Methods for aiding in diagnosing and assessing traumatic brain injury in human subjects using a combination of GFAP and UCH-L1

Abstract

To provide methods to assist in diagnosis and assessment in a subject who has suffered or may have suffered an injury to the head.SOLUTION: For example, in the present disclosure, the combination between the level of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in samples taken at various time points within 48 hours after a subject has sustained or may have sustained a head injury and the level of glial fibrillary acidic protein (GFAP) is detected or measured, whereby proposing methods to assist in diagnosis and assessment in a subject in order to determine whether the subject has suffered a traumatic brain injury (TBI).SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims priority to U.S. Application No. 62 / 596,814, filed December 9, 2017, U.S. Application No. 62 / 610,805, filed December 27, 2017, U.S. Application No. 62 / 663,811, filed April 27, 2018, and U.S. Application No. 62 / 667,227, filed May 4, 2018, the contents of each of which are incorporated herein by reference. Technical Field The present disclosure relates to a method for aiding in the diagnosis and assessment of a subject who has or may have suffered a head injury. For example, the present disclosure provides a method for aiding in the diagnosis and assessment of a subject to determine whether the subject has suffered a traumatic brain injury (TBI) by detecting or measuring a combination of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) levels and glial fibrillary acidic protein (GFAP) levels in samples taken at various time points within 48 hours after the subject has or may have suffered a head injury. [Background technology]

[0002] More than 5 million cases of mild traumatic brain injury (TBI) occur annually in the United States alone. Currently, there are no simple, objective, and accurate measurements available to aid in patient evaluation. In fact, much of the assessment and diagnosis of TBI is based on subjective data. Unfortunately, objective measurements such as head CT and Glasgow Coma Score (GCS) are not very comprehensive or sensitive in assessing mild TBI. Furthermore, head CT scans reveal nothing about mild TBI in most cases, are expensive, and expose patients to unnecessary radiation. In addition, a negative head CT does not mean that the patient is clearly not having a concussion, only that a certain intervention, such as surgery, is not warranted. Patients who have sustained traumatic injuries, such as orthopedic injuries, may also have TBI. Physicians and patients need objective and reliable information to accurately assess this condition and facilitate appropriate triage and recovery. Currently, there are limited data available on the use of UCH-L1 and GFAP in the acute care setting to aid in patient assessment and management. Summary of the Invention [Problem to be solved by the invention]

[0003] Mild TBI or concussion is difficult to detect objectively and presents a daily challenge in emergency rooms around the world. Concussion often does not cause gross pathology, such as bleeding and abnormalities in conventional computed tomography scans of the brain, but causes rapid-onset neurological dysfunction that resolves spontaneously over days to weeks. Approximately 15% of mild TBI patients suffer from persistent cognitive dysfunction. There is an unmet need for orthopedic patients and mild TBI victims in emergency rooms and clinics, hospitals, sports fields, and military activity (e.g., combat) sites to be assessed for their TBI status. [Means for solving the problem]

[0004] Summary of the Invention In one aspect, the disclosure relates to a method for aiding in the diagnosis of or determining whether a subject who has sustained or may have sustained a head injury is suffering from a moderate, severe, or moderate-severe traumatic brain injury (TBI), the method comprising: performing an assay on a sample obtained from the subject within about 48 hours after the actual or suspected injury to measure or detect a combination of levels of glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the sample; and (a) determining that the subject has not suffered a moderate, severe, or moderate-severe TBI if the sample level of GFAP is less than a reference level of about 105 pg / mL for GFAP and the sample level of UCH-L1 is less than a reference level of about 110 pg / mL for UCH-L1; or (b) determining that the subject has not suffered a moderate, severe, or moderate-severe TBI if the sample level of GFAP is equal to a reference level of GFAP of about 105 pg / mL to about 890 pg / mL and the sample level of UCH-L1 is equal to a reference level of UCH-L1 of about 110 pg / mL to about 2000 pg / mL; or (c) determining that the subject is more likely than not to have suffered a moderate, severe, or moderate-severe TBI if the sample level of GFAP is greater than a reference level of about 890 pg / mL for GFAP and the sample level of UCH-L1 is greater than a reference level of about 2000 pg / mL for UCH-L1. Includes.

[0005] In one embodiment of the method described above, the subject may have received a Glasgow Coma Scale (GCS) score before or after the assay is performed. In another embodiment, the subject receiving such a GCS score is suspected to have moderate TBI based on the determined GCS score. In another embodiment, the subject receiving such a GCS score is suspected to have severe TBI. In another embodiment, the subject receiving such a GCS score is suspected to have moderate to severe TBI based on the determined GCS score. In yet another embodiment of the method described above, the reference level of GFAP and the reference level of UCH-L1 correlate or correspond to a Glasgow Coma Scale (GCS) score of 3 to 8 (severe TBI). In yet another aspect, the reference level of GFAP and the reference level of UCH-L1 correlate to a Glasgow Coma Scale (GCS) score of 9 to 12 (moderate TBI). In other embodiments, the baseline levels of GFAP and the baseline levels of UCH-L1 correlate or correspond to a GCS (Glasgow Coma Scale) score of 3-12 (moderate-severe TBI).

[0006] In one embodiment of the method described above, the assay is performed on a sample obtained from the subject within about 0 to about 4 hours after the actual or suspected injury. In another embodiment, the assay is performed on a sample obtained from the subject within about 4 to about 8 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 8 to about 12 hours after the actual or suspected injury. In yet a further embodiment, the assay is performed on a sample obtained from the subject within about 12 to about 16 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 16 to about 20 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 20 to about 24 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 24 to about 28 hours after the actual or suspected injury. In still further embodiments, the assay is performed on a sample obtained from a subject within about 24 hours to about 48 hours after the injury or suspected injury. In yet even further embodiments, the assay is performed on a sample obtained from a subject within about 28 hours to about 32 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 32 hours to about 36 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 36 hours to about 40 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 40 hours to about 44 hours after the actual injury or suspected injury. In still yet another embodiment, the assay is performed on a sample obtained from a subject within about 44 hours to about 48 hours after the actual injury or suspected injury.

[0007] In another embodiment of the methods described above, the baseline level of GFAP and the baseline level of UCH-L1 are determined by an assay having a sensitivity of about 79% or greater and a specificity of about 33% or greater.

[0008] In yet another embodiment of the methods described above, the sample is obtained from the subject within about 8 hours to about 16 hours after the actual or suspected injury.

[0009] In yet another embodiment of the methods described above, the assay has at least 2% greater sensitivity and at least 3% greater specificity compared to assays that measure or detect GFAP or UCH-L1 individually.

[0010] In yet another embodiment of the method described above, a. the sample is obtained from the subject within about 8 hours to about 12 hours after actual or suspected injury; the baseline level of GFAP is about 240 pg / mL and the baseline level of UCH-L1 is about 860 pg / mL; the assay has a sensitivity of 97% or greater and a specificity of 51% or greater; or b. the sample is obtained from the subject within about 12 hours to about 16 hours after the actual or suspected injury; the baseline level of GFAP is about 105 pg / mL and the baseline level of UCH-L1 is about 840 pg / mL; the assay has a sensitivity of 97.5% or greater and a specificity of 36% or greater; or c. the sample is obtained from the subject within about 8 hours to about 12 hours after the actual or suspected injury, the baseline level of GFAP is about 890 pg / mL and the baseline level of UCH-L1 is about 920 pg / mL; the assay has a sensitivity of 90% or greater and a specificity of 79% or greater; or d. The sample is obtained from the subject within about 12 hours to about 16 hours after the actual or suspected injury; the baseline level of GFAP is about 505 pg / mL and the baseline level of UCH-L1 is about 1580 pg / mL; and the assay has a sensitivity of 90% or greater and a specificity of 66% or greater.

[0011] In yet another embodiment of the above-described method, the measurement of the level of GFAP comprises: (a) The samples are subjected to simultaneous or sequential analysis in any order. (1) at least one GFAP capture antibody that binds to an epitope on GFAP or a GFAP fragment to form at least one GFAP capture antibody-GFAP antigen complex; and (2) at least one GFAP detection antibody that contains a detectable label and that binds to an epitope on GFAP not bound by the GFAP capture antibody to form a GFAP antigen-at least one GFAP detection antibody complex; to form at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex; and (b) measuring the amount or concentration of GFAP in the sample based on a signal generated by a detectable label in at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex. Includes.

[0012] In yet another embodiment, the measurement of UCH-L1 in the method identified above comprises: (a) The samples are subjected to simultaneous or sequential analysis in any order. (1) at least one UCH-L1 capture antibody that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form at least one UCH-L1 capture antibody-UCH-L1 antigen complex; and (2) at least one UCH-L1 detection antibody that contains a detectable label and that binds to an epitope on UCH-L1 not bound by the at least one UCH-L1 capture antibody to form a UCH-L1 antigen-at least one UCH-L1 detection antibody complex; to form at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex; and (b) measuring the amount or concentration of UCH-L1 in the sample based on a signal generated by a detectable label in at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex; Includes.

[0013] In one embodiment using the method described above, the subject is evaluated or assessed as having a moderate TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a severe TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a moderate to severe TBI. In yet a further embodiment using the method described above, the subject is evaluated or assessed as not having a TBI.

[0014] The methods described above may further include treating the human subject assessed or assessed as having moderate, severe, or moderate-severe TBI with a treatment for TBI (e.g., surgical treatment, therapeutic treatment, or a combination thereof). Any such treatment known in the art and further described herein may be used. Furthermore, in further embodiments, any subject treated for TBI may also be optionally monitored during or after any course of treatment. Alternatively, the methods may further include monitoring a subject assessed as having moderate, severe, or moderate-severe TBI (such as a subject who may not yet have received treatment).

[0015] In the above described methods, the sample is selected from the group consisting of a whole blood sample, a serum sample, a cerebrospinal fluid sample, and a plasma sample. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a plasma sample. In yet other embodiments, the sample is a serum sample. Such a sample can be obtained in various ways. For example, the sample can be obtained after the subject has suffered a head injury or other type of blunt trauma caused by physical shaking, external mechanical or other forces resulting in closed or open head trauma, one or more falls, blunt impact from an explosion or blast. Alternatively, the sample can be obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin. Examples of chemicals or toxins are fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organometallics, drugs of abuse, or one or more combinations thereof. Still further, the sample may be obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof.

[0016] Any of the methods described above may be performed on any human subject, regardless of factors which may be selected from the group consisting of the clinical condition of the human subject, the laboratory values ​​of the human subject, the classification of the human subject as suffering from mild, moderate, severe or moderate-severe TBI, the presentation of low or high levels of UCH-L1, GFAP and / or UCH-L1 and GFAP by the human subject, and the timing of any event during which the human subject may have suffered a head injury.

[0017] In the methods described above, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In still other embodiments, the assay is a clinical chemistry assay. In still other embodiments, the assay is a single molecule detection assay. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a clinical chemistry assay, and the sample is whole blood. In still further embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a clinical chemistry assay, and the sample is serum. In still further embodiments, the assay is a single molecule detection assay, and the sample is serum. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a clinical chemistry assay, and the sample is plasma. In yet a further embodiment, the assay is a single molecule detection assay, and the sample is plasma.

[0018] In yet another aspect, the disclosure relates to a method for aiding in or determining whether to perform a head computed tomography (CT) scan on a human subject who has sustained or may have sustained a head injury, the method comprising: performing an assay on a sample obtained from the subject within about 48 hours after the actual or suspected injury to measure or detect a combination of levels of glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the sample; and (a) determining that the subject does not require a CT scan if the sample level of GFAP is less than a reference level of about 50 pg / mL for GFAP and the sample level of UCH-L1 is less than a reference level of about 90 pg / mL for UCH-L1; or (b) determining that the subject does not require a CT scan if the sample level of GFAP is equal to a reference level of GFAP of about 50 pg / mL to about 975 pg / mL and the sample level of UCH-L1 is equal to a reference level of UCH-L1 of about 90 pg / mL to about 2000 pg / mL; or (c) determining that the subject is more likely than not to need a CT scan if the sample level of GFAP is greater than a baseline level of about 975 pg / mL for GFAP and the sample level of UCH-L1 is greater than a baseline level of about 2000 pg / mL for UCH-L1; Includes.

[0019] In one embodiment of the methods described above, the subject undergoes a CT scan before or after the assay is performed, and the subject is suspected of having a TBI based on the CT scan results, hi another embodiment, the baseline levels of GFAP and the baseline levels of UCH-L1 correlate with a negative CT scan result.

[0020] More specifically, in the above-described methods for determining or assessing whether to perform a head CT scan, the subject may be presumed to have a traumatic brain injury based on a CT scan that has been performed or has already been performed (meaning before the assay is performed). For example, depending on the subject's medical condition (such as when the patient is unconscious), a CT scan may be performed to evaluate and / or assess whether the subject has a TBI immediately after the subject arrives at an emergency room, trauma center, or other facility. Such a CT scan may be performed before the assay is performed to confirm and determine whether the subject has a mild or moderate-severe TBI. After the assay is performed, one or more subsequent CT scans may be performed based on the results of the assay as part of the physician's (or other medical personnel's) management of the TBI (e.g., to determine whether surgical and / or pharmacological intervention may be required).

[0021] In certain embodiments of the above method, the subject can be inferred to have traumatic brain injury based on CT scan. For example, the subject can be inferred to have mild TBI based on CT scan. Alternatively, the subject can be inferred to have moderate TBI based on CT scan. Alternatively, the subject can be inferred to have severe TBI based on CT scan. Alternatively, the subject can be inferred to have moderate to severe TBI based on CT scan. Still further, the subject can be inferred to not have TBI based on CT scan.

[0022] In certain embodiments of the above method, the reference level used is correlated with or corresponds to a positive head computed tomography. For example, the reference level can be correlated with or corresponds to a subject with a positive head computed tomography (such as through an increase or decrease in the reference level). Alternatively, the reference level can be correlated with or corresponds to a subject with a negative head computed tomography (such as through an increase or decrease in the reference level). Still further alternatively, the reference level can be correlated with or corresponds to a subject with intracerebral hemorrhage or an improved or worsening intracerebral hemorrhage (such as through an increase or decrease in the reference level). In other embodiments of the above method, the reference level is correlated with or corresponds to a control subject that does not suffer from TBI.

[0023] In one embodiment of the method described above, the assay is performed on a sample obtained from the subject within about 0 to about 4 hours after the actual or suspected injury. In another embodiment, the assay is performed on a sample obtained from the subject within about 4 to about 8 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 8 to about 12 hours after the actual or suspected injury. In yet a further embodiment, the assay is performed on a sample obtained from the subject within about 12 to about 16 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 16 to about 20 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 20 to about 24 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 24 to about 28 hours after the actual or suspected injury. In still further embodiments, the assay is performed on a sample obtained from a subject within about 24 hours to about 48 hours after the injury or suspected injury. In yet even further embodiments, the assay is performed on a sample obtained from a subject within about 28 hours to about 32 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 32 hours to about 36 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 36 hours to about 40 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 40 hours to about 44 hours after the actual injury or suspected injury. In still yet another embodiment, the assay is performed on a sample obtained from a subject within about 44 hours to about 48 hours after the actual injury or suspected injury.

[0024] In yet another embodiment of the methods described above, the baseline level of GFAP and the baseline level of UCH-L1 are determined by an assay having a sensitivity of about 54% or greater and a specificity of about 32% or greater.

[0025] In still yet another embodiment of the methods described above, the sample is obtained from the subject within about 4 hours to about 16 hours after the actual or suspected injury.

[0026] In yet another embodiment of the methods described above, the assay has at least 2% greater sensitivity and at least 4% greater specificity compared to assays that measure or detect GFAP or UCH-L1 individually.

[0027] In yet another embodiment of the method described above, a. the sample is obtained from the subject within about 4 hours to about 8 hours after actual or suspected injury; the baseline level of GFAP is about 110 pg / mL and the baseline level of UCH-L1 is about 2000 pg / mL; the assay has a sensitivity of 95% or greater and a specificity of 62% or greater; b. the sample is obtained from the subject within about 8 hours to about 12 hours after the actual or suspected injury; the baseline level of GFAP is about 240 pg / mL and the baseline level of UCH-L1 is about 300 pg / mL; the assay has a sensitivity of 91.5% or greater and a specificity of 52% or greater; or c. The sample is obtained from the subject within about 12 hours to about 16 hours after actual or suspected injury; the baseline level of GFAP is about 190 pg / mL and the baseline level of UCH-L1 is about 90 pg / mL; and the assay has a sensitivity of 99% or greater and a specificity of 36% or greater.

[0028] In yet another embodiment of the above-described method, the measurement of the level of GFAP comprises: (a) The samples are subjected to simultaneous or sequential analysis in any order. (1) at least one GFAP capture antibody that binds to an epitope on GFAP or a GFAP fragment to form at least one GFAP capture antibody-GFAP antigen complex; and (2) at least one GFAP detection antibody that contains a detectable label and that binds to an epitope on GFAP not bound by the GFAP capture antibody to form a GFAP antigen-at least one GFAP detection antibody complex; to form at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex; and (b) measuring the amount or concentration of GFAP in the sample based on a signal generated by a detectable label in at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex. Includes.

[0029] In yet another embodiment, the measurement of UCH-L1 in the method identified above comprises: (a) The samples are subjected to simultaneous or sequential analysis in any order. (1) at least one UCH-L1 capture antibody that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form at least one UCH-L1 capture antibody-UCH-L1 antigen complex; and (2) at least one UCH-L1 detection antibody that contains a detectable label and that binds to an epitope on UCH-L1 not bound by the at least one UCH-L1 capture antibody to form a UCH-L1 antigen-at least one UCH-L1 detection antibody complex; to form at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex; and (b) measuring the amount or concentration of UCH-L1 in the sample based on a signal generated by a detectable label in at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex; Includes.

[0030] In one embodiment using the method described above, the subject is evaluated or assessed as having a mild TBI. In one embodiment using the method described above, the subject is evaluated or assessed as having a moderate TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a severe TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a moderate to severe TBI. In yet a further embodiment using the method described above, the subject is evaluated or assessed as not having a TBI.

[0031] The methods described above may further include treating a human subject assessed or assessed as having a TBI (such as mild, moderate, severe, or moderate-severe TBI) with a treatment for TBI (such as a surgical treatment, a therapeutic treatment, or a combination thereof). Any such treatment known in the art and further described herein may be used. Additionally, in further embodiments, any subject treated for TBI may also, optionally, be monitored during or after any course of treatment. Alternatively, the methods may further include monitoring a subject assessed as having a moderate, severe, or moderate-severe TBI (such as a subject who may not yet have received treatment).

[0032] In the above described methods, the sample may be selected from the group consisting of a whole blood sample, a serum sample, a cerebrospinal fluid sample, and a plasma sample. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a plasma sample. In yet other embodiments, the sample is a serum sample. Such a sample may be obtained in various ways. For example, the sample may be obtained after the subject has suffered a head injury or other type of blunt trauma caused by physical shaking, external mechanical or other forces resulting in closed or open head trauma, one or more falls, blunt impact from an explosion or blast. Alternatively, the sample may be obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin. Examples of chemicals or toxins are fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organometallics, drugs of abuse, or one or more combinations thereof. Still further, the sample may be obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof.

[0033] Any of the methods described above may be performed on any human subject, regardless of factors selected from the group consisting of the clinical condition of the human subject, the laboratory values ​​of the human subject, the classification of the human subject as suffering from mild, moderate, severe or moderate-severe TBI, the presentation of low or high levels of UCH-L1, GFAP and / or UCH-L1 and GFAP by the human subject, and the timing of any event during which the human subject may have suffered a head injury.

[0034] In the methods described above, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In still other embodiments, the assay is a clinical chemistry assay. In still other embodiments, the assay is a single molecule detection assay. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a clinical chemistry assay, and the sample is whole blood. In still further embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a clinical chemistry assay, and the sample is serum. In still further embodiments, the assay is a single molecule detection assay, and the sample is serum. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a clinical chemistry assay, and the sample is plasma. In yet a further embodiment, the assay is a single molecule detection assay, and the sample is plasma.

[0035] In yet another aspect, the disclosure relates to a method for aiding in or determining whether a human subject who has suffered a head injury has suffered a traumatic brain injury (TBI), comprising the steps of: performing an assay on a sample obtained from the subject within about 48 hours after the injury to measure or detect a combination of levels of glial fibrillary acidic protein (GFAP) and levels of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the sample; (a) determining that the subject has not suffered a TBI if the sample level of GFAP is less than a baseline level of about 15 pg / mL for GFAP and the sample level of UCH-L1 is less than a baseline level of about 70 pg / mL for UCH-L1; or (b) determining that the subject is more likely to have suffered a TBI than not if the sample level of GFAP is equal to a reference level of GFAP of about 15 pg / mL to about 40 pg / mL and the sample level of UCH-L1 is equal to a reference level of UCH-L1 of about 70 pg / mL to about 150 pg / mL; or (c) determining that the subject is more likely than not to have suffered a TBI if the sample level of GFAP is greater than a baseline level of about 40 pg / mL for GFAP and the sample level of UCH-L1 is greater than a baseline level of about 150 pg / mL for UCH-L1; Includes.

[0036] In one embodiment of the method described above, the assay is performed on a sample obtained from the subject within about 0 to about 4 hours after injury. In another embodiment, the assay is performed on a sample obtained from the subject within about 4 to about 8 hours after injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 8 to about 12 hours after injury. In yet a further embodiment, the assay is performed on a sample obtained from the subject within about 12 to about 16 hours after injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 16 to about 20 hours after injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 20 to about 24 hours after injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 24 to about 28 hours after injury. In yet a further embodiment, the assay is performed on a sample obtained from the subject within about 24 to about 48 hours after injury or suspected injury. In still yet further embodiments, the assay is performed on a sample obtained from the subject within about 28 hours to about 32 hours after injury. In still yet further embodiments, the assay is performed on a sample obtained from the subject within about 32 hours to about 36 hours after injury. In still yet further embodiments, the assay is performed on a sample obtained from the subject within about 36 hours to about 40 hours after injury. In still yet further embodiments, the assay is performed on a sample obtained from the subject within about 40 hours to about 44 hours after injury. In still yet another embodiment, the assay is performed on a sample obtained from the subject within about 44 hours to about 48 hours after injury.

[0037] In another embodiment of the methods described above, the baseline level of GFAP and the baseline level of UCH-L1 are determined by an assay having a sensitivity of about 90% or greater and a specificity of about 35% or greater.

[0038] In yet another embodiment of the methods described above, a sample may be obtained from a subject within about 4 hours to about 16 hours after injury.

[0039] In still another embodiment, the assay of the methods described above has at least 3% greater sensitivity and at least 17% greater specificity compared to assays that measure or detect GFAP or UCH-L1 individually.

[0040] In yet another embodiment of the method described above, the sample is obtained from the subject within about 8 hours to about 12 hours after injury; the baseline level of GFAP is about 30 pg / mL and the baseline level of UCH-L1 is about 110 pg / mL; the assay has a sensitivity of 92% or greater and a specificity of 99% or greater; or the sample is obtained from the subject within about 12 hours to about 16 hours after injury; the baseline level of GFAP is about 30 pg / mL and the baseline level of UCH-L1 is about 110 pg / mL; the assay has a sensitivity of 90% or greater and a specificity of 99% or greater; or the sample is obtained from the subject within about 4 hours to about 8 hours after injury; the baseline level of GFAP is about 40 pg / mL and the baseline level of UCH-L1 is about 100 pg / mL; the method has a sensitivity of 90% or greater and a specificity of 94% or greater; or the sample is obtained from the subject within about 8 hours to about 12 hours after injury; the baseline level of GFAP is about 15 pg / mL and the baseline level of UCH-L1 is about 150 pg / mL; the assay has a sensitivity of 95% or greater and a specificity of 82% or greater; or Samples are obtained from subjects within about 12 hours to about 16 hours after injury; baseline levels of GFAP are about 20 pg / mL and baseline levels of UCH-L1 are about 60 pg / mL; and the assay has a sensitivity of 95% or greater and a specificity of 65% or greater.

[0041] In the methods described above, the levels of GFAP and UCH-L1 can be measured or detected using immunoassays or clinical chemistry assays. Alternatively, in the methods described above, the levels of GFAP and UCH-L1 can be measured or detected using single molecule detection assays.

[0042] In yet another embodiment of the above-described method, the measurement of the level of GFAP comprises: (a) The samples are subjected to simultaneous or sequential analysis in any order. (1) at least one GFAP capture antibody that binds to an epitope on GFAP or a GFAP fragment to form at least one GFAP capture antibody-GFAP antigen complex; and (2) at least one GFAP detection antibody that contains a detectable label and that binds to an epitope on GFAP not bound by the GFAP capture antibody to form a GFAP antigen-at least one GFAP detection antibody complex; to form at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex; and (b) measuring the amount or concentration of GFAP in the sample based on a signal generated by a detectable label in at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex. Includes.

[0043] In yet another embodiment, the measurement of UCH-L1 in the method identified above comprises: (a) The samples are subjected to simultaneous or sequential analysis in any order. (1) at least one UCH-L1 capture antibody that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form at least one UCH-L1 capture antibody-UCH-L1 antigen complex; and (2) at least one UCH-L1 detection antibody that contains a detectable label and that binds to an epitope on UCH-L1 not bound by the at least one UCH-L1 capture antibody to form a UCH-L1 antigen-at least one UCH-L1 detection antibody complex; to form at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex; and (c) measuring the amount or concentration of UCH-L1 in the sample based on a signal generated by a detectable label in at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex. Includes.

[0044] In one embodiment using the method described above, the subject is evaluated or assessed as having a mild TBI. In one embodiment using the method described above, the subject is evaluated or assessed as having a moderate TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a severe TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a moderate to severe TBI. In yet a further embodiment using the method described above, the subject is evaluated or assessed as not having a TBI.

[0045] The methods described above may further include treating a human subject assessed or assessed as having a TBI (such as mild, moderate, severe, or moderate-severe TBI) with a treatment for TBI (such as a surgical treatment, a therapeutic treatment, or a combination thereof). Any such treatment known in the art and further described herein may be used. Additionally, in further embodiments, any subject treated for TBI may also, optionally, be monitored during or after any course of treatment. Alternatively, the methods may further include monitoring a subject assessed as having a moderate, severe, or moderate-severe TBI (such as a subject who may not yet have received treatment).

[0046] In the above described methods, the sample may be selected from the group consisting of a whole blood sample, a serum sample, a cerebrospinal fluid sample, and a plasma sample. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a plasma sample. In yet other embodiments, the sample is a serum sample. Such a sample may be obtained in various ways. For example, the sample may be obtained after the subject has suffered a head injury or other type of blunt trauma caused by physical shaking, external mechanical or other forces resulting in closed or open head trauma, one or more falls, blunt impact from an explosion or blast. Alternatively, the sample may be obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin. Examples of chemicals or toxins are fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organometallics, drugs of abuse, or one or more combinations thereof. Still further, the sample may be obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof.

