Novel biomarkers and methods for diagnosing and assessing traumatic brain injury

Abstract

To provide a method for diagnosing and assessing a subject who has or may have suffered a head injury, such as a traumatic brain injury (TBI).SOLUTION: A variety of biomarkers in which their detection and / or differential expression may be used to assess presence or absence of TBI in a subject and which may be used as a basis for diagnosing a subject as having a specific type of TBI (for example, a subclass of severe TBI or slight TBI) are identified. The variety of TBI biomarkers may be detected individually or in combination and can be used as an important diagnostic tool, prognosis tool, and / or TBI risk stratification tool as part of assessing TBI status of the subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 611,778, filed on 29 December 2017, and U.S. Provisional Application No. 62 / 630,704, filed on 14 February 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] This disclosure relates to methods for diagnosing and assessing subjects who have suffered or may have suffered head injuries, such as traumatic brain injury (TBI). In particular, this disclosure identifies a variety of biomarkers whose detection and / or differential expression may be used to assess the presence or absence of TBI in a subject and which may be used as a basis for diagnosing a subject as having a particular type of TBI (e.g., a subclass of severe or mild TBI). The variety of TBI biomarkers may be detected individually or in combination and may be used as important diagnostic tools, prognostic tools and / or TBI risk stratification tools as part of assessing the TBI status of a subject. [Background technology]

[0003] In the United States alone, more than 5 million cases of mild traumatic brain injury (mTBI) occur annually. Currently, there are no simple, objective, and accurate measurement methods available to aid in patient assessment. In fact, much of the assessment and diagnosis of TBI is based on subjective data. Unfortunately, objective measurements such as head CT and the Glasgow Coma Score (GCS) are not comprehensive or highly sensitive in assessing mild TBI. Furthermore, head CT often reveals nothing about mTBI, is expensive, and exposes patients to unnecessary radiation. In addition, a negative head CT does not necessarily mean that the patient does not have a concussion; it merely indicates that a particular intervention, such as surgery, is not justified. Physicians and patients need objective and reliable information to accurately assess this condition and facilitate appropriate triage and recovery. Currently, the data available for the use of biomarkers to aid in the diagnosis, assessment, and management of patients is limited.

[0004] While the majority of TBIs are mild, the ability to accurately characterize the disease state or "disease signature" of different TBIs is largely inadequate, resulting in poor diagnostic and treatment of mTBIs. This inadequacy generally stems from challenges associated with the ambiguity of clinically presented symptoms and the ineffectiveness of imaging methods. Consequently, researchers are beginning to explore cell-based and molecular-based approaches to improve both diagnostic and prognostic assessments. This encounters various challenges, including the difficulty of correlating biological markers with current clinical symptoms and overcoming the lack of a fundamental understanding of the pathophysiology of mTBIs. However, the adoption of recent techniques, such as high-throughput technologies and computational biology, provides means to more accurately determine and characterize the disease state of TBIs. [Overview of the project] [Problems that the invention aims to solve]

[0005] Because the underlying pathophysiology of TBI remains unclear, effective and efficient diagnostic tools, prognostic tools, risk stratification tools, and / or therapeutic tools are still unavailable, particularly in clinical settings. The shortcomings of current brain imaging techniques and the inadequacies of clinical diagnostic methods have increased the appeal of identifying immune and damage-related brain-peripheral signaling using peripheral blood, leading researchers to explore TBI at the cellular and molecular levels. The ultimate goal of this approach is to discover a single TBI biomarker or biomarker panel that can aid in early detection and diagnosis, effectively distinguish between diverse TBI disease states (e.g., mTBI in contrast to severe TBI or sTBI), and help predict patient outcomes. Furthermore, these methods may help elucidate underlying biological mechanisms and provide a broader perspective on therapeutic strategies. [Means for solving the problem]

[0006] (Summary of the invention) Multiple embodiments of this disclosure include methods for measuring or detecting at least one biomarker. According to these embodiments, the method includes the steps of: obtaining a sample from a subject after an actual or suspected head injury; and measuring or detecting in the sample at least one biomarker or fragment selected from the group consisting of AL9A1, ATPG, C1RL, CAND1, EPIPL, GLO2, IGHA2, PZP, SYTC, SYYC, or any combination thereof; and / or measuring or detecting in the sample at least one biomarker or fragment selected from the group consisting of ABHEB, AL9A1, DNM1L, FCN2, INF2, K22E, M3K5, NCOR1, SBSN, SYEP, TPP2, or any combination thereof. In some embodiments, the measurement or detection of at least one biomarker indicates that the subject has suffered or may have suffered a traumatic brain injury (TBI).

[0007] Multiple embodiments of the present disclosure also include methods for measuring or detecting at least one biomarker, comprising the steps of: obtaining a sample from a subject after an actual or suspected head injury; and measuring or detecting in the sample at least one biomarker or fragment thereof selected from the group consisting of 1433G, ACK1, ACY1, AKA12, ARGI1, CADH5, CLH1, COPG2, DPOD2, DSG2, HV307, IQGA2, K1C14, K1C19, KV105, LAMC1, MDHM, NQO2, PERM, PLST, PNCB, PTPRC, SEPT7, SYRC, TRXR2, TXNL1, UGGG1, WDR1, or any combination thereof. In some embodiments, the measurement or detection of at least one biomarker indicates that the subject has suffered or may have suffered a mild traumatic brain injury (mTBI).

[0008] Multiple embodiments of the present disclosure also include a biomarker panel for determining a subject's traumatic brain injury (TBI) status. According to these embodiments, the panel includes at least one of the following biomarkers: TPP2, CAND1, NCOR1, K22E, AL9A1, ABHEB, DNM1L, INF2, or any combination thereof; in this case, the measurement or detection of at least one biomarker indicates that the subject has suffered or may have suffered a mild TBI of subclass 4.

[0009] Multiple embodiments of the present disclosure also include a biomarker panel for determining a subject's traumatic brain injury (TBI) status, comprising at least one of the following biomarkers: TPP2, NCOR1, HV103, INF2, IGHD, CK054, M3K5, ABHEB, AL9A1, DNM1L, or any combination thereof, wherein the measurement or detection of at least one biomarker indicates that the subject has suffered or may have suffered a subclass 3 mild TBI.

[0010] Multiple embodiments of the present disclosure also include a biomarker panel for determining a subject's traumatic brain injury (TBI) status, comprising at least one of the following biomarkers: NCOR1, TPP2, K22E, ABHEB, INF2, SBSN, AL9A1, MA2B2, or any combination thereof, wherein the measurement or detection of at least one biomarker indicates that the subject has suffered or may have suffered a subclass 2 mild TBI.

[0011] Multiple embodiments of the present disclosure also include a biomarker panel for determining a subject's traumatic brain injury (TBI) status, comprising at least one of the following biomarkers: K22E, DNM1L, DIAP1, ABHEB, PLOD1, SYEP, KV133, AL9A1, EPHB4, or any combination thereof, wherein the measurement or detection of at least one biomarker indicates that the subject has suffered or may have suffered a subclass 1 mild TBI.

[0012] Multiple embodiments of the present disclosure also include a biomarker panel for determining a subject's traumatic brain injury (TBI) status, comprising at least one of the following biomarkers: CAND1, NCOR1, K22E, ABHEB, DNM1L, SBSN, GLO2, SYEP, or any combination thereof, wherein the measurement or detection of a high level of at least one biomarker in a subject, compared to the level of at least one biomarker in a healthy subject, indicates that the subject has suffered or may have suffered a mild subclass 4 TBI.

[0013] Multiple embodiments of the present disclosure also include a biomarker panel for determining a subject's traumatic brain injury (TBI) status, comprising at least one of the following biomarkers: NCOR1, HV103, IGHD, ABHEB, DNM1L, ALBU, THIM, IGHA2, KV139, or any combination thereof, wherein the measurement or detection of a high level of at least one biomarker in a subject, compared to the level of at least one biomarker in a healthy subject, indicates that the subject has suffered or may have suffered a mild subclass 3 TBI.

[0014] Multiple embodiments of the present disclosure also include a biomarker panel for determining a subject's traumatic brain injury (TBI) status, comprising at least one of the following biomarkers: NCOR1, K22E, ABHEB, SBSN, DNM1L, DIAP1, DYL1, PSA, EPHB4, or any combination thereof, wherein the measurement or detection of a high level of at least one biomarker in a subject, compared to the level of at least one biomarker in a healthy subject, indicates that the subject has suffered or may have suffered a subclass 2 mild TBI.

[0015] Multiple embodiments of the present disclosure also include a biomarker panel for determining a subject's traumatic brain injury (TBI) status, comprising at least one of the following biomarkers: K22E, DNM1L, DIAP1, ABHEB, PLOD1, SYEP, EPHB4, FBLN3, or any combination thereof, wherein the measurement or detection of a high level of at least one biomarker in a subject, compared to the level of at least one biomarker in a healthy subject, indicates that the subject has suffered or may have suffered a mild subclass 1 TBI.

[0016] Multiple embodiments of the present disclosure also include a biomarker panel for determining whether a subject has suffered traumatic brain injury (TBI), comprising at least one of the following biomarkers: ACTBL, ALDH2, ANXA5, CAMP, CPNE3, CRAC1, CYTC, DNPEP, EIF3I, GSHB, ICAM1, HV323, HNRPD, KVD33, FA9, FHR4, FRPD1, HS90B, MA2A1, PCYOX, PNPH, PROC, RL3, SH3L3, SRRM2, TBB1, TENA, TRAP1, or any combination thereof, wherein the measurement or detection of at least one biomarker in the subject indicates that the subject has not suffered TBI. [Brief explanation of the drawing]

[0017] [Figure 1A]This figure includes representative graphs obtained from unsupervised clustering analysis of proteome discovery data obtained by quantitative mass spectrometry from plasma samples acquired from subjects classified as healthy or with mild TBI based on clinical physiological information, as well as from pooled plasma from subjects with severe TBI (i.e., CT-positive). All data points are displayed according to their cluster positions in Figure 1A. Figure 1A is a representative phylogenetic plot obtained from an unsupervised clustering analysis of total proteome profile data showing two major clusters (control cluster and TBI cluster (TBI subclass 1=TBI-1; TBI subclass 2=TBI-2; TBI subclass 3=TBI-3 and TBI subclass 4=composite TBI)) with retrospectively extracted, relevant patient and sample characteristics, including age; sex; normalized GFAP MS peak area (using a targeted mass spectrometry-based GFAP assay based on quantification of individual prototype peptides); CT scan (positive / negative; gray indicates no data); and ELISA-based GFAP biomarker score (positive / negative; gray indicates no data). The major pathological clusters of the mild TBI subclasses include subclass 1 (TBI-1), subclass 2 (TBI-2), subclass 3 (TBI-3), and subclass 4 (complex TBI), also referred to herein as “complex mild TBI.” [Figure 1B] This figure includes representative graphs obtained from unsupervised clustering analysis of proteome discovery data obtained by quantitative mass spectrometry from plasma samples obtained from subjects classified as healthy or with mild TBI based on clinical physiological information, as well as from pooled plasma from subjects with severe TBI (i.e., CT-positive). All data points are displayed according to their cluster positions in Figure 1A. Figure 1B includes the results of principal component analysis for the entire proteome profile. [Figure 1C]A figure including a representative graph obtained from unsupervised clustering analysis of proteome discovery data by quantitative mass spectrometry, derived from plasma samples obtained from subjects classified as healthy or having mild TBI, as well as pooled plasma from severe (i.e., CT-positive) subjects. All data points are displayed according to their cluster positions in Figure 1A. Figure 1C includes a representative phylogenetic clustering depicted as a constellation plot. [Figure 2A] A figure including a representative clustering for common and / or unique proteins among sample clusters based on unsupervised analysis. The major pathological clusters of the mild TBI subclass include subclass 1 (TBI-1), subclass 2 (TBI-2), subclass 3 (TBI-3), and subclass 4 or combined mild TBI (complex TBI); a healthy control cluster (control) is also shown. Each of the five main sample subclusters derived from unsupervised clustering analysis has at least five uniquely expressed proteins. Figure 2A is a representative Venn diagram including common and / or unique proteins among the five identified subgroup clusters. [Figure 2B] A figure including a representative clustering for common and / or unique proteins among sample clusters based on unsupervised analysis. The major pathological clusters of the mild TBI subclass include subclass 1 (TBI-1), subclass 2 (TBI-2), subclass 3 (TBI-3), and subclass 4 or combined mild TBI (complex TBI); a healthy control cluster (control) is also shown. Each of the five main sample subclusters derived from unsupervised clustering analysis has at least five uniquely expressed proteins. Figure 3B includes a numerical count for all proteins and / or unique proteins quantified within each subgroup cluster. [Figure 2C]A figure including representative clustering for common proteins and / or unique proteins among sample clusters based on unsupervised analysis. The major pathological clusters of the mild TBI subclass include subclass 1 (TBI-1), subclass 2 (TBI-2), subclass 3 (TBI-3) and subclass 4 or combined mild TBI (Compound TBI); a healthy control cluster (Control) is also shown. Each of the five main sample subclusters derived from unsupervised clustering analysis has at least five uniquely expressed proteins. Figure 3C includes a list of Uniprot IDs and short names for the unique proteins quantified within each subgroup cluster. [Figure 3] A figure including a representative Venn plot for proteins that are common or unique between healthy samples (left circles) and TBI patient samples (including pooled severe samples; right circles). Only proteins detected in all samples within each group were included in this analysis (Figure 3A). Figure 3B includes a list of eight proteins uniquely expressed in all TBI samples, including their Uniprot IDs and protein names. AL9A1 and SYTC are expressed in the brain, while FA5 is related to coagulation. (All notations are based on information annotated by UniProt (uniprot.org).) [Figure 4A] A figure including a representative schematic diagram for bootstrap forest analysis as an alternative approach to conventional supervised analysis for quantitative data-independent acquisition mass spectrometry (DIA-MS). The contributions of individual proteins were analyzed and grouped into five individual decision trees that meet the criteria of the prediction model (Figures 4A - 4E). This analysis determined a quantitative cutoff, along with the associated diagnostic probability, for the ability of a given protein to be used as a biomarker capable of discriminating patients with TBI from their healthy counterparts. If a single protein is not sufficiently capable of making this distinction, combinations of multiple proteins were identified and defined. [Figure 4B]This figure includes a representative schematic of bootstrap forest analysis as an alternative to conventional supervised analysis for quantitatively independent acquired mass spectrometry (DIA-MS) data. The contributions of individual proteins were analyzed and compiled into five separate decision trees that met the criteria of a predictive model (Figures 4A-4E). This analysis defined a quantitative cutoff for the ability of a given protein to be used as a biomarker capable of distinguishing patients with TBI from their healthy counterparts, along with the associated diagnostic probability. Where a single protein was not sufficiently capable of making this distinction, combinations of multiple proteins were identified and defined. [Figure 4C] This figure includes a representative schematic of bootstrap forest analysis as an alternative to conventional supervised analysis for quantitatively independent acquired mass spectrometry (DIA-MS) data. The contributions of individual proteins were analyzed and compiled into five separate decision trees that met the criteria of a predictive model (Figures 4A-4E). This analysis defined a quantitative cutoff for the ability of a given protein to be used as a biomarker capable of distinguishing patients with TBI from their healthy counterparts, along with the associated diagnostic probability. Where a single protein was not sufficiently capable of making this distinction, combinations of multiple proteins were identified and defined. [Figure 4D] This figure includes a representative schematic of bootstrap forest analysis as an alternative to conventional supervised analysis for quantitatively independent acquired mass spectrometry (DIA-MS) data. The contributions of individual proteins were analyzed and compiled into five separate decision trees that met the criteria of a predictive model (Figures 4A-4E). This analysis defined a quantitative cutoff for the ability of a given protein to be used as a biomarker capable of distinguishing patients with TBI from their healthy counterparts, along with the associated diagnostic probability. Where a single protein was not sufficiently capable of making this distinction, combinations of multiple proteins were identified and defined. [Figure 4E]This figure includes a representative schematic of bootstrap forest analysis as an alternative to conventional supervised analysis for quantitatively independent acquired mass spectrometry (DIA-MS) data. The contributions of individual proteins were analyzed and compiled into five separate decision trees that met the criteria of a predictive model (Figures 4A-4E). This analysis defined a quantitative cutoff for the ability of a given protein to be used as a biomarker capable of distinguishing patients with TBI from their healthy counterparts, along with the associated diagnostic probability. Where a single protein was not sufficiently capable of making this distinction, combinations of multiple proteins were identified and defined. [Modes for carrying out the invention]

[0018] This disclosure relates to methods for diagnosing and assessing subjects who have suffered or may have suffered head injuries, such as traumatic brain injury (TBI). In particular, this disclosure identifies a variety of biomarkers whose detection and / or differential expression may be used to assess the presence or absence of TBI in a subject and which may be used as a basis for diagnosing a subject as having a specific type of TBI (e.g., a subclass of severe TBI or mTBI). The variety of TBI biomarkers may be detected individually or in combination and may be used as important diagnostic and therapeutic tools for assessing the TBI status of a subject.

[0019] The subheadings used in this section and throughout the entire disclosure of this specification are for structural purposes only and are not intended to be restrictive.

[0020] definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. In case of any discrepancy, the definitions in this document shall prevail. Methods and materials similar to or equivalent to those described herein may also be used in the implementation or testing of this disclosure, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. The materials, methods, and examples disclosed herein are illustrative and not intended to be limiting.

[0021] The terms “comprise(s),” “include(s),” “have,” “have,” “may be,” and “contain,” as used herein, and their variations, are intended to be open-ended transitional phrases, transitional terms, or transitional words that do not exclude the possibility of further acts or structures. The singular forms “are” and “it” include multiple referents unless the context clearly indicates otherwise. This disclosure also assumes other embodiments, whether expressly or unexpressed, that “include,” “consist of,” or “be essentially derived from” the embodiments or elements presented herein.

[0022] In this specification, in order to enumerate numerical ranges, each number with the same precision that lies between them is explicitly assumed. For example, for the range 6 to 9, the numbers 7 and 8 are assumed in addition to 6 and 9, and for the range 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 explicitly assumed.

[0023] As used herein, “absolute amount” refers to the absolute value of change or difference between at least two assay results taken or sampled at different time points and associated with or linked to a variety of clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression, non-progression, or improvement of disease), as well as reference levels. As used herein, “absolute value” refers to the magnitude of a real number, regardless of its sign, i.e., whether it is positive or negative (e.g., the magnitude of the difference between two compared levels (e.g., a level taken at a first time point and a level taken at a second time point)).

[0024] This disclosure presents exemplary reference levels and absolute amounts (e.g., calculated by comparing reference levels at different time points). However, reference levels and absolute amounts may vary depending on the nature of the immunoassay (e.g., the antibody used, reaction conditions, sample purity, etc.), and it is well known that assays can be compared and standardized. Furthermore, it is well within the art to adapt this disclosure to other immunoassays and to obtain immunoassay-specific reference levels and absolute amounts for these other immunoassays based on the descriptions presented herein. While the exact values ​​of reference levels and absolute amounts may vary between assays, the findings described herein are generally applicable and can be extrapolated to other assays.

[0025] In this specification, "affinity-mature antibody" refers to the affinity of an antibody against a target antigen (i.e., K D , k d or k aThe term "affinity-mature antibody" is used to refer to an antibody that has one or more modifications within one or more CDRs, resulting in an improvement compared to an unmodified parent antibody. Exemplary affinity-mature antibodies have nanomolar or picomolar affinity for a target antigen. Various procedures for producing affinity-mature antibodies are known in the art, including screening combinatorial antibody libraries prepared using biodisplay methods. For example, Marks et al., BioTechnology, 10:779-783 (1992) describe affinity maturation by domain shuffling of VH and VL. Random mutagenesis of CDR residues and / or framework residues is 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 at selective mutagenesis sites and contact or hypermuta sites associated with activity-enhancing amino acid residues is described in U.S. Patent No. 6,914,128B1.

[0026] As used herein, "antibody" and "antibodies" refer to monoclonal antibodies, single-specific antibodies (which may be monoclonal antibodies or may also be produced by means other than those used to produce them from common germline cells), multispecific antibodies, human antibodies, humanized antibodies (fully humanized or partially humanized antibodies), avian (e.g., duck or goose) antibodies, shark antibodies, whale antibodies, and non-primate (e.g., cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, etc.) antibodies or non-human antibodies. Mammalian antibodies, including primate antibodies (e.g., monkeys, chimpanzees, etc.), but not limited to animal antibodies, recombinant antibodies, chimeric antibodies, single-chain Fv ("scFv"), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fv ("sdFv"), and anti-idiotype ("anti-Id") antibodies, dual-domain antibodies, dual-variable domain (DVD) or triple-variable domain (TVD) antibodies (the contents of each of these are incorporated herein by reference, Wu, C. et al., Nature). This refers to a domain antibody (dAb) (as described in Biotechnology, 25(11):1290-1297 (2007) and PCT international application WO2001 / 058956) (for example, a dAb as described in Holt et al. (2014), Trends in Biotechnology, 21:484-490), and includes, for example, a naturally occurring single-domain antibody sdAb in cartilaginous fish and camelids, or a synthetic sdAb, for example, a nanobody sdAb, VHH or other domain structure and a functionally active epitope-binding fragment of any of the above. In particular, the antibody includes an immunoglobulin molecule and an immunoactive fragment of an immunoglobulin molecule, i.e., a molecule containing an analyte-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0027] As used herein, “antibody fragment” refers to a portion of an intact antibody that includes an antigen-binding site or a variable region. The portion does not include the heavy chain constant 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 the light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing three CDRs of the heavy chain variable region.

[0028] In this specification, “beads” and “particles” are used interchangeably and refer to substantially spherical solid supports. An example of beads or particles is microparticles. Microparticles that may be used herein may be any type known in the art. For example, beads or particles may be magnetic beads or magnetic particles. Magnetic beads / particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or magnetic fluids. 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). Beads may have a solid core portion that is magnetic and surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion may be a layer surrounding the non-magnetic core. The microparticles may be of any size, for example, about 0.75 to about 5 nm, or about 1 to about 5 nm, or about 1 to about 3 nm, and work in the methods described herein.

[0029] In this specification, “binding protein” is used to mean a monomeric or polymeric protein that binds to a binding partner, such as a polypeptide, antigen, compound or other molecule or any type of substrate, and forms a complex with it. Binding proteins bind specifically to their binding partners. Binding proteins include antibodies, as well as antigen-binding fragments and other diverse forms and derivatives thereof that are known in the art and described below in this specification, and other molecules containing one or more antigen-binding domains that bind to an antigen molecule or a specific site (epitope) on an antigen molecule. Therefore, binding proteins include, but are not limited to, antibodies that are tetrameric immunoglobulins, IgG molecules, IgG1 molecules, monoclonal antibodies, chimeric antibodies, CDR graft antibodies, humanized antibodies, affinity-mature antibodies, and any fragments of such antibodies that retain the ability to bind to an antigen.

[0030] In this specification, “bispecific antibody” is used to refer to a full-length antibody produced 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 method or similar technique introducing mutations within the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14):6444-6448 (1993)), resulting in multiple different immunoglobulin molecular species, of which only one is a functional bispecific antibody. A bispecific antibody binds to one antigen (or epitope) in one of its two binding arms (one pair of HC / LC sequences) and to a different antigen (or epitope) in its second arm (a different pair of HC / LC sequences). By this definition, a bispecific antibody has two distinctly different antigen-binding arms (both specific and CDR sequences) and is monovalent for each antigen it binds to.

[0031] In this specification, "CDR" is used to refer to the "complementarity-determining region" within the variable sequence of an antibody. There are three CDRs in each of the variable regions of the heavy chain and the light chain. Starting from the N-terminus of the heavy chain or light chain, these regions are denoted as "CDR1," "CDR2," and "CDR3" for each variable region. 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. Therefore, an antigen-binding site may contain six CDRs, including the CDR sets derived from each of the heavy chain 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 confirms that the amino acid residues of the CDRs form a broad contact area with the bound antigen, and in this case, the broadest antigen contact area is with heavy chain CDR3. Therefore, molecular recognition units can primarily contribute to the specificity of antigen-binding sites. In general, CDR residues are directly and very substantially involved in the influence of antigen binding.

[0032] The precise boundaries of these CDRs are defined in different ways, according to different systems. The system described by Kabat et al., "Sequences of Proteins of Immunological Interest" (National Institutes of Health, Bethesda, Md. (1987) and (1991)), not only presents a clear residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define 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)) described Kabat We found that certain subregions within the CDR adopt nearly identical peptide backbone conformations despite exhibiting significant diversity at the amino acid sequence level. These subregions were designated as "L1," "L2," and "L3" or "H1," "H2," and "H3," where "L" and "H" designate the light chain and heavy chain regions, respectively. These regions are sometimes referred to as "Chothia CDRs," and they have boundaries that overlap with Kabat CDRs. For other boundaries defining CDRs that overlap with Kabat CDRs, see Padlan, FASEB. This is described in J., 9:133-139 (1995); and MacCallum, J.Mol.Biol., 262(5):732-745 (1996). Furthermore, the definitions of other CDR boundaries may not strictly adhere to the definitions of the systems herein, and may be shorter or longer in light of predictions or experimental findings that a particular residue or group of residues, or the entire CDR, does not significantly affect binding to the antigen, but still overlap with Kabat CDRs. The methods used herein may use CDRs defined according to any of these systems, but certain embodiments use CDRs defined by Kabat or CDRs defined by Chothia.

[0033] The "coefficient of variation" (CV), also known as "relative variability," is synonymous with the standard deviation of a distribution, divided by its mean.

[0034] "Component," "components," or "at least one component" generally refers to capture antibodies, detection or conjugates, calibrators, controls, sensitivity panels, containers, buffers, diluents, salts, enzymes, cofactors for enzymes, detection reagents, pretreatment reagents / solutions, substrates (e.g., as solutions), stop solutions, etc., which may be included in a kit for an assay of a test sample such as a patient's 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 an assay.

[0035] As used herein, “control” generally refers to a reagent whose purpose is to assess the performance of the measurement system in order to ensure that the measurement system continues to produce results within acceptable boundaries (e.g., boundaries ranging from a scale appropriate for an assay for research use at one end to an analytical boundary established by quality specifications for a commercially available assay at the other end). To achieve this, the control should show patient results and, optionally, should evaluate in some way the impact of errors (e.g., errors due to reagent stability, variability in calibrators, or variability in the measuring instrument) on the measurement. As used herein, “control subject” refers to one or more subjects who have not suffered traumatic brain injury (TBI). As used herein, “orthopedic control” refers to samples or information derived from (e.g., based on) one or more subjects who have suffered orthopedic injuries but do not have apparent TBI. As used herein, “orthopedic control subject” refers to one or more subjects who have suffered orthopedic injuries but do not have apparent TBI. In some cases, “orthopedic control subjects” refer to adult orthopedic patients with a simplified injury score of ≤4 (not fatal) for their limb injuries and / or pelvic injuries and / or rib fractures. As used herein, “healthy control subjects” refer to samples or information derived from (e.g., based thereon) one or more subjects considered healthy and without apparent TBI or orthopedic injury. As used herein, “healthy control subjects” refer to one or more subjects considered healthy and without apparent TBI or orthopedic injury. As used herein, “TBI control subjects” refer to samples or information derived from (e.g., based thereon) one or more subjects with head injury but without apparent TBI. As used herein, “TBI control subjects” refer to one or more subjects with head injury but without apparent TBI.

[0036] In this specification, "correlated with" means "compared to".

[0037] As used herein, “CT scan” refers to computed tomography (CT) scans. A CT scan combines a series of X-ray images obtained from different angles and uses computer processing to create cross-sectional images or sections of bones, blood vessels, and soft tissues within the body. CT scans may use X-ray CT, positron emission tomography (PET), single-photon emission computed tomography (SPECT), axial computed tomography (CAT scan), or computer-assisted tomography. CT scans can be conventional CT scans or spiral / helical CT scans. In conventional CT scans, scans are obtained section by section, and after scanning each section, there is a pause and movement to the next section, for example, from the upper abdomen to the pelvis. Conventional CT scans require the patient to hold their breath to avoid motion artifacts. Spiral / helical CT scans are continuous scans, obtained in a spiral pattern, and the scanned images are continuous, making the process much faster.

[0038] As used herein, the term "derivative" of an antibody may refer to an antibody having one or more modifications to its amino acid sequence compared to a pure antibody or parent antibody, and may exhibit a modified domain structure. Derivatives may not only employ amino acid sequences capable of specifically binding to a target (antigen), but may also employ typical domain configurations found in natural antibodies. Typical examples of antibody derivatives are antibodies coupled to other polypeptides, rearranged antibody domains, or fragments of antibodies. Derivatives may also include at least one further compound, such as 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. Further domains present in the antibody-containing fusion protein may preferably be linked by a mobile linker, which is advantageous as a peptide linker, in which case the peptide linker comprises a plurality of 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. Antibodies can be ligated to effector molecules that have a conformation suitable for biological activity or that selectively bind to, for example, a solid support, a biologically active substance (e.g., cytokine or growth hormone), a chemical agent, a peptide, a protein, or a drug.

[0039] In this specification, “DIA-MS” or “data-independent acquisition mass spectrometry” is used to refer to molecular structure determination methods in which all ions within a selected m / z range are fragmented and analyzed in the second stage of tandem mass spectrometry. Typically, complex protein mixtures (e.g., plasma samples) are digested into peptides, which are then analyzed by mass spectrometry. Tandem mass spectra are generally collected for each peptide by fragmenting all ions entering the mass spectrometer at a given time point (i.e., DIA-MS) or by sequentially isolating and fragmenting the m / z range (i.e., data-dependent acquisition-MS or DDA-MS). DIA-MS is an alternative to DDA-MS in which a certain number of precursor ions are selected and analyzed by tandem mass spectrometry, but very often yields the same protein information. Prototype peptides (peptides specific to the identification of particular proteins) are used to quantify each protein (see Holewinski, RJ et al., Methods Mol Biol., 2016;1410:165~279; Kirk, JA et al., Sci Transl Med., December 23, 2015;7(319), 319ra and Parker, SJ et al., Proteomics., August 2016;16(15~16):2221~2237).

