Systems and methods for TBI adjudication

Abstract

The present disclosure relates to methods diagnosing or aiding in the diagnosis of a traumatic brain injury (TBI) or adjudicating a TBI diagnosis of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head. The methods involve (a) receiving diagnostic criteria of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head; (b) executing a TBI adjudication algorithm by compiling the diagnostic criteria; and (c) adjudicating a TBI diagnosis based on the diagnostic criteria compiled in step (b). The diagnostic criteria includes, inter alia, the presence or absence of (i) a trauma mechanism; (ii) a neuroimaging intracranial abnormality; (iii) one or more clinical signs indicative of TBI; (iv) one or more confounding factors; and (v) a level of glial fibrillary acidic protein (GFAP) and / or a level of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) measured in a sample obtained from the subject. The present disclosure also relates to systems and kits for implementing the methods.

Description

Docket No. 15893; 43644.601SYSTEMS AND METHODS FOR TBI ADJUDICATIONRELATED APPLICATION INFORMATION

[0001] This application claims priority to provisional applications 63 / 724,425, filed November 25, 2024, 63 / 724,427, filed November 25, 2024, and 63 / 867110, filed August 20, 2025; each of which is herein incorporated by reference in their entirety.SEQUENCE LISTING STATEMENT

[0002] The contents of the electronic sequence listing titled ABBTL-43644-601-ST26.xml (size: 7,801 bytes; and date of creation: November 24, 2025) are herein incorporated by reference in their entirety.TECHNICAL FIELD

[0003] The present disclosure relates to methods diagnosing or aiding in the diagnosis of a traumatic brain injury (TBI) or adjudicating a TBI diagnosis of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head. The methods involve (a) receiving diagnostic criteria of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head; (b) executing a TBI adjudication algorithm by compiling the diagnostic criteria; and (c) adjudicating a TBI diagnosis based on the diagnostic criteria compiled in step (b). The diagnostic criteria includes, inter alia, the presence or absence of (i) a trauma mechanism; (ii) a neuroimaging intracranial abnormality; (iii) one or more clinical signs indicative of TBI; (iv) one or more confounding factors; and (v) a level of glial fibrillary acidic protein (gfap) and / or a level of ubiquitin carboxy-terminal hydrolase 11 (uch-11) measured in a sample obtained from the subject. The present disclosure also relates to systems and kits for implementing the methods.BACKGROUND

[0004] More than 5 million mild traumatic brain injuries (TBIs) occur each year in the United States alone. Currently, there is no simple, objective, accurate measurement available to help in patient assessment. In fact, much of TBI evaluation and diagnosis is based on subjective data. Unfortunately, objective measurements such as head CT and Glasgow Coma Score (GCS) are not very comprehensive or sensitive in evaluating mild TBI. Moreover, head CT is unrevealing for the vast majority of the time for mild TBI, is expensive, and exposes the patient to unnecessary radiation. Additionally, a negative head CT does not mean theDocket No. 15893; 43644.601 patient has been cleared from having a concussion; rather it just means certain interventions, such as surgery, are not warranted. Clinicians and patients need objective, reliable information to accurately evaluate this condition to promote appropriate triage and recovery. To date, limited data have been available for the use biomarkers of intracranial injury, such as UCH-L1 and GFAP, in the acute care setting to aid in patient evaluation and management.

[0005] Mild TBI or concussion is much harder to objectively detect and presents an everyday challenge in emergency care units globally. Concussion usually causes no gross pathology, such as hemorrhage, and no abnormalities on conventional computed tomography scans of the brain, but rather rapid-onset neuronal dysfunction that resolves in a spontaneous manner over a few days to a few weeks. Approximately 15% of mild TBI patients suffer persisting cognitive dysfunction. There is an unmet need for mild TBI victims on scene, in emergency rooms and clinics, in the sports area and in military activity (e.g., combat).

[0006] Current algorithms for assessment of the severity of brain injury include Glasgow Coma Scale score and other measures. These measures may at times be adequate for relating acute severity but are insufficiently sensitive for subtle pathology which can result in persistent deficit. GCS and other measures also do not enable differentiation among types of injury and may not be adequate. Thus, patients grouped into a single GCS level entering a clinical trial may have vastly heterogeneous severity and type of injury. Because outcomes also vary accordingly, inappropriate classification undermines the integrity of a clinical trial. Improved classification of injury will enable more precise delineation of disease severity and type for TBI patients in clinical trials.

[0007] Additionally, current brain injury trials rely on outcome measures such as Glasgow Outcome Scale Extended, which capture global phenomena but fail to assess for subtle differences in outcome. Thus 30 consecutive trials for brain injury therapeutics have failed.

[0008] Thus, there is a long felt need for sensitive outcome measures to consistently and reliably diagnose TBI, as well as recovery from brain injury to test therapeutics and prophylactics.SUMMARY

[0009] In one embodiment, the present disclosure relates to a method of adjudicating a traumatic brain injury (TBI) diagnosis. The method comprises the steps of:

[0010] (a) receiving diagnostic criteria of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head, wherein the diagnostic criteria comprises:

[0011] (i) the presence or absence of a trauma mechanism;Docket No. 15893; 43644.601

[0012] (ii) the presence or absence of a neuroimaging intracranial abnormality;

[0013] (iii) the presence or absence of one or more clinical signs of a TBI; and

[0014] (iv) the presence or absence of one or more confounding factors;

[0015] (b) executing a TBI adjudication algorithm by compiling the diagnostic criteria; and

[0016] (c) adjudicating a TBI diagnosis based on the diagnostic criteria compiled in step (b), wherein a subject is diagnosed as having a TBI when:

[0017] (i) a trauma mechanism and a neuroimaging intracranial abnormality are present; or

[0018] (ii) a trauma mechanism is present, a neuroimaging intracranial abnormality is absent, one or more clinical signs indicative of TBI is present, and one or more confounding factors is absent.

[0019] In one aspect of the above method, the subject is diagnosed as not having a TBI when a trauma mechanism is absent.

[0020] In another aspect of the above method, the subject is diagnosed as not having a TBI when:

[0021] (i) a trauma mechanism is present;

[0022] (ii) a neuroimaging intracranial abnormality is absent; and

[0023] (iii) one more clinical signs of a TBI is absent.

[0024] In still yet another aspect of the above method, the subject is diagnosed as not having a TBI diagnosis when:

[0025] (i) a trauma mechanism is present;

[0026] (ii) a neuroimaging intracranial abnormality is absent;

[0027] (iii) one more clinical signs of a TBI is present; and

[0028] (iv) the one or more clinical signs of TBI is better accounted for by the presence of the one or more confounding factors.

[0029] In some embodiments, the present disclosure relates to a method of diagnosing or aiding in a diagnosis of a traumatic brain injury (TBI) diagnosis. In some aspects, the method comprises:

[0030] (a) receiving diagnostic criteria of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head, wherein the diagnostic criteria comprises:

[0031] (i) the presence or absence of a trauma mechanism; and

[0032] (ii) a level of glial fibrillary acidic protein (GFAP) and / or a level of ubiquitin carboxy -terminal hydrolase LI (UCH-L1) measured in a sample obtained from the subject; andDocket No. 15893; 43644.601

[0033] (b) diagnosing the subject as having a TBI or aiding in the diagnosis that the subject is more likely than not to have a TBI when:

[0034] (i) a trauma mechanism is present; and

[0035] (ii) the level of GFAP is greater than or equal to about 80 pg / mL, the level of UCH- L1 is greater than or equal to about 500 pg / mL, or the level of GFAP is greater than or equal to about 80 pg / mL and the level of UCH-L1 is greater than or equal to about 500 pg / mL.

[0036] In some aspects in the above method, a TBI is diagnosed in the subject or the subject is determined to more likely than not to have a TBI when:

[0037] (i) a trauma mechanism is present; and

[0038] (ii) the level of GFAP is greater than or equal to about 80 pg / mL, the level of UCH- L1 is greater than or equal to about 1000 pg / mL, or the level of GFAP is greater than or equal to about 80 pg / mL and the level of UCH-L1 is greater than or equal to about 1000 pg / mL.

[0039] In other aspects of the above method, a TBI is diagnosed in the subject or the subject is determined to more likely than not to have a TBI when:

[0040] (i) a trauma mechanism is present; and

[0041] (ii) the level of GFAP is greater than or equal to about 100 pg / mL, the level of UCH-L1 is greater than or equal to about 500 pg / mL, or the level of GFAP is greater than or equal to about 100 pg / mL and the level of UCH-L1 is greater than or equal to about 500 pg / mL.

[0042] In still further aspects of the above method, a TBI is diagnosed in the subject or the subject is determined to more likely than not to have a TBI when:

[0043] (i) a trauma mechanism is present; and

[0044] (ii) the level of GFAP is greater than or equal to about 100 pg / mL, the level of UCH-L1 is greater than or equal to about 1000 pg / mL, or the level of GFAP is greater than or equal to about 100 pg / mL and the level of UCH-L1 is greater than or equal to about 1000 pg / mL.

[0045] In still further aspects of the above method, a TBI is diagnosed in the subject or the subject is determined to more likely than not to have a TBI when:

[0046] (i) a trauma mechanism is present; and

[0047] (ii) the level of GFAP is greater than or equal to about 120 pg / mL, the level of UCH-L1 is greater than or equal to about 500 pg / mL, or the level of GFAP is greater than or equal to about 120 pg / mL and the level of UCH-L1 is greater than or equal to about 500 pg / mL.Docket No. 15893; 43644.601

[0048] In still further aspects of the above method, a TBI is diagnosed in the subject or the subject is determined to more likely than not to have a TBI when:

[0049] (i) a trauma mechanism is present; and

[0050] (ii) the level of GFAP is greater than or equal to about 120 pg / mL, the level of UCH-L1 is greater than or equal to about 1000 pg / mL, or the level of GFAP is greater than or equal to about 120 pg / mL and the level of UCH-L1 is greater than or equal to about 1000 pg / mL.

[0051] In still further aspects of the above method, a TBI is diagnosed in the subject or the subject is determined to more likely than not to have a TBI when:

[0052] (i) a trauma mechanism is present; and

[0053] (ii) the level of GFAP is greater than between about 80 pg / mL to about 120 pg / mL, the level of UCH-L1 is greater than between about 500 pg / mL to about 1000 pg / mL, or the level of GFAP is greater than between about 80 pg / mL to about 120 pg / mL and the level of UCH-L1 is greater than between about 500 pg / mL to about 1000 pg / mL.

[0054] In still further aspects of the above method, the subject is diagnosed as not having or likely not to have a TBI when a trauma mechanism is absent.

[0055] In still further aspect the subject is diagnosed as not having or likely not to have a TBI when:

[0056] (i) a trauma mechanism is present and the level of GFAP is less than about 80 pg / mL, the level of UCH-L1 is less than about 500 pg / mL, the level of GFAP is less than about 80 pg / mL, the level of UCH-L1 is less than about 500 pg / mL;

[0057] (ii) a trauma mechanism is present and the level of GFAP is less than about 80 pg / mL, the level of UCH-L1 is less than about 1000 pg / mL, or the level of GFAP is less than about 80 pg / mL and the level of UCH-L1 is less than about 1000 pg / mL;

[0058] (iii) a trauma mechanism is present and the level of GFAP is less than about 100 pg / mL, the level of UCH-L1 is less than about 500 pg / mL, or the level of GFAP is less than about 100 pg / mL and the level of UCH-L1 is less than about 500 pg / mL;

[0059] (iv) a trauma mechanism is present and the level of GFAP is less than about 100 pg / mL, the level of UCH-L1 is less than about 1000 pg / mL, or the level of GFAP is less than about 100 pg / mL and the level of UCH-L1 is less than about 1000 pg / mL;

[0060] (v) a trauma mechanism is present and the level of GFAP is less than about 120 pg / mL, the level of UCH-L1 is less than about 500 pg / mL, or the level of GFAP is less than about 120 pg / mL and the level of UCH-L1 is less than about 500 pg / mL; orDocket No. 15893; 43644.601

[0061] (vi) a trauma mechanism is present and the level of GFAP is less than about 120 pg / mL, the level of UCH-L1 is less than about 1000 pg / mL, or the level of GFAP is less than about 120 pg / mL and the level of UCH-L1 is less than about 1000 pg / mL.

[0062] In still further aspects of the above method, the subject is diagnosed as not having or likely not to have a TBI when:

[0063] (i) a trauma mechanism is present; and

[0064] (ii) the level of GFAP is less than between about 80 pg / mL to about 120 pg / mL, the level of UCH-L1 is less than between about 500 pg / mL to about 1000 pg / mL, or level of GFAP is less than between about 80 pg / mL to about 120 pg / mL and the level of UCH-L1 is less than between about 500 pg / mL to about 1000 pg / mL.

[0065] In still yet further aspects of the above method, the diagnostic criteria further comprises:

[0066] (iii) the presence or absence of one or more clinical signs of a TBI.

[0067] In still yet further aspects of the above method, the subject is diagnosed as having a TBI or more likely than not to have a TBI when:

[0068] (i) a trauma mechanism is present, the level of GFAP is greater than about 80 pg / mL and / or the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is absent;

[0069] (ii) a trauma mechanism is present, the level of GFAP is greater than about 80 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is absent;

[0070] (iii) a trauma mechanism is present, the level of GFAP is greater than about 100 pg / mL and / or the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is absent;

[0071] (iv) a trauma mechanism is present, the level of GFAP is greater than about 100 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is absent;

[0072] (v) a trauma mechanism is present and the level of GFAP is greater than about 120 pg / mL and the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is absent; or

[0073] (vi) a trauma mechanism is present and the level of GFAP is greater than about 120 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is absent.Docket No. 15893; 43644.601

[0074] In yet still further aspects of the above method, the subject is diagnosed as having a TBI or more likely than not to have a TBI when:

[0075] (i) a trauma mechanism is present, the level of GFAP is greater than about 80 pg / mL and / or the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is present;

[0076] (ii) a trauma mechanism is present, the level of GFAP is greater than about 80 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is present;

[0077] (iii) a trauma mechanism is present, the level of GFAP is greater than about 100 pg / mL and / or the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is present;

[0078] (iv) a trauma mechanism is present, the level of GFAP is greater than about 100 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is present;

[0079] (v) a trauma mechanism is present and the level of GFAP is greater than about 120 pg / mL and / or the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is present;

[0080] (vi) a trauma mechanism is present and the level of GFAP is greater than about 100 pg / mL and / or the level of UCH-L1 is greater than about 700 pg / mL, and the one or more clinical signs of a TBI is present; or

[0081] (vii) a trauma mechanism is present and the level of GFAP is greater than about 120 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is present.

[0082] In still further aspects of the above method, the subject is diagnosed as having a TBI or more likely than not to have a TBI when:

[0083] (i) a trauma mechanism is present;

[0084] (ii) the level of GFAP is greater than between about 80 pg / mL to about 120 pg / mL and / or the level of UCH-L1 is greater than between about 500 pg / mL to about 1000 pg / mL; and

[0085] (iii) the one or more clinical signs of a TBI is absent.

[0086] In yet still further aspects of the above method, the subject is diagnosed as having a TBI or more likely than not to have a TBI when:

[0087] (i) a trauma mechanism is present;Docket No. 15893; 43644.601

[0088] (ii) the level of GFAP is greater than between about 80 pg / mL to about 120 pg / mL and / or the level of UCH-L1 is greater than between about 500 pg / mL to about 1000 pg / mL; and

[0089] (iii) the one or more clinical signs of a TBI is present.

[0090] In still yet further aspects of the above method, the diagnostic criteria further comprises:

[0091] (iv) the presence or absence of one or more confounding factors.

[0092] In still yet further aspects of the above method, the subject is diagnosed as not having or likely not to have a TBI when:

[0093] (i) a trauma mechanism is present;

[0094] (ii) one more clinical signs of a TBI is present; and

[0095] (iv) the one or more clinical signs of TBI is better accounted for by the presence of the one or more confounding factors.

[0096] In still yet a further aspect of the above method, the trauma mechanism is selected from the group consisting of an injury involving:

[0097] (i) impact of an object on the subject’s head;

[0098] (ii) the subject’s head striking a hard object or surface;

[0099] (iii) acceleration or deceleration of the subject’s head;

[0100] (iv) a force generated from a blast or explosion; and

[0101] (v) combinations of (i)-(iv).

[0102] In still yet a further aspect of the above method, the neuroimaging is selected from the group consisting of a computed tomography (CT) scan of the subject’s head and a magnetic resonance imaging of the subject’s head.

[0103] In still yet a further aspect of the above method, the one or more clinical signs is selected from the group consisting of:

[0104] (i) loss of consciousness;

[0105] (ii) post-traumatic or peri-traumatic amnesia;

[0106] (iii) alteration of mental status; and

[0107] (iv) combinations of (i)-(iii).

[0108] In still yet another aspect of the above method, executing the TBI adjudication algorithm further comprises compiling an injury severity indicator based on the one or more clinical signs, wherein the one or more clinical signs are selected from the group consisting of:

[0109] (i) a duration of the subject’s loss of consciousness;Docket No. 15893; 43644.601

[0110] (ii) a duration of the subject’s post-traumatic or peri-traumatic amnesia; and

[0111] (iii) a Glasgow Coma Scale (GCS) score, a Ranchos Los Amigos Scale score, and / or a Rivermead Post-Concussion Symptoms Questionnaire score of the subject after a duration of time.

[0112] In still yet a further aspect of the above method, the one or more confounding factors is selected from the group consisting of:

[0113] (i) acute musculoskeletal pain;

[0114] (ii) psychological stress;

[0115] (iii) use of a drug of abuse;

[0116] (iv) pulmonary / circulatory disruption;

[0117] (v) syncopy prior to a fall; and

[0118] (vi) combinations of (i)-(vi).

[0119] In still yet a further aspect of the above method, the diagnostic criteria further comprises the presence of an acute symptom selected from the group consisting of:

[0120] (i) a subjective alteration in mental status selected from the group consisting of feeling confused, feeling disoriented, feeling dazed, and combinations thereof;

[0121] (ii) a physical symptom selected from the group consisting of a headache, nausea, dizziness, balance problems, vision problems, light sensitivity, noise sensitivity, and combinations thereof;

[0122] (iii) a cognitive symptom selected from the group consisting of feeling slowed down, mental fog, difficulty concentrating, memory problems, and combinations thereof; and

[0123] (iv) an emotional symptom selected from the group consisting of unusual emotional lability, emotional irritability, and combinations thereof.

[0124] In still yet a further aspect of the above method, the diagnostic criteria further comprises the presence of an impairment on acute clinical examination or the presence of an elevated biomarker indicative of an intracranial injury.

[0125] In still yet a further aspect of the above method, the impairment on acute clinical examination is selected from the group consisting of:

[0126] (i) a cognitive impairment on acute clinical examination;

[0127] (ii) a balance impairment on acute clinical examination; and

[0128] (iii) an oculomotor impairment or symptom provocation in response to vestibular- oculomotor challenge on acute clinical examination.

[0129] In still yet a further aspect of the above method, the elevated biomarker indicative of an intracranial injury is selected from the group consisting of APOA1, ADAMI 0, brainDocket No. 15893; 43644.601 derived nerve growth factor (BDNF), calcium binding protein (SI 00b), C-reactive protein (CRP), glial fibrillary acidic protein (GFAP), glial fibrillary acidic protein breakdown products (GFAP-BDP), neuron specific enolase (NSE), NF-L, peroxi dredoxin 6 (PRDX6), Tau, p-Tau, ubiquitin carboxy -terminal hydrolase LI (UCH-L1), and combinations thereof.

[0130] In still yet a further aspect of the above method, executing the TBI adjudication algorithm further comprises compiling the presence of the acute symptom, the presence of the impairment on acute clinical examination, and the presence of the elevated biomarker indicative of an intracranial injury.

[0131] In still yet another aspect of the above method, a subject is diagnosed as having a TBI when:

[0132] (i) a mechanism of trauma is present;

[0133] (ii) at least two acute symptoms are present;

[0134] (iii) either at least one impairment on acute clinical examination is present or at least one elevated biomarker indicative of an intracranial injury is present; and

[0135] (iv) the at least two acute symptoms and either the at least one impairment on acute clinical examination or at least one elevated biomarker indicative of the intracranial injury are not better accounted for by the one or more confounding factors.

[0136] In still yet another aspect of the above method the diagnosis is a mild TBI.

[0137] In still yet another aspect of the above method, a moderate, moderate-to-severe, or severe TBI is diagnosed when:

[0138] (i) a duration of the subject’s loss of consciousness exceeds 30 minutes;

[0139] (ii) a duration of the subject’s post-traumatic or peri -traumatic amnesia exceeds 24 hours; or

[0140] (iii) a GCS score of the subject after 30 minutes is less than 13.

[0141] In yet another aspect, the above method is a computer-implemented method.

[0142] In still yet another aspect of the above method, the diagnostic criteria is received from a database comprising electronic medical records of the subject.

[0143] In still yet another aspect, the above method further comprises storing the adjudicated TBI diagnosis in a database comprising an electronic medical record of the subject.