[0047] Any of the methods described above may be performed on any human subject, regardless of factors selected from the group consisting of the clinical condition of the human subject, the laboratory values ​​of the human subject, the classification of the human subject as suffering from mild, moderate, severe or moderate-severe TBI, the presentation of low or high levels of UCH-L1, GFAP and / or UCH-L1 and GFAP by the human subject, and the timing of any event during which the human subject may have suffered a head injury.

[0048] In the methods described above, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In still other embodiments, the assay is a clinical chemistry assay. In still other embodiments, the assay is a single molecule detection assay. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a clinical chemistry assay, and the sample is whole blood. In still further embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a clinical chemistry assay, and the sample is serum. In still further embodiments, the assay is a single molecule detection assay, and the sample is serum. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a clinical chemistry assay, and the sample is plasma. In yet a further embodiment, the assay is a single molecule detection assay, and the sample is plasma.

[0049] In yet another aspect, the disclosure relates to a method for aiding in or determining whether to perform a head magnetic resonance imaging (MRI) procedure on a human subject who has sustained or may have sustained a head injury, the method comprising: performing an assay on a sample obtained from the subject within about 48 hours after the actual or suspected injury to measure or detect a combination of levels of glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the sample; and (a) determining that the subject does not require an MRI procedure if the sample level of GFAP is less than a baseline level of about 15 pg / mL for GFAP and the sample level of UCH-L1 is less than a baseline level of about 50 pg / mL for UCH-L1; or (b) determining that the subject is more likely than not to require an MRI procedure if the sample level of GFAP is equal to a reference level of GFAP of about 15 pg / mL to about 1000 pg / mL and the sample level of UCH-L1 is equal to a reference level of UCH-L1 of about 50 pg / mL to about 2000 pg / mL; or (c) determining that the subject is more likely than not to require an MRI procedure if the sample level of GFAP is greater than a baseline level of about 1000 pg / mL for GFAP and the sample level of UCH-L1 is greater than a baseline level of about 2000 pg / mL for UCH-L1; Includes.

[0050] In the methods described above, the subject may undergo an MRI after the assay is performed, and the subject is suspected of having TBI based on the MRI results. In yet another embodiment, the baseline levels of GFAP and the baseline levels of UCH-L1 correlate with a negative MRI result.

[0051] In one embodiment of the method described above, the assay is performed on a sample obtained from the subject within about 0 to about 4 hours after the actual or suspected injury. In another embodiment, the assay is performed on a sample obtained from the subject within about 4 to about 8 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 8 to about 12 hours after the actual or suspected injury. In yet a further embodiment, the assay is performed on a sample obtained from the subject within about 12 to about 16 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 16 to about 20 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 20 to about 24 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 24 to about 28 hours after the actual or suspected injury. In still further embodiments, the assay is performed on a sample obtained from a subject within about 24 hours to about 48 hours after the injury or suspected injury. In yet even further embodiments, the assay is performed on a sample obtained from a subject within about 28 hours to about 32 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 32 hours to about 36 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 36 hours to about 40 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 40 hours to about 44 hours after the actual injury or suspected injury. In still yet another embodiment, the assay is performed on a sample obtained from a subject within about 44 hours to about 48 hours after the actual injury or suspected injury.

[0052] In the above-described methods for determining or assessing whether to perform an MRI, the subject may be presumed to have a traumatic brain injury based on an MRI or CT scan that has been performed or has already been performed (meaning before the assay is performed). For example, depending on the subject's medical condition (such as when the patient is unconscious), an MRI or CT scan may be performed to evaluate and / or assess whether the subject has a TBI immediately after the subject arrives at an emergency room, trauma center, or other facility. Such an MRI or CT scan may be performed before the assay is performed to confirm and determine whether the subject has a mild, moderate, or moderate-severe TBI. After the assay is performed, one or more subsequent MRI (or CT scans) may be performed based on the results of the assay as part of the physician's (or other medical personnel's) management of the TBI (e.g., to determine whether surgical and / or pharmacological intervention may be required).

[0053] In certain embodiments of the above method, the subject may be presumed to have traumatic brain injury based on MRI. For example, the subject may be presumed to have mild TBI based on MRI. Alternatively, the subject may be presumed to have moderate TBI based on MRI. Alternatively, the subject may be presumed to have severe TBI based on MRI. Alternatively, the subject may be presumed to have moderate to severe TBI based on MRI. Still further, the subject may be presumed not to have TBI based on MRI.

[0054] In yet another embodiment of the methods described above, the baseline level of GFAP and the baseline level of UCH-L1 are determined by an assay having a sensitivity of about 80% to about 98% and a specificity of about 30% to about 85%.

[0055] In a further embodiment of the methods described above, the subject may have received a negative CT scan result before the assay is performed.

[0056] In one embodiment using the method described above, the subject is evaluated or assessed as having a mild TBI. In one embodiment using the method described above, the subject is evaluated or assessed as having a moderate TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a severe TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a moderate to severe TBI. In yet a further embodiment using the method described above, the subject is evaluated or assessed as not having a TBI.

[0057] The methods described above may further include treating a human subject assessed or assessed as having a TBI (such as mild, moderate, severe, or moderate-severe TBI) with a treatment for TBI (such as a surgical treatment, a therapeutic treatment, or a combination thereof). Any such treatment known in the art and further described herein may be used. Additionally, in further embodiments, any subject treated for TBI may also, optionally, be monitored during or after any course of treatment. Alternatively, the methods may further include monitoring a subject assessed as having a moderate, severe, or moderate-severe TBI (such as a subject who may not yet have received treatment).

[0058] In the above described methods, the sample may be selected from the group consisting of a whole blood sample, a serum sample, a cerebrospinal fluid sample, and a plasma sample. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a plasma sample. In yet other embodiments, the sample is a serum sample. Such a sample may be obtained in various ways. For example, the sample may be obtained after the subject has suffered a head injury or other type of blunt trauma caused by physical shaking, external mechanical or other forces resulting in closed or open head trauma, one or more falls, blunt impact from an explosion or blast. Alternatively, the sample may be obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin. Examples of chemicals or toxins are fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organometallics, drugs of abuse, or one or more combinations thereof. Still further, the sample may be obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof.

[0059] Any of the methods described above may be performed on any human subject, regardless of factors selected from the group consisting of the clinical condition of the human subject, the laboratory values ​​of the human subject, the classification of the human subject as suffering from mild, moderate, severe or moderate-severe TBI, the presentation of low or high levels of UCH-L1, GFAP and / or UCH-L1 and GFAP by the human subject, and the timing of any event during which the human subject may have suffered a head injury.

[0060] In the methods described above, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In still other embodiments, the assay is a clinical chemistry assay. In still other embodiments, the assay is a single molecule detection assay. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a clinical chemistry assay, and the sample is whole blood. In still further embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a clinical chemistry assay, and the sample is serum. In still further embodiments, the assay is a single molecule detection assay, and the sample is serum. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a clinical chemistry assay, and the sample is plasma. In yet a further embodiment, the assay is a single molecule detection assay, and the sample is plasma.

[0061] In yet another aspect, the disclosure relates to a method for aiding in or determining whether to perform a head magnetic resonance imaging (MRI) procedure on a human subject who has sustained or may have sustained a head injury, the method comprising: performing an assay on a sample obtained from the subject within about 48 hours after the actual or suspected injury to measure or detect a combination of levels of glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the sample; and (a) determining that the subject does not require an MRI procedure if the sample level of GFAP is equal to a reference level of GFAP of about 0 pg / mL to about 68 pg / mL or the sample level of UCH-L1 is equal to a reference level of UCH-L1 of about 0 pg / mL to about 99 pg / mL; or (b) determining that the subject is more likely than not to require an MRI procedure if the sample level of GFAP is greater than a baseline level of about 68 pg / mL for GFAP and the sample level of UCH-L1 is greater than a baseline level of about 99 pg / mL for UCH-L1; Includes.

[0062] In one embodiment of the method described above, the assay is performed on a sample obtained from the subject within about 0 to about 4 hours after the actual or suspected injury. In another embodiment, the assay is performed on a sample obtained from the subject within about 4 to about 8 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 8 to about 12 hours after the actual or suspected injury. In yet a further embodiment, the assay is performed on a sample obtained from the subject within about 12 to about 16 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 16 to about 20 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 20 to about 24 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 24 to about 28 hours after the actual or suspected injury. In still further embodiments, the assay is performed on a sample obtained from a subject within about 24 hours to about 48 hours after the injury or suspected injury. In yet even further embodiments, the assay is performed on a sample obtained from a subject within about 28 hours to about 32 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 32 hours to about 36 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 36 hours to about 40 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 40 hours to about 44 hours after the actual injury or suspected injury. In still yet another embodiment, the assay is performed on a sample obtained from a subject within about 44 hours to about 48 hours after the actual injury or suspected injury.

[0063] In the methods described above, the subject may undergo an MRI after the assay is performed, and the subject is suspected of having TBI based on the MRI results. In yet another embodiment, the baseline level of GFAP or the baseline level of UCH-L1 correlates with a negative MRI result.

[0064] In the above-described methods for determining or assessing whether to perform an MRI, the subject may be presumed to have a traumatic brain injury based on an MRI or CT scan that has been performed or has already been performed (meaning before the assay is performed). For example, depending on the subject's medical condition (such as when the patient is unconscious), an MRI or CT scan may be performed to evaluate and / or assess whether the subject has a TBI immediately after the subject arrives at an emergency room, trauma center, or other facility. Such an MRI or CT scan may be performed before the assay is performed to confirm and determine whether the subject has a mild or moderate-severe TBI. After the assay is performed, one or more subsequent MRI (or CT scans) may be performed based on the results of the assay as part of the physician's (or other medical personnel's) management of the TBI (e.g., to determine whether surgical and / or pharmacological intervention may be required).

[0065] In certain embodiments of the above method, the subject may be presumed to have traumatic brain injury based on MRI. For example, the subject may be presumed to have mild TBI based on MRI. Alternatively, the subject may be presumed to have moderate TBI based on MRI. Alternatively, the subject may be presumed to have severe TBI based on MRI. Alternatively, the subject may be presumed to have moderate to severe TBI based on MRI. Still further, the subject may be presumed not to have TBI based on MRI.

[0066] In certain embodiments of the above method, the reference level used is correlated with or corresponds to a positive head computed tomography. For example, the reference level can be correlated with or corresponds to a subject with a positive head computed tomography (such as through an increase or decrease in the reference level). Alternatively, the reference level can be correlated with or corresponds to a subject with a negative head computed tomography (such as through an increase or decrease in the reference level). Still further alternatively, the reference level can be correlated with or corresponds to a subject with intracerebral hemorrhage or an improved or worsening intracerebral hemorrhage (such as through an increase or decrease in the reference level). In other embodiments of the above method, the reference level is correlated with or corresponds to a control subject that does not suffer from TBI.

[0067] In yet another embodiment of the method described above, the reference level of GFAP is determined by an assay having a sensitivity of about 90% to about 95% and a specificity of 31% to about 46%. In yet another embodiment of the method described above, the reference level of UCH-L1 is determined by an assay having a sensitivity of about 81% to about 84% and a specificity of 31% to about 46%.

[0068] In a further embodiment of the methods described above, the subject may have received a negative CT scan result before the assay is performed.

[0069] In one embodiment using the method described above, the subject is evaluated or assessed as having a mild TBI. In one embodiment using the method described above, the subject is evaluated or assessed as having a moderate TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a severe TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a moderate to severe TBI. In yet a further embodiment using the method described above, the subject is evaluated or assessed as not having a TBI.

[0070] The methods described above may further include treating a human subject assessed or assessed as having a TBI (such as mild, moderate, severe, or moderate-severe TBI) with a treatment for TBI (such as a surgical treatment, a therapeutic treatment, or a combination thereof). Any such treatment known in the art and further described herein may be used. Additionally, in further embodiments, any subject treated for TBI may also, optionally, be monitored during or after any course of treatment. Alternatively, the methods may further include monitoring a subject assessed as having a moderate, severe, or moderate-severe TBI (such as a subject who may not yet have received treatment).

[0071] In the above described methods, the sample may be selected from the group consisting of a whole blood sample, a serum sample, a cerebrospinal fluid sample, and a plasma sample. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a plasma sample. In yet other embodiments, the sample is a serum sample. Such a sample may be obtained in various ways. For example, the sample may be obtained after the subject has suffered a head injury or other type of blunt trauma caused by physical shaking, external mechanical or other forces resulting in closed or open head trauma, one or more falls, blunt impact from an explosion or blast. Alternatively, the sample may be obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin. Examples of chemicals or toxins are fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organometallics, drugs of abuse, or one or more combinations thereof. Still further, the sample may be obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof.

[0072] Any of the methods described above may be performed on any human subject, regardless of factors selected from the group consisting of the clinical condition of the human subject, the laboratory values ​​of the human subject, the classification of the human subject as suffering from mild, moderate, severe or moderate-severe TBI, the presentation of low or high levels of UCH-L1, GFAP and / or UCH-L1 and GFAP by the human subject, and the timing of any event during which the human subject may have suffered a head injury.

[0073] In the methods described above, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In still other embodiments, the assay is a clinical chemistry assay. In still other embodiments, the assay is a single molecule detection assay. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a clinical chemistry assay, and the sample is whole blood. In still further embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a clinical chemistry assay, and the sample is serum. In still further embodiments, the assay is a single molecule detection assay, and the sample is serum. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a clinical chemistry assay, and the sample is plasma. In yet a further embodiment, the assay is a single molecule detection assay, and the sample is plasma.

[0074] In yet another aspect, the present disclosure relates to a method for aiding in or predicting the outcome of a human subject who has sustained or may have sustained a head injury, the method comprising: performing an assay on a sample obtained from the subject within about 48 hours after the actual or suspected injury to measure or detect a combination of levels of glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the sample; and (a) predicting a favorable outcome for the subject if the sample level of GFAP is less than a reference level of about 80 pg / mL for GFAP and the sample level of UCH-L1 is less than a reference level of about 130 pg / mL for UCH-L1; or (b) a sample level of GFAP equal to a reference level of GFAP of about 80 pg / mL to about 2000 pg / mL and a sample level of UCH-L1 equal to a reference level of UCH-L1 of about 130 pg / mL to about 2000 pg / mL is more likely to predict an adverse outcome for the subject than is otherwise; or (c) predicting an adverse outcome for a subject when the sample level of GFAP is greater than a reference level of about 2000 pg / mL and the sample level of UCH-L1 is greater than a reference level of about 2000 pg / mL; Includes.

[0075] In another embodiment of the above described method, the subject may receive a GOSE (Extended Glasgow Outcome Scale) score after the method is performed, and the subject is predicted to have a poor outcome based on the GOSE score. In yet another embodiment, the baseline level of GFAP and the baseline level of UCH-L1 correlate with the subject having a poor outcome based on a GOSE score of 1.

[0076] In one embodiment of the method described above, the assay is performed on a sample obtained from the subject within about 0 to about 4 hours after the actual or suspected injury. In another embodiment, the assay is performed on a sample obtained from the subject within about 4 to about 8 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 8 to about 12 hours after the actual or suspected injury. In yet a further embodiment, the assay is performed on a sample obtained from the subject within about 12 to about 16 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 16 to about 20 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 20 to about 24 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 24 to about 28 hours after the actual or suspected injury. In still further embodiments, the assay is performed on a sample obtained from a subject within about 24 hours to about 48 hours after the injury or suspected injury. In yet even further embodiments, the assay is performed on a sample obtained from a subject within about 28 hours to about 32 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 32 hours to about 36 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 36 hours to about 40 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 40 hours to about 44 hours after the actual injury or suspected injury. In still yet another embodiment, the assay is performed on a sample obtained from a subject within about 44 hours to about 48 hours after the actual injury or suspected injury.

[0077] In yet another embodiment of the methods described above, the baseline level of GFAP and the baseline level of UCH-L1 are determined by an assay having a sensitivity of about 80% to about 97% and a specificity of about 30% to about 95%.

[0078] In the above-described methods, the levels of GFAP and UCH-L1 are measured or detected using immunoassays or clinical chemistry assays. Alternatively, in the above-described methods, the levels of GFAP and UCH-L1 are measured or detected using single molecule detection assays.

[0079] In yet another embodiment of the above-described method, the measurement of the level of GFAP comprises: (a) The samples are subjected to simultaneous or sequential analysis in any order. (1) at least one GFAP capture antibody that binds to an epitope on GFAP or a GFAP fragment to form at least one GFAP capture antibody-GFAP antigen complex; and (2) at least one GFAP detection antibody that contains a detectable label and that binds to an epitope on GFAP not bound by the GFAP capture antibody to form a GFAP antigen-at least one GFAP detection antibody complex; to form at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex; and (b) measuring the amount or concentration of GFAP in the sample based on a signal generated by a detectable label in at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex. Includes.

[0080] In yet another embodiment, the measurement of UCH-L1 in the method identified above comprises: (a) The samples are subjected to simultaneous or sequential analysis in any order. (1) at least one UCH-L1 capture antibody that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form at least one UCH-L1 capture antibody-UCH-L1 antigen complex; and (2) at least one UCH-L1 detection antibody that contains a detectable label and that binds to an epitope on UCH-L1 not bound by the at least one UCH-L1 capture antibody to form a UCH-L1 antigen-at least one UCH-L1 detection antibody complex; to form at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex; and (b) measuring the amount or concentration of UCH-L1 in the sample based on a signal generated by a detectable label in at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex; Includes.

[0081] In one embodiment using the method described above, the subject is evaluated or assessed as having a mild TBI. In one embodiment using the method described above, the subject is evaluated or assessed as having a moderate TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a severe TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a moderate to severe TBI. In yet a further embodiment using the method described above, the subject is evaluated or assessed as not having a TBI.

[0082] The methods described above may further include treating a human subject assessed or assessed as having a TBI (such as mild, moderate, severe, or moderate-severe TBI) with a treatment for TBI (such as a surgical treatment, a therapeutic treatment, or a combination thereof). Any such treatment known in the art and further described herein may be used. Additionally, in further embodiments, any subject treated for TBI may also, optionally, be monitored during or after any course of treatment. Alternatively, the methods may further include monitoring a subject assessed as having a moderate, severe, or moderate-severe TBI (such as a subject who may not yet have received treatment).

[0083] In the above described methods, the sample may be selected from the group consisting of a whole blood sample, a serum sample, a cerebrospinal fluid sample, and a plasma sample. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a plasma sample. In yet other embodiments, the sample is a serum sample. Such a sample may be obtained in various ways. For example, the sample may be obtained after the subject has suffered a head injury or other type of blunt trauma caused by physical shaking, external mechanical or other forces resulting in closed or open head trauma, one or more falls, blunt impact from an explosion or blast. Alternatively, the sample may be obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin. Examples of chemicals or toxins are fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organometallics, drugs of abuse, or one or more combinations thereof. Still further, the sample may be obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof.

[0084] Any of the methods described above may be performed on any human subject, regardless of factors selected from the group consisting of the clinical condition of the human subject, the laboratory values ​​of the human subject, the classification of the human subject as suffering from mild, moderate, severe or moderate-severe TBI, the presentation of low or high levels of UCH-L1, GFAP and / or UCH-L1 and GFAP by the human subject, and the timing of any event during which the human subject may have suffered a head injury.

[0085] In the methods described above, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In still other embodiments, the assay is a clinical chemistry assay. In still other embodiments, the assay is a single molecule detection assay. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a clinical chemistry assay, and the sample is whole blood. In still further embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a clinical chemistry assay, and the sample is serum. In still further embodiments, the assay is a single molecule detection assay, and the sample is serum. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a clinical chemistry assay, and the sample is plasma. In yet a further embodiment, the assay is a single molecule detection assay, and the sample is plasma.

[0086] In yet another aspect, the disclosure relates to a method for aiding in the diagnosis of or determining whether a subject who has sustained or may have sustained a head injury is suffering from a moderate, severe, or moderate-severe traumatic brain injury (TBI), the method comprising: performing an assay on a sample obtained from the subject within about 48 hours after the actual or suspected injury to measure or detect a combination of levels of glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the sample; and determining that the subject has not suffered a moderate, severe, or moderate-severe TBI if the sample level of GFAP is equal to a reference level of GFAP of about 105 pg / mL to about 890 pg / mL and the sample level of UCH-L1 is equal to a reference level of UCH-L1 of about 110 pg / mL to about 2000 pg / mL. Includes.

[0087] In one embodiment of the method described above, the assay is performed on a sample obtained from the subject within about 0 to about 4 hours after the actual or suspected injury. In another embodiment, the assay is performed on a sample obtained from the subject within about 4 to about 8 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 8 to about 12 hours after the actual or suspected injury. In yet a further embodiment, the assay is performed on a sample obtained from the subject within about 12 to about 16 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 16 to about 20 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 20 to about 24 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 24 to about 28 hours after the actual or suspected injury. In still further embodiments, the assay is performed on a sample obtained from a subject within about 24 hours to about 48 hours after the injury or suspected injury. In yet even further embodiments, the assay is performed on a sample obtained from a subject within about 28 hours to about 32 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 32 hours to about 36 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 36 hours to about 40 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 40 hours to about 44 hours after the actual injury or suspected injury. In still yet another embodiment, the assay is performed on a sample obtained from a subject within about 44 hours to about 48 hours after the actual injury or suspected injury.

[0088] In one embodiment of the method described above, the subject may have received a Glasgow Coma Scale (GCS) score before or after the assay is performed. In another embodiment, the subject receiving such a GCS score is suspected to have moderate TBI based on the determined GCS score. In another embodiment, the subject receiving such a GCS score is suspected to have severe TBI. In another embodiment, the subject receiving such a GCS score is suspected to have moderate to severe TBI based on the determined GCS score. In yet another embodiment of the method described above, the reference level of GFAP and the reference level of UCH-L1 correlate or correspond to a Glasgow Coma Scale (GCS) score of 3 to 8 (severe TBI). In yet another aspect, the reference level of GFAP and the reference level of UCH-L1 correlate to a Glasgow Coma Scale (GCS) score of 9 to 12 (moderate TBI). In other embodiments, the baseline levels of GFAP and the baseline levels of UCH-L1 correlate or correspond to a GCS (Glasgow Coma Scale) score of 3-12 (moderate-severe TBI).

[0089] In another embodiment of the method described above, (a) the baseline level of GFAP is about 105 pg / mL and the baseline level of UCH-L1 is about 840 pg / mL; (b) the baseline level of GFAP is about 150 pg / mL and the baseline level of UCH-L1 is about 2000 pg / mL; (c) the baseline level of GFAP is about 240 pg / mL and the baseline level of UCH-L1 is about 860 pg / mL; (d) the baseline level of GFAP is about 265 pg / mL and the baseline level of UCH-L1 is about , about 860 pg / mL; (e) the baseline level of GFAP is about 370 pg / mL and the baseline level of UCH-L1 is about 110 pg / mL; (f) the baseline level of GFAP is about 505 pg / mL and the baseline level of UCH-L1 is about 1580 pg / mL; (g) the baseline level of GFAP is about 695 pg / mL and the baseline level of UCH-L1 is about 1570 pg / mL; or (h) the baseline level of GFAP is about 890 pg / mL and the baseline level of UCH-L1 is about 920 pg / mL.

[0090] In yet another embodiment of the method described above, (a) a sample is obtained from a subject within about 8 hours to about 12 hours after actual or suspected injury, and the level of GFAP is about 240 pg / mL and the level of UCH-L1 is about 860 pg / mL; (b) a sample is obtained from a subject within about 8 hours to about 12 hours after actual or suspected injury, and the level of GFAP is about 265 pg / mL and the level of UCH-L1 is about 860 pg / mL; (c) the sample is obtained from the subject within about 8 hours to about 12 hours after the actual or suspected injury, and the level of GFAP is about 890 pg / mL and the level of UCH-L1 is about 920 pg / mL; (d) the sample is obtained from the subject within about 12 hours to about 16 hours after the actual or suspected injury, and the level of GFAP is about 105 pg / mL and the level of UCH-L1 is about 840 pg / mL; (e) the sample is obtained from the subject within about 12 hours to about 16 hours after the actual or suspected injury, and the level of GFAP is about 105 pg / mL and the level of UCH-L1 is about 840 pg / mL. (f) a sample is obtained from a subject within about 12 hours to about 16 hours after the actual or suspected injury, and the level of GFAP is about 370 pg / mL and the level of UCH-L1 is about 110 pg / mL; (g) a sample is obtained from a subject within about 12 hours to about 16 hours after the actual or suspected injury, and the level of GFAP is about 505 pg / mL. and the level of UCH-L1 is about 1590 pg / mL; (h) the sample is obtained from the subject within about 12 to about 16 hours after the actual or suspected injury, and the level of GFAP is about 695 pg / mL, and the level of UCH-L1 is about 1570 pg / mL; (i) the sample is obtained from the subject within about 12 to about 16 hours after the actual or suspected injury, and the level of GFAP is about 150 pg / mL, and the level of UCH-L1 is about 2000 pg / mL.

[0091] In another embodiment of the methods described above, the baseline level of GFAP and the baseline level of UCH-L1 are determined by an assay having a sensitivity of about 79% or greater and a specificity of about 33% or greater.

[0092] In yet another embodiment of the methods described above, the sample is obtained from the subject within about 8 hours to about 16 hours after the actual or suspected injury.

[0093] In yet another embodiment of the methods described above, the assay has at least 2% greater sensitivity and at least 3% greater specificity compared to assays that measure or detect GFAP or UCH-L1 individually.

[0094] In yet another embodiment of the method described above, e. the sample is obtained from the subject within about 8 hours to about 12 hours after the actual or suspected injury; the baseline level of GFAP is about 240 pg / mL and the baseline level of UCH-L1 is about 860 pg / mL; the assay has a sensitivity of 97% or greater and a specificity of 51% or greater; or f. the sample is obtained from the subject within about 12 hours to about 16 hours after the actual or suspected injury; the baseline level of GFAP is about 105 pg / mL and the baseline level of UCH-L1 is about 840 pg / mL; the assay has a sensitivity of 97.5% or greater and a specificity of 36% or greater; or g. the sample is obtained from the subject within about 8 hours to about 12 hours after the actual or suspected injury; the baseline level of GFAP is about 890 pg / mL and the baseline level of UCH-L1 is about 920 pg / mL; the assay has a sensitivity of 90% or greater and a specificity of 79% or greater; or h. The sample is obtained from the subject within about 12 hours to about 16 hours after the actual or suspected injury; the baseline level of GFAP is about 505 pg / mL and the baseline level of UCH-L1 is about 1580 pg / mL; and the assay has a sensitivity of 90% or greater and a specificity of 66% or greater.