[0040] In this specification, the term "bispecific antibody" is used to refer to a full-length antibody (see PCT Publication WO02 / 02773) capable of binding to two different antigens (or epitopes) in each of its two binding arms (HC / LC pairs). Therefore, 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.

[0041] In this specification, “dual variable domain” is used to refer to two or more antigen-binding sites on a binding protein that may be a bivalent binding protein (two antigen-binding sites), a tetravalent binding protein (four antigen-binding sites), or a polyvalent binding protein. DVDs may be monospecific, i.e., capable of binding to one antigen (or one specific epitope), or polyspecific, i.e., capable of binding to two or more antigens (e.g., 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 “DVD immunoglobulin” or “DVD-Ig.” Thus, such a DVD-Ig-binding protein is a tetramer and is similar to the IgG molecule, but provides more antigen-binding sites than the IgG molecule. Therefore, each half of the tetrameric DVD-Ig molecule is similar to the other half of the IgG molecule, containing a heavy-chain DVD polypeptide and a light-chain DVD polypeptide. However, unlike the heavy-chain and light-chain pair of the IgG molecule, which results in a single antigen-binding domain, the heavy-chain and light-chain pair of DVD-Ig results in two or more antigen-binding sites.

[0042] Each antigen-binding site of a DVD-Ig-binding protein may originate from a donor ("parent") monoclonal antibody. Therefore, each antigen-binding site may contain a total of six CDRs, including heavy chain variable domains (VH) and light chain variable domains (VL) with CDRs involved in binding to the antigen. Consequently, a DVD-Ig-binding protein that binds to two different epitopes (e.g., two different epitopes of two different antigen molecules or two different epitopes of the same antigen molecule) includes an antigen-binding site derived from a first parent monoclonal antibody and an antigen-binding site from a second parent monoclonal antibody.

[0043] The design, expression, and characterization of DVD-Ig binding molecules are described in PCT Publication WO2007 / 024715, U.S. Patent No. 7,612,181, and Wu et al., Nature Biotech, 25:1290-1297 (2007). Preferred examples of such DVD-Ig molecules include a heavy chain having the structural formula: VD1-(X1)n-VD2-C-(X2)n [wherein 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 it is not CH1, X2 is an Fc region, and n is 0 or 1, but preferably 1]; and a light chain having the structural formula: VD1-(X1)n-VD2-C-(X2)n [wherein 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 it is not CH1, X2 does not contain an Fc region, and n is 0 or 1, but preferably 1]. Such DVD-Ig can contain two such heavy chains and two such light chains, in which case each chain contains tandem linked variable domains without interposing constant regions between the variable domains, and the heavy and light chains can associate to form a tandem functional antigen-binding site, and the heavy and light chain pair can associate with another heavy and light chain pair to form a tetrameric binding protein with four functional antigen-binding sites. In another example, the DVD-Ig molecule can contain heavy and light chains each containing three tandem linked variable domains (VD1, VD2, VD3) without interposing constant regions between the variable domains, in which case the heavy and light chain pair can associate to form three antigen-binding sites, and the heavy and light chain pair can associate with another heavy and light chain pair to form a tetrameric binding protein with six antigen-binding sites.

[0044] As used herein, "dynamic range" refers to the range in which the assay readout is proportional to the amount of the target molecule or analyte in the sample being analyzed. The dynamic range may also be the range of linearity of the calibration curve.

[0045] An "epitope" or "epitopes" or "target epitope" refers to any site on a molecule that is recognized and can bind to a complementary site on its specific binding partner. The molecule and the specific binding partner are part of the specific binding pair. For example, an epitope may be located on a polypeptide, protein, hapten, carbohydrate antigen (such as, but not limited to, glycolipids, glycoproteins, or lipopolysaccharides), or polysaccharide. Its specific binding partner may, but is not limited to, an antibody.

[0046] As used herein, “fragment,” “biomarker fragment,” or “biomarker peptide” includes any identification of any fragment of the TBI biomarkers identified and described herein. A “fragment” includes any isoform of any TBI biomarker identified and described herein, including peptides, prototype peptides, proteolytic peptides, SNPs, or post-translational modified forms, and any endogenous or exogenous derived forms. Biomarker peptides may be used to represent the quantity of their representative protein in DIA-MS, DDA-MS, multiple reaction monitoring (MRM; also known as selective reaction monitoring or SRM) mass spectrometry assays, or parallel reaction monitoring (PRM) mass spectrometry assays. Proteolytic peptides(s) can be targeted individually or together with internal standards for MS quantification (see Fu, Q. et al., J Proteome Res., November 2017 (doi:10.1021 / acs.jproteome.7b00623); Fu, Q. et al., Methods Mol Biol., 2016;1410:249~264 and Liu, X. et al., Methods., June 15, 2013;61(3):304~312).

[0047] As used herein, “framework” (FR) or “framework sequence” may mean the remaining sequence of the variable region, excluding the CDRs. Since the precise definition of a CDR sequence can be determined by different systems (see, for example, above), the meaning of the framework sequence is subject to different interpretations accordingly. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 on the light chain and CDR-H1, CDR-H2, and CDR-H3 on the heavy chain) also divide the framework regions on the light and heavy chains into four sub-regions on each chain (FR1, FR2, FR3, and FR4), where CDR1 is located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Unless a specific sub-region is designated as FR1, FR2, FR3, or FR4, as noted by other researchers, the framework region represents a combination of FRs within a single, innate immunoglobulin chain variable region. As used herein, FR represents one of the four subdomains, and FR represents two or more of the four subdomains that constitute the framework domain.

[0048] Human heavy chain and human light chain FR sequences are known in the art and can be used as heavy chain 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 chain and human light chain acceptor sequences are selected from framework sequences listed in publicly available databases such as V-base (hypertext transfer protocol: / / vbase.mrc-cpe.cam.ac.uk / ) or the international ImMunoGeneTics® (IMGT®) information system (hypertext transferprotocol: / / imgt.cines.fr / texts / IMGTrepertoire / LocusGenes / ).

[0049] As used herein, “functional antigen-binding site” may mean a site on a binding protein (e.g., an antibody) that is capable of binding 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 from which the antigen-binding site originates, e.g., the parent antibody; however, the ability to bind to an antigen must be measurable using one of the various known methods for assessing antigen-binding proteins, e.g., antibodies. Furthermore, the antigen-binding affinity of each antigen-binding site in a multivalent protein, e.g., a multivalent antibody as described herein, does not need to be quantitatively the same.

[0050] In this specification, “GFAP” is used to describe glial fibrillary acidic protein. GFAP is a protein encoded by the GFAP gene and can be produced in humans (e.g., by recombinant means in other species). “GFAP status” can mean the level or amount of circulating GFAP at a given point in time (e.g., a point in time with a single measurement of GFAP), the level or amount of GFAP associated with monitoring (e.g., monitoring with repeated tests to identify an increase or decrease in GFAP levels in a subject), the level or amount of GFAP associated with treatment for traumatic brain injury (whether primary or secondary brain injury), or a combination thereof. GFAP was measured by ELISA and targeted mass spectrometry against specific peptides unique to GFAP.

[0051] As used herein, the “Glasgow Coma Scale” or “GCS” refers to a 15-point scale used to estimate and classify the outcome of brain injury based on overall social ability or dependence on others. The test measures motor responses, verbal responses, and eye-opening responses using the following values: I. Motor Response (6: fully obeying instructions; 5: locating the noxious stimulus; 4: withdrawing from the noxious stimulus; 3: abnormal flexion, i.e., decortical posture; 2: stretching response, i.e., decerebrate posture and 1: no response); II. Verbal Response (5: conscious and oriented; 4: confused but coherent speech; 3: inappropriate words and incoherent phrases; 2: incomprehensible sounds 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 injury and poorer prognosis: mild (13-15); moderate physical disability (9-12) (loss of consciousness for more than 30 minutes; may or may not be lost, physical or cognitive impairment; benefit from rehabilitation); severe physical disability (3-8) (stupor: unconscious state; no meaningful response, no spontaneous activity); and vegetative state (less than 3) (sleep-wake cycle; awake but no interaction with the 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 score 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 score of 3-8.

[0052] As used herein, the "Glasgow Outcome Scale" refers to a global scale for functional outcomes that assesses a patient's condition into one of five categories: death, vegetative state, severe disability, moderate disability, or good recovery.

[0053] The "Extended Glasgow Outcome Scale" or "GOSE," used interchangeably in this specification, presents a more detailed classification into eight categories by subdividing the categories of severe physical disability, moderate physical disability, and good recovery into the upper and lower categories shown in Table 1.

[0054] [Table 1]

[0055] In this specification, “humanized antibody” is used to describe an antibody that contains heavy chain variable region sequences 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 sequence and / or VL sequence has been modified to be more “human-like,” i.e., more similar to a human germline variable sequence. 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 substantially having the amino acid sequence of a human antibody and a complementation-determining region (CDR) substantially having the amino acid sequence of a non-human antibody. In the context of CDRs as used herein, the term “substantially” refers to a CDR having 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 a non-human antibody CDR. A humanized antibody contains at least one, but typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), where all or substantially all of the CDR region corresponds to the CDR region of a non-human immunoglobulin (i.e., donor antibody), and all or substantially all of the framework region is substantially the framework region of the human immunoglobulin consensus sequence. In some embodiments, the humanized antibody also contains the immunoglobulin constant region (Fc), typically at least a portion of the Fc region of a human immunoglobulin. In some embodiments, the humanized antibody contains at least variable domains of the heavy chain in addition to the light chain. The antibody may also contain the CH1 region, hinge region, CH2 region, CH3 region, and CH4 region of the heavy chain. In some embodiments, the humanized antibody contains only the humanized light chain. In some embodiments, the humanized antibody contains only the humanized heavy chain. In specific embodiments, the humanized antibody contains only the humanized variable domains of the light chain and / or the humanized heavy chain.

[0056] Humanized antibodies can be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and, without limitation, any isotype, including IgG1, IgG2, IgG3, and IgG4. Humanized antibodies may contain sequences derived from more than one class or isotype, and specific constant domains can be selected to optimize desired effector function using techniques well known in the art.

[0057] The framework region and CDR of a humanized antibody do not need to correspond precisely to the parent sequence. For example, the CDR or consensus framework of a donor antibody may be mutagenic by substitution, insertion, and / or deletion of at least one amino acid residue so that the CDR residue or framework residue at this site does not correspond to the donor antibody or consensus framework. However, in preferred embodiments, such mutations are not widespread. 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 residues of the parent FR and CDR sequences. As used herein, the term “consensus framework” refers to the framework region within the consensus immunoglobulin sequence. As used herein, the term “consensus immunoglobulin sequence” 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)). Therefore, the "consensus immunoglobulin sequence" may include the "consensus framework region" and / or the "consensus CDR." In the immunoglobulin family, each position within the 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, both may be included within the consensus sequence.

[0058] As used herein in the context of two or more polypeptide sequences or polynucleotide sequences, “identical” or “same” may mean that the amino acid or nucleotide sequences have a specified percentage of residues that are the same across a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing them across a specified region, determining the number of positions in both sequences where identical residues occur, finding the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths or their alignment results in one or more sticky ends, and the specified comparison region contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation but not in the numerator.

[0059] As used herein, “imaging procedure” refers to a medical examination that allows visualization of the inside of the body for the purpose of diagnosing, treating, and monitoring a health condition. Imaging procedures can be non-invasive procedures that enable the diagnosis of disease and injury without harming the body. Examples of imaging procedures include MRI, CT scans, X-rays, positron emission tomography (PET) scans, single-photon emission computed tomography (SPECT) scans, and diffusion tensor imaging (DTI) scans.

[0060] In this specification, “head injury” or “head injury” as used interchangeably means any trauma to the scalp, skull, or brain. Such injury may include only a minor bulge on the skull or a severe brain injury. Such injury includes primary and / or secondary brain injury. Primary brain injury occurs at the time of the initial injury and results from the physical dislocation of the brain’s structure. More specifically, primary brain injury is physical damage to the parenchyma (tissue, blood vessels) that occurs at the time of the traumatic event, resulting in shearing and compression of the surrounding brain tissue. Secondary brain injury occurs after 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) after primary brain injury. Secondary brain injury is a cascade of cellular, chemical, tissue, or vascular changes within the brain, including a cascade that contributes to further destruction of brain tissue.

[0061] Head injuries can be closed or open (penetrating). A closed head injury is an injury to the scalp, skull, or brain in which there is no penetration of the skull by the object that struck the head. An open head injury is an injury to the scalp, skull, or brain in which there is penetration of the skull by the object that struck the head. Head injuries can be caused by physical concussion, blunt force trauma from an external mechanical force or other force resulting in a closed or open head injury (e.g., vehicle accidents such as those involving automobiles, aircraft, or trains; painful blows to the head such as a baseball bat or a firearm), cerebrovascular attacks (e.g., strokes), one or more falls (e.g., in sports or other activities), explosions or blasts (collectively, "explosive injuries"), and other types of blunt force trauma. Alternatively, head injuries can be caused by ingestion 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), organometallic substances (such as methylmercury, tetraethyl lead, and organotin), and / or one or more abused drugs. Alternatively, head injury may be caused as a result of the subject suffering from an autoimmune disease, metabolic disorder, brain tumor, one or more viruses, meningitis, hydrocephalus, hypoxia, or a combination thereof. In some cases, it is impossible to determine whether any such event or injury has occurred or occurred. For example, the patient or subject may have no medical history, the subject may be unable to speak, or the subject may not be aware of or have sufficient information about what event they were exposed to. In this specification, such a situation is described as the subject "may have suffered head injury." In certain embodiments of this specification, closed head injury does not include cerebrovascular events such as stroke, and is explicitly excluded.

[0062] As used herein, “intracranial lesion” refers to an area of ​​damage within the brain. Intracranial lesions can be abnormalities observed in imaging procedures or brain imaging tests, such as MRI scans or CT scans. In CT scans or MRI scans, brain lesions may appear as dark or light-colored spots that do not resemble normal brain tissue.

[0063] As used herein, “isolated polynucleotide” may mean a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or a combination thereof) that, in its origin, is not associated with all or part of any polynucleotides found together in nature; is operably linked to a polynucleotide that is not naturally linked; or does not exist in nature as part of a larger sequence.

[0064] As used herein, “label” and “detectable label” refer to a portion of an antibody or analyte that has been conjugated to the antibody or analyte in such a way that the reaction between the antibody and the analyte is detectable, and such labeled antibodies or analytes are referred to as “detectable label.” The label may produce a signal detectable by visual or measurement means. A variety of labels include signal-generating substances such as chromogens, fluorescent compounds, chemiluminescent compounds, and radioactive compounds. Representative examples of labels include light-producing portions, e.g., acridinium compounds, and fluorescence-producing portions, e.g., fluorescein. Other labels are also described herein. In this regard, a portion itself may not be detectable, but may become detectable when reacting with another portion. The term “detectable label” is intended to encompass such labels.

[0065] Any suitable, detectable label known in the art may be used. For example, detectable labels include radioactive labels (3H, 14C, 32P, 33P, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho and 153Sm, etc.), enzyme labels (horseradish peroxidase, alkaline peroxidase, glucose-6-phosphate dehydrogenase, etc.), chemiluminescent labels (acridinium esters, thioesters or sulfonamides; luminol, isoluminol, phenantridinium ester, etc.), and fluorescent labels (fluorine Labels may include receines (e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, isothiocyanate fluorescein, etc.), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., cadmium selenide capped with zinc sulfide), thermometric labels, or immunopolymerase chain reaction labels. Introductions to labeling, labeling procedures, and the detection of labels can be found in the handbook and catalog combination published by Molecular Probes, Inc., Eugene, Oregon: Polak and Van Noorden, "Introduction to Immunocytochemistry," 2nd edition, Springer Verlag, NY (1997), and Haugland, "Handbook of Fluorescent Probes and Research Chemicals" (1996). Fluorescent labels may be used in FPIAs (see, for example, U.S. Patents No. 5,593,896, No. 5,573,904, No. 5,496,925, No. 5,359,093 and No. 5,352,803, which are incorporated herein by reference in their entirety).Acridinium compounds can be used as detectable labels in homochemical chemiluminescence assays (see, for example, 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)).

[0066] In one embodiment, the acridinium compound is acridinium-9-carboxamide. For methods of preparing acridinium-9-carboxamide, see 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 See Raton, 77-105 (2002); Adamczyk et al., Org. Lett., 5:3779-3782 (2003); and U.S. Patents No. 5,468,646, No. 5,543,524 and No. 5,783,699 (each of which is incorporated herein by reference in whole with respect to its teachings).

[0067] Another example of acridinium compounds is acridinium-9-carboxylate aryl ester. An example of an acridinium-9-carboxylate aryl ester of formula II is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (commercially available from Cayman Chemical, Ann Arbor, MI). Methods for preparing acridinium-9-carboxylate aryl esters 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 whole with respect to its teachings therewith). Such acridinium-9-carboxylate aryl esters are effective chemiluminescent indicators for hydrogen peroxide produced in the oxidation of the analyte by at least one oxidase, in terms of signal intensity and / or signal speed. The chemiluminescence process of acridinium-9-carboxylate aryl esters is rapid, i.e., completed in less than 1 second, while the chemiluminescence of acridinium-9-carboxamide is prolonged to more than 2 seconds. However, acridinium-9-carboxylate aryl esters lose their chemiluminescent properties in the presence of protein. Therefore, their use requires the absence of protein at the time of signal generation and detection. Methods for separating or removing protein from a sample are well known to those skilled in the art and include, but are not limited to, ultrafiltration, extraction, precipitation, dialysis, chromatography and / or digestion (see, e.g., Wells, "High Throughput Bioanalytical Sample Preparation. Methods and Automation Strategies," Elsevier (2003)).The amount of protein removed or separated from the test sample may be approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Further details regarding acridinium-9-carboxylate aryl ester and its use are expressed in U.S. Patent Application No. 11 / 697,835, filed on April 9, 2007. Acridinium-9-carboxylate aryl ester can be dissolved in any suitable solvent, such as degassed anhydrous N,N-dimethylformamide (DMF) or hydrated sodium cholate.

[0068] As used herein, "blank limit (LoB)" refers to the apparent highest concentration of the analyte that is expected to be found when examining a series of blank samples that do not contain the analyte.

[0069] As used herein, “Limit of Detection (LOD)” refers to the lowest concentration of the measured substance (i.e., the quantity intended to be measured) that can be detected at a specified confidence level. The confidence level is typically 95%, with a 5% probability of false negative measurement. The LoD is the lowest analyte concentration at which detection is feasible and which is likely to be reliably distinguishable from the LoB. The LoD can be determined by using both the measured LoB and a series of tests on a sample known to contain a low concentration of the analyte. As used herein, the term LoD is based on the definition in Clinical and Laboratory Standards Institute (CLSI) protocol EP17-A2 ("Protocols for Determination of Limits of Detection and Limits of Quantification; Approved Guideline, 2nd Edition," EP17A2E, James F. Pierson-Perry et al., Clinical and Laboratory Standards Institute, June 1, 2012).

[0070] As used herein, "Limit of Quantitative Quality (LoQ)" refers to the lowest concentration at which the analyte can be reliably detected and which satisfies certain predetermined targets regarding bias and inaccuracy. LoQ may be equivalent to or much higher than LoD.

[0071] "Linearity" refers to how well the actual performance of a method or assay approximates a straight line over a specified working range. Linearity can be measured in terms of deviation or nonlinearity from an ideal straight line. "Deviation from linearity" can be expressed as a percentage of full scale. In some of the methods disclosed herein, a deviation from linearity (DL) of less than 10% is achieved over the dynamic range of the assay. "Linear" means that variation of approximately 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, or 8% or less is observed over (for or over) the listed, exemplary range or values.

[0072] A “linked sequence” or “linked peptide sequence” refers to one or more natural or artificial polypeptide sequences linked to a polypeptide sequence of interest (e.g., a full-length sequence, a sequence fragment, etc.). The term “linked” refers to the conjugation of the linked sequence to the polypeptide sequence of interest. Such polypeptide sequences are preferably linked by one or more peptide bonds. Linked sequences may have a length of about 4 to about 50 amino acids. Preferably, the length of a linked sequence is about 6 to about 30 amino acids. Natural linked sequences may be modified by amino acid substitution, addition, or deletion to create an artificial linked sequence. Linked sequences can be used for many purposes, including use in recombinant Fab. Exemplary linked sequences include, but are not limited to: (i) histidine (His) tags, such as a 6×His tag having the amino acid sequence HHHHHH (SEQ ID NO: 2), are useful as linked sequences that facilitate the isolation and purification of polypeptides and antibodies of interest; (ii) enterokinase cleavage sites, such as His tags, are used in the isolation and purification of proteins and antibodies of interest. Enterokinase cleavage sites, in conjunction with His tags, are frequently used in the isolation and purification of target proteins and antibodies. 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: 2) and its derivatives (e.g., ADDDDK (SEQ ID NO: 3)); (iii) other sequences may also be used to link or ligate the light chain variable region and / or heavy chain variable region of single-chain variable region fragments. Examples of other ligation 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). Ligation sequences may also be modified for further functions, such as drug linkage or linkage to a solid support. In the context of this disclosure, monoclonal antibodies may contain a linking sequence such as a His tag, an enterokinase cleavage site, or both.

[0073] In this specification, “magnetic resonance imaging” or “MRI” as used interchangeably refers to a medical imaging technique used in radiology to form a picture of anatomical and physiological processes within the body, both healthy and diseased. MRI is a form of medical imaging that creates images of internal organs by measuring the response of atomic nuclei of internal tissues to high-frequency radio waves when placed in a strong magnetic field. MRI scanners based on nuclear magnetic resonance (NMR) use a strong magnetic field, radio waves, and a magnetic field gradient to create images of the body.

[0074] As used herein, “monoclonal antibody” refers to an antibody obtained from a population of substantially identical antibodies; that is, the individual antibodies constituting the population are identical except for possible natural mutations that may exist in small amounts. Monoclonal antibodies are highly specific and directed to a single antigen. Furthermore, in contrast to polyclonal antibody preparations, which typically contain different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. Monoclonal antibodies as used herein include, in particular, “chimeric” antibodies in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or antibody subclass, while the rest of the chain is identical or homologous to a corresponding sequence in an antibody derived from a different species or belonging to a different antibody class or antibody subclass, as well as fragments of such antibodies, to the extent that they exhibit the desired biological activity.

[0075] In this specification, “multivalent binding protein” is used to mean a binding protein that contains two or more antigen-binding sites (also referred to herein as “antigen-binding domains”). It is preferable that multivalent binding proteins are engineered to have three or more antigen-binding sites and are generally not natural antibodies. The term “multispecific binding protein” refers to a binding protein capable of binding 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.

[0076] A “point-of-care device” refers to a device used to provide medical diagnostic testing at a point of care (i.e., outside the laboratory) or in its vicinity, at the time and place of patient care (such as a hospital, clinic, emergency care facility or other medical care facility, patient’s home, nursing home and / or long-term care facility and / or hospice). Examples of point-of-care devices include those manufactured by Abbott Laboratories (Abbott Park, IL) (e.g., i-STAT and i-STAT Alinity), Universal Biosensors (Rowville, Australia) (see US2006 / 0134713), Axis-Shield PoC AS (Oslo, Norway), and Clinical Lab Products (Los Angeles, USA).

[0077] In the context of the immunoassays and kits described herein, “quality control reagents” include, but are not limited to, calibrators, controls, and sensitivity panels. A “calibrator” or “standard” (e.g., one or more) is typically used to establish a calibration curve for interpolating the concentration of an analyte, such as an antibody or analyte. Alternatively, a single calibrator located near a reference or control level (e.g., a “low,” “medium,” or “high” level) may also be used. Multiple calibrators (i.e., more than one calibrator or calibrator of variation) may be used in combination to include a “sensitivity panel.”

[0078] "Recombinant antibody" and "recombinant antibodies" refer to antibodies prepared by one or more steps, including the steps of cloning the nucleic acid sequences encoding all or part of one or more monoclonal antibodies into a suitable expression vector by recombinant methods, and then expressing the antibodies in suitable host cells. The term includes, but is not limited to, monoclonal antibodies produced by recombinant methods, chimeric antibodies, humanized antibodies (fully humanized or partially humanized antibodies), polyspecific or polyvalent structures formed from antibody fragments, bifunctional antibodies, heteroconjugate antibodies, DVD-Ig®, and other antibodies described in (i) of this specification (bivariable domain immunoglobulins and methods for producing them are described in Wu, C. et al., Nature Biotechnology, 25:1290-1297 (2007)). As used herein, the term “bifunctional antibody” refers to an antibody comprising a first arm having specificity for one antigenic site and a second arm having specificity for a different antigenic site; that is, a bifunctional antibody has bispecificity.

[0079] As used herein, “reference level” refers to an assay cutoff value used to evaluate diagnostic efficacy, prognostic efficacy, or therapeutic efficacy, and is associated with or linked to a variety of clinical parameters (e.g., presence of disease, stage of disease, severity of disease, disease progression, non-progression, or improvement). This disclosure provides exemplary reference levels. However, reference levels may vary depending on the nature of the immunoassay (e.g., the antibody used, reaction conditions, sample purity, etc.), and it is well known that assays can be compared and standardized. Furthermore, it is well within the art of the art to adapt this disclosure to other immunoassays and to obtain immunoassay-specific reference levels for these other immunoassays based on the descriptions presented herein. While the exact values ​​of reference levels may vary between assays, the findings described herein are generally applicable and extrapolable to other assays.

[0080] As used herein, “risk assessment,” “risk classification,” “risk identification,” or “risk stratification” for a subject (e.g., a patient) refers to an assessment of factors, including biomarkers, for predicting the risk of future events, including the onset or progression of a disease, so that treatment decisions for the subject can be made on a more informed, state-based basis.

[0081] The terms “sample,” “test sample,” “specimen,” “sample derived from subject” and “patient sample” as used herein may be used interchangeably and may be blood samples such as whole blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or placental tissue, endothelial cells, leukocytes or monocytes. Samples may be used directly as obtained from the patient or may be pre-treated to alter the characteristics of the sample in any way discussed herein or in any other way known in the art, such as filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, or addition of reagents.

[0082] Various cell types, tissues, or body fluids may be used to obtain samples. Such cell types, tissues, and body fluids may include tissue sections such as biopsy and autopsy specimens, frozen sections taken for histological purposes, blood (such as whole blood), plasma, serum, red blood cells, platelets, interstitial fluid, and cerebrospinal fluid. Cell types and tissues may also include body fluids recovered by lymph and cerebrospinal fluid. Tissues or cell types may be prepared by extracting cell samples from humans and non-human animals, but they can also be obtained by using pre-isolated cells (e.g., isolated by another person at a different time and / or for a different purpose). In addition, cell types derived from solids, iPSCs or iPSC-derived cell types (e.g., motor neurons), may also be used. Archived tissues, such as tissues with a treatment history or outcome history, may also be used. Isolation and / or purification of proteins or nucleotides may not be necessary.

[0083] As used interchangeably in this specification, “solid phase” or “solid support” means any material that can be used to conjugate 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 may be selected for its intrinsic ability to attract and immobilize capture agents. Alternatively, the solid phase may be accompanied by a binder having the ability to attract and immobilize (1) a capture agent or specific binding partner for capture, or (2) a detection agent or specific binding partner for detection. For example, the binder may include a charged substance that is charged in the opposite direction to the capture agent (e.g., a specific binding partner for capture) or detection agent (e.g., a specific binding partner for detection) itself, or to a charged substance conjugated to (1) a capture agent or specific binding partner for capture, or (2) a detection agent or specific binding partner for detection. Generally, the binder can be any binding partner (preferably a specific binding partner) that is immobilized on (joins to) the solid phase and has the ability to immobilize (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 binder allows for the indirect binding of the capture agent to the solid phase material before or during the assay. For example, the solid phase can be plastic, derivatized plastic, magnetic or non-magnetic metal, glass, or silicon, including, for example, test tubes, microtitration wells, sheets, beads, microparticles, tips, and other configurations known to those skilled in the art.

[0084] As used herein, “specific binding” or “specifically binding to ~” may refer to the interaction of an antibody, protein, or peptide with a second chemical molecular species, in which case the interaction depends on the presence of a specific structure on the chemical molecular species (e.g., an antigenic determinant or epitope); for example, an antibody recognizes and binds to a specific protein structure, rather than to proteins in general. If an antibody is specific to epitope “A”, the presence of a molecule containing epitope A (or free A, unlabeled A) in the reaction product containing labeled “A” and the antibody reduces the amount of labeled A bound to the antibody.

[0085] A "specific binding partner" is a member of a specific binding pair. A specific binding pair consists of two different molecules that bind specifically to each other via chemical or physical means. Therefore, in addition to the specific binding pair of antigen and antibody in a typical immunoassay, other specific binding pairs may include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector molecules and receptor molecules, cofactors and enzymes, enzymes and enzyme inhibitors, aptamers (e.g., RNA aptamers and DNA aptamers), etc. Furthermore, a specific binding pair may include analogs of the original specific binding member, for example, members that are analyte analogs. Specific binding members in immunoreactivity include antigens, antigen fragments, and monoclonal and polyclonal antibodies, as well as antibodies containing these complexes and fragments, whether isolated or recombinant.

[0086] As used interchangeably herein, “subject” and “patient” refer to any vertebrate, including but not limited to mammals and humans. In some embodiments, the subject may be human or non-human. The subject or patient may be undergoing other forms of treatment. In some embodiments, if the subject is human, the subject does not include a human suffering a cerebrovascular event (e.g., stroke). In some embodiments, the subject is presumed to have suffered a head injury. In some embodiments, the subject is known to have suffered a head injury. In some embodiments, the subject is presumed to have mild, moderate, or severe TBI. In some embodiments, the subject is presumed to have mild TBI. In some embodiments, the subject is presumed to have moderate TBI. In some embodiments, the subject is presumed to have severe TBI.