[0144] In still yet another aspect, the above method further comprises recommending a treatment based on the adjudicated TBI diagnosis. In still yet another aspect, the treatment comprises a prescription for a medication. In still yet a further aspects, the prescription is automatically sent to a preferred pharmacy of the subject.Docket No. 15893; 43644.601BRIEF DESCRIPTION OF THE DRAWINGS

[0145] FIG. 1 is a flow diagram illustrating an exemplary method for adjudicating a TBI diagnosis of a subject having or suspected of having a head injury.DETAILED DESCRIPTION

[0146] The present disclosure relates to methods diagnosing or aiding in the diagnosis of a traumatic brain injury (TBI) or adjudicating a TBI diagnosis of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head. The methods involve (a) receiving diagnostic criteria of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head; (b) executing a TBI adjudication algorithm by compiling the diagnostic criteria; and (c) adjudicating a TBI diagnosis based on the diagnostic criteria compiled in step (b). The diagnostic criteria includes, inter alia, the presence or absence of (i) a trauma mechanism; (ii) a neuroimaging intracranial abnormality; (iii) one or more clinical signs indicative of TBI; (iv) one or more confounding factors; and (v) a level of glial fibrillary acidic protein (GFAP) and / or a level of ubiquitin carboxyterminal hydrolase LI (UCH-L1) measured in a sample obtained from the subject. The present disclosure also relates to systems and kits for implementing the methods.

[0147] The methods of the disclosure provide a TBI adjudication algorithm that complies diagnostic criteria useful for adjudicating a consistent and reliable TBI diagnosis regardless of the care setting, such as, for example, sports, and in trauma in civilian and military settings. The TBI adjudication algorithm utilizes an evidence-based approach to improve the quality and consistency of TBI (e.g., mild TBI) research and clinical care.

[0148] The methods of the disclosure further provide diagnostic criteria useful for making consistent and reliable diagnosis of a TBI or likelihood of a TBI, regardless of the care setting, such as, for example, in sports, and in trauma in civilian and military settings. The methods employ an evidence-based approach to improve the quality and consistency of TBI (e.g., mild TBI) research and clinical care.

[0149] An exemplary embodiment of the method includes the steps of (a) receiving diagnostic criteria of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head, wherein the diagnostic criteria comprises: (i) the presence or absence of a trauma mechanism; (ii) the presence or absence of a neuroimaging intracranial abnormality; (iii) the presence or absence of one or more clinical signs of a TBI; and (iv) the presence or absence of one or more confounding factors; (b) executing a TBI adjudicationDocket No. 15893; 43644.601 algorithm by compiling the diagnostic criteria; and (c) adjudicating a TBI diagnosis based on the diagnostic criteria compiled in step (b), wherein a subject is diagnosed as having a TBI when: (i) a trauma mechanism and a neuroimaging intracranial abnormality are present; or (ii) a trauma mechanism is present, a neuroimaging intracranial abnormality is absent, one or more clinical signs indicative of TBI is present, and one or more confounding factors is absent.

[0150] In further embodiments, the method comprises (a) receiving diagnostic criteria of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head, wherein the diagnostic criteria comprises determining: (i) the presence or absence of a trauma mechanism; and (ii) a level of glial fibrillary acidic protein (GFAP) and / or a level of ubiquitin carboxy -terminal hydrolase LI (UCH-L1) measured in a sample obtained from the subject; and (b) diagnosing the subject as having a TBI or aiding in the diagnosis that the subject is more likely than not to have a TBI based when: (i) a trauma mechanism is present; and (ii) the level of GFAP is greater than or equal to about 80 pg / mL, the level of UCH-L1 is greater than or equal to about 500 pg / mL, or the level of GFAP is greater than or equal to about 80 pg / mL and the level of UCH-L1 is greater than or equal to about 500 pg / mL.

[0151] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.1. Definitions

[0152] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. 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 not intended to be limiting.

[0153] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’Docket No. 15893; 43644.601 and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0154] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0155] “Acquired brain injury” or (AB I) as used herein refers to damage to the brain that is caused by events occurring after birth. In other words, acquired brain injuries are not genetic or congenital but are the result neurological conditions and injuries. Acquired brain injuries are often divided into two categories. The first category is acquired traumatic brain injuries (TBI), that occur due to an external force, such as, for example, a sports injury, fall, physical shaking, blunt force trauma, explosion, blast, or exposure to a fire. The second category is non-traumatic acquired brain injuries that in some cases are caused by internal factors and include stroke, tumors, anoxia, infections, metabolic disorders, and others. As used herein, in one embodiment, an acquired brain injury does not include or encompass damage to the brain that is caused by a stroke (including, such as, for example, ischemic stroke, hemorrhagic stroke, or a transient ischemic attack, etc.).

[0156] “Adjudicate” or “adjudicating” refers to making a preliminary or early diagnosis, determination, or decision that a subject has a TBI or does not have a TBI based on certain diagnostic criteria, with the diagnosis, determination, or decision to be confirmed at a future date, e.g., as biomarker cutoffs and / or other means of determination are validated (e.g., eye tracking). In some aspects, the term “adjudicating” refers to stratifying a subject as potentially having a TBI or not having a TBI.

[0157] “Affinity matured antibody” is used herein to refer to an antibody with one or more alterations in one or more CDRs, which result in an improvement in the affinity (i.e., KD, ka or ka) of the antibody for a target antigen compared to a parent antibody, which does not possess the alteration(s). Exemplary affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. A variety of procedures for producing affinity matured antibodies is known in the art, including the screening of a combinatory antibody library that has been prepared using bio-display. For example, Marks et al., BioTechnology, 10: 779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described by Barbas et al., Proc. Nat.Acad. Sci. USA, 91 : 3809-3813 (1994); Schier c / a / ., Gene, 169: 147-155 (1995); Yelton < / al., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol., 154(7): 3310-3319Docket No. 15893; 43644.601(1995); and Hawkins et al, J. Mol. Biol., 226: 889-896 (1992). Selective mutation at selective mutagenesis positions and at contact or hypermutation positions with an activityenhancing amino acid residue is described in U.S. Patent No. 6,914,128 Bl.

[0158] “Antibody” and “antibodies” as used herein refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs (“sdFv”), and anti -idiotypic (“anti-Id”) antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dualvariable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology, 25(11): 1290-1297 (2007) and PCT International Application WO 2001 / 058956, the contents of each of which are herein incorporated by reference), and functionally active epitope-binding fragments of any of the above. Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. For simplicity sake, an antibody against an analyte is frequently referred to herein as being either an “anti-analyte antibody” or merely an “analyte antibody” (e.g., an anti-UCH-Ll antibody or a UCH-L1 antibody).

[0159] “Antibody fragment” as used herein refers to a portion of an intact antibody comprising the antigen-binding site or variable region. The portion does not include the constant heavy chain domains (i.e., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.Docket No. 15893; 43644.601

[0160] The “area under curve” or “AUC” refers to area under a ROC curve. AUC under a ROC curve is a measure of accuracy. An AUC of 1 represents a perfect test, whereas an AUC of 0.5 represents an insignificant test. A preferred AUC may be at least approximately 0.700, at least approximately 0.750, at least approximately 0.800, at least approximately 0.850, at least approximately 0.900, at least approximately 0.910, at least approximately 0.920, at least approximately 0.930, at least approximately 0.940, at least approximately 0.950, at least approximately 0.960, at least approximately 0.970, at least approximately 0.980, at least approximately 0.990, or at least approximately 0.995.

[0161] “Bead” and “particle” are used herein interchangeably and refer to a substantially spherical solid support. One example of a bead or particle is a microparticle. Microparticles that can be used herein can be any type known in the art. For example, the bead or particle can be a magnetic bead or magnetic particle. Magnetic beads / particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic or ferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO2, MnAs, MnBi, EuO, and NiO / Fe. Examples of ferrimagnetic materials include NiFe2O4, CoFe2O4, FesO4 (or FeOEe2O3). Beads can have a solid core portion that is magnetic and is surrounded by one or more non-magnetic layers. Alternately, the magnetic portion can be a layer around a non-magnetic core. The microparticles can be of any size that would work in the methods described herein, e.g., from about 0.75 to about 5 nm, or from about 1 to about 5 nm, or from about 1 to about 3 nm.

[0162] “Binding protein” is used herein 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 chemical compound or other molecule, or a substrate of any kind. A binding protein specifically binds a binding partner. Binding proteins include antibodies, as well as antigen-binding fragments thereof and other various forms and derivatives thereof as are known in the art and described herein below, and other molecules comprising one or more antigen-binding domains that bind to an antigen molecule or a particular site (epitope) on the antigen molecule. Accordingly, a binding protein includes, but is not limited to, an antibody a tetrameric immunoglobulin, an IgG molecule, an IgGl molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, an affinity matured antibody, and fragments of any such antibodies that retain the ability to bind to an antigen.

[0163] “Bispecific antibody” is used herein to refer to a full-length antibody that is generated by quadroma technology (see Milstein et al., Nature, 305(5934): 537-540 (1983)), by chemical conjugation of two different monoclonal antibodies (see, Staerz et al., Nature, 314(6012): 628-631 (1985)), or by knob-into-hole or similar approaches, which introduceDocket No. 15893; 43644.601 mutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14): 6444-6448 (1993)), resulting in multiple different immunoglobulin species of which only one is the functional bispecific antibody. A bispecific antibody binds one antigen (or epitope) on one of its two binding arms (one pair of HC / LC), and binds a different antigen (or epitope) on its second arm (a different pair of HC / LC). By this definition, a bispecific antibody has two distinct antigen-binding arms (in both specificity and CDR sequences) and is monovalent for each antigen to which it binds to.[0164J “CDR” is used herein to refer to the “complementarity determining region” within an antibody variable sequence. There are three CDRs in each of the variable regions of the heavy chain and the light chain. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted "CDR1", "CDR2", and "CDR3", for each of the variable regions. The term "CDR set" as used herein refers to a group of three CDRs that occur in a single variable region that binds the antigen. An antigen-binding site, therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain variable region. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2, or CDR3) may be referred to as a “molecular recognition unit.” Crystallographic analyses of antigen-antibody complexes have demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units may be primarily responsible for the specificity of an antigenbinding site. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.[0165J 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 applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as "Kabat CDRs". Chothia and coworkers (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987); and Chothia et al., Nature, 342: 877-883 (1989)) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These subportions were designated as "LI", "L2", and "L3", or "Hl", "H2", and "H3", where the "L" and the "H" designate the light chain and the heavy chain regions, respectively. These regions may be referred to as "Chothia CDRs", which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have beenDocket No. 15893; 43644.601 described by Padlan, FASEB J., 9: 133-139 (1995), and MacCallum, J. Mol. Biol., 262(5): 732-745 (1996). Still other CDR boundary definitions may not strictly follow one of the herein systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat- or Chothia-defined CDRs.[0166J A “clinically-relevant time frame” refers to a time frame (e.g., seconds, minutes, or hours) during which a careful and prudent medical practitioner (e.g., doctor, nurse, paramedic, or other) would reasonably consider the results of one or more biomarker tests to have bearing on an imaging procedure, such as a head CT scan, or pursuant to guidelines established by an overseeing entity (e.g., a standards-setting body such as the World Health Organization (WHO), physicians review board, regulatory approval authority such as FDA, EMEA or other, etc.).

[0167] “Compiling” refers to a process in which a subject’s diagnostic criteria is organized to facilitate the adjudication of a TBI diagnosis for the subject. In some aspects, the diagnostic criteria can be compiled electronically. In other aspects, the diagnostic criteria can be compiled manually.

[0168] “Component,” “components,” or “at least one component,” refer generally to a capture antibody, a detection or conjugate a calibrator, a control, a sensitivity panel, a container, a buffer, a diluent, a salt, an enzyme, a co-factor for an enzyme, a detection reagent, a pretreatment reagent / solution, a substrate (e.g., as a solution), a stop solution, and the like that can be included in a kit for assay of a test sample, such as a patient urine, whole blood, serum or plasma sample, in accordance with the methods described herein and other methods known in the art. Some components can be in solution or lyophilized for reconstitution for use in an assay.

[0169] “ Correlated to” as used herein refers to compared to.

[0170] “ CT scan” as used herein refers to a computerized tomography (CT) scan. A CT scan combines a series of X-ray images taken from different angles and uses computer processing to create cross-sectional images, or slices, of the bones, blood vessels and soft tissues inside your body. The CT scan may use X-ray CT, positron emission tomography (PET), single-photon emission computed tomography (SPECT), computed axial tomography (CAT scan), or computer aided tomography. The CT scan may be a conventional CT scan or a spiral / helical CT scan. In a conventional CT scan, the scan is taken slice by slice and afterDocket No. 15893; 43644.601 each slice the scan stops and moves down to the next slice, e.g., from the top of the abdomen down to the pelvis. The conventional CT scan requires patients to hold their breath to avoid movement artefact. The spiral / helical CT scan is a continuous scan which is taken in a spiral fashion and is a much quicker process where the scanned images are contiguous.

[0171] A head CT scan is “negative” for a TBI when no intracranial lesion(s) is observed in an image taken from a subject that has sustained, may have sustained or is suspected of sustaining an injury to the head. To further clarify, the head CT scan of a subject is “negative” for a TBI when a lesion is not found or identified; however, in some aspects, the subject may still be experiencing symptoms (e.g., of TBI) even though the head CT is negative. Most subjects will be negative for a TBI on head CT given that not all injuries or lesions can be visualized by head CT. Consequently, the methods and assays described herein can be used to provide an assessment or determination of a subject with a negative head CT that may still have a TBI.

[0172] “Determined by an assay” is used herein to refer to the determination of a reference level by any appropriate assay. The determination of a reference level may, in some embodiments, be achieved by an assay of the same type as the assay that is to be applied to the sample from the subject (for example, by an immunoassay, clinical chemistry assay, a single molecule detection assay, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, or protein immunostaining, electrophoresis analysis, a protein assay, a competitive binding assay, a functional protein assay, or chromatography or spectrometry methods, such as high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS)). The determination of a reference level may, in some embodiments, be achieved by an assay of the same type and under the same assay conditions as the assay that is to be applied to the sample from the subject. As noted herein, this disclosure provides exemplary reference levels (e.g., calculated by comparing reference levels at different time points). It is well within the ordinary skill of one in the art to adapt the disclosure herein for other assays to obtain assayspecific reference levels for those other assays based on the description provided by this disclosure. For example, a set of training samples comprising samples obtained from human subjects known to have sustained an injury to the head (and more particularly, samples obtained from human subjects known to have sustained a (i) mild TBI; and / or (ii) moderate, severe, or moderate to severe TBI and samples obtained from human subjects known not to have sustained an injury to the head may be used to obtain assay-specific reference levels. It will be understood that a reference level “determined by an assay” and having a recited levelDocket No. 15893; 43644.601 of “sensitivity” and / or “specificity” is used herein to refer to a reference level which has been determined to provide a method of the recited sensitivity and / or specificity when said reference level is adopted in the methods of the invention. It is well within the ordinary skill of one in the art to determine the sensitivity and specificity associated with a given reference level in the methods of the invention, for example by repeated statistical analysis of assay data using a plurality of different possible reference levels.

[0173] Practically, when discriminating between a subject as having a traumatic brain injury or not having a traumatic brain injury or a subject as having a mild versus a moderate, severe, or moderate to severe traumatic brain injury, the skilled person will balance the effect of raising a cutoff on sensitivity and specificity. Raising or lowering a cutoff will have a well- defined and predictable impact on sensitivity and specificity, and other standard statistical measures. It is well known that raising a cutoff will improve specificity but is likely to worsen sensitivity (proportion of those with disease who test positive). In contrast, lowering a cutoff will improve sensitivity but will worsen specificity (proportion of those without disease who test negative). The ramifications for detecting traumatic brain injury or determining a mild versus moderate, severe, or moderate to severe traumatic brain injury will be readily apparent to those skilled in the art. In discriminating whether a subject has or does not have a traumatic brain injury or a mild versus a moderate, severe, or moderate to severe traumatic brain injury, the higher the cutoff, specificity improves as more true negatives (i.e., subjects not having a traumatic brain injury, not having a mild traumatic brain injury, not have a moderate traumatic brain injury, not having a severe traumatic brain injury or not having a moderate to severe traumatic brain injury) are distinguished from those having a traumatic brain injury, a mild traumatic brain injury, a moderate traumatic brain injury, a severe traumatic brain injury or a moderate to severe traumatic brain injury. But at the same time, raising the cutoff decreases the number of cases identified as positive overall, as well as the number of true positives, so the sensitivity must decrease. Conversely, the lower the cutoff, sensitivity improves as more true positives (i.e., subjects having a traumatic brain injury, having a mild traumatic brain injury, having a moderate traumatic brain injury, having a severe traumatic brain injury or having a moderate to severe traumatic brain injury) are distinguished from those who do not have a traumatic brain injury, a mild traumatic brain injury, a moderate traumatic brain injury, a severe traumatic brain injury or a moderate to severe traumatic brain injury. But at the same time, lowering the cutoff increases the number of cases identified as positive overall, as well as the number of false positives, so the specificity must decrease.Docket No. 15893; 43644.601

[0174] Generally, a high sensitivity value helps one of skill rule out disease or condition (such as a traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury or moderate to severe traumatic brain injury), and a high specificity value helps one of skill rule in disease or condition. Whether one of skill desires to rule out or rule in disease depends on what the consequences are for the patient for each type of error. Accordingly, one cannot know or predict the precise balancing employed to derive a test cutoff without full disclosure of the underlying information on how the value was selected. The balancing of sensitivity against specificity and other factors will differ on a case-by-case basis. This is why it is sometimes preferable to provide alternate cutoff (e.g., reference) values so a physician or practitioner can choose.

[0175] “Derivative” of an antibody as used herein may refer to an antibody having one or more modifications to its amino acid sequence when compared to a genuine or parent antibody and exhibit a modified domain structure. The derivative may still be able to adopt the typical domain configuration found in native antibodies, as well as an amino acid sequence, which is able to bind to targets (antigens) with specificity. Typical examples of antibody derivatives are antibodies coupled to other polypeptides, rearranged antibody domains, or fragments of antibodies. The derivative may also comprise at least one further compound, e.g., a protein domain, said protein domain being linked by covalent or non- covalent bonds. The linkage can be based on genetic fusion according to the methods known in the art. The additional domain present in the fusion protein comprising the antibody may preferably be linked by a flexible linker, advantageously a peptide linker, wherein said peptide linker comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of the further protein domain and the N-terminal end of the antibody or vice versa. The antibody may be linked to an effector molecule having a conformation suitable for biological activity or selective binding to a solid support, a biologically active substance (e.g., a cytokine or growth hormone), a chemical agent, a peptide, a protein, or a drug, for example.

[0176] “Drug of abuse” is used herein to refer to one or more additive substances (such as a drug) taken for non-medical reasons (such as for, example, recreational and / or mind-altering effects). Excessive overindulgence, use or dependence of such drugs of abuse is often referred to as “substance abuse”. Examples of drugs of abuse include alcohol, barbiturates, benzodiazepines, cannabis, cocaine, hallucinogens (such as ketamine, mescaline (peyote), PCP, psilocybin, DMT and / or LSD), methaqualone, opioids, amphetamines (including methamphetamines), anabolic steroids, inhalants (namely, substances which contain volatileDocket No. 15893; 43644.601 substances that contain psychoactive properties such as, for example, nitrites, spray paints, cleaning fluids, markers, glues, etc.) and combinations thereof.

[0177] “Dual-specific antibody” is used herein to refer to a full-length antibody that can bind two different antigens (or epitopes) in each of its two binding arms (a pair of HC / LC) (see PCT publication WO 02 / 02773). Accordingly, a dual-specific 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.

[0178] “Dual variable domain” is used herein to refer to two or more antigen binding sites on a binding protein, which may be divalent (two antigen binding sites), tetravalent (four antigen binding sites), or multivalent binding proteins. DVDs may be monospecific, z.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 a DVD-Ig binding protein is thus tetrameric and reminiscent of an IgG molecule but provides more antigen binding sites than an IgG molecule. Thus, each half of a tetrameric DVD-Ig molecule is reminiscent of one half of an IgG molecule and comprises a heavy chain DVD polypeptide and a light chain DVD polypeptide, but unlike a pair of heavy and light chains of an IgG molecule that provides a single antigen binding domain, a pair of heavy and light chains of a DVD-Ig provide two or more antigen binding sites.

[0179] Each antigen binding site of a DVD-Ig binding protein may be derived from a donor ("parental") monoclonal antibody and thus comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) with a total of six CDRs involved in antigen binding per antigen binding site. Accordingly, a DVD-Ig binding protein that binds two different epitopes i.e., two different epitopes of two different antigen molecules or two different epitopes of the same antigen molecule) comprises an antigen binding site derived from a first parental monoclonal antibody and an antigen binding site of a second parental monoclonal antibody.