[0095] In yet another embodiment of the above-described method, the measurement of the level of GFAP comprises: (a) The samples are subjected to simultaneous or sequential analysis in any order. (1) at least one GFAP capture antibody that binds to an epitope on GFAP or a GFAP fragment to form at least one GFAP capture antibody-GFAP antigen complex; and (2) at least one GFAP detection antibody that contains a detectable label and that binds to an epitope on GFAP not bound by the GFAP capture antibody to form a GFAP antigen-at least one GFAP detection antibody complex; to form at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex; and (b) measuring the amount or concentration of GFAP in the sample based on a signal generated by a detectable label in at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex. Includes.

[0096] In yet another embodiment, the measurement of UCH-L1 in the method identified above comprises: (a) The samples are subjected to simultaneous or sequential analysis in any order. (1) at least one UCH-L1 capture antibody that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form at least one UCH-L1 capture antibody-UCH-L1 antigen complex; and (2) at least one UCH-L1 detection antibody that contains a detectable label and that binds to an epitope on UCH-L1 not bound by the at least one UCH-L1 capture antibody to form a UCH-L1 antigen-at least one UCH-L1 detection antibody complex; to form at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex; and (b) measuring the amount or concentration of UCH-L1 in the sample based on a signal generated by a detectable label in at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex; Includes.

[0097] In one embodiment using the method described above, the subject is evaluated or assessed as having a moderate TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a severe TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a moderate to severe TBI. In yet a further embodiment using the method described above, the subject is evaluated or assessed as not having a TBI.

[0098] The methods described above may further include treating the human subject assessed or assessed as having moderate, severe, or moderate-severe TBI with a treatment for TBI (e.g., surgical treatment, therapeutic treatment, or a combination thereof). Any such treatment known in the art and further described herein may be used. Furthermore, in further embodiments, any subject treated for TBI may also be optionally monitored during or after any course of treatment. Alternatively, the methods may further include monitoring a subject assessed as having moderate, severe, or moderate-severe TBI (such as a subject who may not yet have received treatment).

[0099] In the above described methods, the sample may be selected from the group consisting of a whole blood sample, a serum sample, a cerebrospinal fluid sample, and a plasma sample. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a plasma sample. In yet other embodiments, the sample is a serum sample. Such a sample may be obtained in various ways. For example, the sample may be obtained after the subject has suffered a head injury or other type of blunt trauma caused by physical shaking, external mechanical or other forces resulting in closed or open head trauma, one or more falls, blunt impact from an explosion or blast. Alternatively, the sample may be obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin. Examples of chemicals or toxins are fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organometallics, drugs of abuse, or one or more combinations thereof. Still further, the sample may be obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof.

[0100] Any of the methods described above may be performed on any human subject, regardless of factors selected from the group consisting of the clinical condition of the human subject, the laboratory values ​​of the human subject, the classification of the human subject as suffering from mild, moderate, severe or moderate-severe TBI, the presentation of low or high levels of UCH-L1, GFAP and / or UCH-L1 and GFAP by the human subject, and the timing of any event during which the human subject may have suffered a head injury.

[0101] In the methods described above, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In still other embodiments, the assay is a clinical chemistry assay. In still other embodiments, the assay is a single molecule detection assay. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a clinical chemistry assay, and the sample is whole blood. In still further embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a clinical chemistry assay, and the sample is serum. In still further embodiments, the assay is a single molecule detection assay, and the sample is serum. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a clinical chemistry assay, and the sample is plasma. In yet a further embodiment, the assay is a single molecule detection assay, and the sample is plasma.

[0102] In yet another aspect, the disclosure relates to a method for aiding in or determining whether to perform a head computed tomography (CT) scan on a human subject who has sustained or may have sustained a head injury, the method comprising: performing an assay on a sample obtained from the subject within about 48 hours after the actual or suspected injury to measure or detect a combination of levels of glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the sample; and determining that the subject does not require a CT scan if the sample level of GFAP is equal to a reference level of about 50 pg / mL to about 975 pg / mL of GFAP and the sample level of UCH-L1 is equal to a reference level of about 90 pg / mL to about 2000 pg / mL of UCH-L1. Includes.

[0103] In one embodiment of the methods described above, the subject undergoes a CT scan before or after the assay is performed, and the subject is suspected of having a TBI based on the CT scan results, hi another embodiment, the baseline levels of GFAP and the baseline levels of UCH-L1 correlate with a negative CT scan result.

[0104] More specifically, in the above-described methods for determining or assessing whether to perform a head CT scan, the subject may be presumed to have a traumatic brain injury based on a CT scan that has been performed or has already been performed (meaning before the assay is performed). For example, depending on the subject's medical condition (such as when the patient is unconscious), a CT scan may be performed to evaluate and / or assess whether the subject has a TBI immediately after the subject arrives at an emergency room, trauma center, or other facility. Such a CT scan may be performed before the assay is performed to confirm and determine whether the subject has a mild or moderate-severe TBI. After the assay is performed, one or more subsequent CT scans may be performed based on the results of the assay as part of the physician's (or other medical personnel's) management of the TBI (e.g., to determine whether surgical and / or pharmacological intervention may be required).

[0105] In certain embodiments of the above method, the subject can be inferred to have traumatic brain injury based on CT scan. For example, the subject can be inferred to have mild TBI based on CT scan. Alternatively, the subject can be inferred to have moderate TBI based on CT scan. Alternatively, the subject can be inferred to have severe TBI based on CT scan. Alternatively, the subject can be inferred to have moderate to severe TBI based on CT scan. Still further, the subject can be inferred to not have TBI based on CT scan.

[0106] In certain embodiments of the above method, the reference level used is correlated with or corresponds to a positive head computed tomography. For example, the reference level can be correlated with or corresponds to a subject with a positive head computed tomography (such as through an increase or decrease in the reference level). Alternatively, the reference level can be correlated with or corresponds to a subject with a negative head computed tomography (such as through an increase or decrease in the reference level). Still further alternatively, the reference level can be correlated with or corresponds to a subject with intracerebral hemorrhage or an improved or worsening intracerebral hemorrhage (such as through an increase or decrease in the reference level). In other embodiments of the above method, the reference level is correlated with or corresponds to a control subject that does not suffer from TBI.

[0107] In one embodiment of the method described above, the assay is performed on a sample obtained from the subject within about 0 to about 4 hours after the actual or suspected injury. In another embodiment, the assay is performed on a sample obtained from the subject within about 4 to about 8 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 8 to about 12 hours after the actual or suspected injury. In yet a further embodiment, the assay is performed on a sample obtained from the subject within about 12 to about 16 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 16 to about 20 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 20 to about 24 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 24 to about 28 hours after the actual or suspected injury. In still further embodiments, the assay is performed on a sample obtained from a subject within about 24 hours to about 48 hours after the injury or suspected injury. In yet even further embodiments, the assay is performed on a sample obtained from a subject within about 28 hours to about 32 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 32 hours to about 36 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 36 hours to about 40 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 40 hours to about 44 hours after the actual injury or suspected injury. In still yet another embodiment, the assay is performed on a sample obtained from a subject within about 44 hours to about 48 hours after the actual injury or suspected injury.

[0108] In yet another embodiment of the above method, (a) the baseline level of GFAP is about 50 pg / mL and the baseline level of UCH-L1 is about 2000 pg / mL; (b) the baseline level of GFAP is about 95 pg / mL and the baseline level of UCH-L1 is about 2000 pg / mL; (c) the baseline level of GFAP is about 110 pg / mL and the baseline level of UCH-L1 is about 2000 pg / mL; (d) the baseline level of GFAP is about 115 pg / mL and the baseline level of UCH-L1 is about 2000 pg / mL. L and the baseline level of UCH-L1 is about 110 pg / mL; (e) the baseline level of GFAP is about 140 pg / mL and the baseline level of UCH-L1 is about 2000 pg / mL; (f) the baseline level of GFAP is about 150 pg / mL and the baseline level of UCH-L1 is about 190 pg / mL; (g) the baseline level of GFAP is about 190 pg / mL and the baseline level of UCH-L1 is about 90 pg / mL; (h) the baseline level of GFAP is about 2 (i) the reference level of GFAP is about 285 pg / mL and the reference level of UCH-L1 is about 190 pg / mL; (j) the reference level of GFAP is about 500 pg / mL and the reference level of UCH-L1 is about 1450 pg / mL; (k) the reference level of GFAP is about 555 pg / mL and the reference level of UCH-L1 is about 810 pg / mL; (l) the reference level of GFAP is about 500 pg / mL and the reference level of UCH-L1 is about 1450 pg / mL; (m) the base level of GFAP is about 840 pg / mL and the base level of UCH-L1 is about 2000 pg / mL; (n) the base level of GFAP is about 880 pg / mL and the base level of UCH-L1 is about 810 pg / mL; or (o) the base level of GFAP is about 975 pg / mL and the base level of UCH-L1 is about 1580 pg / mL.

[0109] In yet another embodiment of the above method, (a) a sample is obtained from a subject within about 4 hours to about 8 hours after the actual or suspected injury, and the level of GFAP is about 50 pg / mL and the level of UCH-L1 is about 2000 pg / mL; (b) a sample is obtained from a subject within about 4 hours to about 8 hours after the actual or suspected injury, and the level of GFAP is about 110 pg / mL and the level of UCH-L1 is about 2000 pg / mL; (c) a sample is obtained from a subject within about 4 hours to about 8 hours after the actual or suspected injury, and the level of GFAP is about 110 pg / mL and the level of UCH-L1 is about 2000 pg / mL. (d) a sample is obtained from a subject within about 4 to about 8 hours after actual or suspected injury, and the level of GFAP is about 140 pg / mL and the level of UCH-L1 is about 2000 pg / mL; (e) a sample is obtained from a subject within about 4 to about 8 hours after actual or suspected injury, and the level of GFAP is about 890 pg / mL and the level of UCH-L1 is about 920 pg / mL. (f) a sample is obtained from a subject within about 12 hours to about 16 hours after the actual or suspected injury, and the level of GFAP is about 105 pg / mL and the level of UCH-L1 is about 840 pg / mL; (g) a sample is obtained from a subject within about 12 hours to about 16 hours after the actual or suspected injury, and the level of GFAP is about 370 pg / mL and the level of UCH-L1 is about 110 pg / mL; (h) a sample is obtained from a subject within about 12 hours to about 16 hours after the actual or suspected injury, and the level of GFAP is about 370 pg / mL and the level of UCH-L1 is about 110 pg / mL. (i) the sample is obtained from the subject within about 12 to about 16 hours after the actual or suspected injury, and the level of GFAP is about 695 pg / mL and the level of UCH-L1 is about 1570 pg / mL; (j) the sample is obtained from the subject within about 12 to about 16 hours after the actual or suspected injury, and the level of GFAP is about 150 pg / mL and the level of UCH-L1 is about 2000 pg / mL.

[0110] In yet another embodiment of the methods described above, the baseline level of GFAP and the baseline level of UCH-L1 are determined by an assay having a sensitivity of about 54% or greater and a specificity of about 32% or greater.

[0111] In still yet another embodiment of the methods described above, the sample is obtained from the subject within about 4 hours to about 16 hours after the actual or suspected injury.

[0112] In yet another embodiment of the methods described above, the assay has at least 2% greater sensitivity and at least 4% greater specificity compared to assays that measure or detect GFAP or UCH-L1 individually.

[0113] In yet another embodiment of the method described above, d. the sample is obtained from the subject within about 4 hours to about 8 hours after actual or suspected injury; the baseline level of GFAP is about 110 pg / mL and the baseline level of UCH-L1 is about 2000 pg / mL; the assay has a sensitivity of 95% or greater and a specificity of 62% or greater; e. the sample is obtained from the subject within about 8 hours to about 12 hours after the actual or suspected injury; the baseline level of GFAP is about 240 pg / mL and the baseline level of UCH-L1 is about 300 pg / mL; the assay has a sensitivity of 91.5% or greater and a specificity of 52% or greater; or f. The sample is obtained from the subject within about 12 to about 16 hours after actual or suspected injury; the baseline level of GFAP is about 190 pg / mL and the baseline level of UCH-L1 is about 90 pg / mL; and the assay has a sensitivity of 99% or greater and a specificity of 36% or greater.

[0114] In yet another embodiment of the above-described method, the measurement of the level of GFAP comprises: (a) The samples are subjected to simultaneous or sequential analysis in any order. (1) at least one GFAP capture antibody that binds to an epitope on GFAP or a GFAP fragment to form at least one GFAP capture antibody-GFAP antigen complex; and (2) at least one GFAP detection antibody that contains a detectable label and that binds to an epitope on GFAP not bound by the GFAP capture antibody to form a GFAP antigen-at least one GFAP detection antibody complex; to form at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex; and (b) measuring the amount or concentration of GFAP in the sample based on a signal generated by a detectable label in at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex. Includes.

[0115] In yet another embodiment, the measurement of UCH-L1 in the method identified above comprises: (a) The samples are subjected to simultaneous or sequential analysis in any order. (1) at least one UCH-L1 capture antibody that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form at least one UCH-L1 capture antibody-UCH-L1 antigen complex; and (2) at least one UCH-L1 detection antibody that contains a detectable label and that binds to an epitope on UCH-L1 not bound by the at least one UCH-L1 capture antibody to form a UCH-L1 antigen-at least one UCH-L1 detection antibody complex; to form at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex; and (b) measuring the amount or concentration of UCH-L1 in the sample based on a signal generated by a detectable label in at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex; Includes.

[0116] In one embodiment using the method described above, the subject is evaluated or assessed as having a mild TBI. In one embodiment using the method described above, the subject is evaluated or assessed as having a moderate TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a severe TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a moderate to severe TBI. In yet a further embodiment using the method described above, the subject is evaluated or assessed as not having a TBI.

[0117] The methods described above may further include treating a human subject assessed or assessed as having a TBI (such as mild, moderate, severe, or moderate-severe TBI) with a treatment for TBI (such as a surgical treatment, a therapeutic treatment, or a combination thereof). Any such treatment known in the art and further described herein may be used. Additionally, in further embodiments, any subject treated for TBI may also, optionally, be monitored during or after any course of treatment. Alternatively, the methods may further include monitoring a subject assessed as having a moderate, severe, or moderate-severe TBI (such as a subject who may not yet have received treatment).

[0118] In the above described methods, the sample may be selected from the group consisting of a whole blood sample, a serum sample, a cerebrospinal fluid sample, and a plasma sample. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a plasma sample. In yet other embodiments, the sample is a serum sample. Such a sample may be obtained in various ways. For example, the sample may be obtained after the subject has suffered a head injury or other type of blunt trauma caused by physical shaking, external mechanical or other forces resulting in closed or open head trauma, one or more falls, blunt impact from an explosion or blast. Alternatively, the sample may be obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin. Examples of chemicals or toxins are fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organometallics, drugs of abuse, or one or more combinations thereof. Still further, the sample may be obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof.

[0119] Any of the methods described above may be performed on any human subject, regardless of factors selected from the group consisting of the clinical condition of the human subject, the laboratory values ​​of the human subject, the classification of the human subject as suffering from mild, moderate, severe or moderate-severe TBI, the presentation of low or high levels of UCH-L1, GFAP and / or UCH-L1 and GFAP by the human subject, and the timing of any event during which the human subject may have suffered a head injury.

[0120] In the methods described above, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In still other embodiments, the assay is a clinical chemistry assay. In still other embodiments, the assay is a single molecule detection assay. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a clinical chemistry assay, and the sample is whole blood. In still further embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a clinical chemistry assay, and the sample is serum. In still further embodiments, the assay is a single molecule detection assay, and the sample is serum. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a clinical chemistry assay, and the sample is plasma. In yet a further embodiment, the assay is a single molecule detection assay, and the sample is plasma.

[0121] In yet another aspect, the disclosure relates to a method for aiding in or determining whether a human subject who has suffered a head injury has suffered a traumatic brain injury (TBI), the method comprising: performing an assay on a sample obtained from the subject within about 48 hours after the actual or suspected injury to measure or detect a combination of levels of glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the sample; and determining that the subject is more likely to have suffered a TBI than not if the sample level of GFAP is equal to a reference level of GFAP of about 15 pg / mL to about 40 pg / mL and the sample level of UCH-L1 is equal to a reference level of UCH-L1 of about 70 pg / mL to about 150 pg / mL; Includes.

[0122] In one embodiment of the method described above, the assay is performed on a sample obtained from the subject within about 0 to about 4 hours after injury. In another embodiment, the assay is performed on a sample obtained from the subject within about 4 to about 8 hours after injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 8 to about 12 hours after injury. In yet a further embodiment, the assay is performed on a sample obtained from the subject within about 12 to about 16 hours after injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 16 to about 20 hours after injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 20 to about 24 hours after injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 24 to about 28 hours after injury. In yet a further embodiment, the assay is performed on a sample obtained from the subject within about 24 to about 48 hours after injury. In still yet further embodiments, the assay is performed on a sample obtained from the subject within about 28 hours to about 32 hours after injury. In still yet further embodiments, the assay is performed on a sample obtained from the subject within about 32 hours to about 36 hours after injury. In still yet further embodiments, the assay is performed on a sample obtained from the subject within about 36 hours to about 40 hours after injury. In still yet further embodiments, the assay is performed on a sample obtained from the subject within about 40 hours to about 44 hours after injury. In still yet another embodiment, the assay is performed on a sample obtained from the subject within about 44 hours to about 48 hours after injury.

[0123] In yet another embodiment of the method described above, (a) the baseline level of GFAP is about 10 pg / mL and the baseline level of UCH-L1 is about 60 pg / mL; (b) the baseline level of GFAP is about 15 pg / mL and the baseline level of UCH-L1 is about 70 pg / mL; (c) the baseline level of GFAP is about 15 pg / mL and the baseline level of UCH-L1 is about 90 pg / mL; (d) the baseline level of GFAP is about 15 pg / mL and the baseline level of UCH-L1 is about 90 pg / mL. (e) the base level of GFAP is about 20 pg / mL and the base level of UCH-L1 is about 60 pg / mL; (f) the base level of GFAP is about 30 pg / mL and the base level of UCH-L1 is about 70 pg / mL; or (g) the base level of GFAP is about 30 pg / mL and the base level of UCH-L1 is about 110 pg / mL.

[0124] In yet another embodiment of the method described above, (a) a sample is obtained from a subject within about 4 hours to about 8 hours after injury, wherein the level of GFAP is about 15 pg / mL and the level of UCH-L1 is about 70 pg / mL; (b) a sample is obtained from a subject within about 4 hours to about 8 hours after injury, wherein the level of GFAP is about 30 pg / mL and the level of UCH-L1 is about 70 pg / mL; (c) a sample is obtained from a subject within about 4 hours to about 8 hours after injury, wherein the level of GFAP is about 40 pg / mL and the level of UCH-L1 is about 100 pg / mL; (d) a sample is obtained from a subject within about 8 hours to about 12 hours after injury, wherein the level of GFAP is about 15 pg / mL and the level of UCH-L1 is about 90 pg / mL; (e) a sample is obtained from a subject within about 8 hours to about 12 hours after injury, wherein the level of GFAP is about 15 pg / mL and the level of UCH-L1 is about 90 pg / mL. (f) a sample is obtained from a subject within about 8 to about 12 hours after injury, wherein the level of GFAP is about 15 pg / mL and the level of UCH-L1 is about 150 pg / mL; (g) a sample is obtained from a subject within about 12 to about 16 hours after injury, wherein the level of GFAP is about 10 pg / mL. and the level of UCH-L1 is about 60 pg / mL; (h) the sample is obtained from the subject within about 12 to about 16 hours after injury, the level of GFAP is about 20 pg / mL, and the level of UCH-L1 is about 60 pg / mL; (i) the sample is obtained from the subject within about 12 to about 16 hours after injury, the level of GFAP is about 30 pg / mL, and the level of UCH-L1 is about 110 pg / mL.

[0125] In another embodiment of the methods described above, the baseline level of GFAP and the baseline level of UCH-L1 are determined by an assay having a sensitivity of about 90% or greater and a specificity of about 35% or greater.

[0126] In yet another embodiment of the methods described above, a sample may be obtained from a subject within about 4 hours to about 16 hours after injury.

[0127] In still another embodiment, the assay of the methods described above has at least 3% greater sensitivity and at least 17% greater specificity compared to assays that measure or detect GFAP or UCH-L1 individually.

[0128] In yet another embodiment of the method described above, the sample is obtained from the subject within about 8 hours to about 12 hours after injury; the baseline level of GFAP is about 30 pg / mL and the baseline level of UCH-L1 is about 110 pg / mL; the assay has a sensitivity of 92% or greater and a specificity of 99% or greater; or the sample is obtained from the subject within about 12 hours to about 16 hours after injury; the baseline level of GFAP is about 30 pg / mL and the baseline level of UCH-L1 is about 110 pg / mL; the assay has a sensitivity of 90% or greater and a specificity of 99% or greater; or the sample is obtained from the subject within about 4 hours to about 8 hours after injury; the baseline level of GFAP is about 40 pg / mL and the baseline level of UCH-L1 is about 100 pg / mL; the method has a sensitivity of 90% or greater and a specificity of 94% or greater; or the sample is obtained from the subject within about 8 hours to about 12 hours after injury; the baseline level of GFAP is about 15 pg / mL and the baseline level of UCH-L1 is about 150 pg / mL; the assay has a sensitivity of 95% or greater and a specificity of 82% or greater; or Samples are obtained from subjects within about 12 hours to about 16 hours after injury; baseline levels of GFAP are about 20 pg / mL and baseline levels of UCH-L1 are about 60 pg / mL; and the assay has a sensitivity of 95% or greater and a specificity of 65% or greater.

[0129] In the methods described above, the levels of GFAP and UCH-L1 can be measured or detected using immunoassays or clinical chemistry assays. Alternatively, in the methods described above, the levels of GFAP and UCH-L1 can be measured or detected using single molecule detection assays.

[0130] In yet another embodiment of the above-described method, the measurement of the level of GFAP comprises: (d) The samples are subjected to simultaneous or sequential analysis in any order. (1) at least one GFAP capture antibody that binds to an epitope on GFAP or a GFAP fragment to form at least one GFAP capture antibody-GFAP antigen complex; and (2) at least one GFAP detection antibody that contains a detectable label and that binds to an epitope on GFAP not bound by the GFAP capture antibody to form a GFAP antigen-at least one GFAP detection antibody complex; to form at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex; and (e) measuring the amount or concentration of GFAP in the sample based on a signal generated by a detectable label in at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex. Includes.

[0131] In yet another embodiment, the measurement of UCH-L1 in the method identified above comprises: (a) The samples are subjected to simultaneous or sequential analysis in any order. (1) at least one UCH-L1 capture antibody that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form at least one UCH-L1 capture antibody-UCH-L1 antigen complex; and (2) at least one UCH-L1 detection antibody that contains a detectable label and that binds to an epitope on UCH-L1 not bound by the at least one UCH-L1 capture antibody to form a UCH-L1 antigen-at least one UCH-L1 detection antibody complex; to form at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex; and (f) measuring the amount or concentration of UCH-L1 in the sample based on a signal generated by a detectable label in at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex; Includes.

[0132] In one embodiment using the method described above, the subject is evaluated or assessed as having a mild TBI. In one embodiment using the method described above, the subject is evaluated or assessed as having a moderate TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a severe TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a moderate to severe TBI. In yet a further embodiment using the method described above, the subject is evaluated or assessed as not having a TBI.

[0133] The methods described above may further include treating a human subject assessed or assessed as having a TBI (such as mild, moderate, severe, or moderate-severe TBI) with a treatment for TBI (such as a surgical treatment, a therapeutic treatment, or a combination thereof). Any such treatment known in the art and further described herein may be used. Additionally, in further embodiments, any subject treated for TBI may also, optionally, be monitored during or after any course of treatment. Alternatively, the methods may further include monitoring a subject assessed as having a moderate, severe, or moderate-severe TBI (such as a subject who may not yet have received treatment).

[0134] In the above described methods, the sample may be selected from the group consisting of a whole blood sample, a serum sample, a cerebrospinal fluid sample, and a plasma sample. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a plasma sample. In yet other embodiments, the sample is a serum sample. Such a sample may be obtained in various ways. For example, the sample may be obtained after the subject has suffered a head injury or other type of blunt trauma caused by physical shaking, external mechanical or other forces resulting in closed or open head trauma, one or more falls, blunt impact from an explosion or blast. Alternatively, the sample may be obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin. Examples of chemicals or toxins are fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organometallics, drugs of abuse, or one or more combinations thereof. Still further, the sample may be obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof.

[0135] Any of the methods described above may be performed on any human subject, regardless of factors selected from the group consisting of the clinical condition of the human subject, the laboratory values ​​of the human subject, the classification of the human subject as suffering from mild, moderate, severe or moderate-severe TBI, the presentation of low or high levels of UCH-L1, GFAP and / or UCH-L1 and GFAP by the human subject, and the timing of any event during which the human subject may have suffered a head injury.

[0136] In the methods described above, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In still other embodiments, the assay is a clinical chemistry assay. In still other embodiments, the assay is a single molecule detection assay. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a clinical chemistry assay, and the sample is whole blood. In still further embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a clinical chemistry assay, and the sample is serum. In still further embodiments, the assay is a single molecule detection assay, and the sample is serum. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a clinical chemistry assay, and the sample is plasma. In yet a further embodiment, the assay is a single molecule detection assay, and the sample is plasma.

[0137] In yet another aspect, the disclosure relates to a method for aiding in or determining whether to perform a head magnetic resonance imaging (MRI) procedure on a human subject who has sustained or may have sustained a head injury, the method comprising: performing an assay on a sample obtained from the subject within about 48 hours after the actual or suspected injury to measure or detect a combination of levels of glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the sample; and (a) determining that the subject does not require an MRI procedure if the sample level of GFAP is less than a baseline level of about 15 pg / mL for GFAP and the sample level of UCH-L1 is less than a baseline level of about 50 pg / mL for UCH-L1; or (b) determining that the subject is more likely than not to require an MRI procedure if the sample level of GFAP is equal to a reference level of GFAP of about 15 pg / mL to about 1000 pg / mL and the sample level of UCH-L1 is equal to a reference level of UCH-L1 of about 50 pg / mL to about 2000 pg / mL; or (c) determining that the subject is more likely than not to require an MRI procedure if the sample level of GFAP is greater than a baseline level of about 1000 pg / mL for GFAP and the sample level of UCH-L1 is greater than a baseline level of about 2000 pg / mL for UCH-L1; Includes.

[0138] In the methods described above, the subject may undergo an MRI after the assay is performed, and the subject is suspected of having TBI based on the MRI results. In yet another embodiment, the baseline levels of GFAP and the baseline levels of UCH-L1 correlate with a negative MRI result.