[0087] As used herein, “mammal” means, without limitation, any member of the class Mammalia, including humans and non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats, llamas, camels and horses; pet mammals such as dogs and cats; and laboratory animals including rodents such as mice, rats, rabbits and guinea pigs. The term does not describe a specific age or sex. Therefore, adult subjects, neonatal subjects and fetal subjects, whether male or female, are intended to be included within the scope of this term.

[0088] In this specification, “to treat,” “to treat,” or “treatment” are each used interchangeably to describe preventing, alleviating, or inhibiting the progression of one or more symptoms of a disease and / or injury or a disease to which such terms apply. Depending on the condition of the subject, the terms may also refer to preventing the disease, including preventing the onset of the disease or preventing symptoms associated with the disease. Treatment may be performed acutely or chronically. The terms may also refer to reducing the severity of a disease or symptoms associated with such a disease prior to the onset of the disease. Such prevention or reduction of the severity of a disease prior to the onset of the disease refers to the administration of a pharmaceutical composition to a subject who is not suffering from the disease at the time of administration. “To prevent” may also refer to preventing the recurrence of one or more symptoms associated with the disease or such a disease. “Treatment” and “therapeutically” refer to the act of treating when “to treat” is as defined above.

[0089] As used herein, the term “single-molecule detection” refers to the detection and / or measurement of a single molecule of an analyte in a test sample at extremely low concentrations (such as pg / mL or femtograms per mL). A number of different single-molecule analyzers or devices are known in the art, including nanopore and nanowell devices. An example of a nanopore device is described in International Patent Publication WO2016 / 161402, which is incorporated herein by reference in its entirety. An example of a nanowell device is described in International Patent Publication WO2016 / 161400, which is incorporated herein by reference in its entirety.

[0090] As used interchangeably herein, “traumatic brain injury” or “TBI” refers to a complex injury with widespread symptoms and physical impairment. Like other injuries, TBI is most often an acute event. TBI can be classified as “mild,” “moderate,” or “severe.” Where “TBI” is used herein, it generally refers to any type of TBI (e.g., mild, moderate, or severe). Causes of TBI are diverse and include, for example, physical shaking caused by human force, car accidents, fire injuries, cerebrovascular attacks (e.g., strokes), falls, explosions or blasts, and other types of blunt trauma. Other causes of TBI include ingestion and / or exposure to one or more chemicals or toxins (e.g., fire, mold, asbestos, pesticides and insecticides, organic solvents, paints, glues, gases (e.g., carbon monoxide, hydrogen sulfide, and cyanide), organometallic substances (e.g., methylmercury, tetraethyl lead, and organotin), one or more abused drugs, or combinations thereof). Alternatively, TBI may occur in subjects with autoimmune diseases, metabolic disorders, brain tumors, hypoxia, one or more viruses, meningitis, hydrocephalus, or a combination thereof. Young adults and the elderly are the age groups at highest risk of TBI. In certain embodiments of this specification, traumatic brain injury or TBI does not include, and is expressly excluded, cerebrovascular events such as stroke.

[0091] As used herein, "mild TBI" refers to a brain injury in which loss of consciousness is brief, typically lasting a few seconds or minutes, and / or confusion and disorientation last for less than one hour. Mild TBI is also referred to as concussion, mild head injury, mild TBI, mild brain injury, and mild head injury. While MRI and CT scans are often normal, individuals with mild TBI may experience cognitive problems such as headache, difficulty thinking, memory problems, attention deficit, mood swings, and frustration.

[0092] 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 number between 13 and 15 (e.g., 13-15 or 14-15). Of those with mild TBI, 15 percent (15%) have symptoms that persist for more than three months. Mild TBI is defined as a result of a forced movement of the head or an impact that causes a brief change in mental state (clouding of consciousness, disorientation, or amnesia) or loss of consciousness for less than 30 minutes. Common symptoms of mild TBI include fatigue, headache, visual disturbances, amnesia, difficulty concentrating, sleep disturbances, dizziness / loss of balance, irritability (affective disturbances), feelings of depression, and seizures. Other symptoms associated with mild TBI include nausea, loss of smell, hypersensitivity to light and sound, mood swings, getting lost or clouding of consciousness and / or slow thinking.

[0093] "Mild TBI subclass 1" (TBI-1) refers to individuals who are classified as having mild TBI and who, unlike controls (e.g., healthy controls or controls without TBI) in each of subclasses 2, 3, or 4, also exhibit a plasma proteome signature that differs from those with moderate to severe TBI and those with mild TBI.

[0094] "Mild TBI subclass 2" (TBI-2) refers to individuals who are classified as having mild TBI and who, unlike controls (e.g., healthy controls or controls without TBI) in each of subclasses 1, 3, or 4, also exhibit a plasma proteome signature that differs from those with moderate to severe TBI and those with mild TBI.

[0095] "Mild TBI subclass 3" (TBI-3) refers to individuals who are classified as having mild TBI and who, unlike controls (e.g., healthy controls or controls without TBI) in each of subclasses 1, 2, or 4, also exhibit a plasma proteome signature that differs from those with moderate to severe TBI and those with mild TBI.

[0096] In this specification, "mild TBI subclass 4" (complex TBI), also referred to as "complex mild TBI," refers to subjects classified as having mild TBI, and who, unlike controls (e.g., healthy controls or controls without TBI) in each of subclasses 1, 2, or 3, exhibit a plasma proteome signature different from subjects with moderate to severe TBI, and different from subjects with mild TBI. In addition, subjects with mild TBI subclass 4 exhibit a proteome signature similar to that obtained from pooled samples of subjects with severe TBI. In the data disclosed herein, mild TBI subclass 4 clusters contain the largest number of subjects with elevated GFAP levels (based on ELISA-based GFAP assays and GFAP mass spectrometry). It included only one subject with a positive CT scan.

[0097] As used herein, “severe TBI” refers to brain injury in which loss of consciousness and / or confusion and disorientation last for 1 to 24 hours, and the subject has a Glasgow Coma Scale number between 9 and 12. Individuals with moderate TBI have abnormal brain imaging results. As used herein, “severe TBI” refers to brain injury in which loss of consciousness lasts for more than 24 hours, memory loss after injury or penetrating cranial injury lasts for more than 24 hours, and the subject has a Glasgow Coma Scale number between 3 and 8. Deficiencies range from high levels of cognitive impairment to comatose states. Survivors may have limited function of the arms or legs, speech or language abnormalities, loss of thinking ability, or emotional problems. Individuals with severe injury may remain in a prolonged refractory state. In many cases with severe TBI, long-term rehabilitation is often required to maximize function and independence.

[0098] Common symptoms of moderate to severe TBI include difficulty with attention, concentration, distractibility, memory, processing speed, confusion, persistence, impulsivity, language processing and / or "executive function," difficulty understanding spoken words (receptive aphasia), difficulty speaking and being understood (expressive aphasia), slurred speech, extremely fast or extremely slow speech, problems with reading, problems with writing, difficulty interpreting touch, temperature, movement, position and fine distinction of the lower limbs, difficulty integrating or patterning sensory impressions into psychologically meaningful data, partial or total vision loss, eye muscle weakness and double vision (diplopia), and blurred vision. Symptoms include eye problems, difficulties judging distance, involuntary eye movements (nystagmus), photophobia (photophobia), hearing problems such as hearing loss or impairment, tinnitus, hypersensitivity to sound, loss or impairment of smell (anosmia), loss or impairment of taste, seizures associated with epilepsy, which may be of several types and may involve disruption of consciousness, sensory perception or motor control, or control of the bowel and bladder, sleep disturbances, loss of energy, changes in appetite, thermoregulation, dysmenorrhea, dependent behaviors, emotional capacity, lack of motivation, irritability, aggression, depression, disinhibition, or cognitive deficits, including denial / absence of consciousness.

[0099] In this specification, “variant” is used to describe peptides or polypeptides that have different amino acid sequences due to amino acid insertions, deletions, or conservative substitutions, but retain at least one biological activity. “SNP” refers to a variant that is a single nucleotide polymorphism. Typical examples of “biological activity” include the ability to be bound by a specific antibody or the ability to promote an immune response. In this specification, variant is also used to describe proteins that have an amino acid sequence substantially identical to a reference protein that has an amino acid sequence that retains at least one biological activity. In the art, conservative amino acid substitution, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree of charge, and distribution of charged area), is typically recognized as involving small changes. As understood in the art, these small changes can be identified in part by examining the hydrophobicity index of the amino acid (Kyte et al., J.Mol.Biol.157:105~132 (1982)). The hydrophobicity index of an amino acid is based on an examination of its hydrophobicity and charge. In the art, it is known that amino acids with similar hydrophobicity indices can be substituted while still retaining protein function. In one embodiment, amino acids with hydrophobicity indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in proteins that retain biological function. In the context of peptides, the study of amino acid hydrophilicity is a useful measure that has been reported to correlate well with antigenicity and immunogenicity, and allows for the calculation of the maximum local mean hydrophilicity of the peptide (U.S. Patent No. 4,554,101, incorporated herein by reference in its entirety). Substitution with 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 carried out with amino acids having hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity and hydrophilicity of amino acids are influenced by the specific side chain of that amino acid.This observation is consistent with the understanding that amino acid substitutions, which are biological functions and compatibility, depend on the relative similarity of amino acids, particularly their side chains, as revealed by their hydrophobicity, hydrophilicity, charge, size, and other properties.

[0100] In this specification, the term "vector" is used to describe a nucleic acid molecule capable of carrying another ligated nucleic acid. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which further DNA segments can be ligated. Another type of vector is a viral vector into which further DNA segments can be ligated into a viral genome. Certain vectors are capable of self-replication within the host cell into which they are introduced (e.g., bacterial vectors having bacterial origins of replication and mammalian episomal vectors). Other vectors (e.g., mammalian non-episomal vectors), upon introduction into a host cell, can be integrated into the host cell's genome and thereby replicated together with the host genome. Furthermore, certain vectors can direct the expression of genes that are operatively ligated. In this specification, such vectors are referred to as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA methods are often in the form of plasmids. Since plasmids are the most commonly used form of vector, the terms "plasmid" and "vector" can be used interchangeably. However, other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), may also be used. In this regard, RNA forms of vectors (including viral RNA vectors) may also be used in the context of this disclosure.

[0101] Unless otherwise specified herein, scientific and technical terms used in connection with this disclosure shall have the same meaning as those generally understood by those skilled in the art. For example, the terminology and techniques used herein in connection with cell culture and tissue culture, molecular biology, immunology, microbiology, genetics, and protein chemistry, nucleic acid chemistry, and hybridization are well-known terminology and techniques and are commonly used in the art. The meaning and scope of terms shall be clear, but in the event of any potential ambiguity, the definitions presented herein shall prevail over any dictionary or external definitions. Furthermore, unless otherwise required by the context, singular terms shall include plural forms, and plural terms shall include singular forms.

[0102] 2. Diagnosis and assessment of whether the subject has suffered traumatic brain injury. This disclosure relates to methods that can assist in the diagnosis, prognosis, risk stratification, and assessment of whether a subject has suffered or may have suffered a head injury, including whether the subject has suffered TBI or various mild forms of TBI. These methods can assist in determining the degree and / or severity of TBI in a human subject with suspected head injury, including, for example, whether the subject has suffered a mild TBI, and if so, whether the subject has suffered a subclass of mild TBI, whether the subject has suffered a moderate to severe TBI, or whether the subject has suffered no TBI. More specifically, the biomarkers of this disclosure can be used in diagnostic tests to determine, quantify and / or evaluate a brain injury status, for example, to diagnose TBI in an individual subject or patient. In some embodiments, the TBI status may include determining the patient's asymptomatic brain injury status or SCI status, for example, to diagnose SCI in an individual subject or patient. Detecting or measuring the biomarkers of this disclosure may help in diagnosing TBI, for example, in generating TBI signatures for subclasses of mild TBI.

[0103] Determining whether a subject has mild TBI (or a subclass of mTBI) or moderate to severe TBI may involve measuring or detecting one or more TBI biomarkers and integrating this information with other information (e.g., clinical evaluation data) to determine that the subject is more likely to have suffered TBI than not, and if so, to determine what type of TBI was suffered. The method may include the steps of performing an assay on a sample obtained from a human subject within approximately 24 hours, such as within approximately 2 hours after suspected head injury, to measure or detect the level of one or more TBI biomarkers in the sample; and determining whether the subject has suffered mild or moderate to severe traumatic brain injury (sTBI). In some embodiments, a subject is determined to have a subclass of TBI and / or mild TBI if the sample level of one or more TBI biomarkers is altered (e.g., high or low expression levels) compared to the reference level of one or more TBI biomarkers (e.g., the level of the TBI biomarker in the control sample). In other embodiments, a subject is determined to have a subclass of TBI and / or mild TBI if the sample level of one or more TBI biomarkers is detected, without the need to confirm the biomarker levels or compare them to a reference or control sample.

[0104] The sample may be a biological sample. As used herein, “sample” may be used interchangeably (e.g., sample, test sample or biological sample) and may be a sample of blood such as whole blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or placental tissue, endothelial cells, lymphocytes or monocytes. In some embodiments, the method may include the steps of obtaining a sample derived from a subject within about 48 hours of suspected injury to the subject and contacting the sample with an antibody against a TBI biomarker to enable the formation of a complex between the antibody and the TBI biomarker. The method may also include the step of detecting the resulting antibody-TBI biomarker complex.

[0105] In some embodiments, subjects may undergo a Glasgow Coma Scale score before or after the level of a TBI biomarker is determined at one or more time points. In certain embodiments, subjects may be inferred to have mild TBI based on their Glasgow Coma Scale score. In certain embodiments, subjects may be inferred to have mild TBI based on an abnormal head CT scan. In some embodiments, subjects undergo a CT scan before or after the assay is performed. In some embodiments, subjects have a normal head CT scan. In some embodiments, the reference level of the TBI biomarker correlates with subjects having TBI. In some embodiments, the reference level of the TBI biomarker correlates with the Glasgow Coma Scale score.

[0106] Generally, reference levels of TBI biomarkers can also be used as benchmarks to evaluate results obtained when test samples are assayed for TBI biomarkers. Generally, when making such comparisons, reference levels of TBI biomarkers are obtained by performing a specific assay a sufficient number of times and under appropriate conditions to establish a correlation or association between the presence, amount, or concentration of the analyte with a specific stage or endpoint of TBI or a specific symptom. Typically, reference levels of TBI biomarkers are obtained by assays on a reference subject (or population of subjects). The TBI biomarker measured may include this fragment, its degradation products, and / or its enzymatic cleavage products. In certain embodiments, the reference level may correlate with a control subject without head injury.

[0107] The nature of the assays used in the methods described herein is not particularly important, and the assays may be any assay known in the art, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, Western blotting or protein immunostaining, or spectroscopic methods such as high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS), DIA-MS, DDA-MS, PRM-MS or SRM / MRM-MS, direct or enriched mass spectrometry assays (e.g., enrichment may be mediated by an antibody against the target protein(s)). Prior to mass spectrometry, the capture reagents used to selectively enrich the sample for candidate biomarker proteins include, but are not limited to, aptamers, antibodies, nucleic acid probes, chimeric bodies, small molecules, F(ab')2 fragments, single-chain antibody fragments, Fv fragments, single-chain Fv fragments, nucleic acids, lectins, ligand-binding receptors, aphibodies, nanobodies, ankyrin, domain antibodies, alternative antibody scaffolds (e.g., diabodies), imprinted polymers, avimers, peptide mimetics, peptoids, peptide nucleic acids, threose nucleic acids, hormone receptors, cytokine receptors, and synthetic receptors, as well as modifications and fragments thereof. Matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF MS or MALDI-TOF) may also be used in conjunction with enrichment. Assays in clinical chemistry formats known to those skilled in the art may also be utilized.

[0108] 3. TBI Biomarker Panel The biomarkers of this disclosure may be used in diagnostic tests (as used interchangeably herein) to assess, determine, and / or quantify a brain injury status in a patient, such as TBI status. The term “brain injury status” includes any manifestation of an identifiable status, including the absence of brain injury. For example, brain injury status includes, but is not limited to, the presence or absence of brain injury in a patient, the risk of developing brain injury, the stage or severity of brain injury, the progression of brain injury (e.g., progression of brain injury over time), the effectiveness of treatment for brain injury or the response thereto (e.g., clinical follow-up and clinical monitoring of brain injury after treatment), and the type of brain injury, such as TBI or a subclass of TBI. Based on this status, further procedures, including further diagnostic tests or treatment procedures or regimens, may be indicated.

[0109] The ability of a diagnostic test to accurately predict a condition is generally measured as the sensitivity, specificity, or area under the receiver operating characteristic ("ROC") curve of the assay. Sensitivity is the percentage of true positives predicted by the test, while specificity is the percentage of true negatives predicted by the test. The ROC curve presents the sensitivity of the test as a function of 1-specificity. A larger area under the ROC curve indicates a stronger predictive value of the test. Other useful measures of the usefulness of a test are the positive predictive value and the negative predictive value. The positive predictive value is the percentage of people who are actually positive after being marked positive by the test. The negative predictive value is the percentage of people who are actually negative after being marked negative by the test.

[0110] Analysis of the data described herein and clinical data from cohorts of TBI patients and controls has resulted in the generation of diverse TBI biomarkers that can be used individually or in combination with other biomarkers, in the form of panels, to diagnose and / or assess brain injury in subjects. A TBI biomarker panel may include any one of the TBI biomarkers disclosed herein and may include more than one and up to 20 different biomarkers corresponding to significantly different proteins. A TBI biomarker panel may also include non-TBI biomarkers (e.g., assay control biomarkers) and biomarkers already identified as being associated with TBI (e.g., GFAP and / or UCH-L1 and / or NSE). In some embodiments of this disclosure, a biomarker panel may show statistically significant differences in different TBI states. Diagnostic tests using these biomarkers may show ROCs of at least 0.6, at least about 0.7, at least about 0.8, or at least about 0.9.

[0111] Since TBI biomarkers may be differentially present / expressed depending on the type or subclass of TBI (e.g., TBI signature), a panel of more than one TBI biomarker may be useful in assisting in the determination of brain injury status. In some embodiments, biomarkers are measured in a patient sample using the methods described herein and correlated with the TBI status, for example, by comparison with a predetermined biomarker level. In some embodiments, the measured(s) may then be compared with a diagnostic(s), cutoff(s), or multivariate model score of involvement that distinguishes positive TBI status from negative TBI status. A diagnostic(s) represents a measured amount of a biomarker(s) above or below which patients are classified as having a particular TBI status. For example, if the biomarker(s) are upregulated at the time of brain injury compared to a control subject (e.g., a subject without TBI), measured(s) above a diagnostic cutoff(s) may indicate a diagnosis of TBI. In addition, if a biomarker(s) are present at the time of brain injury and undetectable in the control, any detectable dose(s) may indicate a diagnosis of brain injury. Alternatively, if a biomarker(s) are downregulated at the time of brain injury, a dose(s) at or below the diagnostic cutoff(s) may indicate a diagnosis of non-brain injury. In addition, if a biomarker(s) are absent at the time of brain injury and detectable in the control, any detectable dose(s) may indicate a diagnosis of non-brain injury. As is well understood in the art, the sensitivity or specificity of a diagnostic assay can be increased according to the diagnostician's preference by adjusting the specific diagnostic cutoff(s) used in the assay. In certain embodiments, a specific diagnostic cutoff may be determined, for example, by measuring the amount of a biomarker in a statistically significant number of samples from patients with different brain injury states and deriving a cutoff suitable for the desired specificity and sensitivity levels.

[0112] Indeed, as those skilled in the art will understand, there are many methods that use measurements of two or more biomarkers to improve the diagnostic problem under investigation. In a very simple, yet often effective, method, a positive result is assumed if the sample is positive for at least one of the markers being investigated.

[0113] Furthermore, in certain embodiments, the measured values ​​for markers in a biomarker panel are mathematically combined, and the combined values ​​are correlated with the underlying diagnostic problem. Biomarker values ​​can be combined using any appropriate modern mathematical method. Known mathematical methods for correlating marker combinations with disease states include discriminant analysis (DA) (e.g., linear DA, quadratic DA, normalized DA), discriminant function analysis (DFA), kernel methods (e.g., SVM), multidimensional scaling (MDS), nonparametric methods (e.g., k-nearest neighbor classifier), PLS (partial least squares), tree-based methods (e.g., logical regression, CART, random forest, boosting / bagging), generalized linear models (e.g., logistic regression), principal component-based methods (e.g., SIMCA), generalized additive models, fuzzy logic-based methods, neural network and gene algorithm-based methods. Those skilled in the art will have no difficulty selecting an appropriate method for assessing the biomarker combinations of the present invention. In one embodiment, a method used to correlate a combination of biomarkers of the present invention, for example, to diagnose brain injury, is selected from DA (e.g., linear discriminant analysis, quadratic discriminant analysis, normalized discriminant analysis), DFA, kernel methods (e.g., SVM), MDS, nonparametric methods (e.g., k-nearest neighbor classifier), PLS (partial least squares method), tree-based methods (e.g., logical regression, CART, random forest method, boosting method), or generalized linear models (e.g., logistic regression) and principal component analysis.For further details on these statistical methods, please refer to the following references: Ruczinski et al., J.OF COMPUTATIONAL AND GRAPHICAL STATISTICS, 475-511 (2003); Friedman, JH, J.OF THE AMERICAN STATISTICAL ASSOCIATION, 165-75 (1989); Hastie, Trevor, Tibshirani, Robert, Friedman, Jerome, "The Elements of Statistical Learning," Springer Series in Statistics (2001); Breiman, L., Friedman, JH, Olshen, RA, Stone, CJ, "Classification and regression trees," California: Wadsworth (1984); Breiman, L., "MACHINE LEARNING," 5-32 (2001); Pepe, MS, "The Statistical Evaluation of Medical Tests for Classification and Prediction," Oxford Statistical Science This is found in Series, 28 (2003) and in Duda, RO, Hart, PE, Stork, DG, "Pattern Classification," Wiley Interscience, 2nd edition (2001).

[0114] 4. Evaluation of TBI characteristics using biomarkers In some embodiments, the Disclosure presents methods for characterizing and / or classifying TBI, including characterizing the severity of TBI and different types of TBI, based on the detection, non-detection, and / or detection levels of one or more TBI biomarkers. Each class or subclass of TBI is likely to have a set of biomarkers at characteristic levels or relative levels (signatures). In one embodiment, the Disclosure presents a method for assessing the progression of a TBI condition over time in a patient, including progression (exacerbation) and regression (improvement). Over time, the amount or relative amount (e.g., pattern or signature) of TBI biomarkers may change. For example, biomarker "X" may increase with brain injury, while biomarker "Y" may decrease with brain injury. Thus, an increase or decrease in these biomarkers over time, indicating a tendency toward brain injury or non-brain injury, indicates the progression of the condition. Therefore, this method involves measuring the level of one or more biomarkers in the patient at at least two different time points (e.g., a first time point and a second time point, and comparing these if there are any changes).

[0115] In some embodiments, classes or subclasses of TBI may be characterized by measuring biomarkers of involvement and then subjecting them to a classification algorithm or by comparing them to a reference quantity (e.g., a predetermined level or pattern of biomarkers associated with a particular class or subclass).

[0116] In some embodiments, data generated using samples such as “known samples” may then be used to “train” a classification model. “Known samples” are samples that have been pre-classified. The data used to form the classification model may be referred to as the “training dataset.” The training dataset used to form the classification model may include raw data or pre-processed data. Once trained, the classification model may recognize patterns in the data generated using unknown samples. The classification model may then be used to classify unknown samples into classes. This may be useful, for example, to predict whether a particular biological sample is associated with a particular biological state (e.g., a diseased state in contrast to a non-disease state).

[0117] Classification models can be formed using any appropriate statistical classification or learning method that attempts to divide the data into classes based on objective parameters present in the data. Classification methods may be supervised or unsupervised. Examples of supervised and unsupervised classification processes, along with their instruction, are described by reference in Jain, "Statistical Pattern Recognition: A Review," IEEE Transactions on Pattern Analysis and Machine Intelligence, Vol. 22, No. 1, January 2000.

[0118] In supervised classification, training data containing examples of known classifications is presented to a learning mechanism that learns one or more sets of relationships that define each of the known classes. Then, new data can be applied to the learning mechanism, which then uses the learned relationships to classify the new data. Examples of supervised classification processes include linear regression processes (e.g., multiple linear regression (MLR), partial least square (PLS) regression, and principal component regression (PCR)), binary decision trees (e.g., recursive partitioning processes such as CART), artificial neural networks such as backpropagation networks, discriminant analysis (e.g., Bayesian classifiers or Fisher analysis), logistic classifiers, and support vector classifiers (support vector machines).

[0119] Another supervised classification method is the recursive partitioning process. The recursive partitioning process uses a recursive partitioning tree to classify data originating from an unknown sample. Further details on the recursive partitioning process are presented by Paulse et al., U.S. Patent Application Publication 2002 / 0138208A1, "Method for analyzing mass spectra".

[0120] In other embodiments, the resulting classification model may be formed using unsupervised learning methods. Unsupervised classification attempts to learn classification based on similarities within the training dataset, without pre-classifying the spectrum from which the training dataset is derived. Unsupervised learning methods include cluster analysis. Cluster analysis attempts to divide the data into "clusters" or groups, each having members that are ideally very similar to one another and very different from members of other clusters. Similarity is then measured using some distance metric, which measures the distance between data items and clusters data items that are close to each other together. Clustering methods include McQueen's K-means algorithm and Kohonen's self-organizing mapping algorithm.

[0121] Learning algorithms implemented for use in the classification of biological information are described, for example, in PCT International Publication WO01 / 31580 (Barnhill et al., "Methods and devices for identifying patterns in biological systems and methods of use thereof"), U.S. Patent Publication 2002 / 0193950 (Gavin et al., "Method or analyzing mass spectra"), U.S. Patent Publication 2003 / 0004402 (Hitt et al., "Process for discriminating between biological states based on hidden patterns from biological data"), and U.S. Patent Publication 2003 / 0055615 (Zhang and Zhang, "Systems and methods for processing biological expression data").

[0122] The classification model can be formed and used on any suitable digital computer. Suitable digital computers include microcomputers, small computers, or large computers using any standard or specialized operating system, such as Unix, Windows®, or Linux®-based operating systems. In embodiments using a mass spectrometer, the digital computer used may be physically separated from or coupled to the mass spectrometer used to generate the desired spectrum.

[0123] Training datasets and classification models according to embodiments of the present invention may be implemented by computer code executed or used by a digital computer. The computer code can be stored on any suitable computer-readable medium, including optical disks or magnetic disks, sticks, tapes, etc., and can be written in any suitable computer programming language, including R, C, C++, Visual Basic, etc.

[0124] The learning algorithm described above is useful for both developing classification algorithms for already discovered biomarkers and for discovering new biomarkers. The classification algorithm forms a basis for diagnostic tests by providing diagnostic values ​​(e.g., cutoff points) for biomarkers that can be used individually or in combination.

[0125] 5. Treatment and monitoring of patients with TBI Subjects identified or assessed as having TBI may be treated or monitored based on assessments that may include detecting or measuring a variety of TBI biomarkers. In some embodiments, the method further includes the step of treating human subjects assessed as having TBI with treatments that may take various forms depending on the severity of the head injury. For example, in the case of subjects with mild TBI, treatments may include one or more of the following: rest, suppression of physical activity such as sports, avoidance of sunlight or wearing sunglasses when going out in the sun, medications to relieve headaches or migraines, anti-nausea medications, etc. Treatment for patients with severe TBI may include the administration of one or more appropriate drugs (e.g., diuretics, anticonvulsants, drugs that sedate the individual and induce drug-induced coma) or other pharmaceuticals or biopharmaceuticals (pharmaceuticals known or to be developed for the treatment of TBI), one or more surgical procedures (e.g., hematoma removal, skull fracture repair, decompressive craniotomy), and one or more treatments (e.g., one or more rehabilitation, cognitive behavioral therapy, anger management, psychological counseling). In some embodiments, the method further includes a step of monitoring a human subject assessed to have traumatic brain injury (e.g., mild or moderate to severe traumatic brain injury). In some embodiments, subjects identified to have traumatic brain injury, such as mild or severe traumatic brain injury, may be monitored by CT scan or MRI.

[0126] In one embodiment, the disclosure presents a method for determining the risk of developing TBI in a patient. Percentages, amounts, or patterns of TBI biomarkers are characteristic of various risk states (e.g., high, moderate, or low). The risk of developing TBI may be determined by measuring the biomarkers involved and then subjecting them to a classification algorithm or by comparing them to a reference amount (e.g., a predetermined level or signature of the biomarker associated with a particular risk level).

[0127] In some embodiments, treating a subject with TBI may involve managing the patient's treatment based on a TBI status established using one or more TBI biomarkers. Such management may include actions by a physician or clinician following the determination of the TBI status. For example, if a physician diagnoses mild TBI, a specific monitoring regime may follow. Subsequently, evaluation of the course of TBI using the method of the Disclosure may require a specific TBI treatment regime. Alternatively, a diagnosis of non-TBI may be followed by further tests to determine any specific disease the patient may have. If the diagnostic tests yield inconclusive results regarding the TBI status, further tests may also be required.

[0128] In another embodiment, the disclosure presents methods for determining the therapeutic efficacy of a drug in the context of TBI treatment. These methods may be useful for conducting clinical trials of a drug, as well as for monitoring patient progression with respect to the drug. Treatment or clinical trials involve the administration of a drug in a specific regimen. The regimen may involve a single dose or multiple doses of the drug over a period of time. A physician or clinical researcher monitors the effect of the drug on a patient or subject over the course of administration. If the drug has a pharmacological effect on a condition, one or more amounts or relative amounts (e.g., pattern or signature) of the TBI biomarkers of the present invention may change to a non-brain injury profile. Thus, the course of one or more TBI biomarkers in a patient can be tracked over the course of treatment.