[0180] A description of the design, expression, and characterization of DVD-Ig binding molecules is provided in PCT Publication No. WO 2007 / 024715, U.S. Patent No. 7,612,181, and Wu et al., Nature Biotech., 25: 1290-1297 (2007). A preferred example of such DVD-Ig molecules comprises a heavy chain that comprises the structural formula VDl-(Xl)n-VD2-C- (X2)n, wherein VD1 is a first heavy chain variable domain, VD2 is a second heavy chainDocket No. 15893; 43644.601 variable domain, C is a heavy chain constant domain, XI is a linker with the proviso that it is not CHI, X2 is an Fc region, and n is 0 or 1, but preferably 1; and a light chain that comprises the structural formula VDl-(Xl)n-VD2-C-(X2)n, wherein VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, XI is a linker with the proviso that it is not CHI, and X2 does not comprise an Fc region; and n is 0 or 1, but preferably 1. Such a DVD-Ig may comprise two such heavy chains and two such light chains, wherein each chain comprises variable domains linked in tandem without an intervening constant region between variable regions, wherein a heavy chain and a light chain associate to form tandem functional antigen binding sites, and a pair of heavy and light chains may 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 may comprise heavy and light chains that each comprise three variable domains (VD1, VD2, VD3) linked in tandem without an intervening constant region between variable domains, wherein a pair of heavy and light chains may associate to form three antigen binding sites, and wherein a pair of heavy and light chains may associate with another pair of heavy and light chains to form a tetrameric binding protein with six antigen binding sites.

[0181] In a preferred embodiment, a DVD-Ig binding protein not only binds the same target molecules bound by its parental monoclonal antibodies, but also possesses one or more desirable properties of one or more of its parental monoclonal antibodies. Preferably, such an additional property is an antibody parameter of one or more of the parental monoclonal antibodies. Antibody parameters that may be contributed to a DVD-Ig binding protein from one or more of its parental monoclonal antibodies include, but are not limited to, antigen specificity, antigen affinity, potency, biological function, epitope recognition, protein stability, protein solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross reactivity, and orthologous antigen binding.

[0182] A DVD-Ig binding protein binds at least one epitope of UCH-L1. Non -limiting examples of a DVD-Ig binding protein include a DVD-Ig binding protein that binds one or more epitopes of UCH-L1, a DVD-Ig binding protein that binds an epitope of a human UCH- L1 and an epitope of UCH-L1 of another species (for example, mouse), and a DVD-Ig binding protein that binds an epitope of a human UCH-L1 and an epitope of another target molecule.

[0183] Dynamic range” as used herein refers to range over which an assay readout is proportional to the amount of target molecule or analyte in the sample being analyzed.Docket No. 15893; 43644.601

[0184] “Epitope,” or “epitopes,” or “epitopes of interest” refer to a site(s) on any molecule that is recognized and can bind to a complementary site(s) 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, glycolipids, glycoproteins or lipopolysaccharides), or a polysaccharide. Its specific binding partner can be, but is not limited to, an antibody.

[0185] “Fragment antigen-binding fragment” or “Fab fragment” as used herein refers to a fragment of an antibody that binds to antigens and that contains one antigen-binding site, one complete light chain, and part of one heavy chain. Fab is a monovalent fragment consisting of the VL, VH, CL and CHI domains. Fab is composed of one constant and one variable domain of each of the heavy and the light chain. The variable domain contains the paratope (the antigen-binding site), comprising a set of complementarity determining regions, at the amino terminal end of the monomer. Each arm of the Y thus binds an epitope on the antigen. Fab fragments can be generated such as has been described in the art, e.g., using the enzyme papain, which can be used to cleave an immunoglobulin monomer into two Fab fragments and an Fc fragment, or can be produced by recombinant means.

[0186] “F(ab')2 fragment” as used herein refers to antibodies generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving intact some of the hinge region. F(ab')2 fragments have two antigen-binding F(ab) portions linked together by disulfide bonds, and therefore are divalent with a molecular weight of about 110 kDa. Divalent antibody fragments (F(ab')2 fragments) are smaller than whole IgG molecules and enable a better penetration into tissue thus facilitating better antigen recognition in immunohistochemistry. The use of F(ab')2 fragments also avoids unspecific binding to Fc receptor on live cells or to Protein A / G. F(ab')2 fragments can both bind and precipitate antigens.

[0187] “Framework” (FR) or “Framework sequence” as used herein may mean the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems (for example, see above), the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3, and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1, FR2, FR3, or FR4, a framework region, as referred byDocket No. 15893; 43644.601 others, represents the combined FRs within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four subregions, and FRs represents two or more of the four sub-regions constituting a framework region.

[0188] Human heavy chain and light chain FR sequences are known in the art that can be used as heavy chain and light chain "acceptor" framework sequences (or simply, "acceptor" sequences) to humanize a non-human antibody using techniques known in the art. In one embodiment, human heavy chain and light chain acceptor sequences are selected from the framework sequences listed in publicly available databases such as V-base (hypertext transfer protocol: / / vbase. mrc-cpe.cam.ac.uk / ) or in the international ImMunoGeneTics® (IMGT®) information system (hypertext transfer protocol: / / imgt.cines.fr / texts / IMGTrepertoire / LocusGenes / ).

[0189] “Functional antigen binding site” as used herein may mean a site on a binding protein (e.g., an antibody) that is capable of binding a target antigen. The antigen binding affinity of the antigen binding site may not be as strong as the parent binding protein, e.g., parent antibody, from which the antigen binding site is derived, but the ability to bind antigen must be measurable using any one of a variety of methods known for evaluating protein, e.g., antibody, binding to an antigen. Moreover, the antigen binding affinity of each of the antigen binding sites of a multivalent protein, e.g., multivalent antibody, herein need not be quantitatively the same.

[0190] “ GFAP” is used herein to describe glial fibrillary acidic protein. GFAP is a protein that is encoded by the GFAP gene in humans, and which can be produced (e.g., by recombinant means, in other species).

[0191] “ GFAP status” can mean either the level or amount of GFAP at a point in time (such as with a single measure of GFAP), the level or amount of GFAP associated with monitoring (such as with a repeat test on a subject to identify an increase or decrease in GFAP amount), the level or amount of GFAP associated with treatment for traumatic brain injury (whether a primary brain injury and / or a secondary brain injury) or combinations thereof.“Glasgow Coma Scale” or “GCS” as used herein refers to a 15-point scale (e.g., described in 1974 by Graham Teasdale and Bryan Jennett, Lancet 1974; 2:81-4) that provides a practical method for assessing impairment of conscious level in patients who have suffered a brain injury. The test measures the best motor response, verbal response and eye opening response with these values: I. Best Motor Response (6 - obey 2-part request; 5 - brings hand above clavicle to stimulus on head neck; 4 - bends arm at elbow rapidly but features notDocket No. 15893; 43644.601 predominantly abnormal; 3 - bends arm at elbow, features clearly predominantly abnormal; 2 - extends arm at elbow; 1- no movement in arms / legs, no interfering factor; NT - paralyzed or other limiting factor); II. Verbal Response (5 - correctly gives name, place and date; 4 - not orientated but communication coherently; 3 - intelligible single words; 2 - only moans / groans; 1- no audible response, no interfering factor; NT - factor interfering with communication); and III. Eye Opening (4 - open before stimulus; 3 - after spoken or shouted request; 2 - after fingertip stimulus; 1 - no opening at any time, no interfering factor; NT - closed by local factor). The final score is determined by adding the values of 1+11+111. A subject is considered to have a mild TBI if the GCS score is 13-15. A subject is considered to have a moderate TBI if the GCS score is 9-12. A subject is considered to have a severe TBI if the GCS score is 8 or less, typically 3-8.

[0192] “Glasgow Outcome Scale” as used herein refers to a global scale for functional outcome that rates patient status into one of five categories: Dead, Vegetative State, Severe Disability, Moderate Disability or Good Recovery. “Extended Glasgow Outcome Scale” or “GOSE” as used interchangeably herein provides more detailed categorization into eight categories by subdividing the categories of severe disability, moderate disability and good recovery into a lower and upper category as shown in Table 1.Table 1Docket No. 15893; 43644.601

[0193] “Health care practitioner” refers to any medical professional that has been trained to observe, determine, and document one or more diagnostic criteria of a subject having or suspected of having a head injury. The health care practitioner may also be trained to adjudicate a TBI diagnosis based on the diagnostic criteria, or to use a system that implements a computer-implemented version of the method to adjudicate a TBI diagnosis of the subject. Examples of such health care practitioners include a paramedic, a nurse (e.g., a triage nurse), a nurse practitioner, a physician’s assistant, a physician, a medical student operating under the supervision of a licensed physician, a specialist, a clinical trial investigator, a principal investigator, etc. Other individuals, such as, for example, a radiologist, a phlebotomist, a laboratory technician may be involved together with a health care practitioner in observing, determining, and / or documenting one or more diagnostic criteria of the subject having or suspected of having a head injury.

[0194] “Humanized antibody” is used herein to describe an antibody that comprises heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and / or VL sequence has been altered to be more “humanlike,” i.e., more similar to human germline variable sequences. A "humanized antibody" is an antibody or a variant, derivative, analog, or fragment thereof, which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having 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 having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and 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 anDocket No. 15893; 43644.601 embodiment, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In some embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and / or humanized heavy chain.

[0195] A humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including without limitation IgGl, IgG2, IgG3, and IgG4. A 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.

[0196] The framework regions and CDRs of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework may be mutagenized by substitution, insertion, and / or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. In a preferred embodiment, such mutations, however, will not be extensive. Usually, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in the consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, 1987)). A "consensus immunoglobulin sequence" may thus comprise a "consensus framework region(s)" and / or a "consensus CDR(s)". In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

[0197] “ Identical” or “identity,” as used herein in the context of two or more polypeptide or polynucleotide sequences, can mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region,Docket No. 15893; 43644.601 determining the number of positions at which the identical residue occurs in both sequences to yield 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 yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of the single sequence are included in the denominator but not the numerator of the calculation.[0198J “Injury to the head” or “head injury” as used interchangeably herein, refers to any injury to the scalp, skull, or brain. Such injuries may include only a minor bump on the skull or may be a serious brain injury. Such injuries include primary injuries to the brain and / or secondary injuries to the brain. Primary brain injuries occur during the initial insult and result from displacement of the physical structures of the brain. More specifically, a primary brain injury is the physical damage to parenchyma (tissue, vessels) that occurs during the traumatic event, resulting in shearing and compression of the surrounding brain tissue. Secondary brain injuries occur subsequent to the primary injury and may involve an array of cellular processes. More specifically, a secondary brain injury refers to the changes that evolve over a period of time (from hours to days) after the primary brain injury. It includes an entire cascade of cellular, chemical, tissue, or blood vessel changes in the brain that contribute to further destruction of brain tissue.

[0199] For TBI, an injury to the head can be either closed or open (penetrating). A closed head injury refers to an injury to the scalp, skull or brain where there is no penetration of the skull resulting from the impact of a striking object. An open head injury refers an injury to the scalp, skull or brain where there is penetration of the skull results from the impact of a striking object. An injury to the head may be caused by physical shaking of a person, by blunt impact by an external mechanical or other force that results in a closed or open head trauma (e.g., vehicle accident such as with an automobile, plane, train, etc.; blow to the head such as with a baseball bat, or from a firearm), a cerebral vascular accident (e.g., stroke), one or more falls (e.g., as in sports or other activities), explosions or blasts (collectively, “blast injuries”) and by other types of blunt force injury. Alternatively, for a non-TBI ABI an injury to the head may be caused by the ingestion and / or exposure to a fire, chemical, toxin or a combination of a chemical and toxin. Examples of such chemicals and / or toxins include molds, asbestos, pesticides and insecticides, organic solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organic metals (such as methyl mercury, tetraethyl lead and organic tin) and / or one or more drugs of abuse. Alternatively, an injury toDocket No. 15893; 43644.601 the head may be caused as a result of a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection (e.g., SARS-CoV-2), a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combinations thereof. In some cases, it is not possible to be certain whether any such event or injury has occurred or taken place. For example, there may be no history on a patient or subject, the subject may be unable to speak, the subject may be aware of what events they were exposed to, etc. Such circumstances are described herein as the subject “may have sustained an injury to the head.” In certain embodiments herein, the closed head injury does not include and specifically excludes a cerebral vascular accident, such as stroke.

[0200] “ Intracranial lesion” as used herein refers to an area of injury within the brain. An intracranial lesion can be an abnormality seen on a CT scan or brain-imaging test, such as magnetic resonance imaging (MRI). On CT or MRI scans, brain lesions can appear as dark or light spots that do not look like normal brain tissue.

[0201] “ Isolated polynucleotide” as used herein may mean a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or a combination thereof) that, by virtue of its origin, the isolated polynucleotide is not associated with all or a portion of a polynucleotide with which the “isolated polynucleotide” is found in nature; is operably linked to a polynucleotide that it is not linked to in nature; or does not occur in nature as part of a larger sequence.

[0202] “Label” and “detectable label” as used herein refer to a moiety attached to an antibody or an analyte to render the reaction between the antibody and the analyte detectable, and the antibody or analyte so labeled is referred to as “detectably labeled.” A label can produce a signal that is detectable by visual or instrumental means. Various labels include signal -producing substances, such as chromagens, fluorescent compounds, chemiluminescent compounds, radioactive compounds, and the like. Representative examples of labels include moi eties that produce light, e.g., acridinium compounds, and moi eties that produce fluorescence, e.g., fluorescein. Other labels are described herein. In this regard, the moiety, itself, may not be detectable but may become detectable upon reaction with yet another moiety. Use of the term “detectably labeled” is intended to encompass such labeling.

[0203] Any suitable detectable label as is known in the art can be used. For example, the detectable label can be a radioactive label (such as 3H, 14C, 32P, 33P, 35S, 90Y, 99Tc, U lin, 1251, 1311, 177Lu, 166Ho, and 153Sm), an enzymatic label (such as horseradish peroxidase, alkaline peroxidase, alkaline phosphatase, glucose 6-phosphate dehydrogenase, and the like), a chemiluminescent label (such as acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, and the like), a fluorescent labelDocket No. 15893; 43644.601(such as fluorescein (e.g., 5 -fluorescein, 6-carboxyfluorescein, 3’6-carboxyfluorescein, 5(6)- carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmium selenide), a thermometric label, or an immunopolymerase chain reaction label. An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997), and in Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996), which is a combined handbook and catalogue published by Molecular Probes, Inc., Eugene, Oregon. A fluorescent label can be used in FPIA (see, e.g., U.S. Patent Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are hereby incorporated by reference in their entireties). An acridinium compound can be used as a detectable label in a homogeneous chemiluminescent assay (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., Biorg. Med. Chem. Lett. 14: 3917-3921 (2004); and Adamczyk et al., Org. Lett. 5: 3779-3782 (2003)).

[0204] In one aspect, the acridinium compound is an acridinium-9-carboxamide. Methods for preparing acridinium 9-carboxamides are described in Mattingly, J. Biolumin. Chemilumin. 6: 107-114 (1991); Adamczyk et al., J. Org. Chem. 63: 5636-5639 (1998); Adamczyk et al., Tetrahedron 55: 10899-10914 (1999); Adamczyk et al., Org. Lett. 1 : 779- 781 (1999); Adamczyk et al., Bioconjugate Chem. 11 : 714-724 (2000); Mattingly et al., In Luminescence Biotechnology: Instruments and Applications,' Dyke, K. V. Ed.; CRC Press: Boca Raton, pp. 77-105 (2002); Adamczyk et al., Org. Lett. 5: 3779-3782 (2003); and U.S. Patent Nos. 5,468,646, 5,543,524 and 5,783,699 (each of which is incorporated herein by reference in its entirety for its teachings regarding same).

[0205] Another example of an acridinium compound is an acridinium-9-carboxylate aryl ester. An example of an 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 esters are described in McCapra et al., Photochem. Photobiol. 4: 1111-21 (1965); Razavi et al., Luminescence 15: 245-249 (2000); Razavi et al., Luminescence 15: 239-244 (2000); and U.S. Patent No. 5,241,070 (each of which is incorporated herein by reference in its entirety for its teachings regarding same). Such acridinium-9-carboxylate aryl esters are efficient chemiluminescent indicators for hydrogen peroxide produced in the oxidation of an analyte by at least one oxidase in terms of the intensity of the signal and / or the rapidity of the signal. The course of theDocket No. 15893; 43644.601 chemiluminescent emission for the acridinium-9-carboxylate aryl ester is completed rapidly, i.e., in under 1 second, while the acridinium-9-carboxamide chemiluminescent emission extends over 2 seconds. Acridinium-9-carboxylate aryl ester, however, loses its chemiluminescent properties in the presence of protein. Therefore, its use requires the absence of protein during signal generation and detection. Methods for separating or removing proteins in the sample are well-known to those skilled in the art and include, but are not limited to, ultrafiltration, extraction, precipitation, dialysis, chromatography, and / or digestion (see, e.g., Wells, High Throughput Bioanalytical Sample Preparation. Methods and Automation Strategies, Elsevier (2003)). The amount of protein removed or separated from the test sample 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 ester and its use are set forth in U.S. Patent App. No. 11 / 697,835, filed April 9, 2007. Acridinium-9-carboxylate aryl esters can be dissolved in any suitable solvent, such as degassed anhydrous N,N-dimethylformamide (DMF) or aqueous sodium cholate.

[0206] In some embodiments, the label is alkaline phosphatase.

[0207] “Linking sequence” or “linking peptide sequence” refers to a natural or artificial polypeptide sequence that is connected to one or more polypeptide sequences of interest (e.g., full-length, 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. Linking sequences can have a length of from about 4 to about 50 amino acids. Preferably, the length of the linking sequence is from about 6 to about 30 amino acids. Natural linking sequences can be modified by amino acid substitutions, additions, or deletions to create artificial linking sequences. Linking sequences can be used for many purposes, including in recombinant Fabs. Exemplary linking sequences include, but are not limited to: (i) Histidine (His) tags, such as a 6X His tag, which has an amino acid sequence of HHHHHH (SEQ ID NO: 3), are useful as linking sequences to facilitate the isolation and purification of polypeptides and antibodies of interest; (ii) Enterokinase cleavage sites, like His tags, are used in the isolation and purification of proteins and antibodies of interest. Often, enterokinase cleavage sites are used together with His tags in the isolation and purification of proteins and antibodies of interest. Various enterokinase cleavage sites are known in the art. Examples of enterokinase cleavage sites include, but are not limited to, the amino acid sequence of DDDDK (SEQ ID NO: 4) and derivatives thereof e.g., ADDDDK (SEQ ID NO: 5), etc.); (iii) Miscellaneous sequences canDocket No. 15893; 43644.601 be used to link or connect the light and / or heavy chain variable regions of single chain variable region fragments. 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 also can be modified for additional functions, such as attachment of drugs or attachment to solid supports. In the context of the present disclosure, the monoclonal antibody, for example, can contain a linking sequence, such as a His tag, an enterokinase cleavage site, or both.[0208J “Magnetic resonance imaging” or “MRI” as used interchangeably herein refers to a medical imaging technique used in radiology to form pictures of the anatomy and the physiological processes of the body in both health and disease (e.g., referred to herein interchangeably as “an MRI”, “an MRI procedure” or “an MRI scan”). MRI is a form of medical imaging that measures the response of the atomic nuclei of body tissues to high- frequency radio waves when placed in a strong magnetic field, and that produces images of the internal organs. MRI scanners, which is based on the science of nuclear magnetic resonance (NMR), use strong magnetic fields, radio waves, and field gradients to generate images of the inside of the body.

[0209] “Monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived 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.

[0210] “Multivalent binding protein” is used herein to refer to a binding protein comprising two or more antigen binding sites (also referred to herein as "antigen binding domains"). A multivalent binding protein is preferably engineered to have three or more antigen binding sites and is generally not a naturally occurring antibody. The term "multispecific binding protein" refers to a binding protein that can bind two or more related or unrelated targets,Docket No. 15893; 43644.601 including a binding protein capable of binding two or more different epitopes of the same target molecule.

[0211] “Negative predictive value” or “NPV” as used interchangeably herein refers to the probability that a subject has a negative outcome given that they have a negative test result.

[0212] “Reference level” as used herein refers to an assay cutoff value that is used to assess diagnostic, prognostic, or therapeutic efficacy and that has been linked or is associated herein with various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression, non-progression, or improvement of disease, etc.). An “absolute amount” as used herein refers to the absolute value of a change or difference between at least two assay results taken or sampled at different time points and, which similar to a reference level, has been linked or is associated herein with various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression, non-progression, or improvement of disease, etc.). “Absolute value” as used herein refers to the magnitude of a real number (such as, for example, the difference between two compared levels (such as levels taken at a first time point and levels taken at a second time point)) without regard to its sign, i.e., regardless of whether it is positive or negative.

[0213] This disclosure provides exemplary reference levels and absolute amounts (e.g., calculated by comparing reference levels at different time points). However, it is well-known that reference levels and absolute amounts may vary depending on the nature of the immunoassay (e.g., antibodies employed, reaction conditions, sample purity, etc.) and that assays can be compared and standardized. It further is well within the ordinary skill of one in the art to adapt the disclosure herein for other immunoassays to obtain immunoassay-specific reference levels and absolute amounts for those other immunoassays based on the description provided by this disclosure. Whereas the precise value of the reference level and absolute amount may vary between assays, the findings as described herein should be generally applicable and capable of being extrapolated to other assays.