[0139] In one embodiment of the method described above, the assay is performed on a sample obtained from the subject within about 0 to about 4 hours after the actual or suspected injury. In another embodiment, the assay is performed on a sample obtained from the subject within about 4 to about 8 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 8 to about 12 hours after the actual or suspected injury. In yet a further embodiment, the assay is performed on a sample obtained from the subject within about 12 to about 16 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 16 to about 20 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 20 to about 24 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 24 to about 28 hours after the actual or suspected injury. In still further embodiments, the assay is performed on a sample obtained from a subject within about 24 hours to about 48 hours after the injury or suspected injury. In yet even further embodiments, the assay is performed on a sample obtained from a subject within about 28 hours to about 32 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 32 hours to about 36 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 36 hours to about 40 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 40 hours to about 44 hours after the actual injury or suspected injury. In still yet another embodiment, the assay is performed on a sample obtained from a subject within about 44 hours to about 48 hours after the actual injury or suspected injury.

[0140] In the above-described methods for determining or assessing whether to perform an MRI, the subject may be presumed to have a traumatic brain injury based on an MRI or CT scan that has been performed or has already been performed (meaning before the assay is performed). For example, depending on the subject's medical condition (such as when the patient is unconscious), an MRI or CT scan may be performed to evaluate and / or assess whether the subject has a TBI immediately after the subject arrives at an emergency room, trauma center, or other facility. Such an MRI or CT scan may be performed before the assay is performed to confirm and determine whether the subject has a mild, moderate, or moderate-severe TBI. After the assay is performed, one or more subsequent MRI (or CT scans) may be performed based on the results of the assay as part of the physician's (or other medical personnel's) management of the TBI (e.g., to determine whether surgical and / or pharmacological intervention may be required).

[0141] In certain embodiments of the above method, the subject may be presumed to have traumatic brain injury based on MRI. For example, the subject may be presumed to have mild TBI based on MRI. Alternatively, the subject may be presumed to have moderate TBI based on MRI. Alternatively, the subject may be presumed to have severe TBI based on MRI. Alternatively, the subject may be presumed to have moderate to severe TBI based on MRI. Still further, the subject may be presumed not to have TBI based on MRI.

[0142] In yet another embodiment of the methods described above, the baseline level of GFAP and the baseline level of UCH-L1 are determined by an assay having a sensitivity of about 80% to about 98% and a specificity of about 30% to about 85%.

[0143] In a further embodiment of the methods described above, the subject may have received a negative CT scan result before the assay is performed.

[0144] In one embodiment using the method described above, the subject is evaluated or assessed as having a mild TBI. In one embodiment using the method described above, the subject is evaluated or assessed as having a moderate TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a severe TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a moderate to severe TBI. In yet a further embodiment using the method described above, the subject is evaluated or assessed as not having a TBI.

[0145] The methods described above may further include treating a human subject assessed or assessed as having a TBI (such as mild, moderate, severe, or moderate-severe TBI) with a treatment for TBI (such as a surgical treatment, a therapeutic treatment, or a combination thereof). Any such treatment known in the art and further described herein may be used. Additionally, in further embodiments, any subject treated for TBI may also, optionally, be monitored during or after any course of treatment. Alternatively, the methods may further include monitoring a subject assessed as having a moderate, severe, or moderate-severe TBI (such as a subject who may not yet have received treatment).

[0146] In the above described methods, the sample may be selected from the group consisting of a whole blood sample, a serum sample, a cerebrospinal fluid sample, and a plasma sample. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a plasma sample. In yet other embodiments, the sample is a serum sample. Such a sample may be obtained in various ways. For example, the sample may be obtained after the subject has suffered a head injury or other type of blunt trauma caused by physical shaking, external mechanical or other forces resulting in closed or open head trauma, one or more falls, blunt impact from an explosion or blast. Alternatively, the sample may be obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin. Examples of chemicals or toxins are fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organometallics, drugs of abuse, or one or more combinations thereof. Still further, the sample may be obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof.

[0147] Any of the methods described above may be performed on any human subject, regardless of factors selected from the group consisting of the clinical condition of the human subject, the laboratory values ​​of the human subject, the classification of the human subject as suffering from mild, moderate, severe or moderate-severe TBI, the presentation of low or high levels of UCH-L1, GFAP and / or UCH-L1 and GFAP by the human subject, and the timing of any event during which the human subject may have suffered a head injury.

[0148] In the methods described above, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In still other embodiments, the assay is a clinical chemistry assay. In still other embodiments, the assay is a single molecule detection assay. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a clinical chemistry assay, and the sample is whole blood. In still further embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a clinical chemistry assay, and the sample is serum. In still further embodiments, the assay is a single molecule detection assay, and the sample is serum. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a clinical chemistry assay, and the sample is plasma. In yet a further embodiment, the assay is a single molecule detection assay, and the sample is plasma.

[0149] In yet another aspect, the present disclosure relates to a method for aiding in or predicting the outcome of a human subject who has sustained or may have sustained a head injury, the method comprising: performing an assay on a sample obtained from the subject within about 48 hours after the actual or suspected injury to measure or detect a combination of levels of glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the sample; and a step in which a sample level of GFAP equal to a reference level of GFAP of about 80 pg / mL to about 2000 pg / mL and a sample level of UCH-L1 equal to a reference level of UCH-L1 of about 130 pg / mL to about 2000 pg / mL is more likely to predict an adverse outcome for the subject than not; Includes.

[0150] In another embodiment of the above described method, the subject may receive a GOSE (Extended Glasgow Outcome Scale) score after the method is performed, and the subject is predicted to have a poor outcome based on the GOSE score. In yet another embodiment, the baseline level of GFAP and the baseline level of UCH-L1 correlate with the subject having a poor outcome based on a GOSE score of 1.

[0151] In one embodiment of the method described above, the assay is performed on a sample obtained from the subject within about 0 to about 4 hours after the actual or suspected injury. In another embodiment, the assay is performed on a sample obtained from the subject within about 4 to about 8 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 8 to about 12 hours after the actual or suspected injury. In yet a further embodiment, the assay is performed on a sample obtained from the subject within about 12 to about 16 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 16 to about 20 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 20 to about 24 hours after the actual or suspected injury. In yet another embodiment, the assay is performed on a sample obtained from the subject within about 24 to about 28 hours after the actual or suspected injury. In still further embodiments, the assay is performed on a sample obtained from a subject within about 24 hours to about 48 hours after the injury or suspected injury. In yet even further embodiments, the assay is performed on a sample obtained from a subject within about 28 hours to about 32 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 32 hours to about 36 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 36 hours to about 40 hours after the actual injury or suspected injury. In still even further embodiments, the assay is performed on a sample obtained from a subject within about 40 hours to about 44 hours after the actual injury or suspected injury. In still yet another embodiment, the assay is performed on a sample obtained from a subject within about 44 hours to about 48 hours after the actual injury or suspected injury.

[0152] In yet another embodiment of the methods described above, the baseline level of GFAP and the baseline level of UCH-L1 are determined by an assay having a sensitivity of about 80% to about 97% and a specificity of about 30% to about 95%.

[0153] In the above-described methods, the levels of GFAP and UCH-L1 are measured or detected using immunoassays or clinical chemistry assays. Alternatively, in the above-described methods, the levels of GFAP and UCH-L1 are measured or detected using single molecule detection assays.

[0154] In yet another embodiment of the above-described method, the measurement of the level of GFAP comprises: (c) The samples are subjected to simultaneous or sequential analysis in any order. (1) at least one GFAP capture antibody that binds to an epitope on GFAP or a GFAP fragment to form at least one GFAP capture antibody-GFAP antigen complex; and (2) at least one GFAP detection antibody that contains a detectable label and that binds to an epitope on GFAP not bound by the GFAP capture antibody to form a GFAP antigen-at least one GFAP detection antibody complex; to form at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex; and (d) measuring the amount or concentration of GFAP in the sample based on a signal generated by a detectable label in at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex. Includes.

[0155] In yet another embodiment, the measurement of UCH-L1 in the method identified above comprises: (c) The samples are subjected to simultaneous or sequential analysis in any order. (1) at least one UCH-L1 capture antibody that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form at least one UCH-L1 capture antibody-UCH-L1 antigen complex; and (2) at least one UCH-L1 detection antibody that contains a detectable label and that binds to an epitope on UCH-L1 not bound by the at least one UCH-L1 capture antibody to form a UCH-L1 antigen-at least one UCH-L1 detection antibody complex; to form at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex; and (d) measuring the amount or concentration of UCH-L1 in the sample based on a signal generated by a detectable label in at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex. Includes.

[0156] In one embodiment using the method described above, the subject is evaluated or assessed as having a mild TBI. In one embodiment using the method described above, the subject is evaluated or assessed as having a moderate TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a severe TBI. In another embodiment using the method described above, the subject is evaluated or assessed as having a moderate to severe TBI. In yet a further embodiment using the method described above, the subject is evaluated or assessed as not having a TBI.

[0157] The methods described above may further include treating a human subject assessed or assessed as having a TBI (such as mild, moderate, severe, or moderate-severe TBI) with a treatment for TBI (such as a surgical treatment, a therapeutic treatment, or a combination thereof). Any such treatment known in the art and further described herein may be used. Additionally, in further embodiments, any subject treated for TBI may also, optionally, be monitored during or after any course of treatment. Alternatively, the methods may further include monitoring a subject assessed as having a moderate, severe, or moderate-severe TBI (such as a subject who may not yet have received treatment).

[0158] In the above described methods, the sample may be selected from the group consisting of a whole blood sample, a serum sample, a cerebrospinal fluid sample, and a plasma sample. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a plasma sample. In yet other embodiments, the sample is a serum sample. Such a sample may be obtained in various ways. For example, the sample may be obtained after the subject has suffered a head injury or other type of blunt trauma caused by physical shaking, external mechanical or other forces resulting in closed or open head trauma, one or more falls, blunt impact from an explosion or blast. Alternatively, the sample may be obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin. Examples of chemicals or toxins are fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organometallics, drugs of abuse, or one or more combinations thereof. Still further, the sample may be obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof.

[0159] Any of the methods described above may be performed on any human subject, regardless of factors selected from the group consisting of the clinical condition of the human subject, the laboratory values ​​of the human subject, the classification of the human subject as suffering from mild, moderate, severe or moderate-severe TBI, the presentation of low or high levels of UCH-L1, GFAP and / or UCH-L1 and GFAP by the human subject, and the timing of any event during which the human subject may have suffered a head injury.

[0160] In the methods described above, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In still other embodiments, the assay is a clinical chemistry assay. In still other embodiments, the assay is a single molecule detection assay. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a clinical chemistry assay, and the sample is whole blood. In still further embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a clinical chemistry assay, and the sample is serum. In still further embodiments, the assay is a single molecule detection assay, and the sample is serum. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a clinical chemistry assay, and the sample is plasma. In yet a further embodiment, the assay is a single molecule detection assay, and the sample is plasma. [Brief description of the drawings]

[0161] [Figure 1A] FIG. 1 includes representative graphs depicting mean GFAP levels at various time points within 48 hours post-injury for the moderate / severe TBI group, the mild TBI group, and the mild and moderate / severe TBI groups. [Figure 1B] FIG. 1 includes representative graphs depicting mean UCH-L1 levels at various time points within 48 hours post-injury for the moderate / severe TBI group, the mild TBI group, and the mild and moderate / severe TBI groups. [Figure 2A] FIG. 1 includes representative graphs depicting mean GFAP levels at various time points within 48 hours post-injury for mild TBI, healthy control, and orthopedic injury control groups. [Figure 2B] FIG. 1 includes representative graphs depicting mean UCH-L1 levels at various time points within 48 hours post-injury for mild TBI, healthy control, and orthopedic injury control groups. [Figure 3A] FIG. 1 includes representative plots of median GFAP levels by time point in subjects assigned GCS scores (13-15=mild TBI; 9-12=moderate TBI; 8 or less=severe TBI). [Figure 3B] FIG. 1 includes representative plots of median GFAP levels by time point in subjects assigned GCS scores (13-15=mild TBI; 9-12=moderate TBI; 8 or less=severe TBI). [Figure 3C] FIG. 1 includes representative plots of median GFAP levels by time point in subjects based on imaging results (CT scan results). [Figure 3D] FIG. 1 includes representative plots of median GFAP levels by time point in subjects based on imaging results (MRI results). [Figure 4A] FIG. 1 includes representative plots of median GFAP levels by time point in subjects assigned GCS scores (13-15=mild TBI; 9-12=moderate TBI; 8 or less=severe TBI). [Figure 4B] FIG. 11 includes representative plots of median GFAP levels by time point in subjects assigned GCS scores (13-15=mild TBI; 9-12=moderate TBI; 8 or less=severe TBI). [Figure 4C] FIG. 1 includes representative plots of median GFAP levels by time point in subjects based on imaging results (CT scan results). [Figure 4D] FIG. 1 includes representative plots of median GFAP levels by time point in subjects based on imaging results (MRI results). [Figure 5A] FIG. 1 includes representative box plots for GFAP levels in subjects classified according to GOSE score. [Figure 5B] FIG. 1 includes representative box plots for UCH-L1 levels in subjects classified according to GOSE score. [Figure 6] 1 is a representative graph comparing the sensitivity and specificity of the assay for various GFAP and UCH-L1 levels in subjects diagnosed with TBI based on CT scan results (CT positive) and healthy control subjects (CT negative). [Figure 7] 1 is a representative graph comparing the sensitivity and specificity of the assay for various GFAP and UCH-L1 levels in subjects assigned a GCS score; subjects assigned a GCS score of ≦12 were positive (moderate or severe TBI) and subjects assigned a GCS score >12 were negative (mild TBI or healthy controls). [Figure 8] 1 is a representative graph comparing the sensitivity and specificity of the assay for various GFAP and UCH-L1 levels in subjects diagnosed with TBI based on MRI results (positive MRI) and healthy control subjects (negative MRI). [Figure 9]1 is a representative graph comparing the sensitivity and specificity of the assay for various GFAP and UCH-L1 levels in subjects diagnosed with TBI based on GOSE score (1=TBI / death) and healthy control subjects (8=healthy / recovered). [Figure 10] 13 is a representative graph comparing the sensitivity and specificity of the assay for various GFAP and UCH-L1 levels in subjects diagnosed with TBI based on MRI results (positive MRI) and orthopedic injury control subjects (negative MRI). [Figure 11] 1 is a representative graph comparing the sensitivity and specificity of the assay for various GFAP and UCH-L1 levels in subjects diagnosed with TBI based on GOSE score (1=TBI / death) and orthopedic injury control subjects (8=healthy / recovered). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0162] The present disclosure relates to a method for aiding in the diagnosis and assessment of a subject who has or may have suffered a head injury. In particular, the present disclosure provides a method for aiding in the diagnosis and assessment of a subject to determine whether the subject has suffered a traumatic brain injury (TBI) by detecting or measuring a combination of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and glial fibrillary acidic protein (GFAP) levels in samples taken at various times within 48 hours after the subject has or may have suffered a head injury. Method embodiments also include a method for aiding in the diagnosis and assessment of a subject who has or may have suffered a head injury to determine whether the subject will benefit from and therefore undergo imaging procedures such as magnetic resonance imaging (MRI) or head computed tomography (CT) scans based on the evaluation of the combination of GFAP and UCH-L1 levels. The method may involve detecting GFAP and UCH-L1 levels in one or more samples taken from a human subject within about 48 hours, e.g., 0 to about 12 hours, of a head injury or suspected head injury. Detection of GFAP and UCH-L1 levels above baseline levels within the first 48 hours after a head injury or suspected head injury aids in determining whether a human subject should undergo (i.e., "become") an imaging procedure. For example, a human subject having GFAP and UCH-L1 levels above baseline levels may also be identified as likely to have a positive head CT scan or a positive MRI (e.g., indicating the presence of an intracranial lesion and thus a potential TBI) and may benefit from being subjected to a head CT scan or MRI. Alternatively, certain GFAP and UCH-L1 levels may be used to "rule out" the need for further medical intervention.For example, human subjects having levels of GFAP and UCH-L1 that are lower than reference levels may be identified as likely to have a negative head CT scan or a negative MRI (i.e., no intracranial pathology is present and thus a head CT scan or MRI is not required or performed).

[0163] The present disclosure relates to a method involving detecting GFAP and UCH-L1 levels in one or more samples taken from a human subject at different time points within 48 hours of a head injury or a suspected head injury. Detection of an increased or elevated combination of GFAP and UCH-L1 levels can also aid in the diagnosis of certain types of TBI. For example, GFAP and UCH-L1 levels higher than a certain reference level may indicate that the subject has moderate, severe, or moderate-severe TBI, while GFAP and UCH-L1 levels lower than the reference level may indicate that the subject has mild TBI. In some cases, the combined GFAP and UCH-L1 levels can also aid in determining whether a subject who has suffered an orthopedic injury has also suffered a mild TBI and thus requires further medical intervention to diagnose the presence or absence of mild TBI.

[0164] The section headings used in this section and throughout the disclosure herein are for organizational purposes only and are not intended to be limiting.

[0165] 1.Definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of discrepancy, the present document, including definitions, will prevail. In the practice or testing of this disclosure, methods and materials similar or equivalent to those described herein may be used, but the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0166] As used herein, the terms "comprise(s)," "include(s)," "having," "having," "may be," and "containing" are open-ended transitional phrases, transitional terms, or transitional words that do not exclude additional acts or structures. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise," "consist of," or "consist essentially of" the embodiments or elements presented herein, whether expressly stated or not.

[0167] In this specification, to recite numerical ranges, each intervening number with the same precision is expressly contemplated. For example, for the range of 6 to 9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated, and for the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0168] As used herein, an "affinity matured antibody" refers to an antibody that has been subjected to a process to reduce the affinity (i.e., K D , k d or k aThe term affinity matured antibody is used to refer to an antibody with one or more alterations in one or more CDRs that result in an improvement over a parent antibody without the alterations. Exemplary affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Various procedures for generating affinity matured antibodies are known in the art, including screening combinatorial antibody libraries prepared using BioDisplay technology. For example, Marks et al., BioTechnology, 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues has been described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-3319 (1995) and Hawkins et al., J. Mol. Biol., 226:889-896 (1992). Selective mutagenesis positions and selective mutations at contact or hypermutation positions with activity enhancing amino acid residues are described in U.S. Patent No. 6,914,128 B1.

[0169] As used herein, "antibody" and "antibodies" refer to animal antibodies, such as, but not limited to, monoclonal antibodies, monospecific antibodies (which may be, for example, monoclonal or may also be produced by means other than those of producing them from common germline cells), multispecific antibodies, human antibodies, humanized antibodies (fully humanized or partially humanized), avian (e.g., duck or goose) antibodies, shark antibodies, whale antibodies, and mammalian antibodies, including non-primate (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, etc.) antibodies or non-human primate (e.g., monkey, chimpanzee, etc.) antibodies, recombinant antibodies, chimeric antibodies, single chain Fvs ("scFvs"), single chain antibodies, single domain antibodies, Fab fragments, F(ab') ... 2"anti-Id" refers to antibodies, fragments, disulfide-linked Fvs ("sdFv") and anti-idiotypic ("anti-Id") antibodies, dual domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dAbs) (such as those described in Wu, C. et al., Nature Biotechnology, 25(11):1290-1297 (2007) and PCT International Application No. 2001 / 058956, the contents of each of which are incorporated herein by reference), or domain antibodies (dAbs) (such as those described in Holt et al. (2014), Trends in Biotechnology, 21:484-490), including sdAbs that are natural single domain antibodies, e.g., in cartilaginous fish and camelids, or synthetic, e.g., nanobodies, VHHs, or other domain structures and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of molecule. For simplicity, antibodies against an analyte are often referred to herein as "anti-analyte antibodies" or simply "analyte antibodies" (e.g., anti-GFAP antibodies, GFAP antibodies, anti-UCH-L1 antibodies, or UCH-L1 antibodies).

[0170] As used herein, "antibody fragment" refers to a portion of an intact antibody that contains the antigen-binding site or variable region. The portion does not contain the heavy chain constant domains of the Fc region of the intact antibody (i.e., CH2, CH3 or CH4, depending on the antibody isotype). Examples of antibody fragments include Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab') fragments, and F(ab') fragments. 2These include, but are not limited to, 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 the three CDRs of a light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of a heavy chain variable region.

[0171] "Area under the curve" or "AUC" refers to the area under the ROC curve. The AUC under the ROC curve is a measure of accuracy. An AUC of 1 represents a perfect test, whereas an AUC of 0.5 represents a non-significant test. A preferred AUC may be at least about 0.700, at least about 0.750, at least about 0.800, at least about 0.850, at least about 0.900, at least about 0.910, at least about 0.920, at least about 0.930, at least about 0.940, at least about 0.950, at least about 0.960, at least about 0.970, at least about 0.980, at least about 0.990, or at least about 0.995.

[0172] As used herein, "beads" and "particles" are used interchangeably and refer to a substantially spherical solid support. One example of a bead or particle is a microparticle. Microparticles that may be used herein may be of any type known in the art. For example, the bead or particle may be a magnetic bead or particle. The magnetic beads / particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or magnetic fluid. Exemplary ferromagnetic materials are Fe, Co, Ni, Gd, Dy, CrO 2 , MnAs, MnBi, EuO, and NiO / Fe. Examples of ferrimagnetic materials include NiFe 2 O 4 , CoFe 2 O 4 , Fe 3 O 4 (or FeO.Fe 2 O 3). The 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 around the non-magnetic core. The microparticles may be of any size that works in the methods described herein, for example, from about 0.75 to about 5 nm, or from about 1 to about 5 nm, or from about 1 to about 3 nm.

[0173] As used herein, "binding protein" is used to refer to a monomeric or multimeric protein that binds to and forms a complex with a binding partner, such as, for example, a polypeptide, an antigen, a compound or other molecule, or a substrate of any kind. A binding protein specifically binds to a binding partner. Binding proteins include antibodies, as well as their antigen-binding fragments and various other forms and derivatives thereof known in the art and described herein below, and other molecules that contain one or more antigen-binding domains that bind to an antigen molecule or a specific site (epitope) on an antigen molecule. Thus, binding proteins include, but are not limited to, antibodies that are tetrameric immunoglobulins, IgG molecules, IgG1 molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, affinity matured antibodies, and any such antibody fragments that retain the ability to bind to an antigen.

[0174] As used herein, "bispecific antibodies" are used to refer to full-length antibodies generated by quadroma technology (see Milstein et al., Nature, 305(5934):537-540 (1983)), chemical conjugation of two different monoclonal antibodies (see Staerz et al., Nature, 314(6012):628-631 (1985)), or the knob-into-hole (KIH) method or similar techniques that introduce mutations within the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14):6444-6448 (1993)), resulting in a number of different immunoglobulin species, only one of which is a functional bispecific antibody. A bispecific antibody binds one antigen (or epitope) in one of its two binding arms (one pair of HC / LC) and binds a different antigen (or epitope) in its second arm (a different pair of HC / LC). By this definition, a bispecific antibody has two significantly different antigen-binding arms (both specificity and CDR sequences) and is monovalent for each antigen to which it binds.

[0175] As used herein, "CDR" is used to refer to the "complementarity determining region" in the variable sequence of an antibody. There are three CDRs in each of the heavy and light chain variable regions. From the N-terminus of the heavy or light chain, these regions are designated as "CDR1", "CDR2" and "CDR3" for each 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. Thus, an antigen-binding site may contain six CDRs, including a CDR set from each of the heavy and light chain variable regions. A polypeptide containing a single CDR (e.g., CDR1, CDR2 or CDR3) may be referred to as a "molecular recognition unit". Crystal structure analysis of antigen-antibody complexes supports that the amino acid residues of the CDRs make extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR3. Thus, the molecular recognition units may be primarily responsible for the specificity of an antigen-binding site. In general, the CDR residues are directly and most substantially involved in influencing binding to antigen.

[0176] The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system that is applicable to any variable region of an antibody, but also provides the exact residue boundaries that define the three CDRs. These CDRs may be referred to as "Kabat CDRs." Chothia and colleagues (Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); and Chothia et al., Nature, 342:877-883 (1989)) have described the Kabat CDRs in a numbered system that is applicable to any variable region of an antibody. It was found that certain sub-portions within the CDRs adopt nearly identical peptide backbone conformations despite great diversity at the amino acid sequence level. These sub-portions were designated "L1", "L2" and "L3", or "H1", "H2" and "H3", with "L" and "H" designating the light and heavy chain regions, respectively. These regions are sometimes referred to as "Chothia CDRs", which have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are described in Padlan, FASEB, and others. J., 9:133-139 (1995); and MacCallum, J. Mol. Biol., 262(5):732-745 (1996). Still other CDR boundary definitions may not strictly follow the system defined herein and may be shorter or longer in light of predicted or experimental findings that a particular residue or group of residues or even the entire CDR does not significantly affect binding to the antigen, but still overlap with the Kabat CDRs. The methods used herein may use CDRs defined according to any of these systems, although certain embodiments use the Kabat-defined CDRs or the Chothia-defined CDRs.

[0177] "Component", "components" or "at least one component" generally refers to capture antibodies, detection reagents or detection conjugates, calibrators, controls, sensitivity panels, containers, buffers, diluents, salts, enzymes, cofactors for enzymes, detection reagents, pretreatment reagents / solutions, substrates (e.g., in solution), stop solutions, etc. that may be included in a kit for assaying a test sample, such as a patient urine sample, whole blood sample, serum sample or plasma sample, according to the methods described herein and other methods known in the art. Some components may be in solution or may be lyophilized for reconstitution for use in the assay.

[0178] As used herein, "control" generally refers to a reagent whose purpose is to assess the performance of a measurement system to ensure that the measurement system continues to provide results within acceptable boundaries (e.g., boundaries ranging from a scale appropriate for an assay for research use at one end to analytical boundaries established by quality specifications for a commercially available assay at the other end). To accomplish this, the control should represent patient results and, optionally, should somehow evaluate the effect of errors on the measurement (e.g., errors due to reagent stability, calibrator variability, instrument variability, etc.). As used herein, "control subject" refers to one or more subjects who have not suffered a traumatic brain injury (TBI). As used herein, "orthopedic injury control" refers to (e.g., based on) samples or information derived from one or more subjects who have suffered an orthopedic injury but have not suffered an apparent TBI. As used herein, "orthopedic injury control subject" refers to one or more subjects who have suffered an orthopedic injury but have not suffered an apparent TBI. In some cases, "orthopedic injury control subjects" are adult orthopedic patients with an abbreviated injury score of ≦4 (non-fatal) for their limb injury and / or pelvic injury and / or rib fracture. As used herein, "healthy control" refers to (e.g., based on) samples or information derived from one or more subjects who are considered healthy and have not sustained apparent TBI or orthopedic injury. As used herein, "healthy control subjects" refers to one or more subjects who are considered healthy and have not sustained apparent TBI or orthopedic injury.

[0179] As used herein, "correlated with" refers to "compared to."

[0180] As used herein, "CT scan" refers to a computed tomography (CT) scan. A CT scan combines a series of X-ray images taken from different angles and uses computer processing to create cross-sectional images or slices of bones, blood vessels, and soft tissues within the body. A CT scan may use X-ray CT, positron emission tomography (PET), single photon emission computed tomography (SPECT), computer axial tomography (CAT scan), or computer-assisted tomography. A CT scan may be a conventional CT scan or a spiral / spiral CT scan. In a conventional CT scan, the scan is acquired slice by slice, stopping and moving to the next slice after each slice scan, e.g., from above the abdomen to the pelvis. A conventional CT scan requires the patient to hold their breath to avoid motion artifacts. A spiral / spiral CT scan is a continuous scan, acquired in a spiral, and the scanned images are continuous, making the process much quicker.