[0129] Therefore, this method may involve the steps of measuring one or more TBI biomarkers in a patient receiving drug therapy and correlating the biomarker levels with the patient's TBI status (e.g., by comparing them to predetermined levels of the biomarkers corresponding to different brain injury states). One embodiment of this method may involve the step of determining the levels of one or more TBI biomarkers at at least two different time points during the course of drug therapy (e.g., a first time point and a second time point, and comparing any changes in biomarker levels). For example, the levels of one or more TBI biomarkers may be measured at two different time points before and after or during drug administration. The effectiveness of the treatment is determined based on these comparisons. If the treatment is effective, one or more TBI biomarkers will show a trend toward normal; however, if the treatment is ineffective, one or more TBI biomarkers will show a trend toward signs of brain injury.

[0130] 6. Biomarkers for TBI The methods described herein may be used to identify one or more TBI biomarkers or candidate TBI biomarkers that may be helpful in the diagnosis and assessment of individuals who may have suffered TBI. Examples of TBI biomarkers are described below.

[0131] a. TBI biomarkers for integration The following TBI biomarkers, described below, have been identified as being able to distinguish healthy subjects from subjects with TBI when detected individually or in combination.

[0132] Ezrin (EZR)

[0133] EZR is also known as cytobilin or bilin 2 and encodes a protein in humans, encoded by the EZR gene. The N-terminus of ezrin contains a FERM domain, which is further subdivided into three subdomains. The C-terminus contains an ERM domain. Ezrin is thought to be involved in the connection of major cytoskeletal structures to the cell membrane. In epithelial cells, ezrin is required for the formation of microvilli and membrane folds at the apical pole. Ezrin, along with PLEKHG6, is required for normal microvitation (UniProt accession number: P15311).

[0134] 4-Trimethylaminobutyraldehyde dehydrogenase (ALDH9A1 or AL9A1)

[0135] AL9A1 is also known as 4-trimethylaminobutyraldehyde dehydrogenase, TMABADH, aldehyde dehydrogenase E3 isozyme, aldehyde dehydrogenase family 9 member A1, gamma-aminobutyraldehyde dehydrogenase, and R-aminobutyraldehyde dehydrogenase. ALDH9A1 or AL9A1 converts gamma-trimethylaminobutyraldehyde to gamma-butyrobetaine and catalyzes the irreversible oxidation of a wide range of aldehydes to their corresponding acids in NAD-dependent reactions. ALDH9A1 or AL9A1 is highly expressed in the liver, skeletal muscle, and kidneys of adults, and at low levels in the heart, pancreas, lungs, and brain, and is expressed throughout all regions of the brain. Expression levels are variable within different brain regions, with the highest levels in the spinal cord and the lowest levels in the occipital pole (UniProt accession number: P49189).

[0136] Mitochondrial ATP synthase subunit gamma (ATPG)

[0137] Mitochondrial membrane ATP synthase (F1F0 ATP synthase or complex V) generates ATP from ADP in the presence of a membrane-wide proton gradient generated by the respiratory chain's electron transport complex. The F-type ATPase consists of two structural domains, F1 containing an extramembrane catalytic core and F0 containing a membrane proton channel, which are integrally linked by a central stalk and an outer stalk. During catalysis, ATP synthesis within the catalytic domain of F1 is coupled to proton transfer via a rotational mechanism of the central stalk subunit. ATPG is the central stalk, which is part of the complex's F1 domain and part of the complex's rotational element. The gamma subunit protrudes into the alpha-3-beta-3 catalytic domain. The rotation of the central stalk relative to the surrounding alpha-3-beta-3 subunit results in ATP hydrolysis within three separate catalytic sites on the beta subunit (UniProt accession number: P36542).

[0138] Complement C1r subcomponent-like protein (C1RL)

[0139] C1RL mediates the proteolytic cleavage of HP / haptoglobin within the endoplasmic reticulum. Diseases associated with C1RL include ovarian adenocarcinoma (UniProt accession number: Q9NZP8).

[0140] Karin-related NEDD8 dissociation protein 1 (CAND1)

[0141] CAND1 is a key assembly factor of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which plays a key role in the intracellular repertoire of SCF complexes by promoting the exchange of the substrate-recognizing F-box subunit within the SCF complex. CAND1 acts as an F-box protein exchange factor. The exchange activity of CAND1 is coupled with the NEDD8-mediated conjugation cycle: in the de-NEDD8-mediated state, karin-bound CAND1 binds to CUL1-RBX1, increasing the dissociation of the SCF complex and promoting the exchange of F-box proteins. CAND1 likely plays a similar role in other karin-RING E3 ubiquitin ligase complexes (UniProt accession number: Q86VP6).

[0142] Epiplaquine (EPIPL or EPPK1)

[0143] EPIPL is a cytoskeletal linker protein that, in response to stress, connects to intermediate filaments and controls their reorganization. In response to mechanical stress such as wound healing, EPIPL is associated with cell motility mechanisms by slowing keratinocyte migration and proliferation and accelerating keratin bundling in keratinocyte proliferation, thereby contributing to tissue architecture. However, in corneal epithelial wound healing, EPIPL also positively regulates cell differentiation and proliferation and negatively regulates migration, thereby controlling corneal epithelial morphogenesis and integrity. In response to cellular stress, EPIPL likely plays a role in keratin filament reorganization by protecting keratin filaments from destruction. In liver and pancreatic injury, EPIPL plays a protective role by chaperoning disease-induced intermediate filament reorganization (by similarity) (UniProt accession number: P58107).

[0144] Mitochondrial hydroxyacyl glutathione hydrolase (GLO2 or HAGH)

[0145] GLO2 is a thiol esterase that catalyzes the hydrolysis of SD-lactoyl-glutathione, which forms glutathione and D-lactic acid (UniProt accession number: Q16775).

[0146] Immunoglobulin constant heavy chain alpha-2 (IGHA2)

[0147] IGHA2 is the constant region of the immunoglobulin heavy chain. Immunoglobulins, also known as antibodies, are membrane-bound or secreted glycoproteins produced by B lymphocytes. In the recognition phase of humoral immunity, membrane-bound immunoglobulins act as receptors that, upon binding to specific antigens, induce clonal expansion and differentiation of B lymphocytes into immunoglobulin-secreting plasma cells. Secreted immunoglobulins mediate the effector phase of humoral immunity, resulting in the elimination of the bound antigen. The antigen-binding site is formed by one variable domain of the heavy chain, combined with the variable domain of the light chain to which it associates. Therefore, each immunoglobulin has two antigen-binding sites with significant affinity for a particular antigen. The variable domains are assembled through a process called V-(D)-J rearrangement and then subjected to somatic hypermutation, which enables affinity maturation for a specific antigen after exposure to and selection of the antigen. Ig alpha is the major immunoglobulin class in the body's secretion system (UniProt accession number: P01877).

[0148] Pregnancy-related proteins (PZPs)

[0149] PZPs can inhibit all four classes of proteinases through a unique "trapping" mechanism. PZPs have a chain of peptides called "bait regions" that contain specific cleavage sites for different proteinases. When a proteinase cleaves the bait region, a conformational change is induced within the PZP that traps the proteinase. The incorporated enzyme maintains its activity for low molecular weight substrates (its activity for high molecular weight substrates is significantly reduced). After cleavage within the bait region, the thioester bond is hydrolyzed, mediating the covalent bonding of the PZP to the proteinase (UniProt accession number: P20742).

[0150] Cytoplasmic threonine-tRNA ligase (SYTC or TARS)

[0151] SYTC catalyzes the reaction: ATP + L-threonine + tRNA(Thr) = AMP + diphosphate + L-threonyl-tRNA(Thr). SYTC is inhibited by borellin (BN; IC50 is 7 nM) (UniProt accession number: P26639), which binds to four distinctly different subsites within the protein, preventing binding to all three substrates.

[0152] Cytoplasmic tyrosine-tRNA ligase (SYYC or YARS)

[0153] SYYC catalyzes the conjugation of tyrosine to tRNA(Tyr) in a two-step reaction: tyrosine is first activated by ATP to form Tyr-AMP, which is then transferred to the acceptor end of tRNA(Tyr) (UniProt accession number: P54577).

[0154] Protein ABHD14B (ABHEB or ABHD14B)

[0155] ABHEB exhibits hydrolase activity against p-nitrophenyl butyrate (in vitro) and can activate transcription (UniProt accession number: Q96IU4). Dinamin-1-like protein (DNM1L)

[0156] DNM1L functions in mitochondrial and peroxisome fission. DNM1L mediates membrane fission via oligomerization into membrane-associated tubular structures that encapsulate separation sites, constrict the mitochondrial membrane, and cleave via a GTP hydrolysis-dependent mechanism. Through its function in mitochondrial fission, DNM1L ensures the survival of postmittal neurons, including Purkinje cells, of at least some species, by suppressing oxidative damage. DNM1L is required for normal brain development, including cerebellar development. DNM1L facilitates developmentally regulated apoptosis during neural tube formation. DNM1L is required for normal rate cytochrome c release and caspase activation during apoptosis, a requirement that depends on cell type and physiological apoptosis cue. DNM1L plays a crucial role in mitochondrial fission during mitosis. DNM1L is required for endocytic vesicle formation. It has been proposed that DNM1L regulates synaptic vesicle membrane dynamics through association with Bcl-X(L), an isoform of BCL2L1 that stimulates its GTPase activity within synaptic vesicles. Its function may involve requiring its recruitment to clathrin-containing vesicles via MFF. Furthermore, DNM1L is required for the execution of programmed necrosis (UniProt accession number: 000429).

[0157] Ficolin 2 (FCN2)

[0158] FCN2 may function in innate immunity by activating the lectin complement pathway. FCN2 exhibits calcium-dependent GlcNAc binding to lectins. FCN2 enhances neutrophil phagocytosis of S. typhimurium, suggesting an opsonizing effect mediated by its collagenous domain (UniProt accession number: Q15485).

[0159] Inverted Formin 2 ( INF2 )

[0160] INF2 This process is involved in the cleavage of actin filaments and accelerates their polymerization and depolymerization (UniProt accession number: Q27J81).

[0161] Epidermal keratin type II cytoskeleton 2 (K22E or KRT2)

[0162] K22E is an intermediate protein thought to contribute to terminal keratinization. K22E is associated with the activation, proliferation, and keratinization of keratinocytes (UniProt accession number: P35908).

[0163] Mitogen-activated protein kinase kinase 5 (M3K5 or MAP3K5)

[0164] M3K5 is a serine / threonine kinase that acts as an essential component of the MAP kinase signaling pathway. M3K5 plays a crucial role in the cascade of cellular responses triggered by environmental changes. It mediates signaling for cell fate determination, including differentiation and survival. M3K5 plays a critical role in the apoptotic signaling pathway, mediated by mitochondrial-dependent caspase activation. MAP3K5 / ASK1 is required for innate immune responses essential for host defense against a wide range of pathogens. M3K5 mediates signaling for various stressors, including oxidative stress, as well as signaling via receptor-mediated inflammatory signals such as tumor necrosis factor (TNF) or lipopolysaccharide (LPS). Upon activation, M3K5 acts as an upstream activator of the MKK / JNK signaling cascade and the p38MAPK signaling cascade, mediated by the phosphorylation and activation of several MAP kinases, including MAP2K4 / SEK1, MAP2K3 / MKK3, MAP2K6 / MKK6, and MAP2K7 / MKK7. These MAP2Ks activate p38MAPK and c-jun N-terminal kinase (JNK). Both p38MAPK and JNK regulate transcription factor activator protein 1 (AP-1) (UniProt accession number: Q99683).

[0165] Nuclear receptor corepressor 1 (NCOR1)

[0166] NCOR1 mediates transcriptional repression by certain nuclear receptors. NCOR1 is part of a complex that promotes the formation of repressive chromatin structures that inhibit histone deacetylation and the access of basal transcription factors. NCOR1 participates in transcriptional repressor activity mediated by BCL6 (UniProt accession number: 075376).

[0167] Splabasin (SBSN)

[0168] SBSN is a novel gene expressed in mice and humans that differentiates keratinocytes. SBSN is thought to be secreted from the stratum spinosum of stratified epithelium and can form a novel gene complex on chromosome 2 together with dermokine alpha / dermokine beta and Kdap (UniProt accession number: Q6UWP8).

[0169] Difunctional glutamate / proline-tRNA ligase (SYEP or EPRS) SYEP catalyzes the conjugation of cognitive amino acids to their corresponding tRNAs in a two-step reaction: tyrosine is first activated by ATP to form a covalent intermediate, which is then transferred to the acceptor terminus of the cognitive tRNA. SYEP is a component of the GAIT (gamma interferon-activate inhibitor of translation) complex, which mediates interferon-gamma-induced transcript-selective translation inhibition during inflammatory processes. Upon activation and subsequent phosphorylation of interferon-gamma, SYEP dissociates from the multisynthetase complex and assembles into the GAIT complex, which binds to stem-loop-containing GAIT elements (such as ceruloplasmin) within the 3'-UTR of various inflammatory mRNAs, thereby repressing their translation (UniProt accession number: P07814).

[0170] Tripeptidyl peptidase 2 (TPP2)

[0171] TPP2 is a component of a proteolytic cascade that acts downstream of the 26S proteasome in the ubiquitin-proteasome pathway. Under conditions where the 26S proteasome is inhibited, TPP2 may be able to complement 26S proteasome function to some extent. TPP2 stimulates lipid synthesis (by similarity) (UniProt accession number: P29144).

[0172] Annexin A6 (ANXA6)

[0173] ANXA6 can associate with CD21, and release Ca from its intracellular storage region. 2+ It can regulate the release of [substance]. ANXA6 can be secreted (UniProt accession number: P08133).

[0174] Endoplasmic reticulum aminopeptidase 1 (ERAP1)

[0175] ERAP1 is an aminopeptidase that plays a central role in peptide trimming, a step required for the production of most HLA class I binding peptides. Peptide trimming is essential for customizing longer precursor peptides to the appropriate length required for presentation on MHC class I molecules. Substrates with a length of 9–16 residues are strongly preferred by ERAP1. ERAP1 rapidly degrades to 13–9 residues and then terminates. ERAP1 preferentially hydrolyzes peptides with the residue Leu and a hydrophobic C-terminus, while exhibiting weak activity towards peptides with a charged C-terminus. ERAP1 may play a role in the inactivation of peptide hormones. ERAP1 may be involved in blood pressure regulation in the kidney through the inactivation of angiotensin II and / or the production of bradykinin (UniProt accession number: Q9NZ08).

[0176] Coagulation factor V (FA5 or F5)

[0177] FA5 is a central regulator of hemostasis. FA5 is used as a crucial cofactor for the prothrombinase activity of factor Xa, which results in the activation of prothrombin to thrombin (UniProt accession number: P12259).

[0178] Glucose-6-phosphate isomerase (G6PI or GPI)

[0179] G6PI is a glycolytic enzyme, and in mammals, G6PI can function as a tumor-secreted cytokine and angiogenic factor (AMF) that stimulates endothelial cell motility. In the cytoplasm, the G6PI gene product functions as a glycolytic enzyme (glucose-6-phosphate isomerase) that interconverts glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P). Extracellularly, the encoded G6PI protein (also known as neuroleukin) functions as a neurotrophic factor that promotes the survival of skeletal motor neurons and sensory neurons, and as a lymphokine that induces immunoglobulin secretion. The encoded G6PI protein is also called an autocretory motility factor (AMF) due to its further functions as a tumor-secreted cytokine and angiogenic factor. Deficiency of this gene is a cause of nonspherocytic hemolytic anemia, and severe enzyme deficiency may be associated with fetal hydrops, immediate neonatal death, and neurological dysfunction. Alternative splicing results in multiple transcript variants (UniProt accession number: P06744).

[0180] Smooth muscle myosin light chain kinase (MYLK)

[0181] MYLK is a calcium / calmodulin-dependent myosin light chain kinase involved in smooth muscle contraction via phosphorylation of myosin light chains (MLCs). MYLK also modulates actin-myosin interactions via non-kinase activity. MYLK phosphorylates PTK2B / PYK2 and myosin light chains. MYLK is involved in inflammatory responses (e.g., apoptosis, vascular permeability, lymphocyte leakage), cell motility and morphology, airway hyperresponsiveness, and other activities related to asthma. MYLK is required for tonic airway smooth muscle contraction necessary for physiological and asthmatic airway resistance. MYLK is required for gastrointestinal motility. MYLK is likely involved in regulating vascular permeability, as well as endothelial permeability, through the regulation of cytoskeletal rearrangement. In the nervous system, MYLK has been shown to control the induction of astrocyte proliferation in cultures and to be involved in the release of neurotransmitters at synapses formed between cultured sympathetic ganglion cells. MYLK is a critical participant in the signaling chain that results in fibroblast apoptosis. MYLK plays a role in regulating epithelial cell survival. MYLK is required for actomyosin ring contraction during epithelial wound healing, particularly wound closure by purse-string sutures. MYLK mediates RhoA-dependent membrane bleb formation. MYLK induces TRPC5 channel activity in calcium-dependent signaling by inducing its intracellular localization in the cell membrane. MYLK promotes cell migration (including tumor cells) and tumor metastasis. Phosphorylation-mediated activation of PTK2B / PYK2 mediates ITGB2 activation, which is therefore essential for inducing neutrophil migration during acute lung injury (ALI). MYLK may regulate the migration of astrocytes in the optic nerve head. MYLK is likely involved in regulating mitosis in the cytoskeleton. MYLK likely regulates tight junctions by modulating ZO-1 exchange within the parajunctional actomyosin ring. MYLK mediates burn-induced microvascular barrier damage and induces endothelial contraction in the development of increased microvascular permeability by phosphorylating MLCs. MYLK is essential for intestinal barrier dysfunction.MYLK mediates the reduction of epithelial barrier function in Giardia lamblia-mediated enteric infection, mediated by Giardia species, through the reorganization of F-actin in the cytoskeleton and ZO-1 in tight junctions. MYLK is hypotonic inducible Ca in cervical cancer cells. 2+ It is necessary for entry and subsequent activation of volume-sensitive organic osmolite / anion channels (VSOACs). MYLK contributes to the high proliferative capacity of breast cancer cells through anti-apoptosis (UniProt accession number: Q15746).

[0182] Serum amyloid-P component (SAMP or APCS)

[0183] SAMP can interact with DNA and histones, and may scavenge nuclear material released from damaged circulating cells. SAMP can also function as a calcium-dependent lectin (UniProt accession number: P02743).

[0184] In some embodiments, one or more of the TBI biomarkers listed above may be used to determine whether a subject has TBI based on the detection of one or more of these TBI biomarkers in a sample derived from the subject and the absence of detection of one or more TBI biomarkers in a control subject (e.g., a subject not suffering from TBI). These TBI biomarkers may include one or more of AL9A1, ATPG, C1RL, EPIPL, IGHA2, PZP, SYTC, SYYC, ABHEB, DNM1L, FCN2, INF2, K22E, M3K5, NCOR1, SBSN, SYEP, TPP2, ANXA6, ERAP1, EZRI, FA5, G6PI, MYLK, SAMP, or any combination thereof. Measurement or detection of one or more of these TBI biomarkers in a subject may be sufficient to indicate that the subject is suffering from TBI, independently of the need to detect, measure, compare, and / or quantify the amount, concentration, and / or expression level of one or more TBI biomarkers in a control subject. One or more of these TBI biomarkers may be present in the control subject or in subjects not suffering from TBI, but generally in amounts that are not detectable by conventional means as described herein. Therefore, in some cases, detection of one or more of these TBI biomarkers in a subject may indicate that the subject is suffering from TBI.

[0185] In some embodiments, one or more of the TBI biomarkers listed above may be used to determine that a subject has severe TBI based on the detection of one or more of these TBI biomarkers in a sample derived from the subject and the absence of detection of one or more TBI biomarkers in a control subject (e.g., a subject without TBI) or in a subject with mild TBI. These TBI biomarkers may include one or more of ATPG, C1RL, SYYC, or any combination thereof. Measurement or detection of one or more of these TBI biomarkers in a subject may be sufficient to indicate that the subject has severe TBI, independently of the need to detect, measure, compare, or quantify the amount, concentration, or expression level of one or more TBI biomarkers in a control subject or a subject with mild TBI.

[0186] In some embodiments, one or more of the TBI biomarkers listed above may be used to determine that a subject has TBI based on the detection of one or more of these TBI biomarkers in a sample derived from the subject at a higher level of quantity, concentration, and / or expression than that of the corresponding TBI biomarker in a control subject. These TBI biomarkers may include one or more of CAND1 and GLO2 or any combination thereof. Measurement or detection of an increase in the level of one or more of these TBI biomarkers in the subject compared to a control subject (e.g., a subject without TBI) may be sufficient to indicate that the subject has TBI, and may, in some cases, that the subject has severe TBI.

[0187] In some embodiments, one or more of the TBI biomarkers listed above may be used to determine whether a subject has TBI based on the detection of one or more of these TBI biomarkers in a sample derived from the subject at a higher or lower level of quantity, concentration, and / or expression than the level of the corresponding TBI biomarker in a control subject. These TBI biomarkers may include one or more of ABHEB, AL9A1, DNM1L, or any combination thereof. Measurement or detection of an increase or decrease in the level of one or more of these TBI biomarkers in a subject compared to a control subject (e.g., a subject without TBI) may be sufficient to indicate that the subject has TBI, and in some cases may be sufficient to indicate that the subject has severe TBI. For example, detection or measurement of high levels of ABHED and / or low levels of AL9A1 and / or DNM1L in a potentially TBI-affected subject compared to the level in a control subject may indicate that the subject actually has TBI. The levels of these TBI biomarkers can be detected or measured individually or in combination as part of a TBI panel or signature.

[0188] In some embodiments, one or more of the TBI biomarkers listed above may be used to determine whether a subject has mild TBI based on the detection of one or more of these TBI biomarkers in a sample derived from the subject at a higher or lower level of quantity, concentration and / or expression than the corresponding TBI biomarker in a control subject. These TBI biomarkers may include one or more of M3K5, SBSN, SYEP, or any combination thereof. Measurement or detection of an increase or decrease in the level of one or more of these TBI biomarkers in the subject compared to a control subject (e.g., a subject without TBI) may be sufficient to indicate that the subject has TBI, and in some cases may be sufficient to indicate that the subject has mild TBI. For example, detection or measurement of high levels of SBSN and / or SYEP, and / or low levels of M3K5 in a potentially TBI-affected subject compared to the level in a control subject may indicate that the subject actually has mild TBI. The levels of these TBI biomarkers can be detected or measured individually or in combination as part of a TBI panel or signature.

[0189] In some embodiments, one or more of the TBI biomarkers listed above may be included together with other biomarkers that may or may not be identified as TBI biomarkers. For example, one or more of the TBI biomarkers listed above may be included in a panel of biomarkers that may include one or more of ANXA6, ERAP1, EZRI, FA5, G6PI, MYLK, SAMP, or combinations thereof. Depending on the circumstances, a panel of TBI biomarkers, including other TBI biomarkers and non-TBI biomarkers, may be more helpful in diagnosing TBI than any single biomarker alone.

[0190] b. Mild TBI biomarkers for inclusion

[0191] The following TBI biomarkers, described below, were identified as being able to distinguish healthy subjects from subjects with mild TBI when detected individually or in combination.

[0192] Poly(rC)-binding protein 2 (alpha-CP2) (PCBP2)

[0193] Poly(rC)-binding protein 2 is a protein encoded by the PCBP2 gene in humans. This gene-encoded protein is considered to be multifunctional. PCBP2, along with PCBP-1 and hnRNPK, corresponds to the major cellular poly(rC)-binding proteins. PCBP2 contains three K-homologous (KH) domains that may be involved in RNA binding. This encoded protein, in conjunction with PCBP-1, also functions as a translational co-activator of poliovirus RNA via sequence-specific interactions with the stem-loop IV of IRES, promoting poliovirus RNA replication by binding to the cloverleaf structure at its 5' end. PCBP2 is also involved in the regulation of translation of 15-lipoxygenase mRNA, 16L2 human papillomavirus mRNA, and hepatitis A virus RNA. The encoded protein has also been suggested to play a role in the formation of the sequence-specific alpha-globin mRNA complex, which is associated with the stability of alpha-globin mRNA. This multi-exon mRNA structure is thought to be retrotransposed to produce a similarly functional PCBP-1 intron-free gene. This gene and PCBP-1 have paralogs PCBP3 and PCBP4, which are thought to have arisen as a result of the replication event of the entire gene. PCBP2 also has two processed pseudogenes, PCBP2P1 and PCBP2P2. Currently, two alternatively spliced ​​transcript variants of this gene have been described. In humans, the PCBP2 gene overlaps with TUC338, a transcribed superconserved element involved in hepatocellular carcinoma (UniProt accession number: Q15366).

[0194] Mitochondrial thioredoxin reductase 2 (TRXR2)

[0195] Thioredoxin reductase (TR, TrxR) (EC 1.8.1.9) is the only enzyme known to reduce thioredoxin (Trx). Two classes of thioredoxin reductase have been identified: one in bacteria and some eukaryotes, and one in animals. Both classes are flavoproteins that function as homodimers. Each monomer contains an active site containing an FAD prosthetic group, an NADPH-binding domain, and a redox-active disulfide bond. Thioredoxin reductase is the only enzyme known to catalyze the reduction of thioredoxin and is therefore a central component of the thioredoxin system. Together with thioredoxin (Trx) and NADPH, the most common description of this system is as a method for forming reduced disulfide bonds within cells. Electrons are obtained from NADPH via TrxR and transferred to the active site of Trx, leading to the reduction of protein disulfides or other substrates. The Trx system is present in all living cells, has an evolutionary history linked to DNA as the genetic material, and performs redox signaling using molecules such as hydrogen peroxide and nitric oxide to protect against oxidative damage caused by oxygen metabolism (UniProt accession number: Q9NNW7).

[0196] 14-3-3 Protein Gamma (1433G or YWHAG)

[0197] 1433G is an adapter protein involved in a broad spectrum of modulation across both general and specialized signaling pathways. 1433G typically binds to numerous partners, usually by recognition of phosphoserine or phosphothreonine motifs. Binding generally results in modulation of the activity of the binding partner (UniProt accession number: P61981).

[0198] Activated CDC42 kinase 1 (ACK1 or TKN2)

[0199] ACK1 is a non-receptor tyrosine-protein and serine / threonine-protein kinase involved in cell diffusion and migration, cell survival, and cell growth and proliferation. ACK1 transmits extracellular signals to cytosolic and nuclear effectors. ACK1 phosphorylates AKT1, AR, MCF2, WASL, and WWOX. ACK1 is involved in trafficking and clathrin-mediated endocytosis via binding to epidermal growth factor receptor (EGFR) and clathrin. ACK1 binds to both polyubiquitin and monoubiquitin, regulating ligand-induced degradation of EGFR, thereby contributing to EGFR accumulation at the boundary membrane of early endosomes. ACK1 is a downstream effector of CDC42, mediating CDC42-dependent cell migration via BCAR1 phosphorylation. ACK1 may be involved in both adult synaptic function and plasticity, as well as brain development. ACK1 activates AKT1 by phosphorylation at "Tyr-176". ACK1 phosphorylates AR at "Tyr-267" and "Tyr-363", thereby promoting its recruitment to androgen-responsive enhancers (AREs). ACK1 phosphorylates WWOX at "Tyr-287". ACK1 phosphorylates MCF2, thereby enhancing its activity as a guanine nucleotide exchange factor (GEF) to Rho family proteins. ACK1 contributes to the regulation of AXL receptor levels. ACK1 promotes tumor growth by conferring metastatic characteristics to cancer cells, negatively regulating tumor suppressors such as WWOX, and positively regulating survival factors such as AKT1 and AR. ACK1 phosphorylates WASP (UniProt accession number: Q07912).

[0200] Aminoacylase 1 (ACY1)

[0201] ACY1 is involved in the hydrolysis of N-acylated amino acids or N-acetylated amino acids (excluding L-aspartic acid) (UniProt accession number: Q03154). A kinase anchor protein 12 (AKA12 or AKAP12)

[0202] AKA12 is an anchoring protein that mediates the intracellular compartmentalization of protein kinase A (PKA) and protein kinase C (PKC) (UniProt accession number: Q02952).

[0203] Arginase 1 (ARGI1 or ARG1)

[0204] ARGI1 is a key component of the urea cycle, which converts L-arginine to urea and L-ornithine, which are further metabolized into proline and polyamides, respectively, driving collagen synthesis and bioenergy pathways crucial for cell proliferation; the urea cycle primarily occurs in the liver, and to a lesser extent in the kidneys. ARGI1 functions in the homeostasis of L-arginine in tissues other than the liver, characterized by competition between nitric oxide synthase (NOS) and arginase, an available intracellular substrate. Arginine metabolism is a critical regulator of both innate and adaptive immune responses. ARGI1 is involved in the antimicrobial effector pathway within polymorphonuclear granulocytes (PMNs). In PMN cell death, ARGI1 is released from phagolysosomes, depleting arginine in the microenvironment and leading to suppression of T cell and natural killer (NK) cell proliferation and cytokine secretion. In group 2 innate lymphoid cells (ILC2s), ARGI1 promotes acute type 2 inflammation and is involved in optimal ILC2 proliferation in the lung, but is not involved in survival (due to similarity) (UniProt accession number: P05089).