[0214] “Point-of-care device” refers to an device or instrument used to provide medical diagnostic testing at or near the point-of-care (namely, outside of a laboratory), at the time and place of patient care (such as in a hospital, physician’s office, urgent or other medical care facility, a patient’s home, a nursing home and / or a long term care and / or hospice facility). Examples of point-of-care devices include those produced by Abbott Laboratories (Abbott Park, IL) (e.g., i-STAT and i-STAT Alinity, Universal Biosensors (Rowville, Australia) (see US 2006 / 0134713), Axis-Shield PoC AS (Oslo, Norway) and Clinical Lab Products (Los Angeles, USA).Docket No. 15893; 43644.601

[0215] “Point-of-observation” means at the time and place at which diagnostic criteria of a subject having a head injury or suspected head injury is observed by a health care practitioner to determine the presence or absence of the diagnostic criteria. The point-of-observation can be the point-of-care, such as in a hospital, a physician’s office, urgent or other medical care facility, a patient’s home, a nursing home and / or a long-term care and / or hospice facility, or the location at which the subject’s head injury or suspected head injury occurred (e.g., a sporting event, car accident site, etc.). However, the subject need not be present at the point- of-observation. For example, the point-of-observation can be in a laboratory where the results of a laboratory test are used to determine the diagnostic criteria.

[0216] “Positive predictive value” or “PPV” as used interchangeably herein refers to the probability that a subject has a positive outcome given that they have a positive test result.

[0217] “Quality control reagents” in the context of immunoassays and kits described herein, include, but are not limited to, calibrators, controls, and sensitivity panels. A “calibrator” or “standard” typically is used (e.g., one or more, such as a plurality) in order to establish calibration (standard) curves for interpolation of the concentration of an analyte, such as an antibody or an analyte. Alternatively, a single calibrator, which is near a reference level or control level (e.g., “low”, “medium”, or “high” levels), can be used. Multiple calibrators (i.e., more than one calibrator or a varying amount of calibrator(s)) can be used in conjunction to comprise a “sensitivity panel.”

[0218] A “receiver operating characteristic” curve or “ROC” curve refers to a graphical plot that illustrates the performance of a binary classifier system as its discrimination threshold is varied. For example, a ROC curve can be a plot of the true positive rate against the false positive rate for the different possible cutoff points of a diagnostic test. It is created by plotting the fraction of true positives out of the positives (TPR = true positive rate) vs. the fraction of false positives out of the negatives (FPR = false positive rate), at various threshold settings. TPR is also known as sensitivity, and FPR is one minus the specificity or true negative rate. The ROC curve demonstrates the tradeoff between sensitivity and specificity (any increase in sensitivity will be accompanied by a decrease in specificity); the closer the curve follows the left-hand border and then the top border of the ROC space, the more accurate the test; the closer the curve comes to the 45-degree diagonal of the ROC space, the less accurate the test; the slope of the tangent line at a cutoff point gives the likelihood ratio (LR) for that value of the test; and the area under the curve is a measure of test accuracy.

[0219] “Recombinant antibody” and “recombinant antibodies” refer to antibodies prepared by one or more steps, including cloning nucleic acid sequences encoding all or a part of oneDocket No. 15893; 43644.601 or more monoclonal antibodies into an appropriate expression vector by recombinant techniques and subsequently expressing the antibody in an appropriate host cell. The terms include, but are not limited to, recombinantly produced monoclonal antibodies, chimeric antibodies, humanized antibodies (fully or partially humanized), multi-specific or multivalent structures formed from antibody fragments, bifunctional antibodies, heteroconjugate Abs, DVD-Ig®s, and other antibodies as 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 comprises a first arm having a specificity for one antigenic site and a second arm having a specificity for a different antigenic site, i.e., the bifunctional antibodies have a dual specificity.

[0220] “Risk assessment,” “risk classification,” “risk identification,” or “risk stratification” of subjects (e.g., patients) as used herein refers to the evaluation of factors including biomarkers, to predict the risk of occurrence of future events including disease onset or disease progression, so that treatment decisions regarding the subject may be made on a more informed basis.

[0221] “Sample,” “test sample,” “specimen,” “sample from a subject,” “biological sample,” and “patient sample” as used interchangeably herein may be a sample of blood, such as whole blood (including for example, capillary blood, venous blood, dried blood spot, etc.), serum or plasma, or tissue, saliva, urine, , amniotic fluid, an oropharyngeal specimen, a nasopharyngeal specimens, lower respiratory specimens such as, but not limited to, sputum, endotracheal aspirate or bronchoalveolar lavage, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art. Additionally, the sample can be a nasopharyngeal or oropharyngeal sample obtained using one or more swabs that, once obtained, is placed in a sterile tube containing a virus transport media (VTM) or universal transport media (UTM), and retained therein or transferred to another media for testing.

[0222] A variety of cell types, tissue, or bodily fluid may be utilized to obtain a sample. Such cell types, tissues, and fluid may include sections of tissues such as biopsy and autopsy samples, oropharyngeal specimens, nasopharyngeal specimens, frozen sections taken for histologic purposes, blood (such as whole blood, dried blood spots, etc.), plasma, serum,Docket No. 15893; 43644.601 saliva, red blood cells, platelets, interstitial fluid, cerebral spinal fluid, etc. Cell types and tissues may also include lymph fluid, cerebrospinal fluid, or any fluid collected by aspiration. A tissue or cell type may be provided by removing a sample of cells from a human and a nonhuman animal but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and / or for another purpose). Archival tissues, such as those having treatment or outcome history, may also be used. Protein or nucleotide isolation and / or purification may not be necessary. In some embodiments, the sample is a blood sample (e.g., a whole blood sample, a serum sample, or a plasma sample). In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a capillary blood sample. In some embodiments, the sample is a dried blood spot. In some embodiments, the sample is a serum sample. In yet other embodiments, the sample is a plasma sample. In some embodiments, the sample is an oropharyngeal specimen. In other embodiments, the sample is a nasopharyngeal specimen. In other embodiments, the sample is sputum. In other embodiments, the sample is endotracheal aspirate. In still yet other embodiments, the sample is bronchoalveolar lavage. In still yet other embodiments, the sample is a saliva sample.

[0223] “Sensitivity” of an assay as used herein refers to the proportion of subjects for whom the outcome is positive that are correctly identified as positive (e.g., correctly identifying those subjects with a disease or medical condition for which they are being tested). For example, this might include correctly identifying subjects as having a TBI as distinct from those who do not have a TBI, correctly identifying subjects having a moderate, severe, or moderate to severe TBI as distinct from those having a mild TBI, correctly identifying subjects as having a mild TBI as distinct from those having a moderate, severe, or moderate to severe TBI, correctly identifying subjects as having a moderate, severe, or moderate to severe TBI as distinct from those having no TBI or correctly identifying subjects as having a mild TBI as distinct from those having no TBI etc..

[0224] “Specificity” of an assay as used herein refers to the proportion of subjects for whom the outcome is negative that are correctly identified as negative (e.g., correctly identifying those subjects who do not have a disease or medical condition for which they are being tested). For example, this might include correctly identifying subjects not having an TBI as distinct from those who do have a TBI, correctly identifying subjects not having a moderate, severe, or moderate to severe TBI as distinct from those having a mild TBI, correctly identifying subjects as not having a mild TBI as distinct from those having a moderate, severe, or moderate to severe TBI, etc.).Docket No. 15893; 43644.601

[0225] “ Series of calibrating compositions” refers to a plurality of compositions comprising a known concentration of UCH-L1, wherein each of the compositions differs from the other compositions in the series by the concentration of UCH-L1.

[0226] “ Solid phase” or “solid support” as used interchangeably herein, refers to any material that can be used to attach and / or attract and immobilize (1) one or more capture agents or capture specific binding partners, or (2) one or more detection agents or detection specific binding partners. The solid phase can be chosen for its intrinsic ability to attract and immobilize a capture agent. Alternatively, the solid phase can have affixed thereto a linking agent that has the ability to attract and immobilize the (1) capture agent or capture specific binding partner, or (2) detection agent or detection specific binding partner. For example, the linking agent can include a charged substance that is oppositely charged with respect to the capture agent (e.g., capture specific binding partner) or detection agent (e.g., detection specific binding partner) itself or to a charged substance conjugated to the (1) capture agent or capture specific binding partner or (2) detection agent or detection specific binding partner. In general, the linking agent can be any binding partner (preferably specific) that is immobilized on (attached to) the solid phase and that has the ability to immobilize the (1) capture agent or capture specific binding partner, or (2) detection agent or detection specific binding partner through a binding reaction. The linking agent enables the indirect binding of the capture agent to a solid phase material before the performance of the assay or during the performance of the assay. For examples, the solid phase can be plastic, derivatized plastic, magnetic, or non-magnetic metal, glass or silicon, including, for example, a test tube, microtiter well, sheet, bead, microparticle, chip, and other configurations known to those of ordinary skill in the art.

[0227] “Specific binding” or “specifically binding” as used herein may refer to the interaction of an antibody, a protein, or a peptide with a second chemical species, wherein the interaction is dependent upon 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 to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0228] “Specific binding partner” is a member of a specific binding pair. A specific binding pair comprises two different molecules, which specifically bind to each other through chemical or physical means. Therefore, in addition to antigen and antibody specific bindingDocket No. 15893; 43644.601 pairs of common immunoassays, 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 members, for example, an analyte-analog. Immunoreactive specific binding members include antigens, antigen fragments, and antibodies, including monoclonal and polyclonal antibodies as well as complexes and fragments thereof, whether isolated or recombinantly produced.[0229J “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 result of a data set is statistically significant. In statistical hypothesis testing, a statistically significant result is attained whenever the observed - value of a test statistic is less than the significance level defined of the study. The - value is the probability of obtaining results at least as extreme as those observed, given that the null hypothesis is true. Examples of statistical hypothesis analysis include Wilcoxon signed-rank test, t-test, Chi-Square or Fisher’s exact test. “Significant” as used herein refers to a change that has not been determined to be statistically significant (e.g., it may not have been subject to statistical hypothesis testing).

[0230] “Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus or rhesus monkey, chimpanzee, etc.) and a human). In some embodiments, the subject may 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.

[0231] “ Treat,” “treating” or “treatment” are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progress of a disease and / or injury, or one or more symptoms of such disease, to which such term applies. In some aspects, a treatment may be either performed in an acute or chronic way. Depending on the condition of the subject, the term also refers to preventing a disease or injury, and includes preventing the onset of a disease or injury, or preventing the symptoms associated with a disease or injury, "preventing" also refers to preventing the recurrence of a disease or injury or of one or more symptoms associated with such disease or injury. "Treatment" and "therapeutically," refer to the act of treating, as "treating" is defined above. In some aspects, the prevention or treatment of a disease can be done prior to affliction or injury, such as, for example, to reduceDocket No. 15893; 43644.601 the severity of a disease or injury or symptoms associated with a disease or an injury. Such prevention or reduction can include (a) administration of one or more pharmaceutical composition and / or one or more nutritional compositions to a subject; (b) the use of one or more of physical therapy, occupational therapy, and / or counseling; or (c) any combinations of (a) and (b).

[0232] “ Traumatic Brain Injury” or “TBI” as used interchangeably herein refers to a complex injury with a broad spectrum of symptoms and disabilities. TBI, a subset of AB I, is most often an acute event similar to other injuries. TBI can be classified as “mild,” “moderate,” or “severe.” The causes of TBI are diverse and include, for example, physical shaking by a person, a car accident, injuries from firearms, cerebral vascular accidents (e.g., strokes), falls, explosions or blasts and other types of blunt force trauma. Other causes of ABI (e.g, non-TBI ABI include the ingestion and / or exposure to one or more fires, chemicals or toxins (such as molds, asbestos, pesticides and insecticides, organic solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organic metals (such as methyl mercury, tetraethyl lead and organic tin), one or more drugs of abuse or combinations thereof). Alternatively, non-TBI ABI can occur in subjects suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection (e.g., SARS-CoV-2, meningitis, etc.), fungal infection (e.g., meningitis), bacterial infection (e.g., meningitis), or any combinations thereof. Young adults and the elderly are the age groups at highest risk for TBI. In certain embodiments herein, traumatic brain injury or TBI does not include and specifically excludes cerebral vascular accidents such as strokes.

[0233] “Mild TBI” as used herein refers to a head injury where a subject may or may not experience a loss of consciousness. For subjects that experience a loss of consciousness, it is typically brief, usually lasting only a few seconds or minutes. Mild TBI is also referred to as a concussion, minor head trauma, minor TBI, minor brain injury, and minor head injury. While MRI and CT scans are often normal, the individual with mild TBI may have cognitive problems such as headache, difficulty thinking, memory problems, attention deficits, mood swings and frustration.

[0234] Mild TBI is the most prevalent TBI and is often missed at time of initial injury. Typically, a subject has a Glasgow Coma scale number of between 13-15 (such as 13-15 or 14-15). Fifteen percent (15%) of people with mild TBI have symptoms that last 3 months or more. Common symptoms of mild TBI include fatigue, headaches, visual disturbances, memory loss, poor attend on / concentrati on, sleep disturbances, dizziness / loss of balance, irritability-emotional disturbances, feelings of depression, and seizures. Other symptomsDocket No. 15893; 43644.601 associated with mild TBI include nausea, loss of smell, sensitivity to light and sounds, mood changes, getting lost or confused, and / or slowness in thinking.

[0235] “Moderate TBI” as used herein refers to a brain injury where loss of consciousness and / or confusion and disorientation is between 1 and 24 hours and the subject has a Glasgow Coma scale number of between 9-13 (such as 9-12 or 9-13). The individual with moderate TBI may have abnormal brain imaging results. “Severe TBI” as used herein refers to a brain injury where loss of consciousness is more than 24 hours and memory loss after the injury or penetrating skull injury longer than 24 hours and the subject has a Glasgow Coma scale number between 3-8. The deficits range from impairment of higher level cognitive functions to comatose states. Survivors may have limited function of arms or legs, abnormal speech or language, loss of thinking ability or emotional problems. Individuals with severe injuries can be left in long-term unresponsive states. For many people with severe TBI, long-term rehabilitation is often necessary to maximize function and independence.

[0236] “Moderate to severe” TBI as used herein refers to a spectrum of brain injury that includes a change from moderate to severe TBI over time and thus encompasses (e.g., temporally) moderate TBI alone, severe TBI alone, and moderate to severe TBI combined. For example, in some clinical situations, a subject may initially be diagnosed as having a moderate TBI but who, over the course of time (minutes, hours or days), progresses to having a severe TBI (such, as for example, in situations when there is a brain bleed). Alternatively, in some clinical situations, a subject may initially be diagnosed as having a severe TBI but who, over the course of time (minutes, hours or days), progresses to having a moderate TBI. Such subjects would be examples of patients that could be classified as “moderate to severe”. Common symptoms of moderate to severe TBI include cognitive deficits including difficulties with attention, concentration, distractibility, memory, speed of processing, confusion, perseveration, impulsiveness, language processing, and / or “executive functions”, not understanding the spoken word (receptive aphasia), difficulty speaking and being understood (expressive aphasia), slurred speech, speaking very fast or very slow, problems reading, problems writing, difficulties with interpretation of touch, temperature, movement, limb position and fine discrimination, the integration or patterning of sensory impressions into psychologically meaningful data, partial or total loss of vision, weakness of eye muscles and double vision (diplopia), blurred vision, problems judging distance, involuntary eye movements (nystagmus), intolerance of light (photophobia), hearing issues, such as decrease or loss of hearing, ringing in the ears (tinnitus), increased sensitivity to sounds, loss or diminished sense of smell (anosmia), loss or diminished sense of taste, the convulsionsDocket No. 15893; 43644.601 associated with epilepsy that can be several types and can involve disruption in consciousness, sensory perception, or motor movements, problems with control of bowel and bladder, sleep disorders, loss of stamina, appetite changes, problems with regulation of body temperature, menstrual difficulties, dependent behaviors, issues with emotional ability or stability, lack of motivation, irritability, aggression, depression, disinhibition, or denial / lack of awareness. Subjects having a moderate to severe TBI can have a Glasgow Coma scale score from 3-12 (which includes the range of 9-12 for a moderate TBI, and 3-8 for a severe TBI).

[0237] “ Trauma mechanism” as used herein refers to determining or evaluating whether a subject being evaluated for a TBI put himself or herself in a position or situation to have experienced or suffered a TBI. For example, in some aspects, the subject may have tripped and fallen and may not be able to recall if he or she hit his or her head. Alternatively, in another example, the subject may have been in an automobile, motorcycle, motorbike or bicycle accident and may not be able to recall if or be aware of whether he or she hit his or her head.

[0238] “Ubiquitin carboxy-terminal hydrolase LI” or “UCH-L1” as used interchangeably herein refers to a deubiquitinating enzyme encoded by the UCH-L1 gene in humans. UCH- Ll, also known as ubiquitin carboxyl-terminal esterase LI and ubiquitin thiolesterase, is a member of a gene family whose products hydrolyze small C-terminal adducts of ubiquitin to generate the ubiquitin monomer.

[0239] “UCH-L1 status” can mean either the level or amount of UCH-L1 at a point in time (such as with a single measure of UCH-L1), the level or amount of UCH-L1 associated with monitoring (such as with a repeat test on a subject to identify an increase or decrease in UCH-L1 amount), the level or amount of UCH-L1 associated with treatment for traumatic brain injury (whether a primary brain injury and / or a secondary brain injury) or combinations thereof.

[0240] “Variant” is used herein to describe a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Representative examples of “biological activity” include the ability to be bound by a specific antibody or to promote an immune response. Variant is also used herein to describe a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree, andDocket No. 15893; 43644.601 distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, incorporated fully herein by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. “Variant” also can be used to refer to an antigenically reactive fragment of an anti-UCH-Ll antibody that differs from the corresponding fragment of anti-UCH-Ll antibody in amino acid sequence but is still antigenically reactive and can compete with the corresponding fragment of anti-UCH-Ll antibody for binding with UCH-L1. “Variant” also can be used to describe a polypeptide or a fragment thereof that has been differentially processed, such as by proteolysis, phosphorylation, or other post-translational modification, yet retains its antigen reactivity. [0241 J “Vector” is used herein to describe a nucleic acid molecule that can transport 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 may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby areDocket No. 15893; 43644.601 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 of utility in recombinant DNA techniques are often in the form of plasmids.“Plasmid” and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions, can be used. In this regard, RNA versions of vectors (including RNA viral vectors) may also find use in the context of the present disclosure.

[0242] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.2. Methods of Diagnosing, Aiding in the Diagnosis, and / or Adjudicating an Acquired Brain Injury (ABI) Diagnosis

[0243] The present disclosure relates, among other methods, to a method of adjudicating an acquired brain injury (ABI) diagnosis, such as, a traumatic brain injury (TBI) diagnosis. Generally, in one aspect, the method involves the steps of (a) receiving diagnostic criteria of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head; (b) executing an ABI adjudication algorithm, by compiling the diagnostic criteria received in step (a); and (c) adjudicating an ABI diagnosis based on the diagnostic criteria compiled in step (b). In some aspects, the method involves the steps of (a) receiving diagnostic criteria of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head; (b) executing a TBI adjudication algorithm, by compiling the diagnostic criteria received in step (a); and (c) adjudicating a TBI diagnosis based on the diagnostic criteria compiled in step (b). For avoidance of any doubt, the discussion of the “TBI adjudication algorithm” herein is also applicable to an “ABI adjudication algorithm” - although TBI and non-TBI ABI are distinct. The methods can be implemented as part of aDocket No. 15893; 43644.601 system, for example, a computer-implemented method that is implemented on one or more devices (e.g., a point-of-care device) through any type of network, such a local area network (LAN) or a wide area network (WAN), or by an external computer (for example, through the Internet using an Internet Service Provider).

[0244] The present disclosure also relates, among other methods, to a method of diagnosing or aiding in the diagnosis an acquired brain injury (AB I), such as a traumatic brain injury (TBI). Generally, in some aspects, the method involves the steps of: (a) receiving diagnostic criteria of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head; and (b) diagnosing an ABI in a subject or aiding in the diagnosis that a subject is more likely than not to have an ABI based on the diagnostic criteria received in step (a). In other aspects, the method involves the steps of: (a) receiving diagnostic criteria of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head; and (b) diagnosing a TBI in a subject or aiding in the diagnosis that a subject is more likely than not to have an TBI based on the diagnostic criteria received in step (a). For avoidance of any doubt, the discussion of “diagnosing or aiding in the diagnosis of a TBI” herein is also applicable to “diagnosing or aiding in the diagnosis of an ABI” - although TBI and non-TBI ABI are distinct. The methods can be implemented as part of a system, for example, a computer-implemented method that is implemented on one or more devices (e.g., a point-of-care device) through any type of network, such a local area network (LAN) or a wide area network (WAN), or by an external computer (for example, through the Internet using an Internet Service Provider).