[0181] As used herein, a "derivative" of an antibody may refer to an antibody that has one or more modifications to its amino acid sequence when compared to the original or parent antibody, and may exhibit a modified domain structure. A derivative may not only adopt an amino acid sequence capable of specifically binding to a target (antigen), but may further adopt the typical domain configuration found in a natural antibody. Typical examples of antibody derivatives are antibodies coupled to other polypeptides, rearranged antibody domains or antibody fragments. A derivative may also comprise at least one further compound, e.g. a protein domain, which is linked by covalent or non-covalent bonds. Linking may be based on gene fusion according to methods known in the art. The further domain present in a fusion protein comprising an antibody may preferably be linked by a flexible linker, advantageously a peptide linker, which comprises multiple, hydrophilic, peptide-linked amino acids of sufficient length to span the distance between the C-terminus of the further protein domain and the N-terminus of the antibody or vice versa. The antibody may have a conformation suitable for biological activity or may be linked to an effector molecule that selectively binds, for example, a solid support, a biologically active substance (e.g., a cytokine or growth hormone), a chemical agent, a peptide, a protein, or a drug.

[0182] "Assay-determined" is used herein to refer to the determination of the reference level by any suitable assay. In some embodiments, the determination of the reference level can be achieved by the same type of assay as that applied to the sample from the subject (e.g., immunoassay, clinical chemistry assay, single molecule detection assay, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis or protein immunostaining, electrophoretic analysis, protein assay, competitive binding assay, functional protein assay, or chromatographic or spectroscopic methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS)). In some embodiments, the determination of the reference level can be achieved by the same type of assay as that applied to the sample from the subject under the same assay conditions. As mentioned herein, the present disclosure presents exemplary reference levels (e.g., calculated by comparing reference levels at different time points). It is well within the skill of the art to adapt the present disclosure herein to other assays and obtain assay-specific reference levels for these other assays based on the description presented by the present disclosure. For example, a set of training samples including samples obtained from human subjects known to have sustained head injury (and more particularly, samples obtained from human subjects known to have sustained (i) mild TBI and / or (ii) moderate, severe, or moderate-severe TBI) and samples obtained from human subjects known to have not sustained head injury can be used to obtain assay-specific reference levels. It is understood that a reference level "determined by an assay" and having a recited level of "sensitivity" and / or "specificity" is used herein to refer to a reference level that has been determined to result in the method of the present disclosure having the recited sensitivity and / or specificity when employed in the method of the present disclosure. It is well within the skill of one of ordinary skill in the art to determine the sensitivity and specificity associated with a given reference level in the method of the present disclosure, for example, by repeating statistical analyses of the assay data using multiple different possible reference levels.

[0183] In effect, when distinguishing subjects with traumatic brain injury from those without traumatic brain injury, or subjects with mild traumatic brain injury versus moderate, severe, or moderate-severe traumatic brain injury, one skilled in the art will weigh the effect of increasing the cutoff on sensitivity and specificity. Raising or lowering the cutoff has a well-defined and predictable impact on sensitivity and specificity, as well as other standard statistical measures. It is well known that raising the cutoff improves specificity but worsens sensitivity (proportion of subjects with disease that test positive). In contrast, lowering the cutoff improves sensitivity but worsens specificity (proportion of subjects without disease that test negative). The threshold for detecting traumatic brain injury or determining mild traumatic brain injury versus moderate, severe, or moderate-severe traumatic brain injury is readily apparent to one skilled in the art. As the cutoff is increased in distinguishing whether a subject has or does not have traumatic brain injury or mild traumatic brain injury versus moderate, severe, or moderate-severe traumatic brain injury, specificity is improved since more true negatives (i.e., subjects with no traumatic brain injury, no mild traumatic brain injury, no moderate traumatic brain injury, no severe traumatic brain injury, or no moderate-severe traumatic brain injury) are differentiated from subjects with traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury. At the same time, however, sensitivity should decrease since increasing the cutoff reduces the overall number of cases identified as positive as well as the number of true positives. Conversely, as the cutoff is lowered, sensitivity is improved since more true positives (i.e., subjects with traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury) are differentiated from subjects without traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury. However, at the same time, specificity should decrease since lowering the cutoff increases the overall number of cases identified as positive as well as the number of false positives.

[0184] In general, high sensitivity values ​​help those skilled in the art to exclude diseases or conditions (such as traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury), and high specificity values ​​help those skilled in the art to include diseases or conditions. Whether a person skilled in the art excludes or includes a disease depends on what the consequences for the patient are for each type of error. Therefore, without full disclosure of the basic information on how the values ​​were selected, the exact balance used to derive the test cutoff cannot be known or predicted. The balance of sensitivity to specificity and other factors will vary from case to case. For this reason, it is often preferable to provide alternative cutoff (e.g., reference) values ​​for the physician or medical practitioner to choose from.

[0185] "Drug of abuse" is used herein to refer to one or more addictive substances (e.g., drugs) taken for non-medical reasons (e.g., recreational and / or mood-altering effects). Excessive indulgence in, use of, or dependence on such drugs of abuse is often referred to as "substance abuse." Examples of drugs of abuse include alcohol, barbiturates, benzodiazepines, cannabis, cocaine, hallucinogens (e.g., ketamine, mescaline (peyote), PCP, psilocybin, DMT, and / or LSD), methaqualone, opioids, amphetamines (including methamphetamine), anabolic steroids, inhalants (i.e., substances containing volatile substances that contain psychoactive properties, such as, for example, nitrates, spray paints, cleaning fluids, markers, glues, etc.), and combinations thereof.

[0186] As used herein, "bispecific antibody" is used to refer to a full-length antibody (see PCT Publication No. 02 / 02773) that can bind to two different antigens (or epitopes) in each of its two binding arms (HC / LC pair). Thus, a bispecific binding protein has two identical antigen-binding arms with identical specificity and identical CDR sequences, and is bivalent for each antigen to which it binds.

[0187] As used herein, "dual variable domain" is used to refer to two or more antigen-binding sites on a binding protein, which may be a bivalent binding protein (two antigen-binding sites), a tetravalent binding protein (four antigen-binding sites), or a multivalent binding protein. A DVD may be monospecific, i.e., capable of binding one antigen (or one specific epitope), or multispecific, i.e., capable of binding two or more antigens (i.e., two or more epitopes of the same target antigen molecule or two or more epitopes of different target antigens). A preferred DVD-binding protein comprises two heavy chain DVD polypeptides and two light chain DVD polypeptides and is referred to as a "DVD immunoglobulin" or "DVD-Ig". Such DVD-Ig binding proteins are thus tetrameric and similar to IgG molecules, but provide more antigen-binding sites than IgG molecules. Thus, each half of a tetrameric DVD-Ig molecule is similar to half of an IgG molecule and contains a heavy chain DVD polypeptide and a light chain DVD polypeptide, but unlike the heavy and light chain pair of an IgG molecule, which results in a single antigen-binding domain, the heavy and light chain pair of a DVD-Ig results in two or more antigen-binding sites.

[0188] Each antigen-binding site of a DVD-Ig binding protein may be derived from a donor ("parent") monoclonal antibody and may comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) with the CDRs involved in binding to the antigen, a total of six CDRs per antigen-binding site. Thus, a DVD-Ig binding protein that binds to two different epitopes (i.e., two different epitopes on two different antigen molecules or two different epitopes on the same antigen molecule) comprises an antigen-binding site derived from a first parent monoclonal antibody and an antigen-binding site of a second parent monoclonal antibody.

[0189] The design, expression and characterization of DVD-Ig binding molecules are described in PCT Publication No. 2007 / 024715, U.S. Patent No. 7,612,181 and Wu et al., Nature Biotech., 25:1290-1297 (2007). A preferred example of such a DVD-Ig molecule comprises a heavy chain comprising the structural formula: VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker, provided that it is not CH1, X2 is an Fc region, and n is 0 or 1, preferably 1; and a light chain comprising the structural formula: VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker, provided that it is not CH1, X2 does not comprise an Fc region, and n is 0 or 1, preferably 1. Such a DVD-Ig can include two such heavy chains and two such light chains, where each chain includes a variable domain linked in tandem with no intervening constant region between the variable domains, where the heavy and light chains can associate to form a functional antigen binding site in tandem, and where a pair of heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein with four functional antigen binding sites. In another example, a DVD-Ig molecule can include heavy and light chains including three variable domains (VD1, VD2, VD3), each linked in tandem with no intervening constant region between the variable domains, where the pair of heavy and light chains can associate to form three antigen binding sites, and where a pair of heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein with six antigen binding sites.

[0190] In a preferred embodiment, a DVD-Ig binding protein not only binds to the same target molecule that its parent monoclonal antibodies bind, but also retains one or more desired properties of one or more of its parent monoclonal antibodies. Preferably, such additional properties are one or more antibody parameters of the parent monoclonal antibodies. Antibody parameters that can be contributed to a DVD-Ig binding protein derived from one or more of its parent monoclonal antibodies include, but are not limited to, antigen specificity, antigen affinity, potency, biological function, epitope recognition, protein stability, protein solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross-reactivity, and binding to orthologous antigens.

[0191] DVD-Ig binding proteins bind to at least one epitope of GFAP and / or UCH-L1. Non-limiting examples of DVD-Ig binding proteins include DVD-Ig binding proteins that bind to one or more epitopes of GFAP and / or UCH-L1, DVD-Ig binding proteins that bind to an epitope of GFAP and / or UCH-L1 and an epitope of GFAP and / or UCH-L1 of another species (e.g., mouse), and DVD-Ig binding proteins that bind to an epitope of human GFAP and / or UCH-L1 and an epitope of another target molecule.

[0192] As used herein, "dynamic range" refers to the range over which the readout of an assay is proportional to the amount of target molecule or analyte in the sample being analyzed.

[0193] "Epitope" or "epitopes" or "epitope of interest" refers to a site on any molecule that can be recognized and bind to a complementary site on its specific binding partner. The molecule and specific binding partner are part of a specific binding pair. For example, an epitope can be on a polypeptide, a protein, a hapten, a carbohydrate antigen (such as, but not limited to, a glycolipid, glycoprotein, or lipopolysaccharide), or a polysaccharide. The specific binding partner can be, but is not limited to, an antibody.

[0194] As used herein, "Fab (fragment antigen-binding) fragment" or "Fab fragment" refers to a fragment of an antibody that binds to an antigen and contains one complete light chain and part of one heavy chain, which is one antigen-binding site. Fab is a monovalent fragment consisting of the VL, VH, CL and CH1 domains. Fab is composed of one constant domain and one variable domain of each heavy and light chain. The variable domain contains a paratope (antigen-binding site) that includes a set of complementarity determining regions at the amino terminus of the monomer. Thus, each arm of the Y binds to an epitope on the antigen. Fab fragments can be produced as described in the art, for example, using papain, an enzyme that can be used to cleave an immunoglobulin monomer into two Fab fragments and an Fc fragment, or can be produced by recombinant means.

[0195] As used herein, "F(ab') 2 "F(ab')" refers to an antibody produced by pepsin digestion of a whole IgG antibody, removing most of the Fc region, while leaving a portion of the hinge region intact. 2 The bivalent antibody fragment (F(ab')) has two antigen-binding F(ab) portions linked together by disulfide bonds, giving it a molecular weight of approximately 110 kDa. 2 F(ab') fragments are smaller than whole IgG molecules, allowing better tissue penetration and thus facilitating better antigen recognition in immunohistochemistry.2 The use of F(ab') fragments also avoids non-specific binding to Fc receptors or protein A / G on live cells. 2 The fragments are capable of binding to and precipitating the antigen.

[0196] As used herein, "framework" (FR) or "framework sequence" may refer to the remaining sequence of the variable region, excluding the CDRs. The exact definition of the CDR sequence may be determined by different systems (see, for example, above), and the meaning of the framework sequence is subject to different interpretations accordingly. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) also divide the framework regions on the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. As mentioned by others, if a particular subregion is not specified as FR1, FR2, FR3, or FR4, the framework region represents the combination of FRs in the variable region of a single, naturally occurring immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FR refers to two or more of the four subregions that make up a framework region.

[0197] Human heavy and light chain FR sequences are known in the art that can be used as heavy and light chain "acceptor" framework sequences (or simply, "acceptor" sequences) to humanize non-human antibodies using techniques known in the art. In one embodiment, the human heavy and light chain acceptor sequences are selected from framework sequences listed in publicly available databases such as V-base (hypertext transferprotocol: / / vbase.mrc-cpe.cam.ac.uk / ) or the international ImMunoGeneTics® (IMGT®) information system (hypertext transferprotocol: / / imgt.cines.fr / texts / IMGTrepertoire / LocusGenes / ).

[0198] As used herein, a "functional antigen-binding site" can refer to a site on a binding protein (e.g., an antibody) that can bind to a target antigen. The antigen-binding affinity of an antigen-binding site may not be as strong as that of the parent binding protein, e.g., the parent antibody, from which the antigen-binding site is derived, but the ability to bind to an antigen must be measurable using any one of a variety of known methods for assessing antigen-binding proteins, e.g., antibodies. Furthermore, the antigen-binding affinity of each of the antigen-binding sites of a multivalent protein, e.g., a multivalent antibody herein, does not need to be quantitatively the same.

[0199] "GFAP" is used herein to describe glial fibrillary acidic protein, a protein that is encoded by the GFAP gene in humans and can be made (e.g., by recombinant means, in other species).

[0200] "GFAP status" can mean the level or amount of GFAP at a point in time (such as a point in time with a single measurement of GFAP), the level or amount of GFAP associated with monitoring (such as monitoring with repeated testing to identify increases or decreases in the amount of GFAP in a subject), the level or amount of GFAP associated with a treatment for traumatic brain injury (whether primary brain injury and / or secondary brain injury), or a combination thereof.

[0201] As used herein, "GCS (Glasgow Coma Scale)" or "GCS" refers to a 15-point scale for estimating and grading the outcome of brain injury based on overall social ability or dependency on others. The test measures motor response, language response and eye opening response with these values: I. Motor response (6: completely follows instructions; 5: locates noxious stimuli; 4: withdraws from noxious stimuli; 3: abnormal flexion, i.e., decorticate posture; 2: extension response, i.e., decorticate posture and 1: no response); II. Language response (5: clear and oriented; 4: confused but coherent speech; 3: incoherent sentences consisting of inappropriate words and words; 2: unintelligible speech and 1: no speech) and III. Eye opening (4: spontaneous eye opening; 3: eye opening in response to speech; 2: eye opening in response to pain and 1: no eye opening). The final score is determined by adding the values ​​of I+II+III. The final score may be categorized into four possible levels of survival, with lower numbers indicating more severe damage and poorer prognosis: mild (13-15); moderate disability (9-12) (loss of consciousness for more than 30 minutes; physical or cognitive impairment that may or may not be lost; would benefit from rehabilitation); severe disability (3-8) (stupor: unconscious state: no meaningful response, no spontaneous activity); and vegetative state (less than 3) (sleep-wake cycle; awakening but no interaction with environment; unlocalized response to pain). Moderate brain injury is defined as brain injury resulting in loss of consciousness for 20 minutes to 6 hours and a Glasgow Coma Scale (GCS) of 9-12. Severe brain injury is defined as brain injury resulting in loss of consciousness for more than 6 hours and a Glasgow Coma Scale (GCS) of 3-8.

[0202] As used herein, "Glasgow Outcome Scale (GOS)" refers to a global scale for functional outcome that assesses a patient's condition into one of five categories: death, vegetative state, severe disability, moderate disability, or good recovery.

[0203] The "Extended Glasgow Outcome Scale" or "GOSE", used interchangeably herein, provides a more detailed categorization into eight categories by subdividing the severely disabled, moderately disabled and well recovered categories into upper and lower categories, as shown in Table 1.

[0204] [Table 1]

[0205] As used herein, "humanized antibody" is used to describe an antibody that contains heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but where at least a portion of the VH and / or VL sequences have been altered to be more "human-like", i.e., more similar to human germline variable sequences. A "humanized antibody" is an antibody or variant, derivative, analog or fragment thereof that immunospecifically binds to an antigen of interest and contains a framework (FR) region that has substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) that has substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR that has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of the non-human antibody CDR. A humanized antibody comprises substantially all of at least one, but typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In certain embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically the Fc region of a human immunoglobulin. In some embodiments, the humanized antibody contains at least the variable domain of the heavy chain as well as the light chain. The antibody may also contain the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only a humanized light chain. In some embodiments, the humanized antibody contains only a humanized heavy chain. In specific embodiments, the humanized antibody contains only humanized variable domains of the light chain and / or humanized heavy chain.

[0206] The humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including, without limitation, IgG1, IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions, using techniques well known in the art.

[0207] The framework regions and CDRs of a humanized antibody need not correspond exactly to the parental sequences, for example, the donor antibody CDR or consensus framework may be mutagenized by substitution, insertion and / or deletion of at least one amino acid residue such that the CDR or framework residue at this site does not correspond to the donor antibody or consensus framework. However, in a preferred embodiment, such mutations are not extensive mutations. Typically, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues correspond to the residues of the parental FR and CDR sequences. The term "consensus framework" as used herein refers to a framework region within a consensus immunoglobulin sequence. The term "consensus immunoglobulin sequence" as used herein refers to a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, for example, Winnaker, "From Genes to Clones" (Verlagsgesellschaft, Weinheim, 1987)). Thus, a "consensus immunoglobulin sequence" may comprise a "consensus framework region" and / or a "consensus CDR." Within a family of immunoglobulins, each position within a consensus sequence is occupied by the amino acid that occurs most frequently at that position within the family. If two amino acids occur equally frequently, either may be included within the consensus sequence.

[0208] "Identical" or "identity" as used herein in the context of two or more polypeptide or polynucleotide sequences may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions in both sequences where identical residues occur to determine the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to determine the percentage of sequence identity. If the two sequences are of different length or the alignment results in one or more sticky ends and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator of the calculation, but not in the numerator.

[0209] As used herein, "imaging procedure" refers to a medical test that allows a view inside the body to diagnose, treat, and monitor a health condition. Imaging procedures can be non-invasive procedures that allow diagnosis of disease and injury without being invasive. Examples of imaging procedures include MRI, CT scans, X-rays, positron emission tomography (PET) scans, single photon emission computed tomography (SPECT), and diffusion tensor imaging (DTI) scans.

[0210] "Head injury" or "head injury", used interchangeably herein, refers to any trauma to the scalp, skull, or brain. Such injury may include only a minor bump on the skull, or may be a severe brain injury. Such injury includes primary injury to the brain and / or secondary injury to the brain. Primary brain injury occurs during the initial insult and results from the displacement of the brain's physical structures. More specifically, primary brain injury is physical damage to the parenchyma (tissue, blood vessels) that occurs during the traumatic event, resulting in shearing and compression of the surrounding brain tissue. Secondary brain injury occurs subsequent to the primary injury and may involve a series of cellular processes. More specifically, secondary brain injury refers to changes that develop over a period of time (hours to days) following the primary brain injury. Secondary brain injury includes a whole cascade of cellular, chemical, tissue, or vascular changes in the brain that contribute to further destruction of brain tissue.

[0211] Head injuries can be closed or open (penetrating). Closed head injuries refer to trauma to the scalp, skull, or brain where the impacting object does not penetrate the skull. Open head injuries refer to trauma to the scalp, skull, or brain where the impacting object penetrates the skull. Head injuries can be caused by physical concussion of a person, blunt blows from an external machine, or other forces that result in closed or open head injuries (e.g., vehicular accidents, such as vehicular accidents from automobiles, airplanes, trains, etc.; blows to the head, such as blows from a baseball bat or blows from a firearm), cerebrovascular accidents (e.g., strokes), one or more falls (e.g., during sports or other activities), explosions or blasts (collectively, "blast injuries"), and other types of blunt trauma. Alternatively, head injuries can be caused by ingestion of and / or exposure to chemicals, toxins, or combinations of chemicals and toxins. Examples of such chemicals and / or toxins include fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organometallics (such as methylmercury, tetraethyl lead, and organotins), and / or one or more drugs of abuse. Alternatively, head injury may be caused as a result of the subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, one or more viruses, meningitis, hydrocephalus, hypoxia, or a combination thereof. In some cases, it is not possible to ascertain whether any such event or injury has occurred or occurred. For example, the patient or subject may not have a medical history, the subject may not be able to speak, the subject may be aware of what event the subject has been exposed to, etc. Such situations are described herein as the subject "may have suffered a head injury." In certain embodiments herein, closed head injury does not include and specifically excludes cerebrovascular accidents such as strokes.

[0212] As used herein, "intracranial lesion" refers to an area of ​​damage in the brain. An intracranial lesion may be an abnormality seen in an imaging procedure or brain imaging test, such as an MRI scan or a CT scan. In a CT scan or an MRI scan, a brain lesion may appear as a dark or light spot that does not look like normal brain tissue.

[0213] As used herein, an "isolated polynucleotide" can mean a polynucleotide that, by its origin, is not associated with all or a portion of a polynucleotide with which the "isolated polynucleotide" is found in nature; that is operably linked to a polynucleotide with which it is not linked in nature; or that does not exist in nature as part of a larger sequence (e.g., by genomic, cDNA, or synthetic origin, or a combination of portions thereof).

[0214] As used herein, "label" and "detectable label" refer to a moiety attached to an antibody or analyte such that the reaction of the antibody with the analyte is detectable, and the antibody or analyte so labeled is said to be "detectably labeled." The label may provide a signal detectable by visual or instrumental means. Various labels include signal generators such as chromogens, fluorescent compounds, chemiluminescent compounds, radioactive compounds, and the like. Representative examples of labels include moieties that provide light, e.g., acridinium compounds, and moieties that provide fluorescence, e.g., fluorescein. Other labels are also described herein. In this regard, the moiety itself may not be detectable, but may become detectable upon reaction with yet another moiety. The term "detectably labeled" is intended to encompass such labels.

[0215] Any suitable detectable label known in the art may be used. For example, detectable labels include radioactive labels (such as 3H, 14C, 32P, 33P, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm), enzyme labels (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase), chemiluminescent labels (acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, etc.), fluorescent labels (fluorescein, ... The label may be a fluorescent label (e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, etc.), rhodamine, phycobiliprotein, R-phycoerythrin, quantum dots (e.g., cadmium selenide capped with zinc sulfide), a calorimetric label, or an immunopolymerase chain reaction label. An introduction to labels, labeling procedures, and detection of labels can be found in Polak and Van Noorden, "Introduction to Immunocytochemistry," 2nd ed., Springer Verlag, NY (1997), and Haugland, "Handbook of Fluorescent Probes and Research Chemicals" (1996), a combination handbook and catalogue published by Molecular Probes, Inc., Eugene, Oregon. Fluorescent labels can be used in FPIA (see, e.g., U.S. Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are incorporated by reference herein in their entireties).Acridinium compounds can be used as detectable labels in homogeneous chemiluminescent assays (see, e.g., Adamczyk et al., Bioorg. Med. Chem. Lett., 16:1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett., 4:2313-2317 (2004); Adamczyk et al., Bioorg. Med. Chem. Lett., 14:3917-3921 (2004) and Adamczyk et al., Org. Lett., 5:3779-3782 (2003)).

[0216] In one embodiment, the acridinium compound is acridinium-9-carboxamide. Methods for preparing acridinium 9-carboxamide are described in Mattingly, J. Biolumin. Chemilumin., 6:107-114 (1991); Adamczyk et al., J. Org. Chem., 63:5636-5639 (1998); Adamczyk et al., Tetrahedron, 55:10899-10914 (1999); Adamczyk et al., Org. Lett., 1:779-781 (1999); Adamczyk et al., Bioconjugate Chem., 11:714-724 (2000); Mattingly et al., Luminescence Biotechnology: Instruments and Applications, edited by Dyke, KV, CRC Press: Boca Raton, 77-105 (2002); Adamczyk et al., Org. Lett., 5:3779-3782 (2003); and U.S. Pat. Nos. 5,468,646, 5,543,524, and 5,783,699, each of which is incorporated by reference in its entirety for its teachings herein.

[0217] Another example of an acridinium compound is acridinium-9-carboxylate aryl ester. An example of the acridinium-9-carboxylate aryl ester of formula II is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available from Cayman Chemical, Ann Arbor, MI). Methods for preparing acridinium-9-carboxylate aryl ester are described in McCapra et al., Photochem. Photobiol., 4:1111-21 (1965); Razavi et al., Luminescence, 15:245-249 (2000); Razavi et al., Luminescence, 15:239-244 (2000) and U.S. Patent No. 5,241,070 (each of which is incorporated herein by reference in its entirety for its teachings therein). Such acridinium-9-carboxylate aryl esters are effective chemiluminescent indicators for hydrogen peroxide generated in the oxidation of an analyte by at least one oxidase in terms of signal intensity and / or signal rapidity. The chemiluminescence process of acridinium-9-carboxylate aryl esters is completed quickly, i.e., in less than one second, whereas the chemiluminescence of acridinium-9-carboxamides lasts for more than two seconds. However, acridinium-9-carboxylate aryl esters lose their chemiluminescent properties in the presence of proteins. Therefore, their use requires the absence of proteins at the time of signal generation and detection. Methods for separating or removing proteins in a sample are well known to those skilled in the art and include, but are not limited to, ultrafiltration, extraction, precipitation, dialysis, chromatography and / or digestion (see, for example, Wells, "High Throughput Bioanalytical Sample Preparation. Methods and Automation Strategies", Elsevier (2003)).The amount of protein removed or separated from the test sample can be about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95%. Further details regarding acridinium-9-carboxylate aryl esters and their uses are set forth in U.S. Patent Application No. 11 / 697,835, filed April 9, 2007. The acridinium-9-carboxylate aryl esters can be dissolved in any suitable solvent, such as degassed anhydrous N,N-dimethylformamide (DMF) or aqueous sodium cholate.