[0205] Cadherin 5 (CADH5 or CDH5)

[0206] CADH5 is a cadherin; cadherins are calcium-dependent cell adhesion proteins. Cadherins interact preferentially and homophilically with each other when connecting cells, thus contributing to the sorting of heterogeneous cell types. This cadherin can play a crucial role in endothelial cell biology by regulating the tightness and organization of intercellular junctions. CADH5 associates with alpha-catenin to form ligation to the cytoskeleton. CADH5 acts cooperatively with KRIT1 to establish and maintain proper endothelial cell polarity and vascular lumen. These effects are mediated by the recruitment and activation of the Par polarity complex and RAP1B. CADH5 is required for the activation of PRKCZ and the localization of phosphorylated PRKCZ, PARD3, TIAM1, and RAP1B to cell junctions (UniProt accession number: P33151).

[0207] Clathrin heavy chain 1 (CLH1 or CLTC)

[0208] CLH1 is the major protein responsible for the polyhedral coating of pitted vesicles and vesicles. Two different adapter protein complexes link the clathrin lattice to the cell membrane or the trans-Golgi network. CLH1 acts as a component of the TACC3 / ch-TOG / clathrin complex, which has been proposed to contribute to the stabilization of kinetochore fibers in the mitotic spindle by acting as an intermicrotubule crosslinker. The TACC3 / ch-TOG / clathrin complex is required for maintaining the tension of kinetochore fibers. CLH1 also plays a role in the formation of early autophagosomes (UniProt accession number: Q00610).

[0209] Courtmar Subunit Gamma 2 (COPG2)

[0210] COPG2 is a cytosolic protein complex that binds to a dilysine motif and reversibly associates with non-clathrin-coated vesicles in the Golgi apparatus, further mediating the transport of biosynthetic proteins from the ER through the Golgi apparatus to the trans-Golgi network. The coatmer complex is required for budding from the Golgi membrane and is essential for the reverse transport of dilysine-tagged proteins between the Golgi apparatus and the ER. In mammals, the coatmer is recruited only by the membrane to associate with ADP-ribosyl factor (ARF), a small GTP-binding protein; the complex also affects the structural integrity of the Golgi apparatus as well as the processing, activity, and endocytic recycling of LDL receptors (by similarity) (UniProt accession number: Q9UBF2).

[0211] DNA polymerase delta subunit 2 (DPOD2 or POLD2)

[0212] As components of the trimer and tetramer DNA polymerase delta complexes (Pol delta 3 and Pol delta 4, respectively), DPOD2 plays a role in high-fidelity genome replication, including lagging strand synthesis and repair. Pol delta 3 and Pol delta 4 are characterized by the absence or presence of POLD4. Pol delta 3 and Pol delta 4 exhibit differences in catalytic activity. Most notably, Pol delta 3 shows higher proofreading activity than Pol delta 4. Both Pol delta 3 and Pol delta 4 process Okazaki fragments in vitro, but Pol delta 3 is also better suited to this task than Pol delta 4 because it exhibits almost no strand displacement activity and stalls when it encounters a blocking oligonucleotide at the 5' end. The idling process of Pol delta 3 can avoid gap formation while maintaining a nick that can be easily ligated. Together with DNA polymerase kappa, DNA polymerase delta carries out approximately half of the nucleotide excision repair (NER) synthesis after UV irradiation. Under DNA replication stress conditions, DPOD2 is required for the repair of cleaved replication forks via break-induced replication (BIR). DPOD2 is involved in damage-overcoming synthesis (TLS) of templates containing O6-methylguanine or non-basic sites, carried out by Pol delta 4, independently of DNA polymerase zeta (REV3L) or DNA polymerase eater (POLH). DPOD2 facilitates bypass of non-basic sites by DNA polymerase delta by promoting elongation from nucleotides inserted on the opposite side of the damage. DPOD2 is also involved in TLS as a component of the POLZ complex. Together with POLD3, DPOD2 dramatically increases the efficiency and productivity of DNA synthesis by a minimal DNA polymerase zeta complex consisting only of REV3L and REV7 (UniProt accession number: P49005).

[0213] Desmograin 2 (DSG2)

[0214] DSG2 is a component of intercellular desmosome junctions. DSG2 is involved in the interaction between plaque proteins and intermediate filaments that mediate intercellular adhesion (UniProt accession number: Q14126).

[0215] Immunoglobulin variable heavy chain 3-7 (HV307 or IGHV3-7)

[0216] HV307 is the V region of the variable domain of the immunoglobulin heavy chain, which participates in antigen recognition. Immunoglobulins, also known as antibodies, are membrane-bound or secreted glycoproteins produced by B lymphocytes. In the recognition phase of humoral immunity, membrane-bound immunoglobulins, upon binding to a specific antigen, act as receptors that induce clonal expansion and differentiation of B lymphocytes into immunoglobulin-secreting plasma cells. The secreted immunoglobulin mediates the effector phase of humoral immunity, resulting in the elimination of the bound antigen. The antigen-binding site is formed by one variable domain of the heavy chain, combined with the variable domain of the light chain to which it associates. Therefore, each immunoglobulin has two antigen-binding sites with significant affinity for a particular antigen. The variable domain is assembled through a process called V-(D)-J rearrangement and then subjected to somatic hypermutation, which enables affinity maturation to a specific antigen after exposure to and selection (UniProt accession number: P01780).

[0217] Ras GTPase-like activator protein IQGAP2 (IQGA2 or IQGAP2)

[0218] IQGA2 binds to activated CDC42 and RAC1, but is not thought to stimulate their GTPase activity. IQGA2 associates with calmodulin (UniProt accession number: Q13576).

[0219] Keratin type I cytoskeleton 14 (K1C14 or KRT14)

[0220] K1C14 is an intermediate filament protein whose non-helical tail domain is involved in promoting the self-assembly of KRT5-KRT14 filaments into larger bundles, thereby enhancing the mechanical properties involved in the restorative force of keratin intermediate filaments in vitro (UniProt accession number: P02533).

[0221] Keratin type I cytoskeleton 19 (K1C19 or KRT19)

[0222] K1C19 is involved in the organization of muscle fibers. Together with KRT8, K1C19 helps to link the contractile apparatus to dystrophin in the costamere of striated muscle (UniProt accession number: P08727).

[0223] Immunoglobulin Kappa Variable 1-5 (KV105 or IGKV1-5)

[0224] KV105 is the V region of the variable domain of the immunoglobulin light chain, which participates in antigen recognition. Immunoglobulins, also known as antibodies, are membrane-bound or secreted glycoproteins produced by B lymphocytes. In the recognition phase of humoral immunity, membrane-bound immunoglobulins, upon binding to a specific antigen, act as receptors that induce clonal expansion and differentiation of B lymphocytes into immunoglobulin-secreting plasma cells. The secreted immunoglobulin mediates the effector phase of humoral immunity, resulting in the elimination of the bound antigen. The antigen-binding site is formed by one variable domain of the heavy chain, combined with the variable domain of the light chain to which it associates. Therefore, each immunoglobulin has two antigen-binding sites with significant affinity for a particular antigen. The variable domain is assembled through a process called V-(D)-J rearrangement and then subjected to somatic hypermutation, which enables affinity maturation to a specific antigen after exposure to and selection (UniProt accession number: P01602).

[0225] Laminin subunit gamma 1 (LAMC1)

[0226] Binding to cells via the high-affinity receptor LAMC1 is thought to mediate cell joining, migration, and organization during embryonic development through interactions with other extracellular matrix components (UniProt accession number: P11047). Mitochondrial malate dehydrogenase (MDHM or MDH2)

[0227] MDHM catalyzes the reversible oxidation of malate to oxaloacetate using the NAD / NADH cofactor system within the citric acid cycle. The protein encoded by this gene is localized in mitochondria and can play a crucial role in the malate-aspartate shuttle, which operates in the metabolic cooperation between the cytosol and mitochondria. Several transcript variants encoding different isoforms of this gene have been identified (UniProt accession number: P40926).

[0228] Ribosyl dihydronicotinamide dehydrogenase [quinone] (NQO2)

[0229] NQO2 is used as a quinone reductase involved in the detoxification pathway, specifically in the conjugation reaction of hydroquinones, as well as in biosynthetic processes such as the vitamin K-dependent gamma-carboxylation of glutamate residues in prothrombin synthesis (UniProt accession number: P16083).

[0230] Myeloperoxidase (PERM or MPO)

[0231] PERM is part of the host defense system mediated by polymorphonuclear leukocytes. PERM contributes to the broad-spectrum bactericidal activity of PMN. Within stimulated PMN, MPO catalyzes the production of hypohalite, and under physiological conditions, primarily hypochlorous acid, and other toxic intermediates that significantly enhance the bactericidal properties of PMN (UniProt accession number: P05164).

[0232] Plastin 3 (PLST or PLS3)

[0233] PLST is an actin-bundling protein found in intestinal microvilli, immobile hair cells, and filopopods of fibroblasts. PLST may play a role in regulating bone development (UniProt accession number: P13797).

[0234] Nicotinate phosphoribosyltransferase (PNCB or NAPRT)

[0235] PNCB catalyzes the conversion of nicotinic acid (NA) to NA mononucleotide (NaMN). PNCB is essential for NA to increase cellular NAD levels and prevent cellular oxidative stress. PNCB catalyzes the synthesis of beta-nicotinic acid D-ribonucleotide from nicotinic acid and 5-phospho-D-ribose 1-phosphate, consuming ATP (UniProt accession number: Q6XQN6).

[0236] Receptor tyrosine protein phosphatase C (PTPRC)

[0237] PTPRC is a tyrosine protein phosphatase required for T cell activation via antigen receptors. When PTPRC binds to DPP4, it acts as a positive regulator of T cell co-activation. The first PTPase domain possesses enzymatic activity, while the second PTPase domain is thought to affect the substrate specificity of the first PTPase domain. Upon T cell activation, PTPRC recruits SKAP1 and FYN and dephosphorylates them. PTPRC also dephosphorylates LYN, thereby modulating LYN activity (by similarity) (UniProt accession number: P08575).

[0238] Septin 7 (SEPT7)

[0239] SEPT7 is a cytoskeletal GTPase that forms filaments. SEPT7 is required for the normal organization of the actin cytoskeleton. SEPT7 is required for the normal progression of mitosis. SEPT7 is involved in cytokinesis. SEPT7 is required for the normal association of CENPE with the kinetochore. SEPT7 plays a role in ciliary body formation and collective cell movement. SEPT7, along with SEPT12, SEPT6, and SEPT2, forms filamentous structures and, possibly, along with SEPT4, forms filamentous structures in the sperm ring, which is required for the structural integrity and motility of sperm tails during post-meiotic differentiation (UniProt accession number: Q16181).

[0240] Cytoplasmic arginine tRNA ligase (STRC or RARS)

[0241] SYRC forms part of a macromolecular complex that catalyzes the binding of specific amino acid cognitives to tRNA during protein synthesis. SYRC modulates AIMP1 secretion and may be involved in the production of the inflammatory cytokine EMAP2 from AIMP1 (UniProt accession number: P54136).

[0242] Thioredoxin-like protein 1 (TXNL1)

[0243] TXNL1 is an active thioredoxin with a redox potential of approximately -250mV (UniProt accession number: 043396).

[0244] UDP glucose: glycoprotein glucosyltransferase 1 (UGGG1 or UGGT1)

[0245] UGGG1 recognizes glycoproteins with small folding defects. UGGG1 reglycosylates a single N-glycan near the misfolded region of the protein, thus providing quality control for protein folding within the endoplasmic reticulum. The reglycosylated protein is recognized by calreticulin for recycling into the endoplasmic reticulum and for refolding or degradation (UniProt accession number: Q9NYU2).

[0246] WD Repeat-containing protein 1 (WDR1)

[0247] WDR1, along with ADF / cofilin family proteins, induces actin filament disassembly. WDR1 enhances cofilin-mediated actin cleavage (by similarity). WDR1 is involved in cytokinesis. WDR1 is involved in chemotactic cell migration by restricting lamellar pseudopodal membrane projections. WDR1 is involved in the organization of cardiac sarcomeres. WDR1 is required for the proliferation and maintenance of cardiomyocytes (by similarity). WDR1 is involved in megakaryotic cell maturation and platelet release. WDR1 is required for the establishment of in-planar cell polarity (PCP) during follicular epithelial development and for changes in cell morphology during PCP; its function is thought to be involved in synergistic action with CFL1 and / or DSTN / ADF. WDR1 is involved in the generation / maintenance of cortical tension (by similarity). WDR1 is involved in the assembly and maintenance of epithelial apical cell junctions and plays a role in the organization of the parajunctional actomyosin belt (UniProt accession number: 075083).

[0248] Neuroblast differentiation-related protein AHNAK (AHNK or AHNAK)

[0249] AHNK is thought to be required for the differentiation of nerve cells (UniProt accession number: Q09666).

[0250] Retinal dehydrogenase 1 (AL1A1 or ALDH1A1)

[0251] AL1A1 converts / oxidizes retinaldehyde to retinoic acid. AL1A1 binds to free retinal and retinal bound to cellular retinol-binding proteins (due to similarity). AL1A1 can exhibit broader specificity and oxidizes other aldehydes in vivo (UniProt accession number: P00352).

[0252] Aminopeptidase N (AMPN or ANPEP)

[0253] AMPN is a broadly specific aminopeptidase that plays a role in the final digestion of peptides produced from protein hydrolysis by gastric and pancreatic proteases. AMPN is also involved in the processing of a variety of peptides, including angiotensin III and IV, neuropeptides, and peptide hormones such as chemokines. AMPN may also be involved in the cleavage of peptides bound to major histocompatibility complex class II molecules in antigen-presenting cells. AMPN can play a role in angiogenesis and may promote cholesterol crystallization. AMPN acts as a receptor for human coronavirus 229E / HCoV-229E. In human coronavirus 229E (HCoV-229E) infection, AMPN is used as a receptor for the HCoV-229E spike glycoprotein. AMPN also mediates human cytomegalovirus (HCMV) infection (UniProt accession number: P15144).

[0254] F-actin capping protein subunit beta (CAPZB)

[0255] CAPZB is Ca at the rapidly proliferating end (the curved end) of actin filaments. 2+CAPZB is an F-actin capping protein that binds independently, thereby blocking the exchange of subunits at these ends. Unlike other capping proteins (such as gelsolin and sevelin), these proteins do not cleave actin filaments. CAPZB plays a role in regulating cell morphology and organizing the cytoskeleton (UniProt accession number: P47756).

[0256] Cathepsin D (CATD or CTSD)

[0257] CATD is an acid protease active in intracellular protein degradation. CATD plays a role in APP processing after cleavage and activation by ADAM30, which leads to APP degradation. CATD is involved in the pathogenesis of several diseases, including breast cancer and possibly Alzheimer's disease (UniProt accession number: P07339).

[0258] CAP-Gly domain-containing linker protein 2 (CLIP2)

[0259] CLIP2 connects microtubules to dendritic lamellar bodies (DLBs), which are membranous organelles primarily found within the bulbous dendritic appendages of neurons, linked by interdendritic gap junctions. CLIP2 may play a role in regulating the migration of brain-specific organelles (due to similarity) (UniProt accession number: Q9UDT6).

[0260] Chromogranin A (CMGA or CHGA)

[0261] Pancreastatin: Potently inhibits glucose-induced insulin release from the pancreas. Catestatin: By acting as a non-competitive nicotinic cholinergic antagonist, it inhibits the release of catecholamines from chromaffin cells and noradrenergic neurons. Catestatin exhibits antibacterial activity against Gram-positive bacteria such as S. aureus and M. luteus, as well as Gram-negative bacteria such as E. coli and P. aeruginosa. Catestatin can induce mast cell migration, degranulation, and cytokine and chemokine production. In vitro, catestatin acts as a potent free radical scavenger. Catestatin may play a role in regulating cardiac function and blood pressure. Serpinin regulates the in vivo development of granules in endocrine cells by upregulating the transcription of the protease nexin 1 (SERPINE2) via the cAMP-PKA-SP1 pathway. This inhibits the degradation of granule proteins within the Golgi complex, thereby promoting granule formation (UniProt accession number: P10645).

[0262] Fascin (FSCN1)

[0263] FSCN1 organizes filamentous actin into bundles, maintaining an actin / fasin ratio of at least 4.1:1. FSCN1 plays a role in the organization of actin filament bundles and the formation of microprojections, membrane folds, and stress fibers. FSCN1 is important for the formation of diverse sets of cell projections such as filopopods, as well as for cell motility and migration (UniProt accession number: Q16658).

[0264] GMP reductase 2 (GMPR2)

[0265] GMPR2 catalyzes the irreversible NADPH-dependent deamination of GMP to IMP. GMPR2 functions in the conversion of nucleobases, nucleoside derivatives, and nucleotide derivatives from G nucleotides to A nucleotides, as well as in maintaining the intracellular balance between A nucleotides and G nucleotides. GMPR2 plays a role in modulating cell differentiation (UniProt accession number: Q9P2T1).

[0266] 78kDa glucose-regulating protein (GRP78 or HSPA5)

[0267] GRP78 plays a role in facilitating the assembly of multimeric protein complexes within the endoplasmic reticulum. GRP78 is likely involved in the proper folding of proteins and the degradation of misfolded proteins through its interaction with DNAJC10, facilitating its release from its substrate (due to similarity). GRP78 is thought to be a secreted protein (UniProt accession number: P11021).

[0268] Glutamate cysteine ​​ligase catalytic subunit (GSH1 or GCLC)

[0269] GSH1, also known as gamma-glutamylcysteine ​​synthetase, is the first rate-limiting enzyme in glutathione synthesis. The enzyme consists of two subunits: a heavy-chain catalytic subunit and a light-chain regulatory subunit. The GSH1 locus encodes the catalytic subunit, while the regulatory subunit originates from a different gene located on chromosomes 1p22-p21. Mutations at the GSH1 locus are associated with hemolytic anemia due to gamma-glutamylcysteine ​​synthetase deficiency and are associated with a higher risk of myocardial infarction (UniProt accession number: P48506).

[0270] Cytoplasmic isocitrate dehydrogenase [NADP] (IDHC or IDH1)

[0271] IDHC is an enzyme encoded by the IDH1 gene on chromosome 2 in humans. Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate. These enzymes belong to two significantly different subclasses, one of which uses NAD + as an electron acceptor and the other uses NADP + as an electron acceptor. Five isocitrate dehydrogenases have been reported: three NAD + -dependent isocitrate dehydrogenases localized in the mitochondrial matrix and two NADP + -dependent isocitrate dehydrogenases, one of which is mitochondrial and the other is mainly cytosolic. Each NADP + -dependent isozyme is a homodimer. The protein encoded by the IDHC gene is the NADP + -dependent isocitrate dehydrogenase found in the cytoplasm and peroxisomes. IDHC contains a PTS (peroxisomal targeting signal)-1 sequence. The presence of this enzyme in peroxisomes suggests a role in the regeneration of NADPH for peroxisomal reduction such as the conversion of 2,4-dienoyl-CoA to 3-enoyl-CoA and peroxisomal reactions that consume 2-oxoglutarate, i.e., in the alpha-hydroxylation of phytanic acid. Cytosolic IDHC plays a prominent role in the production of NADPH in the cytoplasm. Alternative spliced transcript variants encoding the same protein have also been found for the IDHC gene (UniProt basic accession number: O75874).

[0272] Keratin type I cytoskeletal 20 (K1C20 or KRT20)

[0273] K1C20 plays a prominent role in maintaining the organization of keratin filaments in intestinal epithelial cells. When phosphorylated, K1C20 plays a role in the secretion of mucus in the small intestine (by similarity) (UniProt primary accession number: P35900).

[0274] KRR1 small subunit processome component homolog (KRR1)

[0275] KRR1 is required for the biogenesis of the 40S ribosome. Kv is involved in the processing of pre-18S ribosomal RNA by nucleoli and ribosome assembly (by similarity) (UniProt primary accession number: Q13601).

[0276] Mannose-binding protein C (MBL2)

[0277] MBL2 is a calcium-dependent lectin involved in innate immune defense. MBL2 binds to mannose, fucose and N-acetylglucosamine in different microorganisms and activates the lectin complement pathway. MBL2 binds to late apoptotic cells as well as apoptotic bodies and necrotic cells, but does not bind to early apoptotic cells, facilitating their uptake by macrophages. MBL2 can bind to DNA (UniProt primary accession number: P11226).

[0278] Nuclear transport factor 2 (NTF2 or NUTF2)

[0279] NTF2 mediates the import of GDP-bound RAN, which is essential for the function of RAN in cargo receptor-mediated nuclear-cytoplasmic transport, from the cytoplasm into the nucleus. Thereby, NTF2 indirectly plays a general role in cargo receptor-mediated nuclear-cytoplasmic transport. NTF2 interacts with GDP-bound RAN in the cytosol and mobilizes GDP-bound RAN to the nuclear pore complex via its interaction with nucleoporins, promoting its nuclear import (UniProt primary accession number: P61970).

[0280] Phosphoglycerate kinase 1 (PGK1)

[0281] In addition to its role as a glycolytic enzyme, PGK-1 acts as a polymerase alpha cofactor protein (primer recognition protein). PGK1 may play a role in sperm motility (UniProt accession number: P00558).

[0282] Serum amyloid A-1 protein (SAA1)

[0283] SAA1 is a protein encoded by the SAA1 gene in humans. SAA1 is a major acute-phase protein primarily produced by hepatocytes, and it responds to infection, tissue damage, and malignant tumors. When released into the bloodstream, SAA1 exists as an apolipoprotein that associates with high-density lipoprotein (HDL). SAA1 is a major precursor to amyloid A (AA), whose deposits lead to inflammatory amyloidosis (UniProt accession number: P0DJI8).

[0284] Transferrin receptor protein 1 (TFR1 or TFRC)

[0285] Iron uptake into cells occurs via receptor-mediated endocytosis of ligand-occupied transferrin receptors to specialized endosomes. Acidification of the endosomes leads to iron release. The apotransferrin-receptor complex is then recycled to the cell surface, along with the return to neutral pH and the associated loss of apotransferrin's affinity for its receptor. The transferrin receptor is required for the development of red blood cells and the nervous system (due to similarity). A second ligand, the hereditary hemochromatosis protein HFE, competes with transferrin for binding to its overlapping C-terminal binding site. TFR1 positively regulates T cell and B cell proliferation through iron uptake (UniProt accession number: P02786).

[0286] In some embodiments, one or more of the TBI biomarkers listed above may be used to determine that a subject has TBI based on the detection of one or more of these TBI biomarkers in a sample derived from the subject and the absence of detection of one or more TBI biomarkers in a control subject (e.g., a subject without TBI) or, optionally, the absence of detection in a subject with severe TBI. These TBI biomarkers may include one or more of the following: 1433G, ACK1, ACY1, AKA12, ARGI1, CADH5, CLH1, COPG2, DPOD2, DSG2, HV307, IQGA2, K1C14, K1C19, KV105, LAMC1, MDHM, NQO2, PERM, PLST, PNCB, PTPRC, SEPT7, SYRC, TRXR2, TXNL1, UGGG1, WDR1, AHNK, AL1A1, AMPN, CAPZB, CATD, CLIP2, CMGA, FSCN1, GMPR2, GRP78, GSH1, IDHC, K1C20, KRR1, MBL2, NTF2, PCBP2, PGK1, SAA1, TFR1, or any combination thereof. Measurement or detection of one or more of these TBI biomarkers in a subject may be sufficient to indicate that the subject has mild TBI, independently of the need to detect, measure, compare and / or quantify the amount, concentration and / or expression level of one or more TBI biomarkers in a control subject or a subject with severe TBI. One or more of these TBI biomarkers may be present in a control subject or a subject without TBI, but generally in amounts that are not detectable by conventional means as described herein. Therefore, in some cases, detection of one or more of these TBI biomarkers in a subject may indicate that the subject has mild TBI.

[0287] In some embodiments, one or more of the TBI biomarkers listed above may be used to determine that a subject has mild TBI of a particular subclass. Without being bound by any particular theory, underlying etiology, or disease mechanism, one or more of these TBI biomarkers may be used to classify a subject with mild TBI into one of four subclasses (e.g., subclass 1, subclass 2, subclass 3, or subclass 4). For example, detection or measurement of one or more of ACK1, ACY1, PLST, PNCB, PTPRC, UGGG1, or any combination thereof, in a subject potentially suffering from TBI may indicate that the subject actually has mild TBI of subclass 1. Detection or measurement of one or more of AKA12, HV307, PERM, KV105, NQO2, SEPT7, SYRC, TRXR2, or any combination thereof, in a subject potentially suffering from TBI may indicate that the subject actually suffered from subclass 2 mild TBI. Furthermore, detection or measurement of one or more of 1433B, ARGI1, CADH5, CLH1, COPG2, DPOD2, DSG2, IQGA2, K1C14, LAMC1, MDHM, TXNL1, or any combination thereof, in a subject potentially suffering from TBI may indicate that the subject actually suffered from subclass 3 mild TBI. The levels of these TBI biomarkers may be detected or measured individually or in combination as part of a panel or signature of mild TBI.

[0288] In some embodiments, one or more of the TBI biomarkers listed above may be included together with other biomarkers that may or may not be identified as TBI biomarkers. For example, one or more of the TBI biomarkers listed above may be included in a panel of biomarkers that may include one or more of the following: AHNK, AL1A1, AMPN, CAPZB, CATD, CLIP2, CMGA, FSCN1, GMPR2, GRP78, GSH1, IDHC, K1C20, KRR1, MBL2, NTF2, PCBP2, PGK1, SAA1, TFR1, or combinations thereof. Depending on the circumstances, a panel of TBI biomarkers, including other TBI biomarkers and non-TBI biomarkers, may be more helpful in diagnosing TBI than individual biomarkers alone.

[0289] c. Mild TBI signature

[0290] The following mild TBI biomarkers, described below, have been identified as being able to distinguish healthy subjects from subjects with mild TBI and / or specific subclasses of mild TBI, when detected individually or in combination.

[0291] Ephrin receptor 4 (EPHB4)

[0292] Ephrin receptor 4 is a protein encoded by the EPHB4 gene in humans. Ephrin receptors and their ligands, ephrins, mediate numerous developmental processes, particularly in the nervous system. Based on their structural and sequence relationships, ephrins are divided into the ephrin A (EFNA) class, which are membrane-anchored by glycosylphosphatidylinositol linkage, and the ephrin B (EFNB) class, which are transmembrane proteins. The Eph family of receptors is further divided into two groups based on the similarity of their extracellular domain sequences and their affinity for binding to ephrin A and ephrin B ligands. Ephrin receptors constitute the largest subgroup of the receptor tyrosine kinase (RTK) family. The protein encoded by the EPHB4 gene binds to ephrin B2 and plays an essential role in vascular development (UniProt accession number: P54760).

[0293] Immunoglobulin variable heavy chain 1-3 (HV103 or IGHV1-3)

[0294] HV103 is the V region of the variable domain of the immunoglobulin heavy chain, which participates in antigen recognition. Immunoglobulins, also known as antibodies, are membrane-bound or secreted glycoproteins produced by B lymphocytes. In the recognition phase of humoral immunity, membrane-bound immunoglobulins, upon binding to a specific antigen, act as receptors that induce clonal expansion and differentiation of B lymphocytes into immunoglobulin-secreting plasma cells. The secreted immunoglobulin mediates the effector phase of humoral immunity, resulting in the elimination of the bound antigen. The antigen-binding site is formed by one variable domain of the heavy chain, combined with the variable domain of the light chain to which it associates. Therefore, each immunoglobulin has two antigen-binding sites with significant affinity for a particular antigen. The variable domain is assembled through a process called V-(D)-J rearrangement and then subjected to somatic hypermutation, which enables affinity maturation to a specific antigen after exposure to and selection of the antigen (UniProt accession number: A0A0C4DH29).

[0295] Immunoglobulin constant heavy chain delta (IGHD)

[0296] IGHD is the constant region of the immunoglobulin heavy chain. Immunoglobulins, also known as antibodies, are membrane-bound or secreted glycoproteins produced by B lymphocytes. In the recognition phase of humoral immunity, membrane-bound immunoglobulins are used as receptors that induce clonal expansion and differentiation of B lymphocytes into plasma cells that secrete immunoglobulins when specific antigens bind. Secreted immunoglobulins mediate the effector phase of humoral immunity, resulting in the disappearance of the bound antigen. The antigen-binding site is formed by one variable domain of a heavy chain in conjunction with the variable domain of its associated light chain. Thus, each immunoglobulin has two antigen-binding sites with significant affinity for a specific antigen. The variable domains are assembled by a process called V-(D)-J rearrangement and are then subject to somatic hypermutation, which enables affinity maturation for specific antigens after exposure to and selection by the antigen. IgD is the major antigen receptor isotype on the surface of most peripheral B cells, and on the surface of peripheral B cells, IgD is co-expressed with IgM. Membrane-bound IgD (mIgD) induces phosphorylation of CD79A and CD79B by the Src family of protein tyrosine kinases. The serum concentration of soluble IgD (sIgD) is lower than the concentrations of IgG, IgA, and IgM but is much higher than the concentration of IgE. IgM molecules and IgD molecules present on B cells have the same V region and antigen-binding site. After an antigen binds to the B cell receptor, sIgD in its secreted form disappears. IgD is a potent inducer of TNF, IL1B, and IL1RN. IgD also induces the release of IL6, IL10, and LIF from peripheral blood mononuclear cells. Monocytes are thought to be the main cytokine-producing cells in vitro in the presence of IgD (UniProt basic accession number: P01880).

[0297] Esterase C11orf54 (CK054 or C11orf54)

[0298] CK054 is a protein encoded in humans by the C11orf54 gene. The human gene C11orf54 is also known as PTD012 and PTOD12. C11orf54 exhibits hydrolase activity on p-nitrophenyl acetate and acts on ester bonds, but its overall function is not yet fully understood by the scientific community. CK054 is highly conserved to its most distant homolog found in bacteria (UniProt accession number: Q9H0W9).

[0299] Epididymis-specific alpha-mannosidase (MA2B2 or MAN2B2)

[0300] MA2B2 catalyzes the hydrolysis of the terminal non-reducing alpha-D-mannose residue within alpha-D-mannoside (UniProt accession number: Q9Y2E5).