[0245] In some embodiments, the present disclosure relates to a method of both diagnosing or aiding in the diagnosis of an ABI and adjudicating an ABI.

[0246] In some aspects, the present disclosure contemplates evaluating a certain number of inclusion and exclusion criteria to determine or assess whether (or not) a subject would benefit from an adjudication of an ABI diagnosis, such as a TBI diagnosis, or a diagnosis of a ABI, according to the methods of the present disclosure. For example, imaging, such as a computed tomography (CT) scan, magnetic resonance imaging (MRI), or a combination thereof, can used as part of the inclusion or exclusion criteria. In this instance, a determination may be made to include or exclude a subject if no evaluable imaging has been obtained (e.g., the imaging is inconclusive or not performed). Alternatively, a determination may be made to include a subject if a positive CT scan and / or a positive MRI is obtained, or include or exclude a subject if a negative CT scan and / or negative MRI is obtained. The useDocket No. 15893; 43644.601 of imaging to determine the inclusion or exclusion of a subject is merely optional, and / or other criteria, information, and techniques can be used when making such a determination.

[0247] The disclosure contemplates using any diagnostic criteria for diagnosing or aiding in the diagnosis of a ABI and / or adjudicating a ABI known to the skilled artisan. Examples of useful diagnostic criteria include, without limitation: (i) the presence or absence of a trauma mechanism; (ii) the level of glial fibrillary acidic protein (GFAP) and / or level of ubiquitin carboxy -terminal hydrolase LI (UCH-L1) measured in a sample obtained from the subject; (iii) the presence or absence of one or more clinical signs of a TBI; (iv) the presence or absence of one or more confounding factors; (v) the presence or absence of an acute symptom; (vi) the presence or absence of an impairment on acute clinical examination; (vii) the presence or absence of an elevated biomarker indicative of an intracranial injury, and (viii) any combination of (i)-(vii).

[0248] In some embodiments, the diagnostic criteria received comprises: (i) the presence or absence of a trauma mechanism, (ii) the level of glial fibrillary acidic protein (GFAP) and / or level of ubiquitin carboxy -terminal hydrolase LI (UCH-L1) measured in a sample obtained from the subject; and at least one diagnostic criteria selected from the group consisting of items (iii)-(vii), above. In other embodiments, the diagnostic criteria received comprises: (i) the presence or absence of a trauma mechanism, (ii) the level of glial fibrillary acidic protein (GFAP) and / or level of ubiquitin carboxy -terminal hydrolase LI (UCH-L1) measured in a sample obtained from the subject; and at least two diagnostic criteria selected from the group consisting of items (iii)-(vii), above. In still yet other embodiments, the diagnostic criteria received comprises: (i) the presence or absence of a trauma mechanism, (ii) the level of glial fibrillary acidic protein (GFAP) and / or level of ubiquitin carboxy-terminal hydrolase LI (UCH-L1) measured in a sample obtained from the subject; and at least four diagnostic criteria selected from the group consisting of items (iii)-(vii), above. In still yet other embodiments, the diagnostic criteria received comprises: (i) the presence or absence of a trauma mechanism, (ii) the level of glial fibrillary acidic protein (GFAP) and / or level of ubiquitin carboxy-terminal hydrolase LI (UCH-L1) measured in a sample obtained from the subject; and the diagnostic criteria selected of items (iii)-(vii), above.

[0249] The disclosure further contemplates using any diagnostic criteria for adjudicating a TBI diagnosis that is known to the skilled artisan. Examples of useful diagnostic criteria include, without limitation: (i) the presence or absence of a trauma mechanism; (ii) the presence or absence of a neuroimaging intracranial abnormality; (iii) the presence or absence of one or more clinical signs of a TBI; (iv) the presence or absence of one or moreDocket No. 15893; 43644.601 confounding factors; (v) the presence or absence of an acute symptom; (vi) the presence or absence of an impairment on acute clinical examination; (vii) the presence or absence of an elevated biomarker indicative of an intracranial injury; and (viii) any combination of (i)-(vii).

[0250] In some embodiments, the diagnostic criteria received comprises (i) the presence or absence of a trauma mechanism and at least two of the diagnostic criteria selected from the group consisting of (ii)-(vii). In other embodiments, the diagnostic criteria received comprises (i) the presence or absence of a trauma mechanism and at least three of the diagnostic criteria selected from the group consisting of (ii)-(vii). In yet other embodiments, the diagnostic criteria received comprises (i) the presence or absence of a trauma mechanism and at least four of the diagnostic criteria selected from the group consisting of (ii)-(vii). In still other embodiments, the diagnostic criteria received comprises (i) the presence or absence of a trauma mechanism and at least five of the diagnostic criteria selected from the group consisting of (ii)-(vii). In yet still other embodiments, the diagnostic criteria received comprises the diagnostic criteria of (i), (ii), (iii), (iv), (v), (vi), and (vii).

[0251] The subject’s diagnostic criteria can be received in step (a) using any method available to the skilled artisan. In some instances, the diagnostic criteria can be received via a communication to a health care practitioner who is evaluating whether or not the subject has a TBI or is more likely than not to have a TBI and / or executing a TBI adjudication algorithm to diagnose whether or not the subject has a TBI. For example, the diagnostic criteria can be communicated to a practitioner by the subject, a member of the subject’s family, a paramedic, a nurse, a physician’s assistant, another practitioner, a guardian of the subject, etc. In other instances, the diagnostic criteria can be received in a physical format, such as in the subject’s chart, medical record, a report (e.g., accident report), a file, a facsimile, a letter, etc.

[0252] In still other instances, the diagnostic criteria can be received electronically from a device or other apparatus, such as, for example as an e-mail, a file, a notification, an alert, a video, a website, a program, an app, etc. The diagnostic criteria received via a device can be pre-determined diagnostic criteria, for example, that is retrieved from a subject’s electronic medical record, diagnostic criteria that is determined at the point of observation and / or evaluation, or a combination of pre-determined diagnostic criteria and diagnostic criteria that is determined at the point of observation and / or evaluation.

[0253] The diagnostic criteria can be received via one or more user interfaces of one or more devices at one or more points-of-observation and / or evaluation of the subject with the head injury or suspected head injury. In some instances, all the subject’s diagnostic criteria is received via the user interface by a single health care practitioner. For example, a device,Docket No. 15893; 43644.601 such as, for example, a computer, a tablet (e.g., an iPad), or a smart phone, configured as part of a system used to implement the disclosed methods, can display diagnostic criteria for the practitioner to select based on the practitioner’s observations of the subject and / or a first-hand account of the subject’s head injury or suspected head injury (e.g., by the subject or a witness of the subject’s head injury or suspected head injury).

[0254] The diagnostic criteria can be displayed on the user interface in any format that enables the practitioner to perform a comprehensive evaluation of the subject’s diagnostic criteria, including as a list, a questionnaire, a survey, an interactive flow chart or diagram, a table with a draggable list of the diagnostic criteria for the practitioner to drag and drop into “present” or “absent” groupings to indicate the presence or absence of the diagnostic criteria in the subject, etc.

[0255] The diagnostic criteria can be presented on the user interface in a single screen or in a series of screens. For example, the user interface can display trauma mechanisms on a first screen, neuroimaging intracranial abnormalities on a second screen, one or more clinical signs of a TBI on a third screen, one or more confounding factors on a fourth screen, acute clinical symptoms on a fifth screen, impairments on acute clinical examination on a sixth screen, and elevated biomarkers indicative of intracranial injury on a seventh screen. The skilled artisan will appreciate that the diagnostic criteria can be presented in any order. The skilled artisan will also appreciate that a screen can display combinations of the diagnostic criteria from separate categories (e.g., trauma mechanisms and clinical signs of a TBI) as well as combinations of diagnostic criteria within the same category (e.g., a subset of one or more clinical signs of a TBI).

[0256] In other instances, the subject’s diagnostic criteria can be received via a series of user interfaces by multiple health care practitioners operating at different points-of- observation and / or evaluation, as the system can be configured to allow multiple users to operate simultaneously or sequentially to determine and receive the diagnostic criteria. When used in this manner, the system is configured to store the diagnostic criteria in real-time for subsequent retrieval by a health care practitioner (e.g., in a database, e.g., an electronic medical record). For example, a first health care practitioner at a first point-of-observation where a head injury or suspected head injury has occurred can use a first device to receive diagnostic criteria comprising a trauma mechanism (e.g., a computer, a tablet (e.g., iPad), a smart phone), a second health care practitioner at a second point-of-observation at a health care facility can use a second device (e.g., a computer, a tablet (e.g., iPad), a smart phone) to receive diagnostic criteria comprising a neuroimaging intracranial abnormality, a third healthDocket No. 15893; 43644.601 care practitioner at a third point-of-observation at a health care facility can use a third device (e.g., a computer, a tablet (e.g., iPad), a smart phone) to receive diagnostic criteria comprising the level of glial fibrillary acidic protein (GFAP) and / or level of ubiquitin carboxy-terminal hydrolase LI (UCH-L1) measured in a sample obtained from the subject, a fourth health care practitioner at a fourth point-of observation can use a fourth device (e.g., a computer, a tablet (e.g., iPad), a smart phone) to receive diagnostic criteria comprising an elevated biomarker indicative of intracranial injury, and a fifth, sixth, and / or seventh health care practitioner can use a fifth, sixth and / or seventh device (e.g., a computer, a tablet (e.g., iPad), a smart phone) to receive diagnostic criteria comprising one or more clinical signs of a TBI, one or more confounding factors, an acute system, and / or an impairment on acute clinical examination. The system can be configured to sequentially or simultaneously, in real time, integrate the diagnostic criteria received in this way to execute a TBI adjudication algorithm by compiling the diagnostic criteria prior to, while, or after storing the diagnostic criteria in an electronic storage medium of the system (e.g., the subject’s electronic medical record).

[0257] In addition to enabling a health care practitioner to manually input diagnostic criteria as described above, the system can be configured to automatically input diagnostic criteria. For example, a subject having or suspected of having a head injury undergoing a neuroimaging procedure can have the results of the neuroimaging procedure determined by the system and automatically input into the system so that the diagnostic criteria comprising an intracranial abnormality is received automatically. The results of the neuroimaging procedure can also be reviewed by a healthcare practitioner at a point-of-observation and the presence or absence of an intracranial abnormality can be manually received. By way of another example, a healthcare practitioner at a point-of-observation can use a point-of-care device that is electronically connected to a system configured to implement the disclosed methods to obtain a sample from a subject having or suspected of having a head injury and the level of GFAP and / or UCH-L1 can be determined by an assay that is performed using the point-of-care device and the diagnostic criteria comprising the results can be automatically received from the point-of-care device via a networked connection. In another example, a healthcare practitioner at a point-of-observation can use a point-of-care device that is electronically connected to a system configured to implement the disclosed methods to obtain a sample from a subject having or suspected of having a head injury and the presence or absence of an elevated biomarker indicative of an intracranial abnormality can be determined by an assay that is performed using the point-of-care device and the diagnostic criteria comprising the results can be automatically received from the point-of-care device. . TheDocket No. 15893; 43644.601 results of the point-of-care assay can also be displayed on the point-of-care device and the healthcare practitioner can manually input the diagnostic criteria into a device (e.g., a computer, a tablet (e.g., iPad), a smart phone) so that it is received for subsequent processing. The user interface can be configured to enable the healthcare practitioner to enter the levels of GFAP and / or UCH-L1 measured manually e.g., by typing. The user interface can also be configured to enable the healthcare practitioner to select from a list of predetermined ranges of the levels of GFAP and / or UCH-L1, for instance, a level that is below a cut-off level or range that is indicative of the subject not having a TBI, a level that is equal to a cut-off level or within a range of cut-off levels that is indicative of the subject having a TBI, or a level that is above a cut-off level or range of cut-off levels that is indicative of the severity of the TBI.

[0258] The diagnostic criteria can be received in any format that facilitates the compiling of the diagnostic criteria to enable an accurate and timely adjudication of TBI diagnosis of a subject and / or diagnose or aid in the diagnosis of a TBI. For instance, the diagnostic criteria received can be in a table, an image, a graphic, a list, a spreadsheet, a database, etc.

[0259] Aspects of the disclosure involve executing a TBI adjudication algorithm, for example, by compiling a subject’s received diagnostic criteria.

[0260] The TBI adjudication algorithm can be executed by compiling at least one of a subject’s received diagnostic criteria. In one aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism. In an aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a neuroimaging intracranial abnormality. In a further aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of one or more clinical signs of a TBI. In some aspects, executing the TBI adjudication algorithm comprises compiling an injury severity indicator based on the one or more clinical signs of TBI. In further aspects, executing the TBI adjudication algorithm comprises compiling the presence or absence of one or more confounding factors. In yet still further aspects, executing the TBI adjudication algorithm comprises compiling the presence or absence of an acute symptom. In other aspects, executing the TBI adjudication algorithm comprises compiling the presence or absence of an impairment on acute clinical examination. In still yet other aspects, executing the TBI adjudication algorithm comprises compiling the presence of the elevated biomarker indicative of an intracranial injury.

[0261] The TBI adjudication algorithm can be executed by compiling at least two of a subject’s received diagnostic criteria. In another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism and theDocket No. 15893; 43644.601 presence or absence of a neuroimaging intracranial abnormality. In yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism and the presence or absence of one or more clinical signs of a TBI. In still yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism and the presence or absence of one or more confounding factors. In still yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism and the presence or absence of an acute symptom. In still yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism and the presence or absence of an impairment on acute clinical examination. In still yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism and the presence or absence of an elevated biomarker indicative of an intracranial abnormality.

[0262] The TBI adjudication algorithm can be executed by compiling at least three of a subject’s received diagnostic criteria. In some instances, the at least three diagnostic criteria include the presence or absence of a trauma mechanism, and at least two additional diagnostic criteria other than the presence or absence of one or more confounding factors. In another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of a neuroimaging intracranial abnormality, and the presence or absence of one or more clinical signs of a TBI. In still yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of a neuroimaging intracranial abnormality, and the presence or absence of an acute symptom. In still yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of a neuroimaging intracranial abnormality, and the presence or absence of an impairment on acute clinical examination. In still yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of a neuroimaging intracranial abnormality, and the presence or absence of an elevated biomarker indicative of intracranial abnormality.

[0263] In other instances, the at least three diagnostic criteria include the presence or absence of a trauma mechanism, the presence or absence of one or more confounding factors, and at least one additional diagnostic criteria. In yet a further aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism,Docket No. 15893; 43644.601 the presence or absence of a neuroimaging intracranial abnormality, and the presence or absence of one or more confounding factors. In still yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of one or more clinical signs of a TBI, and the presence or absence of one or more confounding factors. In still yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of an acute symptom, and the presence or absence of one or more confounding factors. In still yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of an impairment on acute clinical examination, and the presence or absence of one or more confounding factors, acute clinical examination, and the presence or absence of one or more confounding factors. In still yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of an elevated biomarker indicative of an intracranial abnormality, and the presence or absence of one or more confounding factors.

[0264] The TBI adjudication algorithm can be executed by compiling at least four of a subject’s received diagnostic criteria. In one aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of a neuroimaging intracranial abnormality, the presence or absence of one or more clinical signs of a TBI, and the presence or absence of one or more confounding factors. In yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of a neuroimaging intracranial abnormality, the presence or absence of one or more clinical signs of a TBI, and the presence or absence of an acute symptom. In an aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of a neuroimaging intracranial abnormality, the presence or absence of one or more clinical signs of a TBI, and the presence or absence of an impairment on acute clinical examination. In still yet a further aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of a neuroimaging intracranial abnormality, the presence or absence of one or more clinical signs of a TBI, and the presence or absence of an elevated biomarker indicative of intracranial injury.

[0265] The TBI adjudication algorithm can be executed by compiling at least five, at least six, or at least seven of a subject’s received diagnostic criteria. In another aspect, executingDocket No. 15893; 43644.601 the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of a neuroimaging intracranial abnormality, the presence or absence of one or more clinical signs of a TBI, the presence or absence of one or more confounding factors, and the presence or absence of an acute symptom. In still yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of a neuroimaging intracranial abnormality, the presence or absence of one or more clinical signs of a TBI, the presence or absence of one or more confounding factors, the presence or absence of an acute symptom, and the presence or absence of an impairment on acute clinical examination. In still yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of a neuroimaging intracranial abnormality, the presence or absence of one or more clinical signs of a TBI, the presence or absence of one or more confounding factors, the presence or absence of an acute symptom, the presence or absence of an impairment on acute clinical examination, and the presence or absence of an elevated biomarker indicative of an intracranial injury. In yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of a neuroimaging intracranial abnormality, the presence or absence of one or more clinical signs of a TBI, the presence or absence of one or more confounding factors, the presence of the acute symptom and the presence of the impairment on acute clinical examination. In still yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of a neuroimaging intracranial abnormality, the presence or absence of one or more clinical signs of a TBI, the presence or absence of one or more confounding factors, the presence of the acute symptom and the presence of the elevated biomarker indicative of an intracranial injury. In still yet another aspect, executing the TBI adjudication algorithm comprises compiling the presence or absence of a trauma mechanism, the presence or absence of a neuroimaging intracranial abnormality, the presence or absence of one or more clinical signs of a TBI, the presence or absence of one or more confounding factors, the presence of the impairment on acute clinical examination, and the presence of the elevated biomarker indicative of an intracranial injury. In yet another aspect, executing the TBI adjudication algorithm further comprises compiling the presence or absence of a trauma mechanism, the presence or absence of a neuroimaging intracranial abnormality, the presence or absence of one or more clinical signs of a TBI, the presence or absence of one or more confounding factors, the presence of the acute symptom, the presence of the impairment onDocket No. 15893; 43644.601 acute clinical examination, and the presence of the elevated biomarker indicative of an intracranial injury.

[0266] Additionally, in some embodiments, executing the TBI adjudication algorithm can further include the step of compiling an injury severity indicator based on one or more clinical signs of TBI. The one or more clinical signs that can be used include: (i) if the subject lost consciousness, the duration of the loss of consciousness; (ii) if the subject suffered any post-traumatic or peri -traumatic amnesia, the duration the subject suffered the amnesia; and / or (iii) the subject’s Glasgow Coma Scale (GCS) score, Ranchos Los Amigos Scale score, and / or Rivermead Post-Concussion Symptoms Questionnaire score after a duration of time.

[0267] The disclosure is not limited to any method of compiling the diagnostic criteria, provided that the diagnostic criteria is compiled in a manner that facilitates the adjudication of a TBI diagnosis of a subject. In some cases, the diagnostic criteria are compiled into a flow chart that enables a skilled practitioner to adjudicate a TBI diagnosis based on the presence or absence of the received diagnostic criteria of a subject. In other cases, the diagnostic criteria are compiled into a checklist that enables the skilled practitioner to adjudicate a TBI diagnosis based on the presence or absence of the received diagnostic criteria of a subject. In still other cases, the diagnostic criteria are compiled into a risk assessment that indicates the probability of a TBI diagnosis based on the presence or absence of the received diagnostic criteria of a subject. In yet other cases, the diagnostic criteria are compiled into a balance sheet that separates the received diagnostic criteria of a subject into a first column and second column respectively listing diagnostic criteria indicative and not indicative of a TBI diagnosis.

[0268] The diagnostic criteria received in step (a) can be compiled in step (b) in a physical format or an electronic format. For instance, diagnostic criteria of a subject received electronically (e.g., from an electronic medical record of the subject) can be compiled by a computer program into a flowchart, a checklist, a risk assessment, a balance sheet, or other format that facilitates the adjudication of a TBI diagnosis for a subject by a user of the computer program. In such instances, the compiled diagnostic criteria can be displayed on a user interface to assist the user (e.g., health care practitioner) in adjudicating a TBI diagnosis.

[0269] In one embodiment, the method comprises (a) receiving diagnostic criteria of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head, wherein the diagnostic criteria comprises: (i) the presence or absence of a trauma mechanism; (ii) the presence or absence of a neuroimaging intracranial abnormality; (iii) theDocket No. 15893; 43644.601 presence or absence of one or more clinical signs of a TBI; and (iv) the presence or absence of one or more confounding factors; (b) executing a TBI adjudication algorithm by compiling the diagnostic criteria; and (c) adjudicating a TBI diagnosis based on the diagnostic criteria compiled in step (b), wherein a subject is diagnosed as having a TBI when: (i) a trauma mechanism and a neuroimaging intracranial abnormality are present; or (ii) a trauma mechanism is present, a neuroimaging intracranial abnormality is absent, one or more clinical signs indicative of TBI is present, and one or more confounding factors is absent.[0270J Aspects of the method involve adjudicating a TBI diagnosis based on subject’s received and compiled diagnostic criteria. Adjudicating the TBI diagnosis based on the received and compiled diagnostic criteria can be performed by a healthcare practitioner. When the diagnostic criteria are received and compiled electronically, for example, using a device comprising a system configured to implement a computer-implemented version of the disclosed methods), adjudication of the TBI diagnosis by the practitioner can be assisted by the device.