[0218] A "linking sequence" or "linking peptide sequence" refers to a naturally occurring or artificial polypeptide sequence connected to one or more polypeptide sequences of interest (e.g., full length sequences, sequence fragments, etc.). The term "connected" refers to the joining of the linking sequence to the polypeptide sequence of interest. Such polypeptide sequences are preferably joined by one or more peptide bonds. The linking sequence may have a length of about 4 to about 50 amino acids. Preferably, the length of the linking sequence is about 6 to about 30 amino acids. A naturally occurring linking sequence may be modified by amino acid substitution, addition, or deletion to create an artificial linking sequence. Linking sequences may be used for many purposes, including use in recombinant Fab. Exemplary linking sequences include, but are not limited to: (i) histidine (His) tags, such as a 6xHis tag, having an amino acid sequence of HHHHHH (SEQ ID NO: 3), are useful as linking sequences to facilitate isolation and purification of polypeptides and antibodies of interest; (ii) enterokinase cleavage sites, such as His tags, are used in the isolation and purification of proteins and antibodies of interest. Enterokinase cleavage sites are often used in conjunction with His tags in the isolation and purification of proteins and antibodies of interest. A variety of enterokinase cleavage sites are known in the art. Examples of enterokinase cleavage sites include, but are not limited to, the amino acid sequence of DDDDK (SEQ ID NO: 4) and its derivatives (e.g., ADDDDK (SEQ ID NO: 5) and the like); (iii) other sequences can also be used to link or connect the light chain variable region and / or heavy chain variable region of the single chain variable region fragment. Examples of other linking sequences can be found in Bird et al., Science, 242:423-426 (1988); Huston et al., PNAS USA 85:5879-5883 (1988) and McCafferty et al., Nature, 348:552-554 (1990). Linking sequences can also be modified for additional functions, such as conjugation of drugs or conjugation to solid supports. In the context of the present disclosure, the monoclonal antibody may contain linking sequences such as, for example, a His tag, an enterokinase cleavage site, or both.

[0219] As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical, except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigen. Moreover, in contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies herein include, inter alia, "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological properties.

[0220] "Magnetic resonance imaging" or "MRI," used interchangeably herein, refers to a medical imaging technique used in radiology (e.g., interchangeably referred to herein as "MRI," "MRI procedure," or "MRI scan") to create a picture of the anatomy and physiological processes within the body, both healthy and diseased. MRI is a form of medical imaging that measures the response of atomic nuclei in body tissues to high frequency radio waves when placed within a strong magnetic field, creating pictures of the internal organs. MRI scanners based on nuclear magnetic resonance (NMR) chemistry use strong magnetic fields, radio waves, and magnetic field gradients to create images of the inside of the body.

[0221] As used herein, "multivalent binding protein" is used to refer to a binding protein that contains two or more antigen-binding sites (also referred to herein as "antigen-binding domains"). Multivalent binding proteins are preferably engineered to have three or more antigen-binding sites and are generally not naturally occurring antibodies. The term "multispecific binding protein" refers to binding proteins that can bind to two or more related or unrelated targets, including binding proteins capable of binding to two or more different epitopes of the same target molecule.

[0222] "Negative predictive value" or "NPV," used interchangeably herein, refers to the probability that a subject will have a negative outcome (i.e., the absence of a proposed outcome) if the subject has a negative test result (i.e., a subject who tests negative for a proposed outcome does not have the proposed outcome).

[0223] "Point-of-care device" refers to a device used to provide medical diagnostic testing at or near the point of care (i.e., outside a laboratory) at the time and place of patient care (such as a hospital, clinic, urgent care facility or other medical care facility, the patient's home, a nursing home and / or long-term care facility and / or hospice facility, etc.). Examples of point-of-care devices include point-of-care devices made by Abbott Laboratories (Abbott Park, IL) (e.g., i-STAT and i-STAT Alinity, Universal Biosensors (Rowville, Australia)) (see US2006 / 0134713), Axis-Shield PoC AS (Oslo, Norway) and Clinical Lab Products (Los Angeles, USA).

[0224] "Positive predictive value" or "PPV," used interchangeably herein, refers to the probability that a subject will have a positive outcome (i.e., that a proposed outcome is present) if the subject has a positive test result (i.e., a subject who tests positive for a proposed outcome has the proposed outcome).

[0225] "Quality control reagents" in the context of the immunoassays and kits described herein include, but are not limited to, calibrators, controls, and sensitivity panels. "Calibrators" or "standards" (e.g., a plurality, one or more) are typically used to establish a calibration curve for interpolating the concentration of an analyte, such as an antibody or analyte. Alternatively, a single calibrator that is near a reference or control level (e.g., a "low", "medium", or "high" level) may be used. Multiple calibrators (i.e., more than one calibrator or varying amounts of calibrators) may be used to comprise a "sensitivity panel".

[0226] A "receiver operating characteristic" curve or "ROC" curve refers to a graphical plot illustrating the operation of a binary classifier system as its discrimination threshold is varied. For example, an ROC curve can be a plot of true positive rate against false positive rate for different possible cutoff points of a diagnostic test. An ROC curve is created by plotting the proportion of true positives among positives (TPR=true positive rate) against the proportion of false positives among negatives (FPR=false positive rate) at various threshold situations. TPR is also known as sensitivity, and FPR is [1-specificity or true negative rate]. ROC curves demonstrate a trade-off between sensitivity and specificity (any increase in sensitivity is accompanied by a decrease in specificity); the closer the curve is to the left and then upper boundaries of the ROC space, the more accurate the test; the closer the curve is to the 45 degree diagonal of the ROC space, the less accurate the test; the slope of the tangent at the cutoff point gives the likelihood ratio (LR) for this value of the test and the area under the curve is a measure of the accuracy of the test.

[0227] "Recombinant antibody" and "recombinant antibodies" refer to antibodies prepared by recombinant methods, by one or more steps including cloning a nucleic acid sequence encoding all or a portion of one or more monoclonal antibodies into a suitable expression vector, and then expressing the antibody in a suitable host cell. The terms include, but are not limited to, recombinantly produced monoclonal antibodies, chimeric antibodies, humanized antibodies (fully or partially humanized antibodies), multispecific or multivalent structures formed from antibody fragments, bifunctional antibodies, heteroconjugate Abs, DVD-Ig®, and other antibodies described in (i) herein (dual variable domain immunoglobulins and methods for making them are described in Wu, C. et al., Nature Biotechnology, 25:1290-1297 (2007)). The term "bifunctional antibody" as used herein refers to an antibody that contains a first arm that has specificity for one antigenic site and a second arm that has specificity for a different antigenic site, i.e., a bifunctional antibody has dual specificity.

[0228] As used herein, a "reference level" refers to an assay cutoff value (or level) used to assess diagnostic, prognostic or therapeutic efficacy, and which is herein associated or related to various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression, non-progression or improvement of disease, etc.). As used herein, the term "cutoff" refers to a limit (e.g., number, etc.) above which a certain or specific clinical outcome is seen and below which a different certain or specific clinical outcome is seen.

[0229] This disclosure presents exemplary reference levels. However, it is well known that the reference levels may vary depending on the nature of the immunoassay (e.g., the antibody used, the reaction conditions, the purity of the sample, etc.), and the assays may be compared and standardized. Moreover, it is well within the skill of the artisan to adapt the disclosure herein to other immunoassays and obtain immunoassay-specific reference levels for these other immunoassays based on the descriptions presented by this disclosure. Although the exact values ​​of the reference levels may vary between assays, the findings described herein are generally applicable and may be extrapolated to other assays.

[0230] As used herein, "risk assessment," "risk classification," "risk identification," or "risk stratification" of a subject (e.g., patient) refers to the assessment of factors, including biomarkers, to predict the risk of developing a future event, including disease onset or disease progression, so that treatment decisions for the subject can be made on a more informed, condition-based basis.

[0231] As used herein, "sample," "test sample," "specimen," "sample derived from a subject," and "patient sample" may be used interchangeably and may be a blood sample such as whole blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, white blood cells, or monocytes. The sample may be used directly as obtained from the patient, or may be pretreated to alter the characteristics of the sample, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., as discussed herein or otherwise known in the art.

[0232] A variety of 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 samples, frozen sections taken for histological purposes, blood (such as whole blood), plasma, serum, red blood cells, platelets, interstitial fluid, cerebrospinal fluid, and the like. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a serum sample. In yet other embodiments, the sample is a plasma sample. Cell types and tissues may also include lymphatic fluid, cerebrospinal fluid, and collected body fluids. Tissues or cell types may be prepared by removing cell samples from humans and non-human animals, but may also be arrived at by using previously isolated cells (e.g., isolated by another person, at another time, and / or for another purpose). Archival tissues, such as tissues with treatment history or outcome history, may also be used. Isolation and / or purification of proteins or nucleotides may not be necessary.

[0233] As used herein, the "sensitivity" of an assay refers to the proportion of subjects whose outcome is positive that are correctly identified as positive (e.g., the proportion of subjects that correctly identify subjects with the disease or medical condition for which the subject is being tested). For example, this can include correctly identifying a subject as having a TBI from subjects who do not have TBI, correctly identifying a subject with a moderate, severe, or moderate-severe TBI from subjects who have a mild TBI, correctly identifying a subject as having a mild TBI from subjects who have a moderate, severe, or moderate-severe TBI, correctly identifying a subject as having a moderate, severe, or moderate-severe TBI from subjects who do not have TBI, or correctly identifying a subject as having a mild TBI from subjects who do not have TBI, correctly identifying a subject as having a head CT scan or MRI from subjects who are not likely to benefit from a head CT scan or MRI, etc.

[0234] As used herein, the "specificity" of an assay refers to the proportion of subjects whose outcome is negative who are correctly identified as negative (e.g., the proportion of subjects who correctly identify that they do not have the disease or medical condition for which they are being tested). For example, this can include correctly identifying a subject with a TBI from subjects who do not have TBI, correctly identifying a subject as not having moderate, severe, or moderate-severe TBI from subjects who have mild TBI, correctly identifying a subject as not having mild TBI from subjects who have moderate, severe, or moderate-severe TBI, or correctly identifying a subject as not having any TBI, or correctly identifying a subject as having mild TBI from subjects who do not have TBI, etc.

[0235] A "series of calibration compositions" refers to a plurality of compositions comprising known concentrations of analytes, such as GFAP and UCH-L1, each of which has a different concentration of the analyte, such as GFAP and UCH-L1, than the other compositions in the series.

[0236] As used herein, "single molecule detection" refers to the detection and / or measurement of a single molecule of an analyte in a test sample at very low levels of concentration (such as pg / mL or femtograms per mL). Many different single molecule analyzers or devices are known in the art, including nanopore devices and nanowell devices. Examples of nanopore devices are described in International Patent Publication No. WO 2016 / 161402, which is incorporated herein by reference in its entirety. Examples of nanowell devices are described in International Patent Publication No. WO 2016 / 161400, which is incorporated herein by reference in its entirety.

[0237] As used interchangeably herein, "solid phase" or "solid support" refers to any material that can be attached to and / or attract and immobilize (1) one or more capture agents or specific binding partners for capture, or (2) one or more detection agents or specific binding partners for detection. The solid phase can be selected for its intrinsic ability to attract and immobilize the capture agent. Alternatively, the solid phase has a linking agent attached thereto that has the ability to attract and immobilize (1) the capture agent or specific binding partner for capture, or (2) the detection agent or specific binding partner for detection. For example, the linking agent can include a charged substance that is oppositely charged to the capture agent (e.g., specific binding partner for capture) or detection agent (e.g., specific binding partner for detection) itself, or a charged substance conjugated to (1) the capture agent or specific binding partner for capture, or (2) the detection agent or specific binding partner for detection. In general, the linking agent can be any binding partner (preferably a specific binding partner) that is capable of being immobilized (attached to) a solid phase and immobilizing (1) a capture agent or a specific binding partner for capture, or (2) a detection agent or a specific binding partner for detection, via a binding reaction. The linking agent allows the indirect binding of the capture agent to the solid phase material before the performance of the assay or during the performance of the assay. For example, the solid phase can be plastic, derivatized plastic, magnetic or non-magnetic metal, glass, or silicon, including, for example, test tubes, microtiter wells, sheets, beads, microparticles, chips, and other configurations known to those skilled in the art.

[0238] As used herein, "specific binding" or "specifically binding to" can refer to the interaction of an antibody, protein, or peptide with a second chemical species, where the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure, rather than proteins in general. If an antibody is specific for epitope "A", then the presence of a molecule containing epitope A (or free A, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[0239] A "specific binding partner" is a member of a specific binding pair. A specific binding pair includes two different molecules that specifically bind to each other through chemical or physical means. Thus, in addition to the typical immunoassay specific binding pair of antigen and antibody, other specific binding pairs can include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzymes and enzyme inhibitors, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding member, e.g., analyte analogs. Immunoreactive specific binding members include antigens, antigen fragments, and monoclonal and polyclonal antibodies, as well as antibodies, including complexes and fragments thereof, whether isolated or recombinantly produced.

[0240] "Statistically significant" as used herein refers to the likelihood that a relationship between two or more variables is caused by something other than random chance. Statistical hypothesis testing is used to determine whether the results of a data set are statistically significant. In statistical hypothesis testing, a statistically significant result is reached whenever the observed p-value for the test statistic is less than the significance level defined for the study. The p-value is the probability of obtaining a result at least as extreme as the result observed if the null hypothesis is true. Examples of statistical hypothesis analysis include Wilcoxon's matched rank test, t-test, chi-square test, or Fisher's exact test. "Significant" as used herein refers to a change that has not been determined to be statistically significant (e.g., may not have been subjected to statistical hypothesis testing).

[0241] As used interchangeably herein, "subject" and "patient" refer to any vertebrate, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates (e.g., monkeys such as cynomolgus or rhesus monkeys, chimpanzees, etc.), and humans). In some embodiments, the subject can be a human or a non-human. In some embodiments, the subject is a human. The subject or patient may be undergoing other forms of treatment. In some embodiments, the subject is a human who may be undergoing other forms of treatment. The subject or patient may be undergoing other forms of treatment. In some embodiments, when the subject is a human, the subject does not include a human who has suffered a cerebrovascular accident (e.g., a stroke). In some embodiments, the subject is suspected 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 suspected of having suffered from mild, moderate, severe, or moderate-severe TBI. In some embodiments, the subject is suspected of having suffered from mild TBI. In some embodiments, the subject is suspected of having suffered from moderate TBI. In some embodiments, the subject is suspected of having suffered from severe TBI.

[0242] As used herein, "treating", "treating" or "treatment" are each used interchangeably to describe preventing, alleviating or inhibiting the progression of a disease and / or injury or one or more symptoms of such disease to which such term applies. Depending on the subject's condition, the term also refers to preventing disease, including preventing the onset of a disease or preventing symptoms associated with a disease. Treatment can be performed acutely or chronically. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to contraction of the disease. Such prevention or reduction of the severity of a disease prior to contraction refers to administration of a pharmaceutical composition to a subject who is not affected by the disease at the time of administration. "Preventing" also refers to preventing the recurrence of a disease or one or more symptoms associated with such disease. "Treatment" and "therapeutically" refer to the act of treating, where "treating" is as defined above.

[0243] "Traumatic brain injury" or "TBI", used interchangeably herein, refers to a complex injury with a wide range of symptoms and disability. TBI is most often an acute event, like other injuries. TBI may be classified as "mild", "moderate", or "severe". Causes of TBI are diverse and include man-made physical concussion, motor vehicle accidents, firearm injuries, cerebrovascular accidents (e.g., strokes), falls, explosions or blasts, and other types of blunt trauma. Other causes of TBI include ingestion of and / or exposure to one or more chemicals or toxins (fire, mold, asbestos, pesticides and insecticides, organic solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide, and cyanides), organometallics (such as methylmercury, tetraethyl lead, and organotins), one or more drugs of abuse, or combinations thereof). Alternatively, TBI may occur in a human subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, one or more viruses, meningitis, hydrocephalus, or a combination thereof.Young adults and the elderly are the age groups most at risk for TBI.In certain embodiments herein, traumatic brain injury or TBI does not include and expressly excludes cerebrovascular accidents such as stroke.

[0244] As used herein, "mild TBI" refers to brain injury in which loss of consciousness is brief, usually for a few seconds or minutes, and / or confusion and disorientation lasts for less than an hour. Mild TBI is also referred to as concussion, mild head trauma, mild TBI, mild brain injury, and mild head injury. MRI and CT scans may be normal, but individuals with mild TBI may have cognitive problems such as headaches, difficulty thinking, memory problems, attention deficits, mood swings, and frustration.

[0245] Mild TBI is the most common type of TBI and is often overlooked at the time of initial injury. Typically, subjects have a Glasgow Coma Scale (GCS) score between 13-15 (such as 13-15 or 14-15). Of people with mild TBI, fifteen percent (15%) have symptoms that last for more than three months. Mild TBI is defined as the result of a forceful movement of the head or an impact that causes a brief change in mental status (confusion, disorientation, or memory loss) or loss of consciousness for less than 30 minutes. Common symptoms of mild TBI include fatigue, headache, visual impairment, memory loss, decreased attention / concentration, sleep disorders, vertigo / loss of balance, irritability (emotional disturbance), feelings of depression, and seizures. Other symptoms associated with mild TBI include nausea, loss of smell, sensitivity to light and sound, mood changes, confusion or confusion, and / or slowed thinking.

[0246] "Moderate TBI" as used herein refers to a brain injury in which loss of consciousness and / or confusion and disorientation occurs for 1-24 hours and the subject has a Glasgow Coma Scale score between 9-13 (such as 9-12 or 9-13). Individuals with moderate TBI have abnormal brain imaging results. "Severe TBI" as used herein refers to a brain injury in which loss of consciousness occurs for more than 24 hours, memory loss after injury or penetrating skull injury occurs for more than 24 hours, and the subject has a Glasgow Coma Scale score between 3-8. Deficits range from high level cognitive impairment to coma. Survivors may have limited arm or leg function, speech or language abnormalities, loss of thinking ability, or emotional problems. Individuals with severe injuries may remain in a prolonged state of refractory status. For many people with severe TBI, prolonged rehabilitation is often required to maximize function and independence.

[0247] As used herein, "moderate-severe" TBI refers to a spectrum of brain injury, including moderate-severe, and thus includes moderate TBI alone, severe TBI alone, and moderate-severe TBI combined. A subject suffering from moderate-severe TBI may have a GCS (Glasgow Coma Scale) score between 3 and 13 (such as between 3 and 12 or between 3 and 13). For example, depending on the clinical situation, a subject may initially be diagnosed as having moderate TBI, but over time (minutes, hours, or days), may progress to a subject having severe TBI (such as in the setting of a cerebral hemorrhage). Such a subject is an example of a patient that may be classified as "moderate-severe". Common symptoms of moderate to severe TBI include problems with attention, concentration, distractibility, memory, processing speed, confusion, perseveration, impulsivity, language processing and / or "executive function", not understanding spoken words (receptive aphasia), difficulty speaking and being understood (expressive aphasia), slurred speech, very fast or very slow speech, problems reading, problems writing, difficulties interpreting touch, temperature, movement, position and fine discrimination of the lower extremities, difficulties integrating or patterning sensory impressions into psychologically meaningful data, partial or total loss of vision, weakness and double vision (diplopia), blurred vision, distance problems judging distance, involuntary eye movements (nystagmus), light intolerance (photophobia), hearing problems, such as reduced or lost hearing, ringing in the ears (tinnitus), hypersensitivity to sound, loss or reduced sense of smell (anosmia), loss or reduced sense of taste, convulsions associated with epilepsy which can be of several types and which may involve disruption of consciousness, sensory perception or movement, bowel and bladder control, sleep disorders, loss of energy, changes in appetite, regulation of internal temperature, menstrual difficulties, addictive behaviour, cognitive deficits including emotional capacity, lack of motivation, irritability, aggression, depression, disinhibition or denial / lack of consciousness.

[0248] "Ubiquitin carboxy-terminal hydrolase L1" or "UCH-L1," used interchangeably herein, refers to the deubiquitinating enzyme encoded by the UCH-L1 gene in humans. UCH-L1, also known as ubiquitin carboxyl-terminal esterase L1 and ubiquitin thiolesterase, is a member of a gene family whose products hydrolyze small C-terminal adducts of ubiquitin to generate ubiquitin monomers.

[0249] "UCH-L1 status" can mean the level or amount of UCH-L1 at a point in time (such as a point in time involving a single measurement of UCH-L1), the level or amount of UCH-L1 associated with monitoring (such as monitoring involving repeated testing to identify increases or decreases in the amount of UCH-L1 in a subject), the level or amount of UCH-L1 associated with treatment for traumatic brain injury (whether primary brain injury and / or secondary brain injury), or a combination thereof.

[0250] As used herein, "variant" is used to describe a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or the ability to stimulate an immune response. As used herein, variant is also used to describe a protein with an amino acid sequence that is substantially identical to a reference protein with an amino acid sequence that retains at least one biological activity. It is recognized in the art that conservative substitutions of amino acids, i.e., replacing an amino acid with a different amino acid that has similar properties (e.g., hydrophilicity, degree of charge, and distribution of charged regions), typically involve small changes. As understood in the art, these small changes can be identified, in part, by considering the hydrophobicity index of an amino acid (Kyte et al., J. Mol. Biol. 157:105-132 (1982)). The hydrophobicity index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophilicity indexes may be substituted and still retain protein function. In one embodiment, amino acids with hydrophobicity indexes of ±2 are substituted. The hydrophilicity of amino acids may also be used to identify substitutions that result in proteins that retain biological function. Consideration of the hydrophilicity of amino acids in the context of a peptide allows for the calculation of the maximum local average hydrophilicity of the peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity (U.S. Pat. No. 4,554,101, incorporated herein by reference). Substitution 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 made with amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the particular side chain of the amino acid.Consistent with this observation is the understanding that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size and other properties, and in particular on the side chains of these amino acids. "Variants" may also be used to refer to antigenically reactive fragments of anti-analyte (such as GFAP and / or UCH-L1) antibodies that differ in amino acid sequence from the corresponding fragments of anti-analyte (such as GFAP and / or UCH-L1) antibodies, but are still antigenically reactive and can compete with the corresponding fragments of anti-analyte (such as GFAP and / or UCH-L1) antibodies for binding to the analyte (such as GFAP and / or UCH-L1). "Variants" may also be used to describe polypeptides or fragments thereof that are processed differently, such as by proteolysis, phosphorylation or other post-translational modifications, but that retain their antigenic reactivity.

[0251] As used herein, "vector" is used to describe a nucleic acid molecule that can carry another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., mammalian non-episomal vectors) can be integrated into the genome of the host cell upon introduction into the host cell, and thereby replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Since plasmids are the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions, may also be used. In this regard, RNA forms of vectors, including viral RNA vectors, may also be used in the context of the present disclosure.

[0252] Unless otherwise specified, technical and scientific terms used herein shall have the same meaning as commonly understood by those skilled in the art. For example, the terminology used in connection with and techniques for cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well-known and commonly used in the art. The meaning and scope of the terms shall be clear, but in the unlikely event of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0253] 2. Methods of using a combination of baseline levels of GFAP and baseline levels of UCH-L1 to aid in the diagnosis of subjects who have suffered or may have suffered a head injury. The present disclosure relates, among other methods, to methods for aiding in the diagnosis and assessment of subjects who have or may have suffered a head injury. In particular, the present disclosure provides methods for aiding in the diagnosis and assessment of subjects to determine whether a subject has suffered a traumatic brain injury (TBI), such as, for example, a moderate to severe traumatic brain injury, by detecting or measuring a combination of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) levels and glial fibrillary acidic protein (GFAP) levels in samples taken at various time points within 48 hours after the subject has or may have suffered a head injury. As disclosed herein, the diagnosis and assessment of a subject suspected of having a TBI includes a decision to perform an imaging procedure, along with other medical assessments (e.g., clinical evaluations), based on the levels of GFAP and UCH-L1 in the subject compared to various baseline levels. Such diagnoses and assessments based on a combination of GFAP and UCH-L1 levels can help determine whether a subject is more likely to have a positive MRI scan and / or a positive head CT scan (i.e., the presence of intracranial pathology) than not.

[0254] In some embodiments, the method may help determine whether a subject who has suffered a head injury has suffered a traumatic brain injury based on the levels of GFAP and UCH-L1 compared to baseline levels. According to these embodiments, the method includes performing an assay on a sample obtained from the subject within about 48 hours after injury to measure or detect a combination of a GFAP level in the sample and a UCH-L1 level in the sample; and determining that the subject has not suffered a TBI if (a) the GFAP level in the sample is less than a baseline level of about 15 pg / mL of GFAP and the UCH-L1 level in the sample is less than a baseline level of about 70 pg / mL of UCH-L1; or (b) the GFAP level in the sample is less than a baseline level of about 15 pg / mL of GFAP to about 70 pg / mL of UCH-L1. or (c) determining that the subject is more likely to have suffered a TBI than not when the sample level of GFAP is greater than the reference level of GFAP of about 40 pg / mL and the sample level of UCH-L1 is greater than the reference level of UCH-L1 of about 70 pg / mL to about 150 pg / mL.

[0255] In some embodiments, the methods may include obtaining a sample within about 48 hours of a suspected injury to the subject, and contacting the sample with an antibody to UCH-L1 and / or GFAP to allow formation of a complex between the antibody and UCH-L1 and / or GFAP. The methods also include detecting the resulting antibody-GFAP / UCH-L1 complex. According to these methods, GFAP and UCH-L1 levels may be measured or detected and then correlated with one or more clinical parameters (e.g., GCS score and / or CT scan) before or after the methods are performed to establish baseline levels of GFAP and UCH-L1 that may be used to determine whether a subject has a TBI.

[0256] In some embodiments, the reference level of GFAP and the reference level of UCH-L1 may be used as part of an assay having a specificity of at least about 35% and a sensitivity of at least about 90%, as further described herein. Additionally, in other embodiments, the method when used as an assay has at least 3% higher sensitivity and at least 17% higher specificity than a method or assay that measures or detects GFAP or UCH-L1 separately. In other embodiments, the method when used as an assay has at least 5% higher sensitivity and at least 20% higher specificity than a method or assay that measures or detects GFAP or UCH-L1 separately. In other embodiments, the method when used as an assay has at least 8% higher sensitivity and at least 25% higher specificity than a method or assay that measures or detects GFAP or UCH-L1 separately.

[0257] In some embodiments, the reference levels of GFAP and UCH-L1 are determined by an assay having a sensitivity of at least about 70% to about 100% and a specificity of at least about 30% to about 100%. In some embodiments, the sensitivity is at least about 70% to about 100%, at least about 70% to at least about 99%, at least about 70% to at least about 95%, at least about 70% to at least about 90%, at least about 70% to at least about 85%, at least about 75% to about 100%, at least about 75% to at least about 99%, at least about 75% to at least about 95%, at least about 75% to at least about 90%, at least about 75% to at least about 85%, at least about 80% to at least about 100%, at least about 80% to at least about 9 ... 9%, between at least about 80% and at least about 95%, between at least about 80% and at least about 90%, between at least about 80% and at least about 85%, between at least about 85% and about 100%, between at least about 85% and at least about 99%, between at least about 85% and at least about 95%, between at least about 85% and at least about 90%, between at least about 90% and at least about 99%, between at least about 90% and at least about 95%, between at least about 95% and at least about 100%, or between at least about 95% and at least about 99%. In some embodiments, the sensitivity is at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 87.5%, at least about 90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%.