[0301] Protein diaphanous homolog 1 (DIAP1 or DIAPH1)

[0302] DIAP1 acts in a Rho-dependent manner to recruit PFY1 to the membrane. DIAP1 is required for the assembly of F-actin structures, such as actin cables and stress fibers. DIAP1 nucleates actin filaments. DIAP1 binds to the curved end of actin filaments, slowing actin polymerization and depolymerization. DIAP1 is required for cytokinesis and transcriptional activation of serum response factors. DFR proteins couple Rho with Src tyrosine kinases in the regulation of signal transduction and actin dynamics. DIAP1 functions as a scaffold protein for MAPR1 and APC, stabilizing microtubules and promoting cell migration (by similarity). DIAP1 has neurite outgrowth-promoting activity (by similarity). In hair cells, DIAP1 may play a role in regulating actin polymerization within hair cells. The MEMO1-RHOA-DIAPH1 signaling pathway plays a crucial role in ERBB2-dependent stabilization of microtubules in the cell cortex. DIAP1 regulates the localization of APC and CLASP2 to the cell membrane through the regulation of GSK3B activity. Membrane-bound APC enables the localization of MACF1 to the cell membrane, which is required for microtubule capture and stabilization. DIAP1 plays a role in regulating cell morphology and organizing the cytoskeleton. DIAP1 is required for the control of cell shape. DIAP1 plays a role in brain development (UniProt accession number: O60610).

[0303] Procollagen lysine 2-oxoglutamic acid 5-dioxygenase 1 (PLOD1)

[0304] PLOD1 is part of a complex composed of PLOD1, P3H3, and P3H4 (by similarity) that catalyzes the hydroxylation of lysine residues within the collagen alpha chain and is required for the normal assembly and crosslinking of collagen fibrils. PLOD1 forms hydroxylysine residues within the -Xaa-Lys-Gly- sequence in collagen. These hydroxylysines are used as binding sites to carbohydrate units and are (probably) essential for the stability of intermolecular collagen crosslinks (UniProt accession number: Q02809).

[0305] Immunoglobulin variable kappa 1-33 (KV133 or IGKV1-33)

[0306] KV133 is the V region of the variable domain of the immunoglobulin light chain, which participates in antigen recognition. Immunoglobulins, also known as antibodies, are membrane-bound or secreted glycoproteins produced by B lymphocytes. In the recognition phase of humoral immunity, membrane-bound immunoglobulins, upon binding to a specific antigen, act as receptors that induce clonal expansion and differentiation of B lymphocytes into immunoglobulin-secreting plasma cells. The secreted immunoglobulin mediates the effector phase of humoral immunity, resulting in the elimination of the bound antigen. The antigen-binding site is formed by one variable domain of the heavy chain, combined with the variable domain of the light chain to which it associates. Therefore, each immunoglobulin has two antigen-binding sites with significant affinity for a particular antigen. The variable domain is assembled through a process called V-(D)-J rearrangement and then subjected to somatic hypermutation, which enables affinity maturation to a specific antigen after exposure to and selection (UniProt accession number: P01594).

[0307] As disclosed herein, the following biomarkers: TPP2, CAND1, NCOR1, K22E, AL9A1, ABHEB, DNM1L, INF2, M3K5, SBSN, SYEP, MYLK, and SAMP have also been identified as being able to distinguish healthy subjects from subjects with mild TBI and / or specific subclasses of mild TBI when detected individually or in combination with each other and in combination with other biomarkers described in this section. A description of these biomarkers has been given above and will not be repeated here.

[0308] In some embodiments, one or more of the TBI biomarkers listed above may be used to determine that a subject has mild TBI of a particular subclass. Without being bound by any particular theory, underlying etiology, or disease mechanism, one or more of these TBI biomarkers may be used to classify a subject with mild TBI into one of four subclasses (e.g., subclass 1, subclass 2, subclass 3, or subclass 4). In some embodiments, one or more of the TBI biomarkers listed above may be used to determine that a subject has mild TBI based on the detection of one or more of these TBI biomarkers in a sample derived from the subject at a higher or lower level of quantity, concentration, and / or expression than the corresponding TBI biomarker in a control subject. Measurement or detection of an increase or decrease in the level of one or more of these TBI biomarkers in a subject compared to a control subject (e.g., a subject without TBI) may be sufficient to indicate that the subject has mild TBI, and in some cases may be sufficient to indicate that the subject has a particular subclass of mild TBI.

[0309] For example, detection or measurement of one or more of TPP2, CAND1, NCOR1, K22E, AL9A1, ABHEB, DNM1L, INF2, or any combination thereof, in a subject that may have suffered TBI may indicate that the subject actually suffered mild subclass 4 TBI. In some cases, detection or measurement of high levels of CAND1, NCOR1, K22E, ABHEB, DNM1L, or any combination thereof, and / or low levels of TPP2, AL9A1, INF2, or any combination thereof, in a subject that may have suffered TBI, compared to the levels in a control subject, may indicate that the subject actually suffered mild subclass 4 TBI.

[0310] In some embodiments, detection or measurement of one or more of TPP2, NCOR1, HV103, INF2, IGHD, CK054, M3K5, ABHEB, AL9A1, DNM1L, or any combination thereof, in a subject that may have suffered TBI may indicate that the subject actually suffered a mild subclass 3 TBI. In some cases, detection or measurement of high levels of NCOR1, HV103, IGHD, ABHEB, DNM1L, or any combination thereof, and / or low levels of TPP2, IGHD, CK054, M3K5, AL9A1, or any combination thereof, in a subject that may have suffered TBI, compared to levels in a control subject, may indicate that the subject actually suffered a mild subclass 3 TBI.

[0311] In some embodiments, detection or measurement of one or more of NCOR1, TPP2, K22E, ABHEB, INF2, SBSN, AL9A1, MA2B2, or any combination thereof, in a subject that may have suffered TBI may indicate that the subject actually suffered subclass 2 mild TBI. In some cases, detection or measurement of high levels of NCOR1, K22E, ABHEB, SBSN, or any combination thereof, and / or low levels of TPP2, INF2, AL9A1, MA2B2, or any combination thereof, in a subject that may have suffered TBI, compared to levels in a control subject, may indicate that the subject actually suffered subclass 2 mild TBI.

[0312] In other embodiments, detection or measurement of one or more of K22E, DNM1L, DIAP1, ABHEB, PLOD1, SYEP, KV133, AL9A1, EPHB4, or any combination thereof, in a subject that may have suffered TBI may indicate that the subject actually suffered subclass 1 mild TBI. Depending on the circumstances, detection or measurement of high levels of K22E, DNM1L, DIAP1, ABHEB, PLOD1, SYEP, EPHB4, or any combination thereof, and / or low levels of KV133 and AL9A1, or any combination thereof, in a subject that may have suffered TBI may indicate that the subject actually suffered subclass 1 mild TBI.

[0313] The levels of these TBI biomarkers can be detected or measured individually or in combination as part of a panel or signature of mild TBI.

[0314] In some embodiments, one or more of the TBI biomarkers listed above may be included together with other biomarkers that may or may not be identified as TBI biomarkers. For example, one or more of the TBI biomarkers listed above may be included in a panel of biomarkers that may include one or more of MYLK, SAMP, or combinations thereof. In some cases, a panel of TBI biomarkers, including other TBI biomarkers and non-TBI biomarkers, may be more helpful in diagnosing TBI than individual biomarkers alone.

[0315] The following mild TBI biomarkers, described below, have been identified as being able to distinguish healthy subjects from subjects with mild TBI and / or specific subclasses of mild TBI, when detected individually or in combination. Cytoplasmic dynein light chain 1 (DYL1 or DYNLL1) DYL1 acts as one of several non-catalytic accessory components of the cytoplasmic genein 1 complex, and is thought to be involved in linking genein to cargo and adapter proteins that regulate genein function. Cytoplasmic genein 1 acts as a motor for the intracellular reverse movement of vesicles and organelles along microtubules. DYL1 may play a role in altering or maintaining the spatial distribution of cytoskeletal structures. DYL1 binds to neuronal nitric oxide synthase and inhibits its catalytic activity. DYL1 promotes the transactivation function of ESR1 and plays a role in the nuclear localization of ESR1. DYL1 regulates the apoptotic activity of BCL2L11 by occluding BCL2L11 to microtubules. Upon apoptosis stimulation, the BCL2L11-DYNLL1 complex dissociates from cytoplasmic dynain, translocates to mitochondria, and occludes BCL2, thereby neutralizing its anti-apoptotic activity (UniProt accession number: P63167).

[0316] Puromycin-sensitive aminopeptidase (PSA or NPEPPS)

[0317] PSA is an aminopeptidase with broad substrate specificity for several peptides. PSA is involved in proteolytic events essential for cell proliferation and survival. PSA can act as a regulator of neuropeptide activity. PSA plays a role in the antigen processing pathway for MHC class I molecules. PSA is involved in N-terminal trimming of cytotoxic T cell epitope precursors. PSA digests poly-Q peptides found in many intracellular proteins. PSA digests tau from normal brains more efficiently than tau from Alzheimer's disease brains (UniProt accession number: P55786).

[0318] EGF-containing fibrin-like extracellular matrix protein 1 (FBLN3 or EFEMP1)

[0319] FBLN3 binds to EGFR, an EGF receptor, and induces EGFR autophosphorylation and activation of downstream signaling pathways. FBLN3 may play a role in cell adhesion and migration. FBLN3 may function as a negative regulator of chondrocyte differentiation. In the olfactory epithelium, FBLN3 may regulate glial cell migration, differentiation, and the ability of glial cells to support neuronal neurite outgrowth (UniProt accession number: Q12805).

[0320] Serum albumin (ALBU or ALB)

[0321] ALBU is the main protein in plasma, and is composed of water and calcium. 2+ kaNa + , K + It has good binding ability to fatty acids, hormones, bilirubin, and drugs. Its main function is the regulation of colloidal osmotic pressure in blood. ALBU is the major zinc transporter in plasma and typically binds to about 80% of all plasma zinc (UniProt accession number: P02768).

[0322] Mitochondrial 3-ketoacyl-CoA thiolase (THIM or ACAA2)

[0323] THIM is involved in the elimination of BNIP3-mediated apoptosis and mitochondrial damage (UniProt accession number: P42765). Immunoglobulin variable kappa 1-39 (KV139 or IGKV1-39)

[0324] KV139 is the V region of the variable domain of the immunoglobulin light chain, which participates in antigen recognition. Immunoglobulins, also known as antibodies, are membrane-bound or secreted glycoproteins produced by B lymphocytes. In the recognition phase of humoral immunity, membrane-bound immunoglobulins, upon binding to a specific antigen, act as receptors that induce clonal expansion and differentiation of B lymphocytes into immunoglobulin-secreting plasma cells. Secreted immunoglobulins mediate the effector phase of humoral immunity, resulting in the elimination of the bound antigen. The antigen-binding site is formed by one variable domain of the heavy chain, combined with the variable domain of the light chain to which it associates. Therefore, each immunoglobulin has two antigen-binding sites with significant affinity for a particular antigen. The variable domain is assembled through a process called V-(D)-J rearrangement and then subjected to somatic hypermutation, which enables affinity maturation to a specific antigen after exposure to and selection (UniProt accession number: P01597).

[0325] Mannan-binding lectin serine protease 2 (MASP2)

[0326] MASP2 is a serum protease that plays a crucial role in the activation of the complement system via mannose-binding lectins. Following activation by autocatalytic cleavage, MASP2 cleaves C2 and C4, leading to their activation and the formation of C3 converterase (UniProt accession number: O00187).

[0327] As disclosed herein, the following mild TBI biomarkers: ANXA6, CAND1, NCOR1, K22E, ABHEB, DIAP1, DNM1L, EPHB4, GLO2, HV103, IGHA2, IGHD, PLOD1, SBSN, SYEP, MYLK, and SAMP have also been identified as being able to distinguish healthy subjects from subjects with mild TBI and / or specific subclasses of mild TBI when detected individually, in combination with each other, and in combination with other biomarkers described in this section. A description of these biomarkers has been given above and will not be repeated here.

[0328] In some embodiments, one or more of the TBI biomarkers listed above may be used to determine that a subject has mild TBI of a particular subclass. Without being bound by any particular theory, underlying etiology, or disease mechanism, one or more of these TBI biomarkers may be used to classify a subject with mild TBI into one of four subclasses (e.g., subclass 1, subclass 2, subclass 3, or subclass 4). In some embodiments, one or more of the TBI biomarkers listed above may be used to determine that a subject has mild TBI based on the detection of one or more of these TBI biomarkers in a sample derived from the subject at a higher or lower level of quantity, concentration, and / or expression of the corresponding TBI biomarker in a control subject. Measurement or detection of an increase or decrease in the level of one or more of these TBI biomarkers in a subject compared to a control subject (e.g., a subject without TBI) may be sufficient to indicate that the subject has mild TBI, and in some cases may be sufficient to indicate that the subject has a particular subclass of mild TBI.

[0329] For example, detection or measurement of one or more of CAND1, NCOR1, K22E, ABHEB, DNM1L, SBSN, GLO2, SYEP, or any combination thereof in a subject that may have suffered TBI may indicate that the subject actually suffered mild TBI of subclass 4. In some cases, detection or measurement of high levels of CAND1, NCOR1, K22E, ABHEB, DNM1L, SBSN, GLO2, SYEP, or any combination thereof in a subject that may have suffered TBI may indicate that the subject actually suffered mild TBI of subclass 4.

[0330] In some embodiments, detection or measurement of one or more of NCOR1, HV103, IGHD, ABHEB, DNM1L, ALBU, THIM, IGHA2, KV139, or any combination thereof, in a subject that may have suffered TBI may indicate that the subject actually suffered subclass 3 mild TBI. In some cases, detection or measurement of high levels of NCOR1, HV103, IGHD, ABHEB, DNM1L, ALBU, THIM, IGHA2, KV139, or any combination thereof, in a subject that may have suffered TBI, compared to levels in a control subject, may indicate that the subject actually suffered subclass 3 mild TBI.

[0331] In some embodiments, detection or measurement of one or more of NCOR1, K22E, ABHEB, SBSN, DNM1L, DIAP1, DYL1, PSA, EPHB4, or any combination thereof, in a subject that may have suffered TBI may indicate that the subject actually suffered subclass 2 mild TBI. In some cases, detection or measurement of high levels of NCOR1, K22E, ABHEB, SBSN, DNM1L, DIAP1, DYL1, PSA, EPHB4, or any combination thereof, in a subject that may have suffered TBI, compared to levels in a control subject, may indicate that the subject actually suffered subclass 2 mild TBI.

[0332] In other embodiments, detection or measurement of one or more of K22E, DNM1L, DIAP1, ABHEB, PLOD1, SYEP, EPHB4, FBLN3, or any combination thereof, in a subject that may have suffered TBI may indicate that the subject actually suffered a mild subclass 1 TBI. In some cases, detection or measurement of high levels of K22E, DNM1L, DIAP1, ABHEB, PLOD1, SYEP, EPHB4, FBLN3, or any combination thereof, in a subject that may have suffered TBI, compared to levels in a control subject, may indicate that the subject actually suffered a mild subclass 1 TBI.

[0333] The levels of these TBI biomarkers can be detected or measured individually or in combination as part of a panel or signature of mild TBI.

[0334] In some embodiments, one or more of the TBI biomarkers listed above may be included together with other biomarkers that may or may not be identified as TBI biomarkers. For example, one or more of the TBI biomarkers listed above may be included in a panel of biomarkers that may include one or more of ANXA6, MASP2, MYLK, SAMP, or combinations thereof. In some cases, a panel of TBI biomarkers, including other TBI biomarkers and non-TBI biomarkers, may be more helpful in diagnosing TBI than individual biomarkers alone.

[0335] d. TBI biomarkers for exclusion

[0336] The following TBI biomarkers, described below, have been identified as being able to distinguish healthy subjects from subjects with TBI when detected individually or in combination.

[0337] SH3 domain-binding glutamate-rich-like protein (SH3BGRL)

[0338] SH3-domain-binding glutamate-rich-like protein 3 is a protein encoded in humans by the SH3BGRL3 gene. A 10.5 kDa protein, SH3-domain-binding glutamate-rich-like protein 3 has an isoelectric point of 5.0. The SH3-domain-binding glutamate-rich (SH3BGR) gene is located on human chromosome 21. Two homologous genes, SH3BGRL and SH3BGRL3, are located on chromosomes Xq13.3 and 1p34.3-35, respectively, and encode small proteins similar to the N-terminal region of the SH3BGR protein. The SH3BGRL3 protein shows remarkable similarity to glutaredoxin 1 from E. coli, and all three proteins are predicted to belong to the thioredoxin-like protein family. Glutaredoxin (GRX) is a ubiquitous oxidoreductase that catalyzes the disulfide reduction of many intracellular proteins and plays a crucial role in many redox pathways. However, the SH3BGRL3 protein lacks the enzymatic function of glutaredoxin and may play a role as a regulator of redox activity (UniProt accession number: O75368).

[0339] Beta-actin-like protein 2 (ACTBL or ACTBL2)

[0340] ACTBL is a member of the actin family. Actin is a highly conserved protein involved in various types of cell motility and is ubiquitously expressed in all eukaryotic cells (UniProt accession number: Q562R1).

[0341] Mitochondrial aldehyde dehydrogenase (ALDH2)

[0342] ALDH2 catalyzes the oxidation of aldehydes. Despite the name "dehydrogenase," their oxidation method involves the addition of oxygen rather than the removal of hydrogen (i.e., ALDH2 converts aldehydes (RC(=O)-H) to carboxylic acids (RC(=O)-OH)). Currently, 19 ALDH genes have been identified in the human genome. ALDH genes participate in a wide variety of biological processes, including the detoxification of aldehydes generated exogenously and endogenously (UniProt accession number: P05091).

[0343] Annexin A5 (ANXA5)

[0344] ANXA5 is an anticoagulant protein that acts as an indirect inhibitor of thromboplastin-specific complexes involved in the blood coagulation cascade (UniProt accession number: P08758).

[0345] Cathelicidine antimicrobial peptide (CAMP)

[0346] CAMP binds to bacterial lipopolysaccharide (LPS) and exhibits antibacterial activity (UniProt accession number: P49913).

[0347] Copin 3 (CPNE3)

[0348] CPNE3 is a calcium-dependent phospholipid-binding protein that plays a role in ERBB2-mediated tumor cell migration in response to stimulation by the growth factor helegrin (UniProt accession number: O75131).

[0349] Cartilage acid protein 1 (CRAC1 or CRTAC1)

[0350] CRAC1 encodes a glycosylated extracellular matrix protein found in the interregional matrix of deep articular cartilage. CRAC1 is used as a marker to distinguish chondrocytes from osteoblasts and mesenchymal stem cells in cultures. The presence of FG-GAP and RGD integrin-binding motifs suggests that the CRAC1 protein may be involved in intercellular or cell-matrix interactions. Copy number variations within the CRAC1 gene have been observed in type 1 neurofibromatosis-associated glomus tumors. Alternative splicing results in multiple transcript variants (UniProt accession number: Q9NQ79).

[0351] Cystatin C (CYTC or CST3)

[0352] As a cysteine ​​proteinase inhibitor, CYTC is thought to play an important physiological role as a local regulator of this enzyme activity (UniProt accession number: P01034).

[0353] Aspartylaminopeptidase (DNPEP)

[0354] DNPEP is an aminopeptidase with specificity for acidic amino acids at its N-terminus. DNPEP is likely to play an important role in intracellular protein and peptide metabolism (UniProt accession number: Q9ULA0).

[0355] Eukaryotic translation initiation factor 3 subunit I (EIF3I)

[0356] EIF3I is a component of the eukaryotic translation initiation factor 3 (eIF-3) complex, required for several steps in the initiation of protein synthesis. The eIF-3 complex facilitates the recruitment of eIF-1, eIF-1A, eIF-2:GTP:methionyl-tRNAi, and eIF-5 to associate with the 40S ribosome and form the 43S pre-initiation complex (43S PIC). The eIF-3 complex stimulates the recruitment of mRNA to the 43S PIC and the scanning of mRNA for AUG recognition. The eIF-3 complex is also required for the disassembly and recycling of the ribosome complex after termination, and subsequently prevents immature conjugation between the 40S ribosomal subunit and the 60S ribosomal subunit before initiation. The eIF-3 complex specifically targets subsets of mRNAs involved in cell proliferation, including the cell cycle, differentiation, and apoptosis, initiating their translation and activating or repressing translation using different mechanisms of RNA stem-loop junctioning (UniProt accession number: Q13347).

[0357] Glutathione synthetase (GSHB or GSS)

[0358] GSHB is the second enzyme in the glutathione (GSH) biosynthesis pathway. GSHB catalyzes the condensation of gamma-glutamylcysteine ​​and glycine to form glutathione. Glutathione synthetase is also a potent antioxidant. GSHB is found in numerous species, including bacteria, yeast, mammals, and plants. In humans, GSS deficiency is inherited in an autosomal recessive manner and causes severe metabolic acidosis, 5-oxoprolinuria, increased hemolysis rate, and central nervous system dysfunction. GSS deficiency can similarly cause a spectrum of adverse symptoms in plants and humans. In eukaryotes, GSS is a homodimeric enzyme. The substrate-binding domain has a three-layer alpha / beta / alpha structure. This enzyme then uses and stabilizes an acyl phosphate intermediate to carry out a suitable nucleophilic attack on glycine (UniProt accession number: P48637).

[0359] Intercellular adhesion molecule 1 (ICAM1)

[0360] The ICAM1 protein is a ligand for the leukocyte adhesion protein LFA-1 (integrin alpha-L / beta-2). During transendothelial migration of leukocytes, engagement with ICAM1 promotes the assembly of the endothelial apical cup via the activation of ARHGEF26 / SGEF and RHOG. ICAM1 acts as a receptor for the major receptor group of A- and B rhinovirus capsid proteins. ICAM1 also acts as a receptor for the Coxsackievirus A21 capsid protein. During Kaposi's sarcoma-associated herpesvirus / HHV-8 infection, ICAM1 is likely degraded by the viral E3 ubiquitin ligase MIR2 (UniProt accession number: P05362) to prevent lysis of infected cells by cytotoxic T lymphocytes and NK cells.

[0361] (HV323 or IGHV3-23)

[0362] HV323 is the V region of the variable domain of the immunoglobulin heavy chain, which participates in antigen recognition. Immunoglobulins, also known as antibodies, are membrane-bound or secreted glycoproteins produced by B lymphocytes. In the recognition phase of humoral immunity, membrane-bound immunoglobulins, upon binding to a specific antigen, act as receptors that induce clonal expansion and differentiation of B lymphocytes into immunoglobulin-secreting plasma cells. The secreted immunoglobulin mediates the effector phase of humoral immunity, resulting in the elimination of the bound antigen. The antigen-binding site is formed by one variable domain of the heavy chain, combined with the variable domain of the light chain to which it associates. Therefore, each immunoglobulin has two antigen-binding sites with significant affinity for a particular antigen. The variable domain is assembled through a process called V-(D)-J rearrangement and then subjected to somatic hypermutation, which enables affinity maturation to a specific antigen after exposure to and selection (UniProt accession number: P01764).

[0363] Heteronuclear ribonucleoprotein D0 (HNRPD or HNRNPD)

[0364] HNRPD binds with high affinity to RNA molecules containing AU-rich elements (AREs) found within the 3'-UTR of many proto-oncogenes and cytokine mRNAs. HNRPD also specifically binds to double-stranded and single-stranded DNA sequences and functions as a transcription factor. Each RNA-binding domain can specifically bind only to single-stranded non-monotonous 5'-UUAG-3' sequences and can also weakly bind to single-stranded 5'-TTAGGG-3' telomere DNA repeats. HNRPD binds more tightly to RNA oligonucleotides with 5'-UUAGGG-3' repeats than to single-stranded telomere DNA 5'-TTAGGG-3' repeats. HNRPD's binding to DNA inhibits the formation of DNA quadruple-stranded structures, which can play a role in telomere elongation. HNRPD may be involved in translation-coupled mRNA turnover. HNRPD, along with other RNA-binding proteins in the cytoplasm, is involved in the cytoplasmic deadenylation / translation-degradation interaction of FOS mRNA, mediated by the major coding region determinants of its instability (mCRD) domain. HNRPD may play a role in regulating the rhythmic expression of circadian clock core genes. HNRPD directly binds to the 3'UTR of CRY1 mRNA and induces rhythmic translation of CRY1. HNRPD may also be involved in regulating PER2 translation (UniProt accession number: Q14103).

[0365] Immunoglobulin variable kappa 1D-33 (KVD33 or IGKV1D-33)

[0366] KVD33 is the V region of the variable domain of the immunoglobulin light chain, which participates in antigen recognition. Immunoglobulins, also known as antibodies, are membrane-bound or secreted glycoproteins produced by B lymphocytes. In the recognition phase of humoral immunity, membrane-bound immunoglobulins, upon binding to a specific antigen, act as receptors that induce clonal expansion and differentiation of B lymphocytes into immunoglobulin-secreting plasma cells. The secreted immunoglobulin mediates the effector phase of humoral immunity, resulting in the elimination of the bound antigen. The antigen-binding site is formed by one variable domain of the heavy chain, combined with the variable domain of the light chain to which it associates. Therefore, each immunoglobulin has two antigen-binding sites with significant affinity for a particular antigen. The variable domain is assembled through a process called V-(D)-J rearrangement and then subjected to somatic hypermutation, which enables affinity maturation to a specific antigen after exposure to and selection (UniProt accession number: P01593).

[0367] Coagulation factor IX (FA9 or F9)

[0368] Factor IX is Ca 2+ It is a vitamin K-dependent plasma protein that participates in the intrinsic pathway of blood coagulation by converting factor X to its active form in the presence of ions, phospholipids, and factor VIIIa (UniProt accession number: P00740). Complement factor H-related protein 4 (FHR4 or CFHR4) FHR4 is involved in complement regulation. FHR4 can associate with lipoproteins and may play a role in lipid metabolism (UniProt accession number: Q92496).

[0369] FERM and PDZ domain-containing protein 1 (FRPD1 or FRMPD1)

[0370] FRPD1 stabilizes membrane-bound GPSM1, thereby promoting its interaction with GNAI1 (UniProt accession number: Q5SYB0).

[0371] Heat shock protein HSP90 beta (HS90B or HSP90AB1)

[0372] HS90B is a molecular chaperone that facilitates the maturation, structural maintenance, and proper regulation of specific target proteins, such as those involved in cell cycle control and signal transduction. HS90B undergoes a functional cycle linked to its ATPase activity. This cycle likely induces conformational changes within client proteins, thereby triggering their activation. HS90B dynamically interacts with a variety of co-chaperones that modulate its substrate recognition, ATPase cycle, and chaperone function. HS90B engages with a range of client protein classes through its interactions with various co-chaperone proteins or complexes that act as adapters capable of interacting simultaneously with specific clients and the central chaperone itself. The recruitment of client proteins, following ATP and co-chaperones, forms a functional chaperone. After the chaperoning process is complete, the properly folded client protein and co-chaperone leave the HSP90, which is in a partially open conformation with ADP bound to it. Finally, the ADP is released from the HSP90, and the HSP90 acquires an open conformation for the next cycle. Apart from its chaperone activity, HSP90B also plays a role in regulating the transcription mechanism. HSP90 and its co-chaperone modulate transcription at at least three different levels. First, HSP90 and its co-chaperone alter the steady-state level of certain transcription factors in response to a variety of physiological cues. Second, HSP90 and its co-chaperone modulate the activity of certain epigenetic modifiers, such as histone deacetylase or DNA methyltransferase, thereby responding to changes in the environment. Third, HSP90 and its co-chaperone are involved in the exclusion of histones from the promoter region of certain genes, thereby turning on gene expression. HS90B antagonistizes STUB1-mediated inhibition of TGF beta signaling, mediated by the inhibition of STUB1-mediated ubiquitination and degradation of SMAD3.HS90B promotes cell differentiation by chaperoning BIRC2, thereby protecting BIRC2 from self-ubiquitination and degradation by the proteasome mechanism. Under heat shock, HS90B is a major chaperone involved in the phosphorylation / activation of STAT1 by chaperoning both JAK2 and PRKCE and activating their intrinsic transcription (UniProt accession number: P08238).

[0373] Alpha-mannosidase 2 (MA2A1 or MAN2A1)

[0374] MA2A1 catalyzes the initial involvement step in the biosynthesis of complex N-glycans. MA2A1 controls the final hydrolytic step in the N-glycan maturation pathway, the conversion of high-mannose to complex N-glycans (UniProt accession number: Q16706).

[0375] Prenylcysteine ​​oxidase 1 (PCYOX or PCYOX1)

[0376] PCYOX is involved in the degradation of prenylated proteins. PCYOX cleaves the thioether bond of prenyl-L-cysteine, such as farnesylcysteine ​​and geranylgeranylcysteine ​​(UniProt accession number: Q9UHG3).

[0377] Purine nucleoside phosphorylase (PNPH or PNP)

[0378] PNPH is a purine nucleoside phosphorylase (UniProt accession number: P00491) that catalyzes the phosphorolytic decomposition of the N-glycosidic bond within beta-(deoxy)ribonucleoside molecules, resulting in the formation of the corresponding free purine base and pentose-1-phosphate.

[0379] Vitamin K-dependent protein C (PROC)

[0380] PROC is a vitamin K-dependent serine protease that regulates blood coagulation by inactivating factor Va and factor VIIIa in the presence of calcium ions and phospholipids. PROC has a protective effect on the barrier function of endothelial cells (UniProt accession number: P04070).

[0381] 60S ribosomal protein L3 (RL3 or RPL3)

[0382] RL3 is a component of the large subunit of the cytoplasmic ribosome (UniProt accession number: P39023).

