered a TBI if the level of GFAP in the sample is greater than the baseline level of GFAP and the level of UCH-L1 in the sample is greater than the baseline level of UCH-L1, the baseline level of GFAP being at least about 30 pg / mL and the baseline level of UCH-L1 being about 360 pg / mL; The method includes:

[0375] Clause 40: The method of clause 39, wherein if the level of GFAP in the sample is greater than the reference level of GFAP, and the reference level of GFAP is at least about 50 pg / mL, then it is determined that the subject is more likely to have suffered a TBI than not to have suffered a TBI.

[0376] Clause 41: The method of clause 40, wherein the baseline level of GFAP is at least about 65 pg / mL.

[0377] Clause 42: The method of clause 41, wherein the reference level of GFAP is about 1000 pg / mL.

[0378] Clause 43: The method of clause 39, wherein if the level of UCH-L1 in the sample is greater than the reference level of UCH-L1, and the reference level of UCH-L1 is about 300 pg / mL, then it is determined that the likelihood that the subject has suffered a TBI is greater than the likelihood that the subject has not suffered a TBI.

[0379] Clause 44: The method of clause 39, wherein if the level of GFAP in the sample is greater than the reference level of GFAP and the level of UCH-L1 in the sample is greater than the reference level of UCH-L1, the reference level of GFAP being about 65 pg / mL and the reference level of UCH-L1 being about 360 pg / mL, then it is determined that the subject is more likely to have suffered a TBI than not to have suffered a TBI.

[0380] Clause 45: The method of any one of clauses 39 to 44, wherein the sample is collected within about 48 hours after actual or suspected head injury.

[0381] Clause 46: The method of any one of clauses 39 to 45, wherein the subject has received a GCS (Glasgow Coma Scale) score before or after the assay is performed.

[0382] Clause 47: The method of clause 46, wherein the subject is suspected to have moderate to severe TBI based on the Glasgow Coma Scale (GCS) score or the reference level correlates with the subject having moderate to severe TBI.

[0383] Clause 48: The method of clause 46, wherein the subject is suspected of having mild TBI based on a Glasgow Coma Scale (GCS) score or the reference level correlates with the subject having mild TBI.

[0384] Clause 49:a) the reference level of GFAP is (i) determined by an assay having a sensitivity of at least about 90% and a specificity of at least about 40%; (ii) determined by an assay having a sensitivity of at least about 50% and a specificity of at least about 90%; (iii) determined by an assay having a negative predictive value of at least about 70%; (iv) determined by an assay having a negative predictive value of at least about 90%; (v) determined by an assay having a positive predictive value of at least about 50%; or (vi) determined by an assay having a positive predictive value of at least about 80%; b) the reference level of UCH-L1 is (i) determined by an assay having a sensitivity of at least about 80% and a specificity of at least about 25%; (ii) determined by an assay having a sensitivity of at least about 30% and a specificity of at least about 90%; (iii) determined by an assay having a negative predictive value of at least about 65%; (iv) determined by an assay having a positive predictive value of at least about 40%; or (v) determined by an assay having a positive predictive value of at least about 80%; and / or c) The method of any of clauses 39-48, wherein the baseline level of GFAP and the baseline level of UCH-L1 are (i) determined by an assay having a sensitivity of at least about 70% and a specificity of at least about 10%; (ii) determined by an assay having a sensitivity of at least about 65% and a specificity of at least about 25%; (iii) determined by an assay having a positive predictive value of at least about 35%; (iv) determined by an assay having a negative predictive value of at least about 40%; or (v) determined by an assay having a negative predictive value of at least about 55%.

[0385] Clause 50: The method of any one of clauses 39 to 49, further comprising treating a pediatric subject determined to have TBI with a therapy for TBI, and optionally monitoring the pediatric subject after receiving said therapy.

[0386] Clause 51: The method of any one of clauses 39 to 50, wherein the pediatric subject is a human.

[0387] Clause 52: A method according to any one of clauses 39 to 44, wherein the sample is collected within about 6 hours after an actual or suspected head injury.

[0388] Clause 53: A method according to any one of clauses 39 to 44, wherein the sample is collected within about 12 hours after an actual or suspected head injury.

[0389] Clause 54: A method according to any one of clauses 39 to 44, wherein the sample is collected within about 14 hours after an actual or suspected head injury.

[0390] Clause 55: A method according to any one of clauses 39 to 44, wherein the sample is collected within about 24 hours after an actual or suspected head injury.