[0258] In some embodiments, the specificity is at least between about 30% and about 100%, at least between about 30% and about 99%, at least between about 30% and about 95%, at least between about 30% and about 90%, at least between about 30% and about 85%, at least between about 30% and about 80%, at least about 30% and about 75%, at least about 30% and about 70%, at least about 30% and about 60%, at least about 30% and about 50%, at least about 40% and about 100%, at least about 40% and about 99%, at least about 40% and about 95% between about 40% and about 90%, between about 40% and about 85%, between about 40% and about 80%, between about 40% and about 75%, between about 40% and about 70%, between about 40% and about 60%, between about 40% and about 50%, between about 50% and about 100%, between about 50% and about 99%, between about 50% and about 95%, between about 50% and about 90%, between about 50% and about 85%, between about 50% and about 80%, between about 50% and about 7 between 5%, between at least about 50% and about 70%, between at least about 50% and about 60%, between at least about 60% and about 100%, between at least about 60% and about 99%, between at least about 60% and about 95%, between at least about 60% and about 90%, between at least about 60% and about 85%, between at least about 60% and about 80%, between at least about 60% and about 75%, between at least about 60% and about 70%, between at least about 70% and about 100%, between at least about 70% and about 99%, between at least about 70% and about 95%, at least about 70% at least about 90%, at least about 70% to about 85%, at least about 70% to about 80%, at least about 70% to about 75%, at least about 80% to about 100%, at least about 80% to about 99%, at least about 80% to about 95%, at least about 80% to about 90%, at least about 80% to about 85%, at least about 90% to about 100%, at least about 90% to about 99%, at least about 90% to about 95%, at least about 95% to about 99%, or at least about 95% to about 100%.In some embodiments, the specificity is at least about 30.0%, at least about 31.0%, at least about 32.0%, at least about 33.0%, at least about 34.0%, at least about 35.0%, at least about 36.0%, at least about 37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%, at least about 45.0%, at least about 50.0%, at least about 55.0%, at least about 60.0%, at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 86.0%, at least about 87.0%, at least about 88.0%, at least about 89.0%, at least about 90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%, at least about 94.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 100.0%, at least about 101.0%, at least about 102.0%, at least about 103.0%, at least about 104.0%, at least about 105.0%, at least about 106.0%, at least about 107.0%, at least about 108.0%, at least about 109.0%, at least about 110.0%, at least about 111.0%, at least about 112.0%, at least about 113.0%, at least about 114.0%, at least about 115.0%, at least about 116.0%, at least about 117.0%, at least about 118.0%, at least about 119 5.0%, at least about 90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%, at least about 94.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%. For example, in some embodiments, the sensitivity is at least about 99% and the specificity is at least about 75%, the sensitivity is at least about 99% and the specificity is at least about 99%, or the sensitivity is at least about 100% and the specificity is at least about 100%. In some embodiments, a baseline level of GFAP and a baseline level of UCH-L1 may be used as part of an assay having a specificity of at least about 35% and a sensitivity of at least about 90%.

[0259] In some embodiments, the sample is collected from the human subject within about 48 hours, such as within about 0 to about 4 hours, within about 0 to about 8 hours, within about 0 to about 12 hours, within about 0 to about 16 hours, within about 0 to about 20 hours, within about 0 to about 24 hours, and within about 0 to about 48 hours, of the head injury or suspected injury. In some embodiments, the sample is collected from the human subject within about 4 hours to about 8 hours, within about 8 hours to about 12 hours, within about 12 hours to about 16 hours, within about 16 hours to about 20 hours, within about 20 hours to about 24 hours, and within about 24 hours to about 48 hours. In other embodiments, samples may be collected from a human subject within about 0 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after the injury or suspected injury to the head. In some embodiments, the onset of the presence of the combination of GFAP and UCH-L1 occurs within about 0 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after the head injury. In one embodiment, the sample is obtained from the subject within about 4 hours to about 16 hours after the injury. In another embodiment, the sample is obtained from the subject within about 4 hours to about 8 hours after the injury. In yet another embodiment, the sample is obtained from the subject within about 8 hours to about 12 hours after the injury. In yet another embodiment, the sample is obtained from the subject within about 12 hours to about 16 hours after the injury.

[0260] In some embodiments, the sample is obtained from the subject within about 4 hours to about 8 hours after the injury (or post injury), the baseline level of GFAP is about 40 pg / mL, the baseline level of UCH-L1 is about 100 pg / mL, and the assay has a sensitivity of 90% or greater and a specificity of 94% or greater.

[0261] In some embodiments, the sample is obtained from the subject within about 8 hours to about 12 hours after the injury (or post injury), the baseline level of GFAP is about 15 pg / mL, the baseline level of UCH-L1 is about 150 pg / mL, and the assay has a sensitivity of 95% or greater and a specificity of 82% or greater.

[0262] In some embodiments, the sample is obtained from the subject within about 12 hours to about 16 hours after the injury (or post injury), the baseline level of GFAP is about 20 pg / mL, the baseline level of UCH-L1 is about 60 pg / mL, and the assay has a sensitivity of 95% or greater and a specificity of 65% or greater.

[0263] In some embodiments, the reference level of GFAP can be between about 15 pg / mL and about 40 pg / mL, and the reference level of UCH-L1 can be at least between about 70 pg / mL and about 150 pg / mL. In other embodiments, the reference level of GFAP can be between about 20 pg / mL and about 40 pg / mL, and the reference level of UCH-L1 can be at least between about 80 pg / mL and about 150 pg / mL. In other embodiments, the reference level of GFAP can be between about 25 pg / mL and about 40 pg / mL, and the reference level of UCH-L1 can be at least between about 90 pg / mL and about 150 pg / mL. In other embodiments, the reference level of GFAP can be between about 30 pg / mL and about 40 pg / mL, and the reference level of UCH-L1 can be at least between about 100 pg / mL and about 150 pg / mL. In other embodiments, the reference level of GFAP can be between about 15 pg / mL and about 30 pg / mL, and the reference level of UCH-L1 can be at least between about 70 pg / mL and about 140 pg / mL. In other embodiments, the reference level of GFAP can be between about 15 pg / mL and about 25 pg / mL, and the reference level of UCH-L1 can be at least between about 70 pg / mL and about 130 pg / mL. In some embodiments, the reference level of GFAP is about 40 pg / mL, and the reference level of UCH-L1 is about 100 pg / mL. In other embodiments, the reference level of GFAP is about 15 pg / mL, and the reference level of UCH-L1 is about 150 pg / mL. In still other embodiments, the reference level of GFAP is about 20 pg / mL, and the reference level of UCH-L1 is about 60 pg / mL.

[0264] In some embodiments, the subject undergoes an imaging procedure, such as an MRI or a CT scan, or optionally both, before or after the assay is performed. In some embodiments, the subject is suspected of having a traumatic brain injury based on the imaging procedure. In some embodiments, the baseline levels of UCH-L1 and GFAP correlate with a positive MRI scan and / or a positive head CT scan (i.e., the presence of intracranial lesions). In some embodiments, the baseline levels of GFAP and UCH-L1 can be used to indicate whether the subject needs an MRI procedure, independent of performing a CT scan and independent of a negative CT scan (i.e., indicating no TBI).

[0265] In general, reference levels of biomarkers such as UCH-L1 or GFAP and combinations thereof can be used as a benchmark to evaluate the results obtained when a test sample is assayed for GFAP and UCH-L1. For example, when making such a comparison, reference levels of GFAP and UCH-L1 can be obtained by performing a particular assay a sufficient number of times and under appropriate conditions such that a correlation or association of the presence, amount or concentration of the analyte with a particular stage or endpoint or a particular symptom of TBI can be established. Typically, reference levels of GFAP and UCH-L1 are obtained by performing an assay on a reference subject (or population of subjects). The measured GFAP and UCH-L1 can include fragments thereof, degradation products thereof and / or enzymatic cleavage products thereof.

[0266] In other embodiments, the methods may aid in diagnosing the type of TBI a subject may have suffered (e.g., moderate-severe TBI, etc.) based on the levels of GFAP and UCH-L1 compared to baseline levels. According to these embodiments, the methods include performing an assay on a sample obtained from the subject within about 48 hours after injury to measure or detect a combination of a GFAP level in the sample and a UCH-L1 level in the sample; and determining that the subject has not suffered a moderate, severe, or moderate-severe TBI if the sample level of GFAP is less than a baseline level of about 105 pg / mL of GFAP and the sample level of UCH-L1 is less than a baseline level of about 110 pg / mL of UCH-L1; or (b) determining that the subject has not suffered a moderate, severe, or moderate-severe TBI if the sample level of GFAP is less than a baseline level of about 105 pg / mL of GFAP and the sample level of UCH-L1 is less than a baseline level of about 110 pg / mL of UCH-L1. or (c) determining that the subject is more likely to have suffered a moderate, severe, or moderate-severe TBI than not when the sample level of GFAP is greater than the GFAP reference level of about 890 pg / mL and the sample level of UCH-L1 is greater than the UCH-L1 reference level of about 110 pg / mL to about 2000 pg / mL.

[0267] In some embodiments, the method may include obtaining a sample within about 48 hours of a suspected injury to the subject, and contacting the sample with an antibody against UCH-L1 and / or GFAP to allow formation of a complex between the antibody and UCH-L1 and / or GFAP. The method may also include detecting the resulting antibody-GFAP / UCH-L1 complex. According to these methods, GFAP and UCH-L1 levels may be measured or detected and then correlated with one or more clinical parameters (e.g., GCS score and / or CT scan) before or after the method is performed to establish baseline levels of GFAP and UCH-L1 that may be used to diagnose and assess subjects who have or may have suffered a TBI. For example, in some embodiments, the subject may have received a GCS (Glasgow Coma Scale) score before or after the method is performed. If the GCS score is 12 or less, the subject is likely suspected to have a moderate to severe TBI.

[0268] The baseline levels of GFAP and the baseline levels of UCH-L1 may be used as part of an assay having a specificity of at least about 30% and a sensitivity of at least about 90%, as further described below.

[0269] In some embodiments, the sample is collected from the human subject within about 48 hours, such as within about 0 to about 4 hours, within about 0 to about 8 hours, within about 0 to about 12 hours, within about 0 to about 16 hours, within about 0 to about 20 hours, within about 0 to about 24 hours, and within about 0 to about 48 hours, of the head injury or suspected injury. In some embodiments, the sample is collected from the human subject within about 4 hours to about 8 hours, within about 8 hours to about 12 hours, within about 12 hours to about 16 hours, within about 16 hours to about 20 hours, within about 20 hours to about 24 hours, and within about 24 hours to about 48 hours. In other embodiments, samples may be collected from a human subject within about 0 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after the injury or suspected injury to the head. In some embodiments, the onset of the presence of the combination of GFAP and UCH-L1 occurs within about 0 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after the head injury. In one embodiment, the sample is obtained from the subject within about 8 hours to about 16 hours after the actual or suspected injury. In another embodiment, the sample is obtained from the subject within about 8 hours to about 12 hours after the actual or suspected injury. In yet another embodiment, the sample is obtained from the subject within about 12 hours to about 16 hours after the actual or suspected injury.

[0270] In some embodiments, the subject undergoes an imaging procedure, such as an MRI or CT scan, before or after the assay is performed. In some embodiments, the subject is suspected of having traumatic brain injury based on the imaging procedure. In some embodiments, the baseline levels of UCH-L1 and GFAP correlate with a positive MRI scan and / or a positive head CT scan (i.e., the presence of intracranial lesions). In some embodiments, the baseline levels of GFAP and UCH-L1 can be used to indicate whether the subject needs an MRI procedure, independent of performing a CT scan and independent of a negative CT scan (i.e., indicating no TBI).

[0271] In general, reference levels of biomarkers such as UCH-L1 or GFAP and combinations thereof can be used as a benchmark to evaluate the results obtained when a test sample is assayed for GFAP and UCH-L1. For example, when making such a comparison, reference levels of GFAP and UCH-L1 can be obtained by performing a particular assay a sufficient number of times and under appropriate conditions such that a correlation or association of the presence, amount or concentration of the analyte with a particular stage or endpoint or a particular symptom of TBI can be established. Typically, reference levels of GFAP and UCH-L1 are obtained by performing an assay on a reference subject (or population of subjects). The measured GFAP and UCH-L1 can include fragments thereof, degradation products thereof and / or enzymatic cleavage products thereof.

[0272] In some embodiments, the reference levels of GFAP and UCH-L1 are determined by an assay having a sensitivity of at least about 70% to about 100% and a specificity of at least about 30% to about 100%. In some embodiments, the sensitivity is at least about 70% to about 100%, at least about 70% to at least about 99%, at least about 70% to at least about 95%, at least about 70% to at least about 90%, at least about 70% to at least about 85%, at least about 75% to about 100%, at least about 75% to at least about 99%, at least about 75% to at least about 95%, at least about 75% to at least about 90%, at least about 75% to at least about 85%, at least about 80% to at least about 100%, at least about 80% to at least about 99%, between at least about 80% to at least about 95%, between at least about 80% to at least about 90%, between at least about 80% to at least about 85%, between at least about 85% to about 100%, between at least about 85% to at least about 99%, between at least about 85% to at least about 95%, between at least about 85% to at least about 90%, between at least about 90% to at least about 100%, between at least about 90% to at least about 99%, between at least about 90% to at least about 95%, between at least about 95% to at least about 100%, or between at least about 95% to at least about 99%. In some embodiments, the sensitivity is at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 87.5%, at least about 90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%.

[0273] In some embodiments, the specificity is at least about 30% to about 100%, at least about 30% to about 99%, at least about 30% to about 95%, at least about 30% to about 90%, at least about 30% to about 85%, at least about 30% to about 80%, at least about 30% to about 75%, at least about 30% to about 70%, at least about 30% to about 60%, at least about 30% to about 50%, at least about 40% to about 100%, at least about 40% to about 99%, at least about 40% to about 95%. %, between about 40% and about 90%, between about 40% and about 85%, between about 40% and about 80%, between about 40% and about 75%, between about 40% and about 70%, between about 40% and about 60%, between about 40% and about 50%, between about 50% and about 100%, between about 50% and about 99%, between about 50% and about 95%, between about 50% and about 90%, between about 50% and about 85%, between about 50% and about 80%, between about 50% and about between about 75%, between about 50% and about 70%, between about 50% and about 60%, between about 60% and about 100%, between about 60% and about 99%, between about 60% and about 95%, between about 60% and about 90%, between about 60% and about 85%, between about 60% and about 80%, between about 60% and about 75%, between about 60% and about 70%, between about 70% and about 100%, between about 70% and about 99%, between about 70% and about 95%, % to about 90%, at least about 70% to about 85%, at least about 70% to about 80%, at least about 70% to about 75%, at least about 80% to about 100%, at least about 80% to about 99%, at least about 80% to about 95%, at least about 80% to about 90%, at least about 80% to about 85%, at least about 90% to about 100%, at least about 90% to about 99%, at least about 90% to about 95%, at least about 95% to about 99%, or at least about 95% to about 100%.In some embodiments, the specificity is at least about 30.0%, at least about 31.0%, at least about 32.0%, at least about 33.0%, at least about 34.0%, at least about 35.0%, at least about 36.0%, at least about 37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%, at least about 45.0%, at least about 50.0%, at least about 55.0%, at least about 60.0%, at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%, at least about 94.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%. For example, in some embodiments, the sensitivity is at least about 99% and the specificity is at least about 75%, the sensitivity is at least about 99% and the specificity is at least about 99%, or the sensitivity is at least about 100% and the specificity is at least about 100%. By way of further example, in some embodiments, the baseline level of GFAP and the baseline level of UCH-L1 are determined by an assay having a sensitivity of about 79% or greater and a specificity of about 33% or greater. Additionally, in other embodiments, the method when used as an assay has at least 3% greater sensitivity and at least 17% greater specificity than a method or assay that measures or detects GFAP or UCH-L1 individually. In other embodiments, the method when used as an assay has at least 5% greater sensitivity and at least 20% greater specificity than a method or assay that measures or detects GFAP or UCH-L1 individually.In other embodiments, the method when used as an assay has at least 8% greater sensitivity and at least 25% greater specificity compared to methods or assays that measure or detect GFAP or UCH-L1 individually.

[0274] In some embodiments, the reference level of GFAP can be between about 50 pg / mL and about 2000 pg / mL and the reference level of UCH-L1 can be between at least about 100 pg / mL and about 2000 pg / mL. In some embodiments, the reference level of GFAP or the reference level of UCH-L1 can be between at least about 10 pg / mL and about 500 pg / mL, between at least about 10 pg / mL and about 400 pg / mL, between at least about 10 pg / mL and about 300 pg / mL, between at least about 10 pg / mL and about 200 pg / mL, between at least about 10 pg / mL and about 100 pg / mL, between at least about 10 pg / mL and about 50 pg / mL, between at least about 10 pg / mL and about 40 pg / mL, or at least at least about 10 pg / mL to about 30 pg / mL, at least about 20 pg / mL to about 500 pg / mL, at least about 20 pg / mL to about 400 pg / mL, at least about 20 pg / mL to about 300 pg / mL, at least about 20 pg / mL to about 200 pg / mL, at least about 20 pg / mL to about 100 pg / mL, at least about 20 pg / mL to about 50 pg / mL, at least about 20 pg / mL to about 40 pg / mL, at least about 20 pg / mL to about 30 pg / mL between about 30pg / mL and about 500pg / mL, between about 30pg / mL and about 400pg / mL, between about 30pg / mL and about 300pg / mL, between about 30pg / mL and about 200pg / mL, between about 30pg / mL and about 100pg / mL, between about 30pg / mL and about 50pg / mL, between about 30pg / mL and about 40pg / mL, between about 40pg / mL and about 500pg / mL, between about 40pg / mL and about 500pg / mL, between about 40pg / mL and about 400pg / mL, at least about 40pg / mL and about 300pg / mL, at least about 40pg / mL and about 200pg / mL, at least about 40pg / mL and about 100pg / mL, at least about 40pg / mL and about 50pg / mL, at least about 50pg / mL and about 500pg / mL, at least about 50pg / mL and about 400pg / mL, at least about 50pg / mL and about 300pg / mL, at least about 50pg / mL and about 200pg / mL,Between at least about 50 pg / mL and about 100 pg / mL, between at least about 75 pg / mL and about 500 pg / mL, between at least about 75 pg / mL and about 400 pg / mL, between at least about 75 pg / mL and about 300 pg / mL, between at least about 75 pg / mL and about 200 pg / mL, between at least about 75 pg / mL and about 100 pg / mL, between at least about 100 pg / mL and about 500 pg / mL, between at least about 100 pg / mL and about 400 pg / mL, between at least about 100 pg / mL and about 300 pg / mL g / mL, at least about 100 pg / mL to about 200 pg / mL, at least about 150 pg / mL to about 500 pg / mL, at least about 150 pg / mL to about 400 pg / mL, at least about 150 pg / mL to about 300 pg / mL, at least about 150 pg / mL to about 200 pg / mL, at least about 200 pg / mL to about 500 pg / mL, at least about 200 pg / mL to about 400 pg / mL, or at least about 200 pg / mL to about 300 pg / mL. For example, the reference level for UCH-L1 may be at least about 80 pg / mL to about 150 pg / mL, and the reference level for GFAP may be at least about 20 pg / mL to about 200 pg / mL. By way of further example, in other embodiments, the reference level for GFAP is about 105 pg / mL to about 890 pg / mL, and the reference level for UCH-L1 is about 110 pg / mL to about 2000 pg / mL. In some embodiments, the reference level for GFAP is about 105 pg / mL, and the reference level for UCH-L1 is about 110 pg / mL. In some embodiments, the reference level for GFAP is about 890 pg / mL, and the reference level for UCH-L1 is about 920 pg / mL. In other embodiments, the reference level for GFAP is about 505 pg / mL, and the reference level for UCH-L1 is about 1580 pg / mL.

[0275] In some embodiments, the sample is obtained from the subject within about 8 hours to about 12 hours after the actual or suspected injury; the reference level for GFAP is about 890 pg / mL and the reference level for UCH-L1 is about 920 pg / mL, and the method has a sensitivity of 90% or greater and a specificity of 79% or greater. In another embodiment, the sample is obtained from the subject within about 12 hours to about 16 hours after the actual or suspected injury; the reference level for GFAP is about 505 pg / mL and the reference level for UCH-L1 is about 1580 pg / mL, and the method has a sensitivity of 90% or greater and a specificity of 66% or greater.

[0276] In some embodiments, the method further comprises treating the human subject with a traumatic brain injury treatment and / or monitoring the human subject, as described below.

[0277] The nature of the assay utilized in the methods described herein is not critical, and the test can be any assay known in the art, such as, for example, immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis or protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, or chromatographic or spectroscopic methods, such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). The assay can also be utilized in clinical chemistry formats known to those skilled in the art. Such assays are described in more detail in Sections 5-9 herein. It is known in the art that values ​​(e.g., reference levels, cut-offs, thresholds, specificity, sensitivity, calibrator and / or control concentrations, etc.) used in assays utilizing specific sample types (such as, for example, immunoassays using serum or point-of-care devices using whole blood) can be extrapolated to other assay formats using techniques known in the art, such as assay standardization. For example, one way in which assay standardization can be performed is by applying a factor to the calibrators used in the assay to make the sample concentration readings higher or lower to obtain a slope commensurate with the comparative method. Other methods of standardizing the results obtained in one assay to another are also well known and described in the literature (see, for example, David Wild, "Immunoassay Handbook", 4th Edition, Chapter 3.5, pages 315-322, the contents of which are incorporated herein by reference).

[0278] 3. Methods to aid in the decision about whether to perform imaging on human subjects with head injury The present disclosure relates, among other methods, to methods that aid in the decision of whether to perform an imaging procedure, such as an MRI or CT scan, on a human subject who has sustained or may have sustained a head injury. As used herein, "deciding whether to perform an imaging procedure, such as an MRI or CT scan on a human subject" refers to the fact that the aforementioned methods can be used, along with other information (e.g., clinical evaluation data), to determine whether a subject is more likely to have a positive MRI scan or a positive head CT scan (i.e., the presence of an intracranial lesion) than not.

[0279] In some embodiments, the methods may help determine whether a subject who has suffered a TBI requires a computed tomography (CT) scan based on the levels of GFAP, UCH-L1, or GFAP and UCH-L1 compared to baseline levels. According to these embodiments, the methods include performing an assay on a sample obtained from the subject within about 48 hours after the actual or suspected injury to measure or detect a combination of a GFAP level in the sample and a UCH-L1 level in the sample; and determining that the subject does not require a CT scan if (a) the GFAP level in the sample is less than a baseline level of GFAP of about 50 pg / mL and the UCH-L1 level in the sample is less than a baseline level of UCH-L1 of about 90 pg / mL; or (b) the GFAP level in the sample is less than a baseline level of GFAP of about 50 pg / mL and the UCH-L1 level in the sample is less than a baseline level of UCH-L1 of about 90 pg / mL. determining that the subject does not require a CT scan if the sample level of GFAP is equal to a reference level of about 50 pg / mL to about 975 pg / mL and the sample level of UCH-L1 is equal to a reference level of UCH-L1 of about 90 pg / mL to about 2000 pg / mL; or (c) determining that the subject is more likely to require a CT scan than not if the sample level of GFAP is greater than a reference level of GFAP of about 975 pg / mL and the sample level of UCH-L1 is greater than a reference level of UCH-L1 of about 2000 pg / mL. may include.

[0280] In some embodiments, the reference level of GFAP can be between about 50 pg / mL and about 975 pg / mL, and the reference level of UCH-L1 can be at least between about 90 pg / mL and about 2000 pg / mL. In some embodiments, the reference level of GFAP can be between about 100 pg / mL and about 975 pg / mL, and the reference level of UCH-L1 can be at least between about 100 pg / mL and about 2000 pg / mL. In some embodiments, the reference level of GFAP can be between about 200 pg / mL and about 975 pg / mL, and the reference level of UCH-L1 can be at least between about 200 pg / mL and about 2000 pg / mL. In some embodiments, the reference level of GFAP can be between about 300 pg / mL and about 975 pg / mL, and the reference level of UCH-L1 can be at least between about 300 pg / mL and about 2000 pg / mL. In some embodiments, the reference level of GFAP can be between about 400 pg / mL and about 975 pg / mL, and the reference level of UCH-L1 can be at least between about 400 pg / mL and about 2000 pg / mL. In some embodiments, the reference level of GFAP can be between about 500 pg / mL and about 975 pg / mL, and the reference level of UCH-L1 can be at least between about 500 pg / mL and about 2000 pg / mL. In some embodiments, the reference level of GFAP can be between about 110 pg / mL and about 975 pg / mL, and the reference level of UCH-L1 can be at least between about 90 pg / mL and about 2000 pg / mL. In some embodiments, the reference level of GFAP can be between about 240 pg / mL and about 975 pg / mL, and the reference level of UCH-L1 can be at least between about 300 pg / mL and about 2000 pg / mL. In some embodiments, the reference level of GFAP can be between about 190 pg / mL and about 975 pg / mL, and the reference level of UCH-L1 can be at least between about 90 pg / mL and about 2000 pg / mL.In some embodiments, the baseline level of GFAP is at least 110 pg / mL and the baseline level of UCH-L1 is at least 2000 pg / mL. In other embodiments, the baseline level of GFAP is at least 240 pg / mL and the baseline level of UCH-L1 is at least 300 pg / mL. In other embodiments, the baseline level of GFAP is at least 190 pg / mL and the baseline level of UCH-L1 is at least 90 pg / mL.

[0281] In some embodiments, the sample is collected from the human subject within about 48 hours, such as within about 0 to about 4 hours, within about 0 to about 8 hours, within about 0 to about 12 hours, within about 0 to about 16 hours, within about 0 to about 20 hours, within about 0 to about 24 hours, and within about 0 to about 48 hours, of the head injury or suspected injury. In some embodiments, the sample is collected from the human subject within about 4 hours to about 8 hours, within about 8 hours to about 12 hours, within about 12 hours to about 16 hours, within about 16 hours to about 20 hours, within about 20 hours to about 24 hours, and within about 24 hours to about 48 hours. In other embodiments, samples may be collected from a human subject within about 0 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after the injury or suspected injury to the head. In some embodiments, the onset of the presence of the combination of GFAP and UCH-L1 occurs within about 0 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after the head injury. In one embodiment, the sample is obtained from the subject within about 4 hours to about 16 hours after the actual or suspected injury. In another embodiment, the sample is obtained from the subject within about 4 hours to about 8 hours after the actual or suspected injury. In yet another embodiment, the sample is obtained from the subject within about 8 hours to about 12 hours after the actual or suspected injury. In yet another embodiment, the sample is obtained from the subject within about 12 hours to about 16 hours after the actual or suspected injury.

[0282] In some embodiments, the baseline level of GFAP and the baseline level of UCH-L1 are determined by a method having a sensitivity of about 54% or greater and a specificity of about 32% or greater, as discussed further herein.