[0271] For example, in some instances, the device (e.g., a computer, a tablet, a smart phone) can present the compiled diagnostic criteria on a user interface for the practitioner to use as the basis of the adjudication of the TBI diagnosis. In other instances, the computer program can compile the diagnostic criteria and adjudicate the TBI diagnosis independently of the practitioner. In such instances, the computer program can display the adjudicated TBI diagnosis on a user interface. The adjudicated TBI diagnosis that is displayed on the user interface can be presented as the likelihood or probability that the subject is diagnosed or not diagnosed as having a TBI. The user interface can also present differential diagnoses with the likelihood or probabilities that the subject has a TBI or another condition based on the diagnostic criteria. The user interface can give the practitioner the option to select, based on the practitioner’s professional medical opinion and interpretation of the compiled diagnostic criteria, the adjudicated TBI diagnosis of the subject.

[0272] The adjudication of the TBI diagnosis is performed by analyzing the received and compiled diagnostic criteria of a subject and diagnosing the subject has having a TBI or not having a TBI based on the compiled diagnostic criteria. The disclosure also contemplates providing a differential diagnosis other than a TBI based on the diagnostic criteria when the subject is diagnosed as not having a TBI.

[0273] In one aspect, a subject is diagnosed as not having a TBI when a trauma mechanism is absent. In another aspect, the subject is diagnosed as not having a TBI when: (i) a trauma mechanism is present; (ii) a neuroimaging intracranial abnormality is absent; and (iii) oneDocket No. 15893; 43644.601 more clinical signs of a TBI is absent. In yet another aspect, the subject is diagnosed as not having a TBI diagnosis when: (i) a trauma mechanism is present; (ii) a neuroimaging intracranial abnormality is absent; (iii) one more clinical signs of a TBI is present; and (iv) the one or more clinical signs of TBI is better accounted for by the presence of the one or more confounding factors.

[0274] In some aspects, the diagnostic criteria are presented in a flow chart that enables a healthcare practitioner to make a diagnosis of a TBI or aide in the diagnosis of a TBI based on the presence or absence of the diagnostic criteria in the subject. In other aspects, the diagnostic criteria are presented in a checklist that enables the healthcare practitioner to make a diagnosis of a TBI or aide in the diagnosis of a TBI based on the presence or absence of the diagnostic criteria in the subject. In still other aspects, the diagnostic criteria are presented as a risk assessment that indicates the probability that a subject is likely to have a TBI based on the presence or absence of the diagnostic criteria in the subject. In still yet other aspects, the diagnostic criteria are presented as a balance sheet that separates the diagnostic criteria into a first column and second column respectively listing diagnostic criteria indicative and not indicative of the subject having a TBI.

[0275] Aspects of the methods involve (i) diagnosing a subject as having a TBI or not having a TBI; or (ii) aiding in the diagnosis that a subject is more likely than not to have an TBI or is likely not to have a TBI, or having one or more alternative diagnoses based on subject’s received diagnostic criteria. A healthcare practitioner can use the diagnostic criteria to diagnose the subject as having (or more likely than not as having), or not having (or not likely to have), a TBI with or without the assistance of a device configured to implement a computer-implemented version of the method. For example, in some aspects, the device (e.g., a computer, a tablet (e.g., iPad), a smart phone) can present the diagnostic criteria on a user interface for the practitioner to use as the basis of a diagnosis of TBI (“TBI diagnosis”) or to aid in the diagnosis of a TBI. In other instances, the computer program can analyze the diagnostic criteria and propose a TBI diagnosis or alternative diagnoses for the healthcare practitioner to consider. In such instances, the computer program can display the TBI diagnosis, likelihood of having a TBI, or alternative diagnoses on the user interface. The diagnosis that is displayed on the user interface can be presented as the likelihood or probability that the subject is diagnosed or not diagnosed as having a TBI or the alternative diagnoses. The user interface can give the healthcare practitioner the option to accept the subject’s proposed TBI diagnosis, or to select one of several diagnoses that include TBI and other conditions that are better accounted for by one or more confounding factors.Docket No. 15893; 43644.601

[0276] In one embodiment, a method of diagnosing or aiding in the diagnosis of a traumatic brain injury (TBI) diagnosis comprises: (a) receiving diagnostic criteria of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head, wherein the diagnostic criteria comprises: (i) the presence or absence of a trauma mechanism; and (ii) a level of glial fibrillary acidic protein (GFAP) and / or a level of ubiquitin carboxy -terminal hydrolase LI (UCH-L1) measured in a sample obtained from the subject; and (b) diagnosing the subject as having a TBI or more likely than not to have a TBI when: (i) a trauma mechanism is present; and (ii) the level of GFAP is greater than or equal to about 80 pg / mL, the level of UCH-L1 is greater than or equal to about 500 pg / mL, or the level of GFAP is greater than or equal to about 80 pg / mL and the level of UCH-L1 is greater than or equal to about 500 pg / mL.

[0277] In another embodiment, a subject is diagnosed with a TBI or determined more likely than not to have a TBI when: (i) a trauma mechanism is present; and (ii) the level of GFAP is greater than or equal to about 80 pg / mL, the level of UCH-L1 is greater than or equal to about 1000 pg / mL, or the level of GFAP is greater than or equal to about 80 pg / mL and the level of UCH-L1 is greater than or equal to about 1000 pg / mL.

[0278] In yet another embodiment, the subject is diagnosed with a TBI or determined more likely than not to have a TBI when: (i) a trauma mechanism is present; and (ii) the level of GFAP is greater than or equal to about 100 pg / mL, the level of UCH-L1 is greater than or equal to about 500 pg / mL, or the level of GFAP is greater than or equal to about 100 pg / mL and the level of UCH-L1 is greater than or equal to about 500 pg / mL.

[0279] In still another embodiment, the subject is diagnosed with a TBI or determined more likely than not to have a TBI when: (i) a trauma mechanism is present; and (ii) the level of GFAP is greater than or equal to about 100 pg / mL, the level of UCH-L1 is greater than or equal to about 1000 pg / mL, or the level of GFAP is greater than or equal to about 100 pg / mL and the level of UCH-L1 is greater than or equal to about 1000 pg / mL.

[0280] In yet another embodiment, the subject is diagnosed with a TBI or determined more likely than not to have a TBI when: (i) a trauma mechanism is present; and (ii) the level of GFAP is greater than or equal to about 120 pg / mL, the level of UCH-L1 is greater than or equal to about 500 pg / mL, or the level of GFAP is greater than or equal to about 120 pg / mL and the level of UCH-L1 is greater than or equal to about 500 pg / mL.

[0281] In still another embodiment, the subject is diagnosed with a TBI or determined more likely than not to have a TBI when: (i) a trauma mechanism is present; and (ii) the level of GFAP is greater than or equal to about 120 pg / mL, the level of UCH-L1 is greater than orDocket No. 15893; 43644.601 equal to about 1000 pg / mL, or the level of GFAP is greater than or equal to about 120 pg / mL and the level of UCH-L1 is greater than or equal to about 1000 pg / mL.

[0282] In yet another embodiment, the subject is diagnosed with a TBI or determined more likely than not to have a TBI when: (i) a trauma mechanism is present; and (ii) the level of GFAP is greater than between about 80 pg / mL to about 120 pg / mL, the level of UCH-L1 is greater than between about 500 pg / mL to about 1000 pg / mL, or the level of GFAP is greater than between about 80 pg / mL to about 120 pg / mL and the level of UCH-L1 is greater than between about 500 pg / mL to about 1000 pg / mL.

[0283] In one aspect, a subject is diagnosed as not having a TBI or not likely to have a TBI when a trauma mechanism is absent. In another aspect, the subject is diagnosed as not having a TBI when: (i) a trauma mechanism is present and the level of GFAP is less than about 80 pg / mL, the level of UCH-L1 is less than about 500 pg / mL, or the level of GFAP is less than about 80 pg / mL and the level of UCH-L1 is less than about 500 pg / mL; (ii) a trauma mechanism is present and the level of GFAP is less than about 80 pg / mL, the level of UCH- L1 is less than about 1000 pg / mL, or the level of GFAP is less than about 80 pg / mL and the level of UCH-L1 is less than about 1000 pg / mL; (iii) a trauma mechanism is present and the level of GFAP is less than about 100 pg / mL, the level of UCH-L1 is less than about 500 pg / mL, or the level of GFAP is less than about 100 pg / mL and the level of UCH-L1 is less than about 500 pg / mL; (iv) a trauma mechanism is present and the level of GFAP is less than about 100 pg / mL, the level of UCH-L1 is less than about 1000 pg / mL, or the level of GFAP is less than about 100 pg / mL and the level of UCH-L1 is less than about 1000 pg / mL; (v) a trauma mechanism is present and the level of GFAP is less than about 120 pg / mL, the level of UCH-L1 is less than about 500 pg / mL, or the level of GFAP is less than about 120 pg / mL and the level of UCH-L1 is less than about 500 pg / mL; or (vi) a trauma mechanism is present and the level of GFAP is less than about 120 pg / mL, the level of UCH-L1 is less than about 1000 pg / mL, or the level of GFAP is less than about 120 pg / mL and the level of UCH-L1 is less than about 1000 pg / mL.

[0284] In still yet another aspect, the subject is diagnosed as not having a TBI or not likely to have a TBI: (i) a trauma mechanism is present; and (ii) the level of GFAP is less than between about 80 pg / mL to about 120 pg / mL, the level of UCH-L1 is less than between about 500 pg / mL to about 1000 pg / mL, or level of GFAP is less than between about 80 pg / mL to about 120 pg / mL and the level of UCH-L1 is less than between about 500 pg / mL to about 1000 pg / mL.Docket No. 15893; 43644.601

[0285] The diagnostic criteria used in the method to diagnose or aid in the diagnosis of a TBI in a subject having or suspected of having a head injury can also include: (iii) the presence or absence of one or more clinical signs of a TBI. In one aspect, the subject is diagnosed as having a TBI or more likely than not to have a TBI when: (i) a trauma mechanism is present, the level of GFAP is greater than about 80 pg / mL and / or the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is absent; (ii) a trauma mechanism is present, the level of GFAP is greater than about 80 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is absent; (iii) a trauma mechanism is present, the level of GFAP is greater than about 100 pg / mL and / or the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is absent; (iv) a trauma mechanism is present, the level of GFAP is greater than about 100 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is absent; (v) a trauma mechanism is present and the level of GFAP is greater than about 120 pg / mL and the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is absent; (vi) a trauma mechanism is present and the level of GFAP is greater than about 100 pg / mL and the level of UCH-L1 is greater than about 700 pg / mL, and the one or more clinical signs of a TBI is absent; or (vii) a trauma mechanism is present and the level of GFAP is greater than about 120 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is absent.

[0286] In another aspect, the subject is diagnosed as having a TBI or determined more likely than not to have a TBI when: (i) a trauma mechanism is present, the level of GFAP is greater than about 80 pg / mL and / or the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is present; (ii) a trauma mechanism is present, the level of GFAP is greater than about 80 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is present; (iii) a trauma mechanism is present, the level of GFAP is greater than about 100 pg / mL and / or the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is present; (iv) a trauma mechanism is present, the level of GFAP is greater than about 100 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is present; (v) a trauma mechanism is present and the level of GFAP is greater than about 120 pg / mL and / or the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is present; or (vi) a trauma mechanism is present and the level ofDocket No. 15893; 43644.601GFAP is greater than about 120 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is present.

[0287] In yet another aspect, the subject is diagnosed as having a TBI or determined more likely than not to have a TBI when: (i) a trauma mechanism is present; (ii) the level of GFAP is greater than between about 80 pg / mL to about 120 pg / mL and / or the level of UCH-L1 is greater than between about 500 pg / mL to about 1000 pg / mL; and (iii) the one or more clinical signs of a TBI is absent.

[0288] In still yet another aspect, the subject is diagnosed as having a TBI or determined more likely than not to have a TBI when: (i) a trauma mechanism is present; (ii) the level of GFAP is greater than between about 80 pg / mL to about 120 pg / mL and / or the level of UCH- L1 is greater than between about 500 pg / mL to about 1000 pg / mL; and (iii) the one or more clinical signs of a TBI is present.

[0289] The diagnostic criteria used in the method to diagnose or aid in the diagnosis of a TBI in a subject having or suspected of having a head injury can also include (iv) the presence or absence of one or more confounding factors. In one aspect, the subject is diagnosed as not having a TBI when (i) a trauma mechanism is present; (ii) one more clinical signs of a TBI is present; and (iv) the one or more clinical signs of TBI is better accounted for by the presence of the one or more confounding factors.

[0290] The methods described herein contemplate accounting for a variety of trauma mechanisms. Examples of useful trauma mechanisms for adjudicating a TBI diagnosis in the methods of the disclosure include, without limitation, traumas resulting from (i) impact of an object on the subject’s head (e.g., subject’s head is struck by an object); (ii) the subject’s head striking a hard object or surface; (iii) acceleration or deceleration of the subject’s head; (iv) a force generated from a blast or explosion; and (v) combinations of (i)-(iv). In an embodiment, the trauma mechanism is an injury involving: (i) impact of an object on the subject’s head (e.g., subject’s head is struck by an object). In another embodiment, the trauma mechanism is an injury involving: (ii) the subject’s head striking a hard object or surface. In yet another embodiment, the trauma mechanism is an injury involving: (iii) acceleration or deceleration of the subject’s head. In still another embodiment, the trauma mechanism is an injury involving: (iv) a force generated from a blast or explosion. The trauma mechanism can include any combinations of (i)-(iv).

[0291] Aspects of the methods disclosed herein involve receiving neuroimaging results of a subject’s head, for example, to assist in the adjudication of a TBI diagnosis based on the presence or absence of an intracranial abnormality of the subject. The disclosure not limitedDocket No. 15893; 43644.601 by the neuroimaging procedure that is used to determine the presence or absence of an intracranial abnormality. In some aspects, the presence or absence of the intracranial abnormality is determined based on a computed tomography (CT) scan of the subject’s head. In other aspects, the presence or absence of the intracranial abnormality is based on a magnetic resonance imaging of the subject’s head. In some aspects, the neuroimaging procedure is performed within a clinically-relevant time frame, such as for example, within about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, or about 124 hours, of the injury or suspected injury to the subject’s head.

[0292] Aspects of the adjudicating methods of the disclosure involve receiving diagnostic criteria comprising the presence or absence of one or more clinical signs of a TBI. The methods are not limited to any particular clinical TBI signs. Examples of useful clinical signs of a TBI include, without limitation, (i) loss of consciousness; (ii) post-traumatic or peri- traumatic amnesia; (iii) alteration of mental status; and (iv) combinations of (i)-(iii). In one aspect, the one or more clinical signs of a TBI is loss of consciousness. In another aspect, the one or more clinical signs of a TBI is post-traumatic or peri-traumatic amnesia. In yet still another aspect, the one or more clinical signs of a TBI is alteration of mental status. In an yet another aspect, the one or more clinical signs is selected from the group consisting of: (i) loss of consciousness; (ii) post-traumatic or peri-traumatic amnesia; (iii) alteration of mental status; and (iv) combinations of (i)-(iii).

[0293] The alteration of mental status post injury due to a trauma mechanism described herein can be evidenced by diminished responsiveness or inappropriate responses to stimuli, slow responses to questions or instructions, agitation, inability to follow directions with multiple parts, and the subject’s disorientation regarding place, situation, or time.

[0294] In still yet other aspects, the alteration of mental status can be determined based on a Glascow Coma Score (GCS). Alternatively, in still other aspects, the alteration of mental status can be determined using the Ranchos Los Amigos Scale. In still yet other aspects, the alteration of mental status can be determined using the Rivermead Post-Concussion Symptoms Questionnaire. In still further aspects, any combination of GCS, the Ranchos Los Amigos Scale and the Rivermead Post-Concussion Symptoms Questionnaire can be used to determine alteration of mental status in a subject.Docket No. 15893; 43644.601

[0295] In still further aspects, other acute neurological signs of a TBI can be taken into consideration by the adjudication methods disclosed herein. For example, lack of motor coordination upon standing, seizure, or tonic posturing immediately following injury can be used to adjudicate a TBI diagnosis.

[0296] In some embodiments, executing the TBI adjudication algorithm further includes compiling an injury severity indicator based on one or more clinical signs. For example, the one or more clinical signs that can be selected from the group consisting of: (i) a duration of the subject’s loss of consciousness; (ii) a duration of the subject’s post-traumatic or peri- traumatic amnesia; and (iii) a Glasgow Coma Scale (GCS) score, a Ranchos Los Amigos Scale score, and / or a Rivermead Post-Concussion Symptoms Questionnaire score of the subject after a duration of time.

[0297] Aspects of the methods disclosed herein involve receiving and compiling diagnostic criteria comprising the presence or absence of one or more confounding factors. The skilled practitioner will appreciate that a variety of confounding factors can be useful to adjudicate a TBI diagnosis for a subject. Examples of particularly useful confounding factors include, without limitation, (i) acute musculoskeletal pain; (ii) psychological stress; (iii) use of a drug of abuse; (iv) pulmonary / circulatory disruption; (v) syncopy prior to a fall; and (vi) combinations of (i)-(vi). In one aspect, the one or more confounding factors is (i) acute musculoskeletal pain. In another aspect, the one or more confounding factors is (ii) psychological stress. In still yet another aspect, the one or more confounding factors is (iii) use of a drug of abuse. In still yet another aspect, the one or more confounding factors is (iv) pulmonary or circulatory disruption. In still yet another aspect, the one or more confounding factors is (v) syncopy prior to a fall. The disclosure also contemplates (vi) any combinations of (i)-(vi). In yet a further aspect, the one or more confounding factors is selected from the group consisting of: (i) acute musculoskeletal pain; (ii) psychological stress; (iii) use of a drug of abuse; (iv) pulmonary / circulatory disruption; (v) syncopy prior to a fall; and (vi) any combinations of (i)-(vi).

[0298] Aspects of the methods disclosed herein involve receiving and compiling diagnostic criteria comprising the presence or absence of an acute symptom to adjudicate a TBI diagnosis of a subject. The skilled practitioner will appreciate that a variety of acute symptoms can be used to adjudicate a TBI diagnosis of a subject. Examples of useful acute symptoms include, without limitation, a subjective alteration in the subject’s mental status, physical symptoms, cognitive symptoms, emotional symptoms, and combinations thereof. In one aspect, the diagnostic criteria received and compiled comprises (i) a subjective alterationDocket No. 15893; 43644.601 in mental status selected from the group consisting of feeling confused, feeling disoriented, feeling dazed, and combinations thereof. In yet another aspect, the diagnostic criteria received and compiled comprises (ii) a physical symptom selected from the group consisting of a headache, nausea, dizziness, balance problems, vision problems, light sensitivity, noise sensitivity, and combinations thereof. In still yet another aspect, the diagnostic criteria received and compiled comprises (iii) a cognitive symptom selected from the group consisting of feeling slowed down, mental fog, difficulty concentrating, memory problems, and combinations thereof. In still yet further embodiments, the diagnostic criteria received and compiled comprises (iv) an emotional symptom selected from the group consisting of unusual emotional lability, emotional irritability, and combinations thereof. In a further aspect, the diagnostic criteria further comprises the presence of an acute symptom selected from the group consisting of: (i) a subjective alteration in mental status selected from the group consisting of feeling confused, feeling disoriented, feeling dazed, and combinations thereof; (ii) a physical symptom selected from the group consisting of a headache, nausea, dizziness, balance problems, vision problems, light sensitivity, noise sensitivity, and combinations thereof; (iii) a cognitive symptom selected from the group consisting of feeling slowed down, mental fog, difficulty concentrating, memory problems, and combinations thereof; and (iv) an emotional symptom selected from the group consisting of unusual emotional lability, emotional irritability, and combinations thereof.