Claims

1. 1. A method of assessing a pediatric subject for head injury, comprising: a) performing an assay on a sample obtained from said subject following actual or suspected head injury to measure the level of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and / or the level of glial fibrillary acidic protein (GFAP) in said sample; and b) i. the level of GFAP in the sample is greater than a reference level of GFAP, the reference level of GFAP being at least about 30 pg / mL; ii. the level of UCH-L1 in the sample is greater than a reference level of UCH-L1, the reference level of UCH-L1 being at least about 55 pg / mL; or iii. the level of GFAP in the sample is greater than a baseline level of GFAP and the level of UCH-L1 in the sample is greater than a baseline level of UCH-L1, the baseline level of GFAP being at least about 30 pg / mL and the baseline level of UCH-L1 being about 360 pg / mL. determining that the subject has suffered a traumatic brain injury (TBI) if

2. The method of claim 1, wherein the subject is determined to have suffered a TBI if the level of GFAP in the sample is greater than a reference level of GFAP, the reference level of GFAP being at least about 50 pg / mL.

3. 3. The method of claim 2, wherein the reference level of GFAP is at least about 65 pg / mL.

4. 4. The method of claim 3, wherein the reference level of GFAP is about 1000 pg / mL.

5. 2. The method of claim 1, wherein the subject is determined to have suffered a TBI if the level of UCH-L1 in the sample is greater than a reference level of UCH-L1, the reference level of UCH-L1 being about 300 pg / mL.

6. 2. The method of claim 1, wherein the subject is determined to have suffered a TBI if the level of GFAP in the sample is greater than a baseline level of GFAP and the level of UCH-L1 in the sample is greater than a baseline level of UCH-L1, the baseline level of GFAP being about 65 pg / mL and the baseline level of UCH-L1 being about 360 pg / mL.

7. 10. The method of claim 1, wherein the sample is collected within (a) about 12 hours; (b) about 24 hours; (c) about 36 hours; or about 48 hours after the actual or suspected head injury.

8. 2. The method of claim 1, wherein the subject has a Glasgow Coma Scale (GCS) score before or after the assay is performed.

9. 9. The method of claim 8, wherein the subject is suspected of having moderate to severe TBI based on the Glasgow Coma Scale (GCS) score or the reference level correlates with a subject having moderate to severe TBI.

10. 9. The method of claim 8, wherein the subject is suspected of having mild TBI based on the Glasgow Coma Scale (GCS) score or the reference level correlates with subjects having mild TBI.

11. a) the reference level of GFAP is (i) determined by an assay having a sensitivity of at least about 90% and a specificity of at least about 40%; (ii) determined by an assay having a sensitivity of at least about 50% and a specificity of at least about 90%; (iii) determined by an assay having a negative predictive value of at least about 70%; (iv) determined by an assay having a negative predictive value of at least about 90%; (v) determined by an assay having a positive predictive value of at least about 50%; or (vi) determined by an assay having a positive predictive value of at least about 80%; b) the reference level of UCH-L1 is (i) determined by an assay having a sensitivity of at least about 80% and a specificity of at least about 25%; (ii) determined by an assay having a sensitivity of at least about 30% and a specificity of at least about 90%; (iii) determined by an assay having a negative predictive value of at least about 65%; (iv) determined by an assay having a positive predictive value of at least about 40%; or (v) determined by an assay having a positive predictive value of at least about 80%; and / or c) The method of claim 1, wherein the reference level of GFAP and the reference level of UCH-L1 are (i) determined by an assay having a sensitivity of at least about 70% and a specificity of at least about 10%; (ii) determined by an assay having a sensitivity of at least about 65% and a specificity of at least about 25%; (iii) determined by an assay having a positive predictive value of at least about 35%; (iv) determined by an assay having a negative predictive value of at least about 40%; or (v) determined by an assay having a negative predictive value of at least about 55%.

12. 10. The method of claim 1, further comprising treating a pediatric subject determined to have TBI with a therapy for TBI, and optionally monitoring the pediatric subject after receiving the therapy.

13. 10. The method of claim 1, wherein the pediatric subject is a human.

14. 1. A method for assessing whether to perform a head computed tomography (CT) scan in a pediatric subject, comprising: a) performing an assay on a sample obtained from the subject following actual or suspected head injury to measure the level of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and / or the level of glial fibrillary acidic protein (GFAP) in the sample; and b) i. the level of GFAP in the sample is greater than a reference level of GFAP, the reference level of GFAP being at least about 30 pg / mL; ii. the level of UCH-L1 in the sample is greater than a reference level of UCH-L1, wherein the reference level of UCH-L1 is at least about 55 pg / mL; or iii. the level of GFAP in the sample is greater than a baseline level of GFAP and the level of UCH-L1 in the sample is greater than a baseline level of UCH-L1, the baseline level of GFAP being at least about 30 pg / mL and the baseline level of UCH-L1 being at least about 360 pg / mL. determining that a head CT scan should be performed on the pediatric subject if The method includes:

15. The method of claim 14, wherein if the level of GFAP in the sample is greater than a reference level of GFAP, and the reference level of GFAP is at least about 50 pg / mL, it is determined that a head CT scan should be performed.

16. 16. The method of claim 15, wherein the reference level of GFAP is at least about 65 pg / mL.

17. 17. The method of claim 16, wherein the reference level of GFAP is about 1000 pg / mL.