[0283] In some embodiments, the baseline levels of GFAP and UCH-L1 are determined by an assay having a sensitivity of at least about 50% to about 100% and a specificity of at least about 30% to about 100%. In some embodiments, the sensitivity is between at least about 50% to about 100%, between at least about 50% to at least about 99%, between at least about 50% to at least about 95%, between at least about 50% to at least about 90%, between at least about 50% to at least about 85%, between at least about 55% to about 100%, between at least about 55% to at least about 99%, between at least about 55% to at least about 95%, between at least about 55% to at least about 90%, between at least about 55% to at least about 85%, between at least about 60% to about 100%, between at least about 60% to at least about 99%, between at least about 60% to at least about 95%, between at least about 60% to at least about 90%, between at least about 60% to at least about 85%, between at least about 70% to about 100%, between at least about 70% to at least about 9 9%, between at least about 70% and at least about 95%, between at least about 70% and at least about 90%, between at least about 70% and at least about 85%, between at least about 80% and about 100%, between at least about 80% and at least about 99%, between at least about 80% and at least about 95%, between at least about 80% and at least about 90%, between at least about 80% and at least about 85%, between at least about 85% and at least about 100%, between at least about 85% and at least about 99%, between at least about 85% and at least about 95%, between at least about 85% and at least about 90%, between at least about 90% and at least about 95%, between at least about 95% and at least about 100%, or between at least about 95% and at least about 99%.In some embodiments, the sensitivity is at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least about 55%, at least about 60%, at least about 65%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 87.5%, at least about 90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%. In some embodiments, the sensitivity of the assay is 54% or greater.

[0284] In some embodiments, the specificity is at least about 30% to about 100%, at least about 30% to about 99%, at least about 30% to about 95%, at least about 30% to about 90%, at least about 30% to about 85%, at least about 30% to about 80%, at least about 30% to about 75%, at least about 30% to about 70%, at least about 30% to about 60%, at least about 30% to about 50%, at least about 40% to about 100%, at least about 40% to about 99%, at least about 40% to about 95%. %, between about 40% and about 90%, between about 40% and about 85%, between about 40% and about 80%, between about 40% and about 75%, between about 40% and about 70%, between about 40% and about 60%, between about 40% and about 50%, between about 50% and about 100%, between about 50% and about 99%, between about 50% and about 95%, between about 50% and about 90%, between about 50% and about 85%, between about 50% and about 80%, between about 50% and about between about 75%, between about 50% and about 70%, between about 50% and about 60%, between about 60% and about 100%, between about 60% and about 99%, between about 60% and about 95%, between about 60% and about 90%, between about 60% and about 85%, between about 60% and about 80%, between about 60% and about 75%, between about 60% and about 70%, between about 70% and about 100%, between about 70% and about 99%, between about 70% and about 95%, % to about 90%, at least about 70% to about 85%, at least about 70% to about 80%, at least about 70% to about 75%, at least about 80% to about 100%, at least about 80% to about 99%, at least about 80% to about 95%, at least about 80% to about 90%, at least about 80% to about 85%, at least about 90% to about 100%, at least about 90% to about 99%, at least about 90% to about 95%, at least about 95% to about 99%, or at least about 95% to about 100%.In some embodiments, the specificity is at least about 30.0%, at least about 31.0%, at least about 32.0%, at least about 33.0%, at least about 34.0%, at least about 35.0%, at least about 36.0%, at least about 37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%, at least about 45.0%, at least about 50.0%, at least about 55.0%, at least about 60.0%, at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%, at least about 94.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%. For example, in some embodiments, the sensitivity is at least about 99% and the specificity is at least about 75%, the sensitivity is at least about 99% and the specificity is at least about 99%, or the sensitivity is at least about 100% and the specificity is at least about 100%.

[0285] In another embodiment, the sample is obtained from the subject within about 4 hours to about 8 hours after the actual or suspected injury; the baseline level of GFAP is about 110 pg / mL and the baseline level of UCH-L1 is about 20,000 pg / mL, and the method has a sensitivity of 95% or greater and a specificity of 62% or greater. In yet another embodiment, the sample is obtained from the subject within about 8 hours to about 12 hours after the actual or suspected injury; the baseline level of GFAP is about 240 pg / mL and the baseline level of UCH-L1 is about 300 pg / mL, and the method has a sensitivity of 91.5% or greater and a specificity of 52% or greater. In yet a further embodiment, the sample is obtained from the subject within about 12 hours to about 16 hours after the actual or suspected injury; the baseline level of GFAP is about 190 pg / mL, the baseline level of UCH-L1 is about 90 pg / mL, and the method has a sensitivity of 99% or greater and a specificity of 36% or greater.

[0286] In other embodiments, the methods may help determine whether a subject who has suffered a TBI requires an MRI procedure based on the levels of GFAP and UCH-L1 compared to baseline levels. According to these embodiments, the methods include performing an assay on a sample obtained from the subject within about 48 hours after the actual or suspected injury to measure or detect a combination of a sample level of GFAP and a sample level of UCH-L1; and determining that the subject does not require an MRI procedure if (a) the sample level of GFAP is less than a baseline level of about 15 pg / mL of GFAP and the sample level of UCH-L1 is less than a baseline level of about 50 pg / mL of UCH-L1; or (b) the sample level of GFAP is less than a baseline level of about 15 pg / mL of GFAP and the sample level of UCH-L1 is less than a baseline level of about 50 pg / mL of UCH-L1. or (c) determining that the subject is more likely than not to require an MRI procedure if the sample level of GFAP is greater than the GFAP reference level of about 1000 pg / mL and the sample level of UCH-L1 is greater than the UCH-L1 reference level of about 50 pg / mL to about 2000 pg / mL; or (b) determining that the subject is more likely than not to require an MRI procedure if the sample level of GFAP is greater than the GFAP reference level of about 1000 pg / mL and the sample level of UCH-L1 is greater than the UCH-L1 reference level of about 2000 pg / mL.

[0287] In some embodiments, the reference level of GFAP can be between about 10 pg / mL and about 1000 pg / mL, and the reference level of UCH-L1 can be at least between about 40 pg / mL and about 2000 pg / mL. In some embodiments, the reference level of GFAP can be between about 15 pg / mL and about 1000 pg / mL, and the reference level of UCH-L1 can be at least between about 50 pg / mL and about 2000 pg / mL. In some embodiments, the reference level of GFAP can be between about 25 pg / mL and about 1000 pg / mL, and the reference level of UCH-L1 can be at least between about 75 pg / mL and about 2000 pg / mL. In some embodiments, the reference level of GFAP can be between about 50 pg / mL and about 1000 pg / mL, and the reference level of UCH-L1 can be at least between about 100 pg / mL and about 2000 pg / mL. In some embodiments, the reference level of GFAP can be between about 75 pg / mL and about 1000 pg / mL, and the reference level of UCH-L1 can be at least between about 150 pg / mL and about 2000 pg / mL. In some embodiments, the reference level of GFAP can be between about 100 pg / mL and about 1000 pg / mL, and the reference level of UCH-L1 can be at least between about 200 pg / mL and about 2000 pg / mL.

[0288] In some embodiments, the sample is collected from the human subject within about 48 hours, such as within about 0 to about 4 hours, within about 0 to about 8 hours, within about 0 to about 12 hours, within about 0 to about 16 hours, within about 0 to about 20 hours, within about 0 to about 24 hours, and within about 0 to about 48 hours, of the head injury or suspected injury. In some embodiments, the sample is collected from the human subject within about 4 hours to about 8 hours, within about 8 hours to about 12 hours, within about 12 hours to about 16 hours, within about 16 hours to about 20 hours, within about 20 hours to about 24 hours, and within about 24 hours to about 48 hours. In other embodiments, samples may be collected from a human subject within about 0 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after the injury or suspected injury to the head. In some embodiments, the onset of the presence of the combination of GFAP and UCH-L1 occurs within about 0 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after the head injury. In one embodiment, the sample is obtained from the subject within about 4 hours to about 16 hours after the actual or suspected injury. In another embodiment, the sample is obtained from the subject within about 4 hours to about 8 hours after the actual or suspected injury. In yet another embodiment, the sample is obtained from the subject within about 8 hours to about 12 hours after the actual or suspected injury. In yet another embodiment, the sample is obtained from the subject within about 12 hours to about 16 hours after the actual or suspected injury.

[0289] In some embodiments, the baseline levels of GFAP and the baseline levels of UCH-L1 are determined by a method having a sensitivity of about 80% to about 98% and a specificity of about 30% to about 85%, as discussed in more detail below.

[0290] In some embodiments, the reference levels of GFAP and UCH-L1 are determined by an assay having a sensitivity of at least about 70% to about 100% and a specificity of at least about 30% to about 100%. In some embodiments, the sensitivity is at least about 70% to about 100%, at least about 70% to at least about 99%, at least about 70% to at least about 95%, at least about 70% to at least about 90%, at least about 70% to at least about 85%, at least about 75% to about 100%, at least about 75% to at least about 99%, at least about 75% to at least about 95%, at least about 75% to at least about 90%, at least about 75% to at least about 85%, at least about 80% to at least about 100%, at least about 80% to at least about 9 ... 9%, between at least about 80% and at least about 95%, between at least about 80% and at least about 90%, between at least about 80% and at least about 85%, between at least about 85% and about 100%, between at least about 85% and at least about 99%, between at least about 85% and at least about 95%, between at least about 85% and at least about 90%, between at least about 90% and at least about 99%, between at least about 90% and at least about 95%, between at least about 95% and at least about 100%, or between at least about 95% and at least about 99%. In some embodiments, the sensitivity is at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 87.5%, at least about 90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%.

[0291] In some embodiments, the specificity is at least between about 30% and about 100%, at least between about 30% and about 99%, at least between about 30% and about 95%, at least between about 30% and about 90%, at least between about 30% and about 85%, at least between about 30% and about 80%, at least about 30% and about 75%, at least about 30% and about 70%, at least about 30% and about 60%, at least about 30% and about 50%, at least about 40% and about 100%, at least about 40% and about 99%, at least about 40% and about 95% between about 40% and about 90%, between about 40% and about 85%, between about 40% and about 80%, between about 40% and about 75%, between about 40% and about 70%, between about 40% and about 60%, between about 40% and about 50%, between about 50% and about 100%, between about 50% and about 99%, between about 50% and about 95%, between about 50% and about 90%, between about 50% and about 85%, between about 50% and about 80%, between about 50% and about 7 between 5%, between at least about 50% and about 70%, between at least about 50% and about 60%, between at least about 60% and about 100%, between at least about 60% and about 99%, between at least about 60% and about 95%, between at least about 60% and about 90%, between at least about 60% and about 85%, between at least about 60% and about 80%, between at least about 60% and about 75%, between at least about 60% and about 70%, between at least about 70% and about 100%, between at least about 70% and about 99%, between at least about 70% and about 95%, at least about 70% at least about 90%, at least about 70% to about 85%, at least about 70% to about 80%, at least about 70% to about 75%, at least about 80% to about 100%, at least about 80% to about 99%, at least about 80% to about 95%, at least about 80% to about 90%, at least about 80% to about 85%, at least about 90% to about 100%, at least about 90% to about 99%, at least about 90% to about 95%, at least about 95% to about 99%, or at least about 95% to about 100%.In some embodiments, the specificity is at least about 30.0%, at least about 31.0%, at least about 32.0%, at least about 33.0%, at least about 34.0%, at least about 35.0%, at least about 36.0%, at least about 37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%, at least about 45.0%, at least about 50.0%, at least about 55.0%, at least about 60.0%, at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 86.0%, at least about 87.0%, at least about 88.0%, at least about 89.0%, at least about 90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%, at least about 94.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 100.0%, at least about 101.0%, at least about 102.0%, at least about 103.0%, at least about 104.0%, at least about 105.0%, at least about 106.0%, at least about 107.0%, at least about 108.0%, at least about 109.0%, at least about 110.0%, at least about 111.0%, at least about 112.0%, at least about 113.0%, at least about 114.0%, at least about 115.0%, at least about 116.0%, at least about 117.0%, at least about 118.0%, at least about 119 5.0%, at least about 90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%, at least about 94.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%. In some embodiments, the reference level of GFAP and the reference level of UCH-L1 are determined by a method having a sensitivity of about 80% to about 98% and a specificity of about 30% to about 85%.

[0292] In some embodiments, the sample is obtained from the subject within about 24 hours to about 48 hours after injury; the baseline level of GFAP is about 35 pg / mL, and the assay has a sensitivity of 94% or greater and a specificity of 30% or greater. In yet other embodiments, the sample is obtained from the subject within about 24 hours to about 48 hours after injury; the baseline level of GFAP is about 143 pg / mL, and the assay has a sensitivity of 88% or greater and a specificity of 50% or greater. The sample is obtained from the subject within about 24 hours to about 48 hours after injury; the baseline level of GFAP is about 602 pg / mL, and the assay has a sensitivity of 57% or greater and a specificity of 95% or greater.

[0293] In some embodiments, the method is performed on a subject undergoing a CT scan before or after the assay is performed, and optionally the CT scan indicates that no TBI has occurred (i.e., a normal CT scan). In such a case, if the subject's GFAP and UCH-L1 levels indicate, for example, that an MRI procedure is required, the method may include determining that an MRI procedure should be performed independent of the CT scan results to diagnose and evaluate the subject and / or determine what type of TBI the subject has suffered. In some embodiments, the subject does not undergo a CT scan before or after the assay is performed. In such a case, if the subject's GFAP and UCH-L1 levels indicate, for example, that an MRI procedure is required, the method may include determining that an MRI procedure should be performed independent of the absence of a negative CT scan to diagnose and evaluate the subject and / or determine what type of TBI the subject has suffered. In some embodiments, baseline levels of GFAP and UCH-L1 correlate with a positive MRI scan or a positive head CT scan (ie, the presence of intracranial lesions).

[0294] In other embodiments, the methods may help determine whether a subject who has suffered a TBI requires an MRI procedure based on the levels of GFAP or UCH-L1 compared to baseline levels. In accordance with these embodiments, the method may include performing an assay on a sample obtained from the subject within about 48 hours after the actual or suspected injury to measure or detect a combination of the sample level of GFAP and the sample level of UCH-L1; and (a) determining that the subject does not require an MRI procedure if the sample level of GFAP is equal to a reference level of GFAP of about 0 pg / mL to about 68 pg / mL, or the sample level of UCH-L1 is equal to a reference level of UCH-L1 of about 0 pg / mL to about 99 pg / mL; or (b) determining that the subject is more likely than not to require an MRI procedure if the sample level of GFAP is higher than the reference level of GFAP of about 68 pg / mL, or the sample level of UCH-L1 is higher than the reference level of UCH-L1 of about 99 pg / mL.

[0295] In some embodiments, the reference level of GFAP can be between about 1 pg / mL and about 68 pg / mL, or the reference level of UCH-L1 can be at least between about 1 pg / mL and about 99 pg / mL. In some embodiments, the reference level of GFAP can be between about 5 pg / mL and about 68 pg / mL, or the reference level of UCH-L1 can be at least between about 5 pg / mL and about 99 pg / mL. In some embodiments, the reference level of GFAP can be between about 5 pg / mL and about 65 pg / mL, or the reference level of UCH-L1 can be at least between about 5 pg / mL and about 95 pg / mL. In some embodiments, the reference level of GFAP can be between about 5 pg / mL and about 60 pg / mL, or the reference level of UCH-L1 can be at least between about 5 pg / mL and about 90 pg / mL. In some embodiments, the reference level of GFAP can be between about 5 pg / mL and about 55 pg / mL, or the reference level of UCH-L1 can be at least between about 5 pg / mL and about 85 pg / mL. In some embodiments, the reference level of GFAP can be between about 5 pg / mL and about 50 pg / mL, or the reference level of UCH-L1 can be at least between about 5 pg / mL and about 80 pg / mL.

[0296] In some embodiments, the sample is collected from the human subject within about 48 hours, such as within about 0 to about 4 hours, within about 0 to about 8 hours, within about 0 to about 12 hours, within about 0 to about 16 hours, within about 0 to about 20 hours, within about 0 to about 24 hours, and within about 0 to about 48 hours, of the head injury or suspected injury. In some embodiments, the sample is collected from the human subject within about 4 hours to about 8 hours, within about 8 hours to about 12 hours, within about 12 hours to about 16 hours, within about 16 hours to about 20 hours, within about 20 hours to about 24 hours, and within about 24 hours to about 48 hours. In other embodiments, samples may be collected from a human subject within about 0 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 ...

Claims

1. 1. A method for aiding in the decision of whether to perform a head computed tomography (CT) scan on a human subject who has sustained or may have sustained a head injury, comprising: performing an assay on a sample obtained from the subject within 48 hours after the actual or suspected injury to measure a combination of levels of glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the sample; and determining that the subject does not require a CT scan if the sample level of GFAP is equal to a reference level of 50 pg / mL to 975 pg / mL for GFAP and the sample level of UCH-L1 is equal to a reference level of 90 pg / mL to 2000 pg / mL for UCH-L1. The method includes:

2. (a) the reference level of GFAP is 50 pg / mL and the reference level of UCH-L1 is 2000 pg / mL; (b) the reference level of GFAP is 95 pg / mL and the reference level of UCH-L1 is 2000 pg / mL; (c) the reference level of GFAP is 110 pg / mL and the reference level of UCH-L1 is 2000 pg / mL; (d) the reference level of GFAP is 115 pg / mL and the reference level of UCH-L1 is 1 (e) the reference level of GFAP is 140 pg / mL and the reference level of UCH-L1 is 2000 pg / mL; (f) the reference level of GFAP is 150 pg / mL and the reference level of UCH-L1 is 190 pg / mL; (g) the reference level of GFAP is 190 pg / mL and the reference level of UCH-L1 is 90 pg / mL; (h) the reference level of GFAP is 240 pg / mL and the reference level of UCH-L1 is 200 pg / mL; (i) the reference level of GFAP is 285 pg / mL and the reference level of UCH-L1 is 190 pg / mL; (j) the reference level of GFAP is 500 pg / mL and the reference level of UCH-L1 is 1450 pg / mL; (k) the reference level of GFAP is 555 pg / mL and the reference level of UCH-L1 is 810 pg / mL; (l) the reference level of GFAP is 800 pg / mL. and the reference level of UCH-L1 is 900 pg / mL; (m) the reference level of GFAP is 840 pg / mL and the reference level of UCH-L1 is 2000 pg / mL; (n) the reference level of GFAP is 880 pg / mL and the reference level of UCH-L1 is 810 pg / mL; or (o) the reference level of GFAP is 975 pg / mL and the reference level of UCH-L1 is 1580 pg / mL.

3. 3. The method of claim 1 or 2, wherein the baseline level of GFAP and the baseline level of UCH-L1 are determined by an assay having a sensitivity of 54% or greater and a specificity of 32% or greater.

4. The method of any of claims 1 to 3, wherein the sample is obtained from the subject within 4 to 16 hours after the actual or suspected injury.

5. The method of any one of claims 1 to 4, wherein the assay has at least 2% higher sensitivity and at least 4% higher specificity compared to an assay that measures or detects GFAP or UCH-L1 individually.

6. (a) the sample is obtained from the subject within 4 to 8 hours after the actual or suspected injury; the reference level of GFAP is 110 pg / mL and the reference level of UCH-L1 is 2000 pg / mL; and the assay has a sensitivity of 95% or greater and a specificity of 62% or greater; (b) the sample is obtained from the subject within 8 to 12 hours after the actual or suspected injury; the reference level of GFAP is 240 pg / mL and the reference level of UCH-L1 is 300 pg / mL; and the assay has a sensitivity of 91.5% or greater and a specificity of 52% or greater; or (c) the sample is obtained from the subject within 12 to 16 hours after the actual or suspected injury; the reference level of GFAP is 190 pg / mL and the reference level of UCH-L1 is 90 pg / mL; and the assay has a sensitivity of 99% or greater and a specificity of 36% or greater. The method according to any one of claims 1 to 5.

7. 1. A method for aiding in determining whether a human subject who has suffered a head injury has suffered a traumatic brain injury (TBI), comprising: performing an assay on a sample obtained from the subject within 48 hours following injury to measure a combination of levels of glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the sample; and determining that the subject is more likely to have suffered a TBI than not if the sample level of GFAP is equal to a reference level of 15 pg / mL to 40 pg / mL for GFAP and the sample level of UCH-L1 is equal to a reference level of 70 pg / mL to 150 pg / mL for UCH-L1. The method includes:

8. (a) the reference level of GFAP is 10 pg / mL and the reference level of UCH-L1 is 60 pg / mL; (b) the reference level of GFAP is 15 pg / mL and the reference level of UCH-L1 is 70 pg / mL; (c) the reference level of GFAP is 15 pg / mL and the reference level of UCH-L1 is 90 pg / mL; (d) the reference level of GFAP is 15 pg / mL and the reference level of UCH-L1 is 90 pg / mL.

8. The method of claim 7, wherein the reference level is 150 pg / mL; (e) the reference level of GFAP is 20 pg / mL and the reference level of UCH-L1 is 60 pg / mL; (f) the reference level of GFAP is 30 pg / mL and the reference level of UCH-L1 is 70 pg / mL; or (g) the reference level of GFAP is 30 pg / mL and the reference level of UCH-L1 is 110 pg / mL.

9. The method of claim 7 or 8, wherein the baseline level of GFAP and the baseline level of UCH-L1 are determined by an assay having a sensitivity of 90% or greater and a specificity of 35% or greater.

10. 10. The method of any one of claims 7 to 9, wherein the sample is obtained from the subject within 4 to 16 hours after the injury.

11. The method of any one of claims 7 to 10, wherein the assay has at least 3% higher sensitivity and at least 17% higher specificity compared to an assay that measures or detects GFAP or UCH-L1 individually.

12. (a) the sample is obtained from the subject within 8 to 12 hours after the injury; the baseline level of GFAP is 30 pg / mL and the baseline level of UCH-L1 is 110 pg / mL; and the assay has a sensitivity of 92% or greater and a specificity of 99% or greater; (b) the sample is obtained from the subject within 12 to 16 hours after the injury; the baseline level of GFAP is 30 pg / mL and the baseline level of UCH-L1 is 110 pg / mL; and the assay has a sensitivity of 90% or greater and a specificity of 99% or greater; (c) the sample is obtained from the subject within 4 to 8 hours after the injury; the reference level of GFAP is 40 pg / mL and the reference level of UCH-L1 is 100 pg / mL; and the assay has a sensitivity of 90% or greater and a specificity of 94% or greater; (d) the sample is obtained from the subject within 8 to 12 hours after the injury; the baseline level of GFAP is 15 pg / mL and the baseline level of UCH-L1 is 150 pg / mL; the assay has a sensitivity of 95% or greater and a specificity of 82% or greater; or (e) the sample is obtained from the subject within 12 to 16 hours after the injury; the baseline level of GFAP is 20 pg / mL and the baseline level of UCH-L1 is 60 pg / mL; and the assay has a sensitivity of 95% or greater and a specificity of 65% or greater. The method according to any one of claims 7 to 11.

13. 1. A method for aiding in the decision of whether to perform a head magnetic resonance imaging (MRI) procedure on a human subject who has sustained or may have sustained a head injury, comprising: performing an assay on a sample obtained from the subject within 48 hours after the actual or suspected injury to measure a combination of levels of glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the sample; and (a) determining that the subject does not require an MRI procedure if the sample level of GFAP is less than a reference level of 15 pg / mL for GFAP and the sample level of UCH-L1 is less than a reference level of 50 pg / mL for UCH-L1; or (b) determining that the subject is more likely than not to require an MRI procedure if the sample level of GFAP is equal to a reference level of GFAP between 15 pg / mL and 1000 g / mL and the sample level of UCH-L1 is equal to a reference level of UCH-L1 between 50 pg / mL and 2000 g / mL; or (c) determining that the subject is more likely than not to require an MRI procedure if the sample level of GFAP is greater than a reference level of 1000 pg / mL for GFAP and the sample level of UCH-L1 is greater than a reference level of 2000 pg / mL for UCH-L1; The method includes:

14. The method of claim 13, wherein the baseline level of GFAP and the baseline level of UCH-L1 are determined by an assay having a sensitivity of 80% to 98% and a specificity of 30% to 85%.

15. 15. The method of claim 13 or 14, wherein the subject has had a negative CT scan result before the assay is performed.

16. 1. A method for aiding in or predicting an outcome in a human subject who has sustained or may have sustained a head injury, comprising: performing an assay on a sample obtained from the subject within 48 hours after the actual or suspected injury to measure or detect a combination of levels of glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the sample; and a step in which, when the level of GFAP in the sample is equal to a reference level of GFAP of 80 pg / mL to 2000 pg / mL and the level of UCH-L1 in the sample is equal to a reference level of UCH-L1 of 130 pg / mL to 2000 pg / mL, a step in which, when the level of GFAP in the sample is equal to a reference level of UCH-L1 of 130 pg / mL to 2000 pg / mL, a step in which, when the level of UCH-L1 ... The method includes:

17. Measuring the level of GFAP comprises: (a) subjecting the samples to, simultaneously or sequentially, any order; (1) at least one GFAP capture antibody that binds to an epitope on GFAP or a GFAP fragment to form at least one GFAP capture antibody-GFAP antigen complex; and (2) at least one GFAP detection antibody that contains a detectable label and that binds to an epitope on GFAP that is not bound by the GFAP capture antibody; to form at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex; and (b) determining the amount or concentration of GFAP in said sample based on a signal generated by a detectable label in said at least one GFAP capture antibody-GFAP antigen-at least one GFAP detection antibody complex; The method according to any one of claims 1 to 16, comprising:

18. Measuring the level of UCH-L1 (a) subjecting the samples to, simultaneously or sequentially, any order; (1) at least one UCH-L1 capture antibody that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form at least one UCH-L1 capture antibody-UCH-L1 antigen complex; and (2) at least one UCH-L1 detection antibody that contains a detectable label and that binds to an epitope on UCH-L1 that is not bound by said at least one UCH-L1 capture antibody; to form at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex; and (b) determining the amount or concentration of UCH-L1 in said sample based on a signal generated by a detectable label in said at least one UCH-L1 capture antibody-UCH-L1 antigen-at least one UCH-L1 detection antibody complex; The method according to any one of claims 1 to 17, comprising:

19. The method according to any one of claims 1 to 18, wherein the sample is selected from the group consisting of a whole blood sample, a serum sample, a cerebrospinal fluid sample, a tissue sample, a body fluid sample and a plasma sample.

20. The sample comprises: (a) after the subject has suffered injury to the head caused by physical concussion, an external mechanical or other force resulting in a closed or open head injury, one or more falls, blunt impact from an explosion or blast, or other type of blunt trauma; (b) after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin; or (c) from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof. The method according to any one of claims 1 to 19, wherein

21. The method of any one of claims 1 to 20, further comprising the step of monitoring the subject.

22. The method of any one of claims 1 to 21, wherein the sample is obtained after the subject has suffered an orthopedic injury.

23. The method of any one of claims 1 to 22, wherein the assay is (a) an immunoassay; (b) a clinical chemistry assay; or (c) a single molecule detection assay.

Images