[0383] Serine / arginine repeat matrix protein 2 (SRRM2)

[0384] SRRM2 is involved in premRNA splicing. SRRM2 may function in the first catalytic step of splicing, or prior to it, at the catalytic center of the spliceosome. SRRM2 may function by stabilizing the catalytic center or the RNA substrate (by similarity). SRRM2 binds to RNA (UniProt accession number: Q9UQ35).

[0385] Tubulin beta single chain (TBB1 or TUBB1)

[0386] TBBI is a subunit of tubulin, the main component of microtubules. TBBI binds to 2 moles of GTP (due to similarity), with 1 mole at the interchangeable site on the beta chain and 1 mole at the non-interchangeable site on the alpha chain (UniProt accession number: Q9H4B7).

[0387] Tenascin (TENA or TNC)

[0388] TENA is an extracellular matrix protein involved in the induction of migrating neurons, axons during development and synaptic plasticity, and neuronal regeneration. TENA promotes neurite outgrowth from proliferating cortical neurons on monolayer astrocytes. TENA is a ligand for integrins alpha-8 / beta-1, alpha-9 / beta-1, alpha-V / beta-3, and alpha-V / beta-6. In tumors, TENA stimulates angiogenesis through endothelial cell elongation, migration, and sprouting (UniProt accession number: P24821).

[0389] Mitochondrial 75kDa heat shock protein (TRAP1)

[0390] TRAP1 is a chaperone that expresses ATPase activity. Downstream of PINK1 and mitochondrial complex I, TRAP1 is involved in maintaining mitochondrial function and polarization. TRAP1 is a negative regulator of mitochondrial respiration, capable of modulating the equilibrium between oxidative phosphorylation and aerobic glycolysis. The effects of TRAP1 on mitochondrial respiration are likely mediated by modulation of mitochondrial SRCs and inhibition of SDHA (UniProt accession number: Q12931).

[0391] In some embodiments, one or more of the TBI biomarkers listed above may be used to determine that a subject is not infected with TBI based on the detection of one or more of these TBI biomarkers in a sample derived from the subject (TBI biomarkers for exclusion). These TBI biomarkers may include one or more of ACTBL, ALDH2, ANXA5, CAMP, CPNE3, CRAC1, CYTC, DNPEP, EIF3I, GSHB, ICAM1, HV323, HNRPD, KVD33, FA9, FHR4, FRPD1, HS90B, MA2A1, PCYOX, PNPH, PROC, RL3, SH3L3, SRRM2, TBB1, TENA, TRAP1, or any combination thereof. Measurement or detection of one or more of these TBI biomarkers in a subject may be sufficient to indicate that the subject is not suffering from TBI, independently of the need to detect, measure, compare, and / or quantify the amount, concentration, and / or expression level of one or more TBI biomarkers in a control subject. One or more of these TBI biomarkers may be present in subjects suffering from TBI, but generally in amounts that are not detectable by conventional means as described herein. Therefore, in some cases, detection of one or more of these TBI biomarkers in a subject indicates that the subject is not suffering from TBI. The levels of these TBI biomarkers may be detected or measured individually or in combination as part of a panel or signature of mild TBI.

[0392] 7. Methods for measuring TBI biomarker levels

[0393] In the methods described above, TBI biomarker levels may be measured by any means, including 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 high-performance liquid chromatography (HPLC), mass spectrometry or liquid chromatography-mass spectrometry (LC / MS), or capillary electrophoresis (CE)-MS, or chromatographic or spectroscopic methods such as direct injection or any pre-separation step coupled with MS. Assays in clinical chemistry formats known to those skilled in the art may also be used.

[0394] In some embodiments, the step of measuring the level of a TBI biomarker includes contacting a sample with a first specific binding member and a second specific binding member. In some embodiments, the first specific binding member is a capture antibody, and the second specific binding member is a detection antibody. In some embodiments, the step of measuring the level of a TBI biomarker includes contacting the sample simultaneously or sequentially, in any order, with a detection antibody (e.g., a TBI biomarker detection antibody) comprising (1) a capture antibody (e.g., a TBI biomarker capture antibody) that binds to an epitope on a TBI biomarker or TBI biomarker fragment to form a capture antibody-TBI biomarker antigen complex (e.g., a TBI biomarker capture antibody-TBI biomarker antigen complex) and (2) a detectable label that binds to an epitope on a TBI biomarker to which the capture antibody is not bound to form a TBI biomarker antigen-detection antibody complex (e.g., a TBI biomarker antigen-TBI biomarker detection antibody complex), and measuring the amount or concentration of the TBI biomarker in the sample based on the signal generated by the detectable label in the capture antibody-TBI biomarker antigen-detection antibody complex.

[0395] 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 TBI biomarker antibody as described below.

[0396] In some embodiments, the sample is diluted or not diluted. 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 approximately 1 to approximately 150 microliters or less, or approximately 1 to approximately 25 microliters or less.

[0397] Some measuring instruments other than point-of-care devices (e.g., ARCHITECT® measuring instruments from Abbott Laboratories, Abbott Alinity instruments, and other core testing measuring instruments) may be capable of measuring TBI biomarkers at sample levels of approximately 0.032 μg / L with a coefficient of variation of 10% or less. Other detection methods may include or be adapted for use in nanopore devices or nanowell devices. An example of a nanopore device is described in International Patent Publication WO2016 / 161402, which is incorporated entirely herein by reference. An example of a nanowell device is described in International Patent Publication WO2016 / 161400, which is incorporated entirely herein by reference.

[0398] a. Detection by mass spectrometry

[0399] As used herein, “MS data” generally refers to raw MS data obtained from a mass spectrometer, and / or processed MS data in which peptides and their fragments (e.g., transitions and MS peaks) have already been identified, analyzed, and / or quantified. In some embodiments of this disclosure, methods based on MRM-MS or SRM-MS and / or PRM-MS enable the detection and accurate quantification of specific peptides in complex mixtures. SRM / MRM-MS is a technique with potential for reliable and comprehensive quantification of substances with low abundance in complex samples. SRM / MRM-MS is performed on a triple quadrupole-like analyzer, in which case the increased selectivity is obtained via collision-induced dissociation. Non-scanning mass spectrometry is a method that uses two mass spectrometers (Q1 and Q3) as static mass filters to monitor specific fragments of selected precursors. On a triple quadrupole analyzer, a variety of ionization methods can be used, including, but not limited to, electrospray ionization, chemical ionization, electron ionization, atmospheric pressure chemical ionization, and matrix-assisted laser desorption ionization. Both the first mass spectrometer and the collision cell are continuously exposed to ions from the source in a time-dependent manner. Time dependence becomes a factor when ions move to the third mass spectrometer. On a triple quadrupole analyzer, the first quadrupole mass filter, Q1, is a primary m / z sorter after the sample has left the ionization source. Ions with mass-to-charge ratios other than the selected mass-to-charge ratio are not permitted to enter Q1. The collision cell, denoted as "q2" and located between the first quadrupole mass filter Q1 and the second quadrupole mass filter Q3, is where sample fragmentation occurs in the presence of an inert gas such as argon, helium, or nitrogen. Next, upon exiting the collision cell, the fragmented ions move to the second quadrupole mass filter, Q3, where m / z selection may occur again.

[0400] A special pair of mass-to-charge (m / z) values ​​associated with a precursor and selected fragment ion is called a "transition." The detector acts as a counting device for ions matching the selected transition, thereby returning the intensity distribution over time. MRM-MS is the measurement of multiple SRM-MS transitions within the same experiment on a chromatographic timescale by rapidly switching between different precursor / fragment pairs. Typically, a triple quadrupole detector cycles through a series of transitions, recording the signal of each transition as a function of elution time. The method allows for further selectivity by monitoring the chromatographic co-elution of multiple transitions for a given analyte.

[0401] For general references on mass spectrometry and proteomics, see, for example, Salvatore Sechi, "Quantitative Proteomics by Mass Spectrometry" ("Methods in Molecular Biology"), 2nd edition, 2016, Humana Press (New York, NY, 2009); Daniel Martins-de-Souza, "Shotgun Proteomics: Methods and Protocols", 2014 edition, Humana Press (New York, NY, 2014); Jorg Reinders and Albert Sickmann, "Proteomics: Methods and Protocols" ("Methods in Molecular Biology"), 2009 edition, Humana Press (New York, NY, 2009); and Jorg Reinders, "Proteomics in Systems Biology: Methods and Protocols" ("Methods in Molecular Biology"), 1st edition, 2016, Humana Press (New See York, NY, 2009.

[0402] In addition, SRM (Structured Refinement Mass Spectrometer) can be applied using a high-resolution mass spectrometer, with parallel detection of all transitions in a single analysis. PRM-MS quantifies selected peptides (Q1), thus providing high selectivity, sensitivity, and high throughput for protein quantification (MS1). Here again, multiple peptides can be specifically selected for each protein. The PRM-MS method uses the quadrupole of the mass spectrometer to isolate target precursor ions, fragments the targeted precursor ions in a collision cell, and then detects the resulting product ions in an Orbitrap mass spectrometer. Quantification is performed by extracting one or more fragment ions after data acquisition within a mass window of 5–10 ppm. PRM-MS performs peptide / protein quantification using a quadrupole time-of-flight (QTOF) mass spectrometer or a quadrupole-Orbitrap hybrid (QOrbitrap) mass spectrometer. Examples of QTOF include, but are not limited to, the TripleTOF 6600 or 5600 systems (Sciex); the X500R QTOF system (Sciex); the 6500 series Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) (Agilent); or the Xevo G2-XS QTOF Quadrupole Time-of-Flight Mass Spectrometry (Waters). Examples of QObitrap include, but are not limited to, the Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific) or the Orbitrap Fusion Tribrid (Thermo Scientific).

[0403] In some embodiments, the methods developed herein may be applied to the quantification of polypeptides or proteins in biological samples. Any type of biological sample containing polypeptides or proteins can be a starting point and can be analyzed by the methods disclosed herein. In fact, any protein / peptide-containing sample (e.g., tissue, cell) can be used for and analyzed by the methods provided herein. The methods herein may also be used with peptide mixtures obtained by digestion. Digestion of polypeptides or proteins includes any type of cleavage strategy, such as enzymatic cleavage strategies, chemical cleavage strategies, physical cleavage strategies, or combinations thereof. According to some embodiments, the following parameters of the methods presented herein are determined: digestion and removal of peptides by trypsin (or other proteases), the polypeptide with the best response, the protein with the best response, the peptide with the best response, the fragment with the best response, the fragment intensity ratio (increase in high peak intensity and reproducible peak intensity), the optimal collision energy, and all optimal parameters that maximize the sensitivity and / or specificity of the method.

[0404] In other embodiments, quantification of polypeptides and / or corresponding proteins, or activity / modulation of corresponding proteins, is desired. Selected peptides are labeled with stable isotopes and used as internal standards (SILs) to achieve absolute quantification of the target protein. A tag with the quantified stable-labeled peptide analog is added to the peptide sample in known amounts; then, the tag and the target peptide are quantified by mass spectrometry to obtain absolute quantification of the endogenous level of the protein.

[0405] In some embodiments of this disclosure, biomarkers may be detected by mass spectrometry, which is a method utilizing a mass spectrometer that detects gas-phase ions as described above. Examples of mass spectrometers include time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer, and hybrid or combination thereof. In one embodiment, the mass spectrometry method includes matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF MS or MALDI-TOF). In another embodiment, the method includes MALDI-TOF tandem mass spectrometry (MALDI-TOF MS / MS). In yet another embodiment, mass spectrometry may be combined with other suitable methods that can be envisioned by those skilled in the art. For example, MALDI-TOF may be used with trypsin digestion and tandem mass spectrometry or enrichment as described herein. In another embodiment, the mass spectrometry method is multiple reaction monitoring (MRM) or quantitative MRM.

[0406] In some embodiments, mass spectrometry involves a first enrichment by capturing one or more biomarkers on a chromatographic resin having chromatographic properties that bind to the biomarkers. For example, the biomarkers may be captured on a cation exchange resin such as CM Ceramic HyperD F resin, the resin may be washed, the biomarkers may be eluted, and detected by MALDI. Alternatively, prior to this method, the sample may be fractionated on an anion exchange resin before application to the cation exchange resin. In one embodiment, fractionation on an anion exchange resin may be performed and directly detected by MALDI. In another embodiment, the biomarkers may be captured on an immunochromatographic resin containing an antibody that binds to the biomarkers, the resin may be washed to remove unbound material, the biomarkers may be eluted from the resin, and the eluted biomarkers may be detected by MALDI or on another MS analyzer (using any method for quantification).

[0407] The biomarkers of this disclosure may also be detected by other suitable methods. Detection paradigms that may be used for this purpose include optical methods, electrochemical methods (voltage and current measurement techniques), atomic force microscopy, and radio frequency methods, such as multipole resonance spectroscopy. In addition to both confocal and non-confocal microscopy, examples of optical methods include detection of fluorescence, emission, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, polarization analysis, resonant mirror method, grating coupler waveguide method, or interferometry) or by mass spectrometry using any MS detector and any MS method.

[0408] 8. Antibodies that recognize TBI biomarkers The methods described herein may use isolated antibodies that specifically bind to a TBI biomarker or a fragment thereof, referred to as "TBI biomarker antibodies." TBI biomarker antibodies can be used to assess TBI biomarker status as a measure of traumatic brain injury, to detect the presence of a TBI biomarker in a biological sample, to quantify the amount of a TBI biomarker present in a biological sample, or to detect the presence of a TBI biomarker in a biological sample and quantify the amount of a TBI biomarker in a biological sample.

[0409] A TBI biomarker antibody comprises any antibody that binds to a TBI biomarker, a fragment thereof, an epitope of a TBI biomarker, or a variant thereof. The antibody may be a fragment of an anti-TBI biomarker antibody, or a variant or derivative thereof. The antibody may be polyclonal or monoclonal. The antibody may be a chimeric antibody, a single-chain antibody, an affinity-mature 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 an F(ab')2 fragment, an Fv fragment, or an scFv fragment. The antibody derivative may be produced by a peptide mimetic. Furthermore, the techniques described for producing single-chain antibodies may be adapted to produce single-chain antibodies.

[0410] The anti-TBI biomarker antibody may be a chimeric anti-TBI biomarker antibody or a humanized anti-TBI biomarker antibody. In one embodiment, both the humanized antibody and the chimeric antibody are monovalent. In one embodiment, both the humanized antibody and the chimeric antibody contain a single Fab region linked to an Fc region.

[0411] Human antibodies can be derived using phage display technology or transgenic mice expressing human immunoglobulin genes. Human antibodies can be produced and isolated as a result of the in vivo immune response in humans. See, for example, Funaro et al., BMC Biotechnology, 2008(8):85. Therefore, antibodies can be human products and may not be part of the animal repertoire. Because the antibodies are human-derived, the risk of reactivity to autoantigens can be minimized. Alternatively, standard yeast display libraries and yeast display technology can be used to select and isolate human anti-TBI biomarker antibodies. For example, a library of naive human single-chain variable fragments (scFv) can be used to select human anti-TBI biomarker I antibodies. Transgenic animals can be used to express human antibodies.

[0412] A humanized antibody is an antibody molecule derived from a non-human species antibody that binds to a desired antigen, and may be an antibody molecule having one or more complementarity-determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule.

[0413] The antibody is distinguishable from known antibodies in that it possesses a different biological function from antibodies known in the art.

[0414] Antibodies can immunospecifically bind to the TBI biomarker peptide, its fragment, or its variants. Antibodies can 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. Antibodies can immunospecifically recognize and bind to epitopes within the epitope region that have 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.

[0415] 9. Antibody preparation / production

[0416] Antibodies can be prepared by any of a variety of techniques, including those well known to those skilled in the art. Generally, antibodies can be prepared by cell culture methods that include the production of monoclonal antibodies, either via conventional techniques or via transfection of the antibody gene, heavy chain, and / or light chain into a suitable bacterial or mammalian cell host, enabling the production of recombinant antibodies. The diverse forms of the term "transfection" are intended to encompass the wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, and DEAE-dextran transfection. Antibodies can be expressed in prokaryotic or eukaryotic host cells, but intraecuated expression of antibodies is preferred, and most preferred, in mammalian host cells, because such eukaryotic cells (and especially mammalian cells) are more likely to secrete antibodies that are assembled, properly folded, and immunologically active than those of prokaryotic cells.

[0417] Exemplary mammalian host cells for expressing recombinant antibodies include, for example, Chinese hamster ovary (CHO) cells (including dhfr-CHO cells as described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), NS0 myeloma cells, COS cells, and SP2 cells used with a DHFR selection marker, as described in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982). When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a sufficient time to allow antibody expression within the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells grow. The antibody can be recovered from the culture medium using standard protein purification methods.

[0418] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It is understood that variations of the above procedure can also be performed. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of the light and / or heavy chains of an antibody. Recombinant DNA technology can also be used to remove some or all of the DNA encoding one or both of the light and / or heavy chains that are not necessary for binding to the antigen of interest. Molecules expressed from such cleaved DNA molecules can also be incorporated into the antibody. In addition, bifunctional antibodies, in which one heavy chain and one light chain are antibodies (i.e., bound to human troponin I) and the other heavy and light chains are specific to antigens other than human TBI biomarkers, can also be produced by crosslinking an antibody to a second antibody via standard chemical crosslinking methods.

[0419] In a preferred system for recombinant expression of an antibody or its antigen-binding moiety, a recombinant expression vector encoding both the antibody heavy chain and 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 antibody light chain gene is operatively ligated to a CMV enhancer / AdMLP promoter regulatory element to drive high levels of gene transcription. The recombinant expression vector also contains a DHFR gene, which allows selection of vector-transfected CHO cells using methotrexate selection / amplification. Host cells of selected transformants are cultured to enable expression of the antibody heavy and light chains, and intact antibodies are recovered from the culture medium. Standard molecular biology methods are used to prepare the recombinant expression vector, transfect host cells, select transformants, culture host cells, and recover antibodies from the culture medium. Furthermore, recombinant antibodies can be synthesized by culturing host cells in a suitable culture medium until recombinant antibodies are synthesized. The method may further include the step of isolating the recombinant antibody from the culture medium.

[0420] Methods for preparing monoclonal antibodies involve the preparation of immortalized cell lines capable of producing antibodies with desired specificity. Such cell lines can be prepared from spleen cells obtained from immunized animals. Animals can be immunized with a TBI biomarker or its fragment and / or variants. The peptides used to immunize the animals may include human Fc, for example, amino acids encoding the crystallizable region or tail region of a human antibody fragment. The spleen cells can then be immortalized, for example, by fusion with a myeloma cell fusion partner. Various fusion methods can be used. For example, spleen cells and myeloma cells are combined with a nonionic washing agent for several minutes and then seeded at low density on a selective medium that supports the proliferation of hybrid cells but not myeloma cells. One such technique uses hypoxanthine, aminopterin, and thymidine (HAT) selection. Another technique involves electrofusion. After a sufficient amount of time, typically about 1-2 weeks, colonies of the hybrids are observed. A single colony is selected, and its culture supernatant is examined for polypeptide binding activity. Hybridomas with high reactivity and specificity may be used.

[0421] Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. In addition, various techniques can be used to enhance yield, such as intraperitoneal injection of hybridoma cell lines into suitable vertebrate hosts, such as mice. The monoclonal antibodies can then be collected from ascites fluid or blood. Contaminants can be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. Affinity chromatography is an example of a method that can be used in the antibody purification process.

[0422] The protease papain preferentially cleaves IgG molecules, yielding several fragments, each containing two of the F(ab) fragments, a covalent heterodimer, with an intact antigen-binding site. The enzyme pepsin can also cleave IgG molecules, yielding several fragments, including an F(ab')2 fragment containing both antigen-binding sites.

[0423] Fv fragments can be produced by preferential proteolytic cleavage of IgM, but in rare cases, they can also be produced by preferential proteolytic cleavage of IgG or IgA immunoglobulin molecules. Fv fragments can be derived using recombinant methods. Fv fragments contain a non-covalent VH::VL heterodimer that includes an antigen-binding site that retains most of the antigen recognition and antigen-binding ability of the native antibody molecule.

[0424] An antibody, antibody fragment, or derivative may each contain a set of heavy-chain and light-chain complementarity-determining regions ("CDRs") inserted between sets of heavy-chain and light-chain frameworks ("FRs") that provide support to the CDRs and define the spatial relationships of the CDRs relative to each other. A set of CDRs may contain three hypervariable regions, each consisting of either a heavy-chain V region or a light-chain V region.

[0425] Other suitable methods for producing or isolating antibodies with essential specificity may be used, including, but not limited to, methods for selecting recombinant antibodies from peptide libraries or protein libraries (such as display libraries including bacteriophage libraries, ribosome libraries, oligonucleotide libraries, RNA libraries, cDNA libraries, and yeast libraries) that are commercially available from various distributors, such as Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid / Planegg, Del.), Biovation (Aberdeen, Scotland, UK), and 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, which are available. An alternative method is immunization of transgenic animals capable of producing 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), which is known in the art and / or is available available 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 production techniques (e.g., selective lymphocyte antibody assay ("SLAM") (US 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 microdroplet and flow cytometry (Powell et al. (1990), Biotechnol., 8:333-337; One Cell) This includes, but is not limited to, systems (Cambridge, Mass.); Gray et al. (1995), J.Imm.Meth., 182:155~163; Kenny et al. (1995), Bio / Technol., 13:787~790); and B cell selection (Steenbakkers et al. (1994), Molec. Biol. Reports, 19:125~134 (1994)).

[0426] Affinity-matured antibodies can be produced by any one of several procedures known in the art. For example, Marks et al., BioTechnology, 10:779-783 (1992) describe affinity maturation by domain shuffling between VH and VL. Random mutagenesis of CDR residues and / or framework residues is 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 sites and contact or hypermuta sites with activity-enhancing amino acid residues are described in U.S. Patent No. 6,914,128B1.

[0427] Antibody variants may also be prepared by delivering the polynucleotide encoding the antibody to a transgenic animal or a host suitable for producing such antibodies in the milk of mammals such as goats, cattle, horses, and sheep. These methods are known in the art and are described, for example, in U.S. Patents 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362 and 5,304,489.

[0428] Antibody variants can also be prepared by delivering polynucleotides to produce transgenic plants and cultured plant cells (e.g., tobacco, maize, and duckweed) that produce such antibodies, specified parts, or variants in plant parts or cells cultured therefrom. For example, Cramer et al. (1999), Curr. Top. Microbiol. Immunol., 240:95-118 and the references cited in this document describe, for example, the production of transgenic tobacco leaves that express large quantities of recombinant protein using inducible promoters. Transgenic maize has been used to express mammalian proteins with biological activity equivalent to mammalian proteins produced in other recombinant systems or purified from natural sources, at levels of commercial production. For example, see Hood et al., Adv. Exp. Med. Biol. (1999), 464:127-147 and the references cited in this document. Antibody variants are also produced in large quantities from seeds of transgenic plants containing antibody fragments, such as single-chain antibodies (scFv), including tobacco seeds and potato tubers. See, for example, Conrad et al. (1998), Plant Mol. Biol., 38:101-109 and the references cited in this document. Therefore, antibodies can also be produced using transgenic plants according to known methods.

[0429] Antibody derivatives can be produced, for example, by adding exogenous sequences to modify immunogenicity, or by reducing, enhancing, or modifying binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other appropriate characteristics. Generally, the non-human CDR sequence or some or all of the human CDR sequence is maintained while substituting the non-human sequences of the variable and constant regions with human amino acids or other amino acids.

[0430] A small antibody fragment is a diabody having two antigen-binding sites, and the fragment may be a diabody containing a heavy chain variable domain (VH) (VH VL) linked to a light chain variable domain (VL) on the same polypeptide chain. See, for example, EP404,097;WO93 / 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 two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. See also U.S. Patent No. 6,632,926 by Chen et al., which is incorporated herein by reference in its entirety and also discloses antibody variants in which one or more amino acids are inserted into the hypervariable region of the parent antibody, and whose binding affinity to a target antigen is at least about twice as strong as the binding affinity of the parent antibody to that antigen.

[0431] Antibodies can be linear antibodies. Procedures for producing 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 can be bispecific or monospecific.

[0432] Antibodies can be recovered and purified from recombinant cell cultures by known methods, including but not limited to protein A purification, ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion exchange chromatography or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High-performance liquid chromatography ("HPLC") may also be used for purification.

[0433] Labeling antibodies in a detectable manner can be useful. Methods for conjugating antibodies to these drugs are known in the art. For illustrative purposes only, antibodies may be labeled with detectable moieties such as radioactive atoms, chromophores, or fluorophores. Such labeled antibodies may be used for diagnostic methods in vivo or in isolated test samples. Such labeled antibodies may be conjugated to cytokines, ligands, or other antibodies. Drugs suitable for coupling to antibodies to achieve antitumor effects 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, neocarutinostatin, and carboplatin; diphtheria toxin, Pseudomonas aeruginosa exotoxin A, Staphylococcus enterotoxin Liposomes containing lysine A, abrin A toxin, lysine A (deglycosylated lysine A and natural lysine A), TGF-alpha toxin, cytotoxin derived from Taiwanese cobra (Naja naja atra) and geronin (phytoxin), including bacterial toxins, phytotoxins and other toxins; ribosome inactivating proteins derived from plants, bacteria and fungi, including restrictosin (ribosome inactivating protein produced by Aspergillus restrictus), saporin (ribosome inactivating protein derived from Saponaria officinalis) and RNases; tyrosine kinase inhibitors; ly207702 (difluorinated purine nucleoside); anticystic agents (e.g., antisense oligonucleotides, plasmids encoding toxins, methotrexate, etc.), as well as other antibodies or antibody fragments such as F(ab).

[0434] The production of antibodies using hybridoma technology, selective lymphocyte antibody assay (SLAM), transgenic animals, and recombinant antibody libraries is described in more detail below.

[0435] 10. Aptamers that recognize TBI biomarkers The methods of this disclosure include the use of aptamers for detecting or identifying one or more TBI biomarkers. The aptamers are suitable for use in the development of probes having high affinity and selectivity for target molecules such as TBI peptide biomarkers. The aptamers include single-stranded DNA (ssDNA), RNA, or modified nucleic acids, ranging from small organic molecules to proteins and peptides, that have the ability to specifically bind to their targets. The base for target recognition is a tertiary structure formed from single-stranded oligonucleotides, as known in the art. In some embodiments, the aptamers used to detect or identify one or more TBI biomarkers may be obtained via an in vitro selection process known as SELEX, in which the aptamers are selected from a library of random sequences of synthetic DNA or RNA by the repetitive binding of oligonucleotides to the target molecule.

[0436] In some embodiments, the nucleic acids constituting the aptamer library mixture used for screening candidate TBI biomarker capture agents may be single-stranded DNA or RNA with or without chemical modification. The introduction of further chemical entities into the DNA during the selection step may include, for example, the use of 5-alkyne-modified nucleobases (e.g., thymine). In addition, 5-C8-alkyne-modified nucleotide triphosphates, such as deoxythymidine, are commercially available or can be synthesized. Such 5-C8-alkyne-modified nucleobases can be introduced into DNA by PCR. Such modifications can be further derivatized using so-called bioorthogonal chemistry, for example, by using Cu(I)-catalyzed 1,3-dipolar cycloaddition of each azide with an alkyne. In addition to Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), copper-free strain-accelerated azide-alkyne cycloaddition (SPAAC) reactions are also useful. In some embodiments relating to cell systems or biological systems, strain-enhanced azide-alkyne cycloaddition can overcome the toxicity problems associated with the use of Cu(I). Any number of desired chemical modifications can be added to an oligonucleotide library used for screening purposes. Examples of such modifications include, but are not limited to, aliphatic residues, aromatic residues, charged residues, basic residues, acidic residues, heteroaromatic residues, glycosylated residues, metal-containing residues, or peptide residues.

[0437] In some embodiments, the nucleobases modified to contain an azido-alkyne chemical group may include ethynyl dU nucleotide, ethynyl dA nucleotide, ethynyl dC nucleotide or ethynyl dG nucleotide, propynyl dU nucleotide, propynyl dA nucleotide, propynyl dC nucleotide or propynyl dG nucleotide or butynyl dU nucleotide, butynyl dA nucleotide, butynyl dC nucleotide or butynyl dG nucleotide. In other embodiments, the nucleobases modified to contain an azido-alkyne chemical group may be ethynyl dU nucleotide, ethynyl dA nucleotide, ethynyl dC nucleotide or ethynyl dG nucleotide. Nucleonucleotide aptamer libraries with these exemplary modifications can be used in a variety of SELEX-based selection methods to enhance the chemical diversity of DNA aptamer libraries. Nucleic acid starting or candidate mixtures may be modified such that at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% of the members of the mixture are modified, for example, by functionalization introduced by click chemistry. Modifications of less than 100% allow for increased diversity by enabling modification of certain positions within the oligonucleotide while preventing modification of other positions, while 100% modification ensures consistency in the selection process. In some embodiments, different modifications are made at different positions within the oligonucleotide to further enhance diversity.

[0438] Aptamers that recognize TBI biomarkers can be used in a variety of ways to detect the presence or level of one or more TBI biomarkers in a biological sample (e.g., a biological entity of interest, such as a protein, nucleic acid, or microvesicle). Aptamers can function as binders or scavengers to assess the presence or level of cognitive TBI biomarkers. In various embodiments of this disclosure, which relate to diagnostic and / or prognostic diagnostic methods, one or more aptamers may constitute a ligand-target-based assay in which one or more aptamers come into contact with a selected biological sample, allowing one or more aptamers to associate with or bind to their target TBI biomarker molecule. Aptamers can also be used to identify a profile of multiple TBI biomarkers ("biomarker" profile or "biomarker" signature) based on the evaluated biological sample and the detected biomarkers. The biomarker profile of a biological sample is the presence, level, or other characteristics of one or more biomarkers of interest that can be evaluated, including, but not limited to, presence, level, sequence, mutation, rearrangement, translocation, deletion, epigenetic modification, methylation, posttranslational modification, allele, activity, complex partners, stability, half-life, etc.