[0299] Aspects of the methods disclosed herein involve receiving and compiling diagnostic criteria comprising the presence or absence of an impairment on acute clinical examination to adjudicate a TBI diagnosis of a subject. The skilled practitioner will appreciate that a variety of impairments on acute clinical examination can be used to adjudicate a TBI diagnosis of a subject. Examples of useful impairments on acute clinical examination include, without limitation, (i) a cognitive impairment on acute clinical examination; (ii) a balance impairment on acute clinical examination; and (iii) an oculomotor impairment or symptom provocation in response to vestibular-oculomotor challenge on acute clinical examination. In one aspect, the diagnostic criteria received and compiled further comprises (i) a cognitive impairment on acute clinical examination. In another aspect, the diagnostic criteria received and compiled further comprises (ii) a balance impairment on acute clinical examination. In still yet another aspect, the diagnostic criteria received and compiled further comprises (iii) an oculomotor impairment or symptom provocation in response to vestibular-oculomotor challenge on acute clinical examination. In yet another aspect, the received and compiled diagnostic criteria comprises (i) a cognitive impairment onDocket No. 15893; 43644.601 acute clinical examination; (ii) a balance impairment on acute clinical examination; and (iii) an oculomotor impairment or symptom provocation in response to vestibular-oculomotor challenge on acute clinical examination, and (iv) combinations thereof.[0300J Aspects of the methods disclosed herein involve receiving and compiling diagnostic criteria comprising the presence or absence of an elevated biomarker (also referred to interchangeably herein as “analyte of interest”) indicative of an intracranial injury to adjudicate a TBI diagnosis of a subject. The skilled practitioner will appreciate that a variety of elevated biomarker indicative of an intracranial injury can be used to adjudicate a TBI diagnosis of a subject. Examples of such biomarkers include, without limitation, AP0A1, ADAM 10, brain derived nerve growth factor (BDNF), calcium binding protein (SI 00b), C- reactive protein (CRP), glial fibrillary acidic protein (GFAP), glial fibrillary acidic protein breakdown products (GFAP-BDP), neuron specific enolase (NSE), NF-L, peroxidredoxin 6 (PRDX6), Tau, p-Tau, ubiquitin carboxy-terminal hydrolase LI (UCH-L1), and combinations thereof. In some aspects, the biomarker indicative of an intracranial injury is APOA1. In other aspects, the biomarker indicative of an intracranial injury is ADAM10. In still yet further aspects, the biomarker indicative of an intracranial injury is brain derived nerve growth factor (BDNF). In still yet further aspects, the biomarker indicative of an intracranial injury is calcium binding protein (SI 00b). In still yet further aspects, the biomarker indicative of an intracranial injury is C-reactive protein (CRP). In still yet further aspects, the biomarker indicative of an intracranial injury is glial fibrillary acidic protein (GFAP). In still yet further aspects, the biomarker indicative of an intracranial injury is glial fibrillary acidic protein breakdown products (GFAP-BDP). In still yet further aspects, the biomarker indicative of an intracranial injury is neuron specific enolase (NSE). In still yet further aspects, the biomarker indicative of an intracranial injury is NF-L. In still yet further aspects, the biomarker indicative of an intracranial injury is peroxidredoxin 6 (PRDX6). In still yet further aspects, the biomarker indicative of an intracranial injury is Tau. In still yet further aspects, the biomarker indicative of an intracranial injury is p-Tau. In still yet further aspects, the biomarker indicative of an intracranial injury is ubiquitin carboxy-terminal hydrolase LI (UCH-L1). In yet further aspects, the elevated biomarker indicative of an intracranial injury is selected from the group consisting of APO Al, ADAM 10, brain derived nerve growth factor (BDNF), calcium binding protein (SI 00b), C-reactive protein (CRP), glial fibrillary acidic protein (GFAP), glial fibrillary acidic protein breakdown products (GFAP-BDP), neuron specific enolase (NSE), NF-L, peroxidredoxin 6 (PRDX6), Tau, p-Tau, ubiquitin carboxy-terminal hydrolase LI (UCH-L1), and any combinations thereof.Docket No. 15893; 43644.601

[0301] The levels of a biomarker indicative of an intracranial injury can be determined from a sample obtained from a subject having or suspected of having a head injury. In some aspects, the sample is obtained within about 24 hours of a suspected injury to the subject and contacting the sample with an antibody for the biomarker indicative of intracranial injury, such as ubiquitin carboxy -terminal hydrolase LI (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, to allow formation of a complex of the antibody and the biomarker. The method also includes detecting the resulting antibody -biomarker complex.

[0302] In some embodiments, the sample is taken from the subject, such as a human subject, within about 24 hours of injury (e.g., an actual injury) or suspected injury to the head, such as within about 0 to about 6 hours, within about 0 to about 8 hours, within about 0 to about 10 hours, within about 0 to about 12 hours, within about 0 to about 18 hours, within about 6 hours to about 12 hours, within about 6 hours to about 18 hours, or within about 12 hours to about 18 hours. For example, the sample can be taken from the subject, such as 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, 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 of injury or suspected injury to the head. In some embodiments, the onset of the presence of the biomarker, such as UCH-L1, GFAP, or a combination thereof, appears 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, 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 injury or suspected injury to the head.

[0303] Generally, a reference level of the biomarker, such as UCH-L1, GFAP, or a combination thereof, can be employed as a benchmark against which to assess results obtained upon assaying a test sample of the biomarker (e.g., UCH-L1, GFAP, or a combination thereof). Generally, in making such a comparison, the reference level of the biomarker, such as UCH-L1, GFAP, or a combination thereof, is obtained by running a particular assay a sufficient number of times and under appropriate conditions such that a linkage or association of analyte presence, amount or concentration with a particular stage or endpoint of intracranial injury (e.g., TBI) or with particular indicia can be made. Typically,Docket No. 15893; 43644.601 the reference level of the biomarker, such as UCH-L1, GFAP, or a combination thereof, is obtained with assays of reference subjects (or populations of subjects). The biomarker, such as UCH-L1, GFAP, or a combination thereof, measured can include fragments thereof, degradation products thereof, and / or enzymatic cleavage products thereof.

[0304] The nature of the assay employed 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, electrophoresis analysis, a protein assay, a competitive binding assay, a functional protein assay, or chromatography or spectrometry methods, such as high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). Also, the assay can be employed in a clinical chemistry format such as would be known by one of ordinary skill in the art. Such assays are described in further detail herein. It is known in the art that the values (e.g., reference levels, cutoffs, thresholds, specificities, sensitivities, concentrations of calibrators and / or controls etc.) used in an assay that employs specific sample type (e.g., such as an immunoassay that utilizes serum or a point-of-care device that employs whole blood) can be extrapolated to other assay formats using known techniques 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 calibrator employed in the assay to make the sample concentration read higher or lower to get a slope that aligns with the comparator method. Other methods of standardizing results obtained on one assay to another assay are well known and have been described in the literature (See, for example, David Wild, Immunoassay Handbook, 4thedition, chapter 3.5, pages 315-322, the contents of which are herein incorporated by reference).

[0305] In certain aspects, the method comprises diagnosing a subject as having a TBI based on the received and compiled diagnostic criteria of the subject. In an aspect, a subject is diagnosed as having a TBI when: (i) a mechanism of trauma is present; and (ii) at least two acute symptoms are present. In another aspect, a subject is diagnosed as having a TBI when: (i) a mechanism of trauma is present; and (iii) at least one impairment on acute clinical examination is present. In yet another aspect, a subject is diagnosed as having a TBI when: (i) a mechanism of trauma is present; and (iii) at least one elevated biomarker indicative of an intracranial injury is present. In still yet another aspect, a subject is diagnosed as having a TBI when: (i) a mechanism of trauma is present; (ii) at least two acute symptoms are present; (iii) either at least one impairment on acute clinical examination is present or at least one elevated biomarker indicative of an intracranial injury is present. In yet a further aspect, aDocket No. 15893; 43644.601 subject is diagnosed as having a TBI when: (i) a mechanism of trauma is present; (ii) at least two acute symptoms are present; (iii) either at least one impairment on acute clinical examination is present or at least one elevated biomarker indicative of an intracranial injury is present; and (iv) the at least two acute symptoms and either the at least one impairment on acute clinical examination or at least one elevated biomarker indicative of the intracranial injury are not better accounted for by the one or more confounding factors.

[0306] In a further aspect, a subject is diagnosed as having a TBI or determined more likely than not to have a TBI when: (i) a mechanism of trauma is present; (ii) at least two acute symptoms are present; (iii) either at least one impairment on acute clinical examination is present or at least one elevated biomarker indicative of an intracranial injury is present; and (iv) the at least two acute symptoms and either the at least one impairment on acute clinical examination or at least one elevated biomarker indicative of the intracranial injury are not better accounted for by the one or more confounding factors.

[0307] In some aspects, the diagnosis is a mild TBI. In other aspects, the diagnosis is a moderate TBI. In still other aspects, the diagnosis is a moderate to severe TBI. In still yet other aspects, the diagnosis is a severe TBI. In one aspect, a moderate, moderate-to-severe, or severe TBI is diagnosed when: (i) a duration of the subject’s loss of consciousness exceeds 30 minutes; (ii) a duration of the subject’s post-traumatic or peri-traumatic amnesia exceeds 24 hours; or (iii) a GCS score of the subject after 30 minutes is less than 13.

[0308] In some aspects, the methods of adjudicating a TBI diagnosis of a subject can be implemented on a computer, a tablet, a smart phone, or any combination thereof. In such aspects, the diagnostic criteria can be received from a database comprising electronic medical records of the subject. In such aspects, the method further comprises storing the adjudicated TBI diagnosis in a database comprising an electronic medical record of the subject. In such aspects, the method further comprises recommending a treatment based on the adjudicated TBI diagnosis. In such aspects, the method further comprises monitoring a treatment recommended based on the adjudicated TBI diagnosis. In such aspects, the treatment comprises a prescription for a medication. In such aspects, the prescription is automatically sent to a preferred pharmacy of the subject.

[0309] In some embodiments, the method further includes treating the subject, such as a human subject, with a traumatic brain injury treatment and / or monitoring the subject, as described below in Section 4.

[0310] In still further embodiments, the present disclosure relates to an apparatus, device, or instrument. The apparatus, device, or instrument contains software to execute one or moreDocket No. 15893; 43644.601 tasks, including the performance of the methods described in this Section 2. In some aspects, the apparatus, device, or instrument contains software to automatically determine the next appropriate step in the methods described herein. The software may display this determination, such as on a graphical user interface.

[0311] In some aspects, the apparatus, device, or instruments stores software that instructs processor or processor circuitry to execute or instantiate a given task. In some aspects, the software stores machine-readable instructions that cause processor circuity to execute or instantiate a given task. The machine-readable instructions may be one or more executable programs or portion(s) of an executable program for execution by a computer. The programs may be embodied in software stored on a non-transitory computer readable storage medium such as a CD-ROM, a floppy disk, a hard drive, a DVD, a Blu-ray disk, or a memory associated with the processors. Alternatively, the entire programs and / or parts thereof could alternatively be executed by a device other than the processors and / or embodied in firmware or dedicated hardware. Additionally or alternatively, processes may be implemented by one or more hardware circuits (e.g., discrete and / or integrated analog and / or digital circuitry, an FPGA, an ASIC, a comparator, an operational-amplifier (op-amp), a logic circuit, etc.) structured to perform the corresponding operation without executing software or firmware.

[0312] The machine-readable instructions may be stored in one or more of a compressed format, an encrypted format, a fragmented format, a compiled format, an executable format, a packaged format, etc. Machine readable instructions as described herein may be stored as data (e.g., portions of instructions, code, representations of code, etc.) that may be utilized to create, manufacture, and / or produce machine executable instructions. For example, the machine-readable instructions may be fragmented and stored on one or more storage devices and / or computing devices (e.g., servers). The machine-readable instructions may require one or more of installation, modification, adaptation, updating, combining, supplementing, configuring, decryption, decompression, unpacking, distribution, reassignment, compilation, etc. in order to make them directly readable, interpretable, and / or executable by a computing device and / or other machine. For example, the machine-readable instructions may be stored in multiple parts, which are individually compressed, encrypted, and stored on separate computing devices, wherein the parts when decrypted, decompressed, and combined form a set of executable instructions that implement a program such as that described herein.

[0313] In another example, the machine-readable instructions may be stored in a state in which they may be read by a computer, but require addition of a library (e.g., a dynamic link library (DLL)), a software development kit (SDK), an application programming interfaceDocket No. 15893; 43644.601(API), etc. in order to execute the instructions on a particular computing device or other device. In another example, the machine-readable instructions may need to be configured (e.g., settings stored, data input, network addresses recorded, etc.) before the machine- readable instructions and / or the corresponding program(s) can be executed in whole or in part. Thus, the disclosed machine-readable instructions and / or corresponding program(s) are intended to encompass such machine-readable instructions and / or program(s) regardless of the particular format or state of the machine-readable instructions and / or program(s) when stored or otherwise at rest or in transit.

[0314] The machine-readable instructions described herein can be represented by any past, present, or future instruction language, scripting language, programming language, etc. For example, the machine-readable instructions may be represented using any of the following languages: C, C++, Java, C#, Perl, Python, JavaScript, HyperText Markup Language (HTML), Structured Query Language (SQL), Swift, etc.

[0315] The machine-readable instructions may be stored on a non-transitory computer and / or non-transitory machine-readable medium such as a hard disk drive, a flash memory, a read-only memory, a compact disk, a digital versatile disk, a cache, a random-access memory and / or any other storage device or storage disk in which information is stored for any duration (e.g., for extended time periods, permanently, for brief instances, for temporarily buffering, and / or for caching of the information). As used herein, the term “non-transitory computer readable medium” is defined to include any type of computer readable storage device and / or storage disk and to exclude propagating signals and to exclude transmission media.

[0316] In some further aspects, disclosed herein is a system for evaluation (also referred to herein as an “evaluation system”) to adjudicate whether a subject has suffered a traumatic brain injury using the TBI adjudication algorithm and the methods described herein.

[0317] In further aspects, disclosed herein is a computer device that comprises a storage device having machine-readable instructions stored thereon, and a processor. The processor executes the machine-readable instructions to perform the steps of the above computer- implemented method or the steps performed by the above evaluation system.

[0318] In further aspects, disclosed herein is an apparatus, device, or instrument that comprises a storage device having machine-readable instructions stored thereon, and a processor. The processor instantiates or executes the machine-readable instructions to perform the steps of the TBI adjudication algorithm and the methods described herein.Docket No. 15893; 43644.601

[0319] In further aspects, disclosed herein is a non-transitory machine-readable storage medium having machine-readable instructions stored thereon, wherein the machine-readable instructions are configured to be executed by a processor to perform the steps of the above described computer-implemented method or the steps performed by the above one or more systems, such as the evaluation system.

[0320] In yet further aspects, disclosed herein is a non-transitory machine-readable storage medium. The non-transitory machine-readable storage medium can comprise instructions to cause one or processors or processor circuity to performs the steps of the TBI adjudication algorithm and the methods described herein.

[0321] In still yet further embodiments disclosed herein are systems. In some aspects, the system comprises an apparatus, one or more devices, or instrument and certain instructions. With respect to the apparatus, the apparatus, one or more devices, or instrument comprises a storage device having machine readable instructions stored thereon and a processor or processor circuitry which instantiates or executes the machine-readable instructions perform the methods described herein. The instructions that are included in the system can be provided as written instructions, such as in a product insert, product manual, excel document, etc., on a mobile device (e.g., tablet, a smart phone), on a website, in an e-mail, or any combination thereof.3. Apparatus, Non-Transitory Machine-Readable Storage Medium, and Systems

[0322] In some aspects, disclosed herein are apparatus, machine-readable storage mediums and systems for use in implementing or performing the methods described in Section 2. More specifically, the apparatus, machine-readable storage mediums and systems described herein can be used to identify GFAP, UCH-L1, or GFAP and UCH-L1 in one or more biological samples obtained from a subject, determine the level of GFAP, UCH-L1 or GFAP and UCH-L1 in the one or more biological samples, obtain diagnostic criteria, adjudicate a TBI diagnosis, and communicate from an apparatus (e.g., such as a point-of-care, non-point-of-care, or a point-of-care or non-point-of-care apparatus), whether the level of GFAP, UCH-L1 or GFAP and UCH-L1, is greater than, less than, or between one or more reference or other levels. The term “apparatus” is used interchangeably with “device” or “instrument”.

[0323] In another aspect, the present disclosure relates to an apparatus, device, or instrument. The apparatus, device, or instrument contains software to execute one or more tasks, including the performance of the methods described in Section 2. In some aspects, theDocket No. 15893; 43644.601 apparatus, device, or instrument contains software to automatically determine the next appropriate step in a method described herein. For example, the apparatus, device, or instrument may contain software that determines the amount or presence of an analyte of interest. The software may display this determination, such as on a graphical user interface.

[0324] In some aspects, the apparatus, device, or instruments stores software that instructs processor or processor circuitry to execute or instantiate a given task. In some aspects, the software stores machine-readable instructions that cause processor circuity to execute or instantiate a given task. The machine-readable instructions may be one or more executable programs or portion(s) of an executable program for execution by a computer. The programs may be embodied in software stored on a non-transitory computer readable storage medium such as a CD-ROM, a floppy disk, a hard drive, a DVD, a Blu-ray disk, or a memory associated with the processors. Alternatively, the entire programs and / or parts thereof could alternatively be executed by a device other than the processors and / or embodied in firmware or dedicated hardware. Additionally or alternatively, processes may be implemented by one or more hardware circuits (e.g., discrete and / or integrated analog and / or digital circuitry, an FPGA, an ASIC, a comparator, an operational-amplifier (op-amp), a logic circuit, etc.) structured to perform the corresponding operation without executing software or firmware.

[0325] The machine-readable instructions may be stored in one or more of a compressed format, an encrypted format, a fragmented format, a compiled format, an executable format, a packaged format, etc. Machine readable instructions as described herein may be stored as data (e.g., portions of instructions, code, representations of code, etc.) that may be utilized to create, manufacture, and / or produce machine executable instructions. For example, the machine-readable instructions may be fragmented and stored on one or more storage devices and / or computing devices (e.g., servers). The machine-readable instructions may require one or more of installation, modification, adaptation, updating, combining, supplementing, configuring, decryption, decompression, unpacking, distribution, reassignment, compilation, etc. in order to make them directly readable, interpretable, and / or executable by a computing device and / or other machine. For example, the machine-readable instructions may be stored in multiple parts, which are individually compressed, encrypted, and stored on separate computing devices, wherein the parts when decrypted, decompressed, and combined form a set of executable instructions that implement a program such as that described herein.

[0326] In another example, the machine-readable instructions may be stored in a state in which they may be read by a computer, but require addition of a library (e.g., a dynamic link library (DLL)), a software development kit (SDK), an application programming interfaceDocket No. 15893; 43644.601(API), etc. in order to execute the instructions on a particular computing device or other device. In another example, the machine-readable instructions may need to be configured (e.g., settings stored, data input, network addresses recorded, etc.) before the machine- readable instructions and / or the corresponding program(s) can be executed in whole or in part. Thus, the disclosed machine-readable instructions and / or corresponding program(s) are intended to encompass such machine-readable instructions and / or program(s) regardless of the particular format or state of the machine-readable instructions and / or program(s) when stored or otherwise at rest or in transit.

[0327] The machine-readable instructions described herein can be represented by any past, present, or future instruction language, scripting language, programming language, etc. For example, the machine-readable instructions may be represented using any of the following languages: C, C++, Java, C#, Perl, Python, JavaScript, HyperText Markup Language (HTML), Structured Query Language (SQL), Swift, etc.

[0328] The machine-readable instructions may be stored on a non-transitory computer and / or non-transitory machine-readable medium such as a hard disk drive, a flash memory, a read-only memory, a compact disk, a digital versatile disk, a cache, a random-access memory and / or any other storage device or storage disk in which information is stored for any duration (e.g., for extended time periods, permanently, for brief instances, for temporarily buffering, and / or for caching of the information). As used herein, the term “non-transitory computer readable medium” is defined to include any type of computer readable storage device and / or storage disk and to exclude propagating signals and to exclude transmission media.

[0329] In some aspects, the system comprises:

[0330] a. a data-obtaining module to obtain data of a level of glial fibrillary acidic protein (GFAP), a level of ubiquitin carboxy-terminal hydrolase LI (UCH-L1), or a level of GFAP and a level of UCH-L1 from one or more assays performed on at least one or more samples obtained from a subject after an actual or suspected ABI;

[0331] b. an evaluation module to analyze the data to obtain:

[0332] i) a first evaluation result if the subject’s: (a) GFAP level is less than from about 80 pg / mL (e.g., 80, 70, 60, 50, or less); (b) UCH-L1 level is less than about 500 pg / mL (e.g., 500, 400, 300, 200, 100, or less); or (c) GFAP level is less than about 80 pg / mL (e.g., 80, 70, 60, 50, or less) and UCH-L1 level is less than about 500 pg / mL (e.g., 500, 400, 300, 200, 100, or less);Docket No. 15893; 43644.601

[0333] ii) a second evaluation result if the subject’s: (a) GFAP level is greater than about 80 pg / mL (e.g., 80, 90, 100, 120, 150, 200, or greater); (b) UCH-L1 level is greater than about 500 (e.g., 500, 600, 700, 800, 900, 1000 or more); or (c) GFAP level is greater than about 80 pg / mL (e.g., 80, 90, 100, 120, 150, 200, or greater) and UCH-L1 level is greater than about 500 pg / mL (e.g., 500, 600, 700, 800, 900, 1000 or more); and

[0334] c. a data-outputting module to output the evaluation result (e.g., the first evaluation results or the second evaluation result),

[0335] wherein the first evaluation result is associated with determining that the subject has not suffered from an actual ABI; the second evaluation result is associated with determining that the subject sustained an actual ABI.

[0336] In further embodiments, the system comprises:

[0337] a. a data-obtaining module to obtain data of a level of glial fibrillary acidic protein (GFAP), a level of ubiquitin carboxy-terminal hydrolase LI (UCH-L1), or a level of GFAP and a level of UCH-L1 from one or more assays performed on at least one or more samples obtained from a subject after an actual or suspected ABI;

[0338] b. an evaluation module to analyze the data to obtain:

[0339] i) a first evaluation result if the subject’s: (a) GFAP level is less than about 100 pg / mL; (b) UCH-L1 level is less than about 700 pg / mL; or (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than about 700 pg / mL;

[0340] ii) a second evaluation result if the subject’s: (a) GFAP level is greater than about 100 pg / mL; (b) UCH-L1 level is greater than about 700 pg / mL; or (c) GFAP level is greater than about 100 and UCH-L1 level is greater than about 700 pg / mL; and

[0341] c. a data-outputting module to output the evaluation result (e.g., the first evaluation results or the second evaluation result),

[0342] wherein the first evaluation result is associated with determining that the subject has not suffered from an actual ABI; the second evaluation result is associated with determining that the subject sustained an actual ABI.

[0343] In other aspects, the evaluation system comprises:

[0344] a. a data-obtaining module to obtain data of A) a level of glial fibrillary acidic protein (GFAP), a level of ubiquitin carboxy-terminal hydrolase LI (UCH-L1), or a level of GFAP and a level of UCH-L1 from one or more assays performed on at least one or more samples obtained from a subject after an actual or suspected ABI; and B) the presence or absence of a trauma mechanism;

[0345] b. an evaluation module to analyze the data to obtain:Docket No. 15893; 43644.601

[0346] i) a first evaluation result if the subject’s trauma mechanism is absent and: (a) GFAP level is less than from about 80 pg / mL (e.g., 80, 70, 60, 50, or less); (b) UCH-L1 level is less than about 500 pg / mL (e.g., 500, 400, 300, 200, 100, or less); or (c) GFAP level is less than about 80 pg / mL (e.g., 80, 70, 60, 50, or less) and UCH-L1 level is less than about 500 pg / mL (e.g., 500, 400, 300, 200, 100, or less);

[0347] ii) a second evaluation result if the subject’s trauma mechanism is present and: (a) GFAP level is greater than about 80 pg / mL (e.g., 80, 90, 100, 120, 150, 200, or greater); (b) UCH-L1 level is greater than about 500 (e.g., 500, 600, 700, 800, 900, 1000 or more); or (c) GFAP level is greater than about 80 pg / mL (e.g., 80, 90, 100, 120, 150, 200, or greater) and UCH-L1 level is greater than about 500 pg / mL (e.g., 500, 600, 700, 800, 900, 1000 or more); and

[0348] c. a data-outputting module to output the evaluation result (e.g., the first evaluation results or the second evaluation result),

[0349] wherein the first evaluation result is associated with determining that the subject has not suffered from an actual ABI; the second evaluation result is associated with determining that the subject sustained an actual ABI.

[0350] In still other aspects, the evaluation system comprises:

[0351] a. a data-obtaining module to obtain data of A) a level of glial fibrillary acidic protein (GFAP), a level of ubiquitin carboxy-terminal hydrolase LI (UCH-L1), or a level of GFAP and a level of UCH-L1 from one or more assays performed on at least one or more samples obtained from a subject after an actual or suspected ABI; and B) the presence or absence of a trauma mechanism;

[0352] b. an evaluation module to analyze the data to obtain:

[0353] i) a first evaluation result if the subject’s trauma mechanism is absent and: (a) GFAP level is less than from about 100 pg / mL; (b) UCH-L1 level is less than from about 700 pg / mL; (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than from about 700 pg / mL;

[0354] ii) a second evaluation result if the subject’s trauma mechanism is present and: (a) GFAP level is greater than about 100 pg / mL; (b) UCH-L1 level is greater than about 700 pg / mL; or (c) GFAP level is greater than about 100 pg / mL and UCH-L1 level is greater than about 700 pg / mL; and

[0355] c. a data-outputting module to output the evaluation result (e.g., the first evaluation results or the second evaluation result),Docket No. 15893; 43644.601

[0356] wherein the first evaluation result is associated with determining that the subject has not suffered from an actual ABI; the second evaluation result is associated with determining that the subject sustained an actual ABI.

[0357] In certain aspects, the evaluation system comprises:

[0358] a. a data-obtaining module to obtain data of A) a level of glial fibrillary acidic protein (GFAP), a level of ubiquitin carboxy-terminal hydrolase LI (UCH-L1), or a level of GFAP and a level of UCH-L1 from one or more assays performed on at least one or more samples obtained from a subject after an actual or suspected ABI; B) the presence or absence of a trauma mechanism; and C) the presence or absence of one or more clinical signs of a TBI;

[0359] b. an evaluation module to analyze the data to obtain:

[0360] i) a first evaluation result if the subject’s trauma mechanism is absent, the one or more clinical signs of a TBI are absent, and(a) GFAP level is less than from about 80 pg / mL (e.g., 80, 70, 60, 50, or less); (b) UCH-L1 level is less than about 500 pg / mL (e.g., 500, 400, 300, 200, 100, or less); or (c) GFAP level is less than about 80 pg / mL (e.g., 80, 70, 60, 50, or less) and UCH-L1 level is less than about 500 pg / mL (e.g., 500, 400, 300, 200, 100, or less);

[0361] ii) a second evaluation result if the subject’s trauma mechanism is present, the one or more clinical signs of a TBI are absent, and(a) GFAP level is greater than about 80 pg / mL (e.g., 80, 90, 100, 120, 150, 200, or greater); (b) UCH-L1 level is greater than about 500 (e.g., 500, 600, 700, 800, 900, 1000 or more); or (c) GFAP level is greater than about 80 pg / mL (e.g., 80, 90, 100, 120, 150, 200, or greater) and UCH-L1 level is greater than about 500 pg / mL (e.g., 500, 600, 700, 800, 900, 1000 or more); and

[0362] c. a data-outputting module to output the evaluation result (e.g., the first evaluation results or the second evaluation result),

[0363] wherein the first evaluation result is associated with determining that the subject has not suffered from an actual ABI; the second evaluation result is associated with determining that the subject sustained an actual ABI.

[0364] In additional aspects, the evaluation system comprises:

[0365] a. a data-obtaining module to obtain data of A) a level of glial fibrillary acidic protein (GFAP), a level of ubiquitin carboxy-terminal hydrolase LI (UCH-L1), or a level of GFAP and a level of UCH-L1 from one or more assays performed on at least one or more samples obtained from a subject after an actual or suspected ABI; B) the presence or absence of a trauma mechanism; and C) the presence or absence of one or more clinical signs of a TBI;Docket No. 15893; 43644.601

[0366] b. an evaluation module to analyze the data to obtain:

[0367] i) a first evaluation result if the subject’s trauma mechanism is absent, the one or more clinical signs of a TBI are absent, and: (a) GFAP level is less than from about 100 pg / mL; (b) UCH-L1 level is less than from about 700 pg / mL; (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than from about 700 pg / mL;

[0368] ii) a second evaluation result if the subject’s trauma mechanism is present, the one or more clinical signs of a TBI are absent, and: (a) GFAP level is greater than about 100 pg / mL; (b) UCH-L1 level is greater than about 700 pg / mL; or (c) GFAP level is greater than about 100 pg / mL and UCH-L1 level is greater than about 700 pg / mL; and

[0369] c. a data-outputting module to output the evaluation result (e.g., the first evaluation results or the second evaluation result),

[0370] wherein the first evaluation result is associated with determining that the subject has not suffered from an actual ABI; the second evaluation result is associated with determining that the subject sustained an actual ABI.

[0371] In additional aspects, the evaluation system comprises:

[0372] a. a data-obtaining module to obtain data of A) a level of glial fibrillary acidic protein (GFAP), a level of ubiquitin carboxy-terminal hydrolase LI (UCH-L1), or a level of GFAP and a level of UCH-L1 from one or more assays performed on at least one or more samples obtained from a subject after an actual or suspected ABI; B) the presence or absence of a trauma mechanism; and C) the presence or absence of one or more clinical signs of a TBI;

[0373] b. an evaluation module to analyze the data to obtain:

[0374] i) a first evaluation result if the subject’s trauma mechanism is absent, the one or more clinical signs of a TBI are absent, and: (a) GFAP level is less than from about 100 pg / mL; (b) UCH-L1 level is less than from about 700 pg / mL; (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than from about 700 pg / mL;

[0375] ii) a second evaluation result if the subject’s trauma mechanism is present, the one or more clinical signs of a TBI are absent, and: (a) GFAP level is greater than about 100 pg / mL; (b) UCH-L1 level is greater than about 700 pg / mL; or (c) GFAP level is greater than about 100 pg / mL and UCH-L1 level is greater than about 700 pg / mL; and

[0376] c. a data-outputting module to output the evaluation result (e.g., the first evaluation results or the second evaluation result),Docket No. 15893; 43644.601

[0377] wherein the first evaluation result is associated with determining that the subject has not suffered from an actual ABI; the second evaluation result is associated with determining that the subject sustained an actual ABI.

[0378] In still further aspects of the above evaluation system, the GFAP level, UCH-L1 level, or GFAP and UCH-L1 level is correlated with a level associated with: (a) a subject’s Glasgow Coma Scale Score; (b) a subject’s Extended Glasgow Outcome Scale Score; (c) a positive CT scan for a TBI; (d) a negative CT scan for a TBI; or (e) any combinations of (a)- (d).

[0379] In still other aspects of the above evaluation system, measuring the GFAP level, UCH-L1 level, or GFAP level and UCH-L1 level is done by immunoassay. In still further aspects of the above evaluation system, measuring the GFAP level, UCH-L1 level, or GFAP level and UCH-L1 level is done by the GFAP level, UCH-L1 level, or GFAP level and UCH- L1 level is done by a clinical chemistry assay.

[0380] In still further aspects of the above evaluation system, the assay is a point-of-care assay or single molecule detection.

[0381] In still further aspects of the above evaluation system, the sample is selected from the group consisting of a blood sample, a urine sample, a cerebrospinal fluid sample, a tissue sample, a bodily fluid sample, a saliva sample, an oropharyngeal specimen, and a nasopharyngeal specimen.

[0382] In still yet further aspects of the above evaluation system, the actual or suspected ABI is caused by physical shaking, blunt impact by an external mechanical or other force that results in a closed or open head trauma, one or more falls, explosions or blasts or other types of blunt force trauma. Specifically, in some aspects, the actual or suspected ABI is a traumatic brain injury. In yet further aspects, the actual or suspected ABI is a subacute ABI. In other aspects, when the actual or suspected ABI is a traumatic brain injury, the traumatic brain injury is a subacute traumatic brain injury.

[0383] In yet further aspects of the above evaluation system, the actual or suspected ABI is caused by fire exposure, ingestion of a chemical or toxin, or a combination of fire exposure, and ingestion of a chemical or toxin. Specifically, the chemical or toxin is mold, asbestos, a pesticide, an insecticide, an organic solvent, a paint, a glue, a gas, an organic metal, a drug of abuse or one or more combinations thereof.

[0384] In still further aspects of the above evaluation system, the actual or suspected ABI is caused by an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viralDocket No. 15893; 43644.601 infection, a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combinations thereof.

[0385] In yet further aspects of the above evaluation system, the blood sample is whole blood, serum or plasma, either venous blood or capillary blood. In yet further aspects, the blood sample is obtained from venous blood and is whole blood, serum, or plasma. In still yet other aspects, blood sample is obtained from capillary blood and is whole blood, serum, or plasma.

[0386] In still further aspects of the above evaluation system, the subject is a human subject.

[0387] In further aspects, disclosed herein is a computer device that comprises a storage device having machine-readable instructions stored thereon, and a processor. The processor executes the machine-readable instructions to perform the steps of the above computer- implemented method or the steps performed by the above evaluation system.

[0388] In further aspects, disclosed herein is an apparatus, device, or instrument that comprises a storage device having machine-readable instructions stored thereon, and a processor. The processor instantiates or executes the machine-readable instructions to:

[0389] (A) identify GFAP (as a first biomarker), identify UCH-L1 (as a second biomarker), or both GFAP and UCH-L1 (as first and second biomarkers, respectively) in at least one or more biological samples obtained from a subject using at least one assay, where the at least one or more samples is obtained from the subject after an actual or suspected ABI;

[0390] (B) determine a level of GFAP in the one or more samples, determine a level ofUCH-L1 in one or more samples, or determine a level of GFAP in one or more samples and a level of UCH-L1 in one or more samples, and

[0391] (C) determine the presence or absence of a trauma mechanism and / or one or more clinical signs of a TBI; and

[0392] (D) communicate from the apparatus, device, or instrument (e.g., such as displaying on the apparatus, device, or instrument) whether the subject’s level of (a) GFAP is greater than or less than a reference level of (i) about 80 pg / mL (ii) greater than or less than a reference level of 100 pg / mL; or (iii) greater than or less than a reference level of about 120 pg / mL; (b) UCH-L1 is above, below, or between a reference level of (i) about 500 pg / mL (ii) greater than or less than a reference level of about 700 pg / mL; or (iii) greater than or less than a reference level of about 1000 pg / mL; (c) the presence or absence of a trauma mechanism; (d) the presence or absence of one or more clinical signs of a TBI; or any combination of (a) through (d).Docket No. 15893; 43644.601

[0393] In some aspects, the communication from the apparatus, device, or instrument is whether the subject’s:

[0394] (A) (a) GFAP level is less than about 100 pg / mL; (b) UCH-L1 level is less than about 700 pg / mL; or (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than about 700 pg / mL; or

[0395] (B) (a) GFAP level is greater than about 100 pg / mL; (b) UCH-L1 level is greater than about 700 pg / mL; or (c) GFAP level is greater than about 100 pg / mL and UCH-L1 level is greater than about 700 pg / mL;

[0396] In still further aspects, the communication from the apparatus, device, or instrument is whether the subject’s:

[0397] (A) a trauma mechanism is absent and (a) GFAP level is less than about 100 pg / mL; (b) UCH-L1 level is less than about 700 pg / mL; or (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than about 700 pg / mL; or

[0398] (B) a trauma mechanism is present and (a) GFAP level is greater than about 100 pg / mL; (b) UCH-L1 level is greater than about 700 pg / mL; or (c) GFAP level is greater than about 100 pg / mL and UCH-L1 level is greater than about 700 pg / mL.

[0399] In still other aspects, the communication from the apparatus, device, or instrument is whether the subject’s:

[0400] (A) a trauma mechanism is absent, one or more clinical signs of a TBI are absent, and (a) GFAP level is less than about 100 pg / mL; (b) UCH-L1 level is less than about 700 pg / mL; or (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than about 700 pg / mL; or

[0401] (B) a trauma mechanism is present, one or more clinical signs of a TBI are present, and (a) GFAP level is greater than about 100 pg / mL; (b) UCH-L1 level is greater than about 700 pg / mL; or (c) GFAP level is greater than about 100 pg / mL and UCH-L1 level is greater than about 700 pg / mL.

[0402] In further aspects, disclosed herein is a non-transitory machine-readable storage medium having machine-readable instructions stored thereon, wherein the machine-readable instructions are configured to be executed by a processor to perform the steps of the above described computer-implemented method or the steps performed by the above one or more systems, such as the evaluation system.

[0403] In yet further aspects, disclosed herein is a non-transitory machine-readable storage medium. The non-transitory machine-readable storage medium can comprise instructions to cause one or processors or processor circuity to at least:Docket No. 15893; 43644.601

[0404] (A) identify GFAP (as a first biomarker), identify UCH-L1 (as a second biomarker), or both GFAP and UCH-L1 (as first and second biomarkers, respectively) in at least one or more biological samples obtained from a subject using at least one assay, where the at least one or more samples is obtained from the subject after an actual or suspected ABI;

[0405] (B) determine a level of GFAP in the one or more samples, determine a level ofUCH-L1 in one or more samples, or determine a level of GFAP in one or more samples and a level of UCH-L1 in one or more samples, and

[0406] (C) determine the presence or absence of a trauma mechanism and / or one or more clinical signs of a TBI; and

[0407] (D) communicate from the apparatus, device, or instrument (e.g., such as displaying on the apparatus, device, or instrument) whether the subject’s level of (a) GFAP is greater than or less than a reference level of (i) about 80 pg / mL (ii) greater than or less than a reference level of 100 pg / mL; or (iii) greater than or less than a reference level of about 120 pg / mL; (b) UCH-L1 is above, below, or between a reference level of (i) about 500 pg / mL (ii) greater than or less than a reference level of about 700 pg / mL; or (iii) greater than or less than a reference level of about 1000 pg / mL; (c) the presence or absence of a trauma mechanism; (d) the presence or absence of one or more clinical signs of a TBI; or any combination of (a) through (d).

[0408] In some aspects, the communication from the apparatus, device, or instrument is whether the subject’s:

[0409] (A) (a) GFAP level is less than about 100 pg / mL; (b) UCH-L1 level is less than about 700 pg / mL; or (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than about 700 pg / mL; or

[0410] (B) (a) GFAP level is greater than about 100 pg / mL; (b) UCH-L1 level is greater than about 700 pg / mL; or (c) GFAP level is greater than about 100 pg / mL and UCH-L1 level is greater than about 700 pg / mL;

[0411] In still further aspects, the communication from the apparatus, device, or instrument is whether the subject’s:

[0412] (A) a trauma mechanism is absent and (a) GFAP level is less than about 100 pg / mL; (b) UCH-L1 level is less than about 700 pg / mL; or (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than about 700 pg / mL; or

[0413] (B) a trauma mechanism is present and (a) GFAP level is greater than about 100 pg / mL; (b) UCH-L1 level is greater than about 700 pg / mL; or (c) GFAP level is greater than about 100 pg / mL and UCH-L1 level is greater than about 700 pg / mL.Docket No. 15893; 43644.601

[0414] In still other aspects, the communication from the apparatus, device, or instrument is whether the subject’s:

[0415] (A) a trauma mechanism is absent, one or more clinical signs of a TBI are absent, and (a) GFAP level is less than about 100 pg / mL; (b) UCH-L1 level is less than about 700 pg / mL; or (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than about 700 pg / mL; or

[0416] (B) a trauma mechanism is present, one or more clinical signs of a TBI are present, and (a) GFAP level is greater than about 100 pg / mL; (b) UCH-L1 level is greater than about 700 pg / mL; or (c) GFAP level is greater than about 100 pg / mL and UCH-L1 level is greater than about 700 pg / mL.

[0417] In still yet further aspects disclosed herein are systems. In some aspects, the system comprises an apparatus, device, or instrument and certain instructions. With respect to the apparatus, the apparatus, device, or instrument comprises a storage device having machine readable instructions stored thereon and a processor or processor circuitry which instantiates or executes the machine-readable instructions to:

[0418] (A) identify GFAP (as a first biomarker), identify UCH-L1 (as a second biomarker), or both GFAP and UCH-L1 (as first and second biomarkers, respectively) in at least one or more biological samples obtained from a subject using at least one assay, where the at least one or more samples is obtained from the subject within after about twenty -four hours to (i) about one week, (ii) about two weeks, or (iii) about three weeks, after an actual or suspected ABI;

[0419] (B) determine a level of GFAP in the one or more samples, determine a level ofUCH-L1 in one or more samples, or determine a level of GFAP in one or more samples and a level of UCH-L1 in one or more samples, and

[0420] (D) communicate from the apparatus, device, or instrument (e.g., such as displaying on the apparatus, device, or instrument) whether the subject’s level of (a) GFAP is greater than or less than a reference level of (i) about 80 pg / mL (ii) greater than or less than a reference level of 100 pg / mL; or (iii) greater than or less than a reference level of about 120 pg / mL; (b) UCH-L1 is above, below, or between a reference level of (i) about 500 pg / mL (ii) greater than or less than a reference level of about 700 pg / mL; or (iii) greater than or less than a reference level of about 1000 pg / mL; (c) the presence or absence of a trauma mechanism; (d) the presence or absence of one or more clinical signs of a TBI; or any combination of (a) through (d).Docket No. 15893; 43644.601

[0421] In some aspects, the communication from the apparatus, device, or instrument is whether the subject’s:

[0422] (A) (a) GFAP level is less than about 100 pg / mL; (b) UCH-L1 level is less than about 700 pg / mL; or (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than about 700 pg / mL; or

[0423] (B) (a) GFAP level is greater than about 100 pg / mL; (b) UCH-L1 level is greater than about 700 pg / mL; or (c) GFAP level is greater than about 100 pg / mL and UCH-L1 level is greater than about 700 pg / mL;

[0424] In still further aspects, the communication from the apparatus, device, or instrument is whether the subject’s:

[0425] (A) a trauma mechanism is absent and (a) GFAP level is less than about 100 pg / mL; (b) UCH-L1 level is less than about 700 pg / mL; or (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than about 700 pg / mL; or

[0426] (B) a trauma mechanism is present and (a) GFAP level is greater than about 100 pg / mL; (b) UCH-L1 level is greater than about 700 pg / mL; or (c) GFAP level is greater than about 100 pg / mL and UCH-L1 level is greater than about 700 pg / mL.

[0427] In still other aspects, the communication from the apparatus, device, or instrument is whether the subject’s:

[0428] (A) a trauma mechanism is