18. 15. The method of claim 14, wherein if the level of UCH-L1 in the sample is greater than a reference level of UCH-L1, and the reference level of UCH-L1 is about 300 pg / mL, it is determined that a head CT scan should be performed.

19. The method of claim 14, wherein if the level of GFAP in the sample is greater than a reference level of GFAP and the level of UCH-L1 in the sample is greater than a reference level of UCH-L1, the reference level of GFAP being about 65 pg / mL and the reference level of UCH-L1 being about 360 pg / mL, it is determined that a head CT scan should be performed.

20. a) the reference level of GFAP is (i) determined by an assay having a sensitivity of at least about 90% and a specificity of at least about 40%; (ii) determined by an assay having a sensitivity of at least about 50% and a specificity of at least about 90%; (iii) determined by an assay having a negative predictive value of at least about 70%; (iv) determined by an assay having a negative predictive value of at least about 90%; (v) determined by an assay having a positive predictive value of at least about 50%; or (vi) determined by an assay having a positive predictive value of at least about 80%; b) the reference level of UCH-L1 is (i) determined by an assay having a sensitivity of at least about 80% and a specificity of at least about 25%; (ii) determined by an assay having a sensitivity of at least about 30% and a specificity of at least about 90%; (iii) determined by an assay having a negative predictive value of at least about 65%; (iv) determined by an assay having a positive predictive value of at least about 40%; or (v) determined by an assay having a positive predictive value of at least about 80%; and / or c) The method of claim 14, wherein the reference level of GFAP and the reference level of UCH-L1 are (i) determined by an assay having a sensitivity of at least about 70% and a specificity of at least about 10%; (ii) determined by an assay having a sensitivity of at least about 65% and a specificity of at least about 25%; (iii) determined by an assay having a positive predictive value of at least about 35%; (iv) determined by an assay having a negative predictive value of at least about 40%; or (v) determined by an assay having a negative predictive value of at least about 55%.

21. 15. The method of claim 14, wherein the sample is collected within about 48 hours after the actual or suspected head injury.

22. 15. The method of claim 1 or 14, further comprising performing an assay on the sample to measure or detect the level of one or more other biomarkers that are not UCH-L1 or GFAP.

23. 23. The method of claim 22, wherein the one or more other biomarkers are selected from the group consisting of S100beta, neuron-specific enolase (NSE), lipoprotein 1, Tau, C-reactive protein (CRP), free brain-derived neurotrophic factor (BDNF), p-Tau, total BDNF, troponin I (TnI), and combinations thereof.

24. 15. The method of claim 1 or 14, wherein measuring the level of UCH-L1 comprises performing an immunoassay.

25. Measuring the level of UCH-L1 (a) forming a capture antibody-UCH-L1 antigen-detection antibody complex; (1) a capture antibody that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form a capture antibody-UCH-L1 antigen complex; and (2) a detection antibody that contains a detectable label and binds to an epitope of UCH-L1 to which the capture antibody is not bound, thereby forming a UCH-L1 antigen-detection antibody complex; contacting said samples simultaneously or sequentially, in any order; and (b) 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; 25. The method of claim 24, comprising:

26. 15. The method of claim 1 or 14, wherein measuring the level of GFAP comprises performing an immunoassay.

27. Measuring the level of GFAP (a) allowing a capture antibody-GFAP antigen-detection antibody complex to form; (1) a capture antibody that binds to an epitope on GFAP or a GFAP fragment to form a capture antibody-GFAP antigen complex; and (2) a detection antibody that contains a detectable label and binds to an epitope of GFAP to which the capture antibody is not bound, thereby forming a GFAP antigen-detection antibody complex; contacting said samples simultaneously or sequentially, in any order; and (b) measuring the amount or concentration of UCH-L1 in the sample based on the signal generated by the detectable label in the capture antibody-UCH-L1 antigen-detection antibody complex; 27. The method of claim 26, comprising:

28. 15. The method of claim 1 or 14, wherein the sample is a whole blood sample, a serum sample, a cerebrospinal fluid sample, a plasma sample, a tissue sample, a saliva sample, an oropharyngeal sample, a nasopharyngeal sample, a nasal mucus sample, or a body fluid.

29. 15. The method of claim 1 or 14, wherein the sample is obtained after the subject has suffered a head injury caused by physical shaking, an external mechanical or other force resulting in a closed or open head injury, one or more falls, blunt impact from an explosion or blast, or other type of blunt force trauma.

30. 20. The method of claim 1 or 14, wherein the sample is obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin.

31. 31. The method of claim 30, wherein the chemical or toxin is fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organometallics, drugs of abuse, or one or more combinations thereof.

32. 15. The method of claim 1 or 14, wherein the sample is obtained from a pediatric subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof.

33. 15. The method of claim 1 or 14, wherein the assay is an immunoassay or a clinical chemistry assay.

34. 15. The method of claim 1 or 14, wherein the assay is performed using a single molecule detection or point-of-care device.

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