[0439] Biomarker profiles or biomarker signatures may be used to assess diagnostic and / or prognostic criteria, including the presence of disease, disease staging, disease monitoring, disease stratification, or detection of disease, and monitoring for metastasis, recurrence, or progression. For example, the methods of this disclosure may include methods for correlating a TBI biomarker profile with a selected condition or disease, such as severe TBI, mild TBI, or a subclass of mild TBI. Biomarker profiles may also be used clinically when making decisions regarding treatment modalities, including therapeutic interventions. Biomarker profiles based on the detection, identification, and / or quantification of aptamers may also be used clinically to make decisions regarding treatment, including whether to perform imaging procedures (e.g., MRI).

[0440] 11. Variations of Method The disclosed methods for determining the presence or amount of the target analyte (e.g., TBI biomarker) present in a sample may be as described herein. The methods may also be adapted with other methods for analyzing the analyte in mind. Examples of well-known variations include, but are not limited to, enzyme detection (enzyme immunoassay (EIA) or enzyme immunoassay (ELISA)), sandwich immunoassays (e.g., monoclonal-monoclonal sandwich immunoassay, monoclonal-polyclonal sandwich immunoassay), competitive inhibition immunoassays (e.g., forward competitive inhibition immunoassay and reverse competitive inhibition immunoassay), enzyme multiplexing immunoassay (EMIT), competitive binding assays, bioluminescent energy transfer (BRET), one-step antibody detection assays, allochemical assays, heterochemical assays, capture-on-the-fly assays, and others.

[0441] a. Immunoassay The target analyte and / or this peptide or fragment (e.g., the TBI biomarker and / or this peptide or fragment) can be analyzed using a TBI biomarker antibody in an immunoassay. The presence or amount of the analyte (e.g., the TBI biomarker) can be determined by using an antibody and detecting its specific binding to the analyte. For example, the antibody or this antibody fragment can specifically bind to the analyte. 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).

[0442] The presence or amount of analytes (e.g., TBI biomarkers) in a biological sample can be easily determined using immunoassays such as sandwich immunoassays (e.g., detection of radioisotopes (including radioimmunoassays (RIAs), monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonal sandwich immunoassays) and enzymatic detection (enzyme immunoassays (EIAs) or enzyme immunoassays (ELISAs) (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). Other methods that can be used include chemiluminescent microparticle immunoassays, particularly the ARCHITECT® automated analyzer (Abbott Laboratories, Abbott), mentioned as an example. This includes chemiluminescent microparticle immunoassays using Park (IL). Other methods include, for example, mass spectrometry and immunohistochemistry (e.g., with sections derived from tissue biopsy) using antibodies (monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, etc.) or fragments of these antibodies against an analyte (e.g., a TBI biomarker). Other detection methods include, for example, U.S. Patent Nos. 6,143,576; 6,113,855; 6,011,576, each of which is incorporated herein in whole by reference. This includes detection methods described in Nos. 9,944; Nos. 5,985,579; Nos. 5,947,124; Nos. 5,939,272; Nos. 5,922,615; Nos. 5,885,527; Nos. 5,851,776; Nos. 5,824,799; Nos. 5,679,526; Nos. 5,525,524 and Nos. 5,480,792. Specific immunobinding of an antibody to an analyte can be detected via direct labeling such as fluorescent or luminescent tags, metals, and radionuclides conjugated to the antibody, or via indirect labeling such as alkaline phosphatase or horseradish peroxidase.

[0443] The use of immobilized antibodies or fragments of these antibodies can be incorporated into immunoassays. Antibodies can be immobilized on various supports, such as magnetic particles or chromatography matrix particles, the surface of assay plates (e.g., microtitration wells), or small pieces of solid substrate material. Assay strips can be prepared by coating an array of antibodies or multiple antibodies on a solid support. These strips can then be immersed in the test sample and rapidly processed through washing and detection steps to generate a measurable signal, such as a colored spot.

[0444] Homogeneous formats may be used. For example, after the test sample is obtained from the subject, the mixture is prepared. The mixture contains the test sample to be evaluated for an analyte (e.g., a TBI biomarker) and a specific binding partner. The order in which the test sample and the specific binding partner are added to form the mixture is not particularly important. The test sample is in contact with the specific binding partner simultaneously. In some embodiments, the specific binding partner and any TBI biomarker contained in the test sample can form a specific binding partner-analyte (e.g., TBI biomarker) antigen complex. The specific binding partner may be an anti-analyte antibody (e.g., an anti-TBI biomarker antibody that binds to an epitope of the TBI biomarker having an amino acid sequence containing at least three consecutive amino acids). Furthermore, the specific binding partner may be labeled with or contain a detectable label as described above.

[0445] Different formats may be used. For example, after the test sample is obtained from the subject, a first mixture is prepared. The mixture contains the test sample to be evaluated for an analyte (e.g., a TBI biomarker) and a first specific binding partner, in which case the first specific binding partner and any TBI biomarker contained in the test sample form a first specific binding partner-analyte (e.g., TBI biomarker) antigen complex. The first specific binding partner may be an anti-analyte antibody (e.g., an anti-TBI biomarker antibody that binds to an epitope of the TBI biomarker having an amino acid sequence containing at least three consecutive amino acids). The order in which the test sample and the first specific binding partner are added to form the mixture is not particularly important.

[0446] The first specific binding partner may be immobilized on a solid phase. The solid phase used in the immunoassay (for the specific binding partner) may be any solid phase known in the art, including but not limited to magnetic particles, beads, test tubes, microtitration plates, cuvettes, membranes, scaffolding molecules, thin films, filter paper, disks, and chips. In embodiments where the solid phase is beads, the beads may be magnetic beads or magnetic particles. Magnetic beads / magnetic particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or magnetic fluids. 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 surrounding the non-magnetic core. The solid support on which the first specific binding member is immobilized can be stored in a dry or liquid state. The magnetic beads can be placed under a magnetic field before or after contact with the sample containing the magnetic beads on which the first specific binding member is immobilized.

[0447] After a mixture containing a first specific binding partner-analyte (e.g., TBI biomarker) antigen complex is formed, any unbound analytes (e.g., TBI biomarkers) are removed from the complex using any technique known in the art. For example, unbound analytes can be removed by washing. However, it is desirable that the first specific binding partner be present in excess of any analytes present in the test sample so that all analytes present in the test sample bind to the first specific binding partner.

[0448] After any unbound analytes (e.g., TBI biomarkers) have been removed, a second specific binding partner is added to the mixture to form a first specific binding partner-analyte of interest (e.g., TBI biomarker)-second specific binding partner complex. The second specific binding partner may be an anti-analyte antibody (e.g., a TBI biomarker antibody that binds to an epitope of the TBI biomarker having an amino acid sequence containing at least three consecutive amino acids). Furthermore, the second specific binding partner is labeled with or contains the detectable label described above.

[0449] The use of immobilized antibodies or fragments of these antibodies can be incorporated into immunoassays. Antibodies can be immobilized on a variety of supports, such as magnetic particles or chromatography matrix particles (e.g., magnetic beads), latex particles or surface-modified latex particles, polymers or polymer films, plastics or plastic films, planar substrates, the surface of assay plates (e.g., microtitration wells), or small pieces of solid substrate material. Assay strips can be prepared by coating an array of antibodies or multiple antibodies on a solid support. These strips can then be immersed in the test sample and rapidly processed through washing and detection steps to generate a measurable signal, such as a colored spot.

[0450] (1) Sandwich immunoassay A sandwich immunoassay measures the amount of antigen between two layers, each consisting of an antibody (i.e., at least one capture antibody) and a detection antibody (i.e., at least one detection antibody). The capture antibody and detection antibody bind to different epitopes on the target analyte, such as a TBI biomarker. 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 a sandwich immunoassay.

[0451] Generally, at least two antibodies are used to separate and quantify the analyte (e.g., TBI biomarker) in a test sample. More specifically, at least two antibodies bind to a particular epitope of the analyte, forming an immune complex called a "sandwich." One or more antibodies may be used to capture the analyte in the test sample (these antibodies are often referred to as "capture" antibodies or "capture" antibodies), and one or more antibodies may be 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, it is desirable that the binding of an antibody to its epitope is not attenuated 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, upon contact with the test sample presumed to contain the analyte, do not bind to all or part of the epitopes recognized by the second antibody or subsequent antibodies, thereby preventing interference with the ability of one or more second detection antibodies to bind to the analyte.

[0452] The antibody may be used as a first antibody in the immunoassay. The antibody immunospecifically binds to an epitope on the analyte (e.g., a TBI biomarker). In addition to the antibody of this disclosure, the immunoassay may include a second antibody that immunospecifically binds to an epitope that is not recognized by or does not bind to the first antibody.

[0453] A test sample suspected to contain an analyte (e.g., a TBI biomarker) may be contacted simultaneously or sequentially with at least one first capture antibody (or antibody) and at least one second detection antibody. In a sandwich assay format, a test sample suspected to contain an analyte is first contacted with at least one first capture antibody that specifically binds to a particular epitope, under conditions that allow for the formation of a first antibody-analyte antigen complex. If more than one capture antibody is used, multiple first capture antibody-TBI biomarker antigen complexes are formed. In a sandwich assay, the antibody, preferably at least one capture antibody, is used in a molar excess relative to the maximum amount of analyte expected in the test sample. For example, an antibody concentration of approximately 5 μg / ml to approximately 1 mg / ml per 1 ml of microparticle coating buffer may be used.

[0454] i. Anti-TBI biomarker capture antibody Optionally, before contacting the test sample with at least one first capture antibody, at least one first capture antibody may be bound to a solid support that facilitates the separation of the first antibody-analyte (e.g., TBI biomarker) complex from the test sample. Any solid support known in the art may be used, including but not limited to solid supports made from polymer materials in the form of wells, tubes, or beads (such as microparticles). Antibodies may be bound to the solid support by adsorption, by covalent bonding using a chemical coupling agent, or by other means known in the art, provided that such binding does not interfere with the antibody's ability to bind to the analyte. Furthermore, if necessary, the solid support may be derivatized to allow reactivity with a variety of functional groups on the antibody. Such derivatization requires the use of certain coupling agents, including but not limited to maleic anhydride, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0455] Subsequently, the test sample, which is presumed to contain an analyte (e.g., a TBI biomarker), is incubated to allow the formation of a first capture antibody (or multiple antibodies)-analyte complex. Incubation may be carried out at a pH of approximately 4.5 to approximately 10.0 and a temperature of approximately 2°C to approximately 45°C for a duration of at least approximately 1 minute to approximately 18 hours, approximately 2 to 6 minutes, approximately 7 to 12 minutes, approximately 5 to 15 minutes, or approximately 3 to 4 minutes.

[0456] ii. Detected antibodies Subsequently, after the formation of the first / multiple capture antibody-analyte (e.g., TBI biomarker) complexes, the complexes are contacted with at least one second detection antibody (under conditions that allow for the formation of the first / multiple antibody-analyte antigen-second antibody complexes). In some embodiments, the test sample is contacted with the detection antibody simultaneously with the capture antibody. If the first antibody-analyte complex is contacted with more than one detection antibody, the first / multiple capture antibody-analyte-multiple detection antibody complexes are formed. If at least second (and subsequent) antibodies are contacted with the first antibody-analyte complexes, an incubation time similar to that for the first antibody, under the same conditions as described above, is required for the formation of the first / multiple antibody-analyte-second / multiple antibody complexes. Preferably, at least one second antibody contains a detectable label. The detectable label may bind to at least one second antibody either before, simultaneously with, or after the formation of the first / multiple antibody-analyte-second / multiple antibody complex. Any detectable label known in the art may be used.

[0457] The chemiluminescence assay can be performed according to the method described in Adamczyk et al., Anal.Chim.Acta, 579(1):61~67 (2006). Any suitable assay format can be used, but a microplate chemiluminescence analyzer (Mithras LB-940, Berthold Technologies USA, LLC, Oak Ridge, TN) enables rapid assay of multiple small-volume samples. The chemiluminescence analyzer may be equipped with multiple reagent injectors and uses a 96-well black polystyrene microplate (Costar #3792). Each sample may be added to an individual well, followed by the 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 using acridinium aryl esters, such as by acidification. The chemiluminescence response is then recorded for each well. In this regard, the time required to record the chemiluminescence response depends in part on the delay between the addition of the reagent used and the addition of the specific acridinium.

[0458] The order in which the test sample and specific binding partners are added to form a mixture for a chemiluminescence assay is not particularly important. If the first specific binding partner is detectably labeled with an acridinium compound, a detectably labeled first specific binding partner-antigen (e.g., TBI biomarker) complex is formed. Alternatively, if a second specific binding partner is used and the second specific binding partner is detectably labeled with an acridinium compound, a detectably labeled first specific binding partner-analyte-second specific binding partner complex is formed. Any unbound specific binding partners, whether labeled or unlabeled, can be removed from the mixture using any technique known in the art, such as washing.

[0459] Hydrogen peroxide may be generated in the mixture in situ, or may be added to or supplied to the mixture simultaneously with, before, or after, the addition of the acridinium compound described above. Hydrogen peroxide may be generated in situ by a number of methods, including those obvious to those skilled in the art.

[0460] Alternatively, the source of hydrogen peroxide can 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 can simply be added.

[0461] When at least one basic solution is added to the sample, either simultaneously or subsequently, a detectable signal is generated, i.e., a chemiluminescent signal indicating the presence of the analyte (e.g., a TBI biomarker). The basic solution contains at least one base and has a pH of 10 or higher, preferably 12 or higher. Examples of basic solutions include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide, calcium carbonate, and calcium bicarbonate. The amount of basic solution added to the sample depends on the concentration of the basic solution. Based on the concentration of the basic solution used, those skilled in the art can easily determine the amount of basic solution to add to the sample. Other labels besides chemiluminescent labeling may also be used. For example, enzyme labeling (including, but not limited to, alkaline phosphatase) may be used.

[0462] The generated chemiluminescent signal or other signal can be detected using prescribed techniques known to those skilled in the art. Based on the intensity of the generated signal, the amount of the target analyte (e.g., TBI biomarker) in the sample can be quantified. Specifically, the amount of analyte in the sample is proportional to the intensity of the generated signal. The amount of analyte present can be quantified by comparing the amount of light generated with a calibration curve for the analyte or with a reference standard. The calibration curve can be prepared using a series of dilutions or solutions of known concentrations of the analyte by mass spectrometry, gravimetric methods, and other techniques known in the art.

[0463] (2) Forward competitive inhibition assay In a forward competition format, aliquots of a labeled target analyte (e.g., a TBI biomarker) at known concentrations, having a fluorescent label, a cleavable linker, and a conjugated tag, are used to compete for binding to the target analyte in the test sample with the target analyte antibody (e.g., a TBI biomarker antibody).

[0464] In a forward competitive assay, an immobilized specific binding partner (such as an antibody) may be brought into contact sequentially or simultaneously with the test sample and the labeled analyte of interest, a fragment of the analyte of interest, or a variant of the analyte of interest. The peptide of the analyte of interest, a fragment of the analyte of interest, or a variant of the analyte of interest may be labeled with any detectable label, including a detectable label consisting of a cleavable linker and a conjugated tag. In this assay, the antibody may be immobilized on a solid support. Alternatively, the antibody may be coupled to an antibody, such as an anti-molecular-weight antibody, immobilized on a solid support such as microparticles or a planar substrate.

[0465] 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 different molecular species of antibody-analyte of interest complexes may then be produced. Specifically, one of the produced antibody-analyte of interest complexes may contain a detectable label (e.g., a fluorescent label), while the other antibody-analyte of interest complex may not contain a detectable label. The antibody-analyte of interest complex may, but may not, be separated from the remaining test sample before quantification of the detectable label. The amount of detectable label within the antibody-analyte of interest complex is then quantified, regardless of whether the antibody-analyte of interest complex is separated from the remaining test sample. Next, the concentration of the target analyte (such as the target analyte associated with the membrane, the soluble target analyte, fragments of the soluble target analyte, variants of the target analyte (the target analyte associated with the membrane or the soluble target analyte), or any combination thereof) in the test sample can be determined, for example, as described above.

[0466] (3) Reverse competitive inhibition assay In a reverse competitive assay, the immobilized analyte of interest (e.g., a TBI biomarker) may be brought into contact with the test sample and at least one labeled antibody sequentially or simultaneously.

[0467] The target analyte can be bound to a solid support, such as the solid support discussed above in relation to the sandwich assay format.

[0468] The immobilized analyte of interest, the test sample, and at least one labeled antibody are incubated under conditions similar to those described above, in relation to the sandwich assay format. Two different molecular species of analyte of interest-antibody complexes may then be produced. Specifically, one of the produced analyte of interest-antibody complexes is immobilized and contains a detectable label (e.g., a fluorescent label), while the other analyte of interest-antibody complex is not immobilized and contains a detectable label. The unimmobilized analyte of interest-antibody complex and the remaining test sample are removed from the presence of the immobilized analyte of interest-antibody complex by techniques known in the art, such as washing. After the removal of the unimmobilized analyte of interest-antibody complex, the amount of detectable label in the immobilized analyte of interest-antibody complex is quantified after tag cleavage. The concentration of the analyte of interest in the test sample can then be determined by comparing the quantities of detectable label as described above.

[0469] (4) One-step immunoassay or "Capture-on-the-Fly" assay In a capture-on-the-fly immunoassay, the solid substrate is pre-coated with an immobilizer. The capture agent, analyte (e.g., TBI biomarker), 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 and includes a ligand for the immobilizer. The capture agent and detection agent may be an antibody or any other part that is capable of being captured or detected, as described herein or known in the art. The ligand may include a peptide tag, and the immobilizer may include an anti-peptide-tagged antibody. Alternatively, the ligand and immobilizer may be any pair of agents capable of binding together for use in a capture-on-the-fly assay (e.g., a specific binding pair and another pair known in the art). More than one analyte may be measured. In some embodiments, the solid substrate may be coated with an antigen, and the analyte analyzed is an antibody. This method can also be coupled with detection and quantification by MS.

[0470] In certain and other embodiments of a one-step immunoassay or "capture-on-the-fly" method, a solid support (such as microparticles) pre-coated with an immobilizing agent (such as biotin or streptavidin), along with at least a first specific binding member and a second specific binding member (each functioning as a capture reagent and a detection reagent, respectively), are used. The first specific binding member contains a ligand for the immobilizing agent (for example, if the immobilizing agent on the solid support is streptavidin, the ligand on the first specific binding member may be biotin) and also binds to the analyte of interest (e.g., a TBI biomarker). The second specific binding member contains a detectable label and binds to the analyte of interest. The solid support and the first and second specific binding members may be added to the test sample (sequentially or simultaneously). 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 target analyte present in the sample binds to a solid support / first specific binding member complex to form a solid support / first specific binding member / analyte complex. A second specific binding member binds to the solid support / first specific binding member / analyte complex, and a detectable label is detected. An optional washing step may be used before detection. In certain embodiments of the one-step assay, more than one analyte may be measured. In certain other embodiments, more than two specific binding members may be used. In certain other embodiments, multiple detectable labels may be added. In certain other embodiments, multiple targets may be detected, or their quantities, levels, or concentrations may be measured, determined, or evaluated, including by the use of mass spectrometry.

[0471] The use of capture-on-the-fly assays can be carried out in various formats described herein and known in the art. For example, the format may be a sandwich assay as described above, but alternatively, a competitive assay is also possible, and other variants may be used, such as monospecific binding members or known variants. This method can also be coupled with detection and quantification by MS.

[0472] 12. Other factors The diagnostic methods, prognostic methods, risk stratification methods, and / or evaluation methods described above may further include the use of other factors for diagnosis, prognosis, and evaluation. In some embodiments, traumatic brain injury may be diagnosed using the Glasgow Coma Scale or the Extended Glasgow Outcome Scale (GOSE). Other tests, scales, or indicators may also be used alone or in combination with the Glasgow Coma Scale. An example is the Ranchos Los Amigos scale, which measures the level of consciousness, cognition, behavior, and interaction with the environment. The Ranchos Los Amigos scale includes levels I: no response; level II: general response; level III: local response; level IV: confusion (excitement); level V: confusion (inappropriate); level VI: confusion (appropriate); level VII: automatic (appropriate); and level VIII: intentional (appropriate).

[0473] 13. Sample In some embodiments, the sample is obtained after a human subject has suffered a head injury caused by a physical shake, an external mechanical or other force resulting in a closed or open head injury, one or more falls, an explosion or blast, or other type of blunt force trauma. In some embodiments, the sample is obtained after a human subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin. 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), organometallic substances (such as methylmercury, tetraethyl lead, and organotin), and / or one or more drugs of abuse. In some embodiments, the sample is obtained from a human subject suffering from an autoimmune disease, metabolic disorder, brain tumor, hypoxia, one or more viruses, meningitis, hydrocephalus, or a combination thereof.

[0474] In yet another embodiment, the methods described herein may also be used to determine whether a subject has or is at risk of developing mild traumatic brain injury by determining the level of the TBI biomarker in the subject using the anti-TBI biomarker antibody or a fragment of the antibody described below. Thus, in certain embodiments, the disclosure also presents methods, which are discussed herein and known in the art, for determining whether a subject has or is at risk of developing traumatic brain injury is a candidate for treatment or procedure. Generally, subjects are those who have at least (i) suffered head injury; (ii) ingested and / or been exposed to one or more chemicals and / or toxins; (iii) suffer from autoimmune disease, metabolic disorder, brain tumor, hypoxia, one or more viruses, meningitis, hydrocephalus, or any combination thereof; or (iv) any combination of (i) to (iii) or subjects who have TBI or have been actually diagnosed as being at risk of TBI (e.g., subjects suffering from autoimmune disease, metabolic disorder, brain tumor, hypoxia, one or more viruses, meningitis, hydrocephalus, or any combination thereof) and / or subjects exhibiting undesirable (i.e., clinically undesirable) concentrations or amounts of TBI biomarkers or TBI biomarker fragments as described herein.

[0475] a. Test subject or biological sample As used herein, “sample,” “test sample,” and “biological sample” refer to a bodily fluid sample containing or presumed to contain a TBI biomarker. Samples may originate from any suitable source. Samples may include liquids, fluid particulate solids, or suspensions of solid particles. Samples may be processed before the analysis described herein. For example, a sample may be separated or purified from its source before analysis, although in certain embodiments, an unprocessed sample containing a TBI biomarker may be assayed directly. In certain examples, a source containing a TBI biomarker is a human bodily substance (e.g., bodily 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, tissues, organs, etc.). Tissues may 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 may be a liquid sample or a liquid extract of a solid sample. In certain cases, the source of the sample may be tissue, such as an organ or biopsy sample, which can be solubilized by tissue degradation / cell lysis.

[0476] A wide range of body fluid samples can be analyzed. In a few exemplary embodiments, sample volumes may be approximately 0.5 nL, 1 nL, 3 nL, 0.01 μL, 0.1 μL, 1 μL, 5 μL, 10 μL, 100 μL, 1 mL, 5 mL, 10 mL, etc. Depending on the case, the volume of the body fluid sample may be between approximately 0.01 μL and 10 mL, between approximately 0.01 μL and 1 mL, between approximately 0.01 μL and 100 μL, or between approximately 0.1 μL and 10 μL.

[0477] In some cases, body fluid samples may be diluted before use in the assay. For example, in embodiments where the source containing the TBI biomarker 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). Body fluid samples may be diluted approximately 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 10-fold, or more than 100-fold before use. In other cases, body fluid samples are not diluted before use in the assay.

[0478] In some cases, samples may undergo pretreatment for analysis. Pretreatment can provide further functionality, such as removal of nonspecific proteins and / or mixed functionality, which is effective but inexpensive to implement. Common pretreatment methods may include the use of electrokinetic trapping, AC electrokinetic phenomena, surface acoustic waves, isokinetic electrophoresis, dielectrophoresis, electrophoresis, or other preconcentration methods known in the art. In some cases, body fluid samples may be concentrated before use in the assay. For example, in embodiments where the source containing the TBI biomarker is human body fluid (e.g., blood, serum), the body fluid may be concentrated by precipitation, evaporation, filtration, centrifugation, or a combination thereof. Body fluid samples may be concentrated about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 10-fold, or more than 100-fold before use.

[0479] b. Contrast It may be desirable to include control samples. The controls may be analyzed synchronically with the samples derived from the subject, as described above. Results obtained from the subject samples may be compared with results obtained from the control samples. Calibration curves may be prepared for comparison of assay results for samples. Such calibration curves present the marker levels as a function of assay units, i.e., the fluorescence signal intensity if fluorescent labeling is used. Calibration curves may be prepared using samples from multiple donors for reference levels of TBI biomarkers in normal healthy tissue and for levels of "hazardous" TBI biomarkers in tissues from donors that may have one or more of the characteristics specified above.

[0480] Accordingly, with the above in mind, a method is provided for determining the presence, amount, or concentration of a TBI biomarker in a test sample. The method includes, for example, the step of assaying the test sample for a TBI biomarker by immunoassay using at least one capture antibody that binds to an epitope on the TBI biomarker and at least one detection antibody that binds to an epitope on the TBI biomarker that is different from the epitope for the capture antibody; and optionally including a detectable label, the step of comparing the signal generated by the detectable label as a direct or indirect indicator of the presence, amount, or concentration of the TBI biomarker in the test sample with the signal generated as a direct or indirect indicator of the presence, amount, or concentration of the TBI biomarker in a calibrator. The calibrator is preferably, optionally, part of a series of calibrators in which each calibrator has a different concentration of the TBI biomarker than other calibrators in the series.

[0481] 14. Kit This specification presents kits that can be used to assay or evaluate a test sample for one or more TBI biomarkers and / or fragments thereof. The kit includes at least one component for assaying the test sample for the TBI biomarker and instructions for assaying the test sample for the TBI biomarker. For example, the kit may include instructions for assaying the test sample for the TBI biomarker by immunoassay (e.g., chemiluminescent microparticle immunoassay) or by mass spectrometry assay (e.g., PRM-MS or MRM / SRM-MS). Instructions included in the kit may be affixed to the packaging material or included as accompanying documentation in the package. Instructions may typically be, but are not limited to, documents or printed materials. Any medium capable of storing such instructions and communicating them to end users is envisioned in this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips) and optical media (e.g., CD-ROMs). As used herein, the term “instructions” may include the address of an internet site that provides instructions.

[0482] At least one component may comprise at least one composition comprising one or more isolated antibodies or fragments thereof that specifically bind to a TBI biomarker. The antibodies may be TBI biomarker detection antibodies and / or TBI biomarker capture antibodies.

[0483] Alternatively, or in addition, the kit may include a calibrator or control (e.g., a purified and optionally lyophilized TBI biomarker) and / or at least one container (e.g., a tube, microtitration plate, or strip, which may be pre-coated with an anti-TBI biomarker antibody) for performing the assay, and / or a buffer such as an assay buffer or wash buffer, one of which may be provided as a concentrate, a substrate solution for detectable labeling (e.g., enzyme labeling), or a stop solution. Preferably, the kit includes all the components necessary to perform the assay, namely reagents, standards, buffers, diluents, etc. Instructions may also include instructions for creating a calibration curve.

[0484] The kit may further include a reference standard for quantifying TBI biomarkers. The reference standard can be used to establish calibration curves for interpolating and / or extrapolating TBI biomarker concentrations. The standard can contains stable N2 for protein or peptide fragments or various analytes composed of amino acid residues. 15 The reference material includes isotope-labeled protein or peptide fragments, as well as a standard material for sample preparation, which includes a standard material containing a spike substance in the protein and a quantification peptide. In some embodiments, the reference material for the TBI biomarker corresponds to the 99th percentile derived from a healthy reference population. Such reference materials can be determined using prescribed techniques known in the art.

[0485] ...

Claims

1. A method comprising the step of measuring or detecting GFAP in a sample obtained from a subject having CT-negative findings in the brain, wherein a higher level of measured or detected GFAP compared to a level of GFAP obtained from a healthy subject indicates that the subject has or may have suffered a subclass 4 mild traumatic brain injury.

2. The method according to claim 1, wherein the subject is a human.

3. The method according to any one of claims 1 to 2, further comprising the step of measuring or detecting at least one or a fragment thereof of a biomarker, which is TPP2, CAND1, NCOR1, K22E, AL9A1, ABHEB, DNM1L, INF2, or any combination thereof, in the sample.

4. The levels of CAND1, NCOR1, K22E, ABHEB, and DNMI1L are higher and / or higher than the levels of CAND1, NCOR1, K22E, ABHEB, and DNMI1L in samples obtained from healthy subjects. The levels of TTP2, AL9A1, and INF2 are lower compared to the levels of TTP2, AL9A1, and INF2 in samples obtained from healthy subjects. The method according to claim 3.

5. A biomarker panel for determining the status of traumatic brain injury (TBI), comprising GFAP, Measurement or detection of GFAP in a sample obtained from a subject with CT-negative findings in the brain indicates that the subject has or may have had subclass 4 mild TBI. panel.

6. The biomarker panel according to claim 5, wherein the level of GFAP is higher than that obtained from healthy subjects.

7. The biomarker panel according to any one of claims 5 to 6, wherein the subject is human.

8. The biomarker panel according to any one of claims 5 to 7, further comprising the step of measuring or detecting at least one or a fragment thereof of a biomarker, which is TPP2, CAND1, NCOR1, K22E, AL9A1, ABHEB, DNM1L, INF2, or any combination thereof, in the sample.

9. The levels of CAND1, NCOR1, K22E, ABHEB, and DNMI1L are higher and / or higher than the levels of CAND1, NCOR1, K22E, ABHEB, and DNMI1L in samples obtained from healthy subjects. The levels of TTP2, AL9A1, and INF2 are lower compared to the levels of TTP2, AL9A1, and INF2 in samples obtained from healthy subjects. The biomarker panel according to claim 8.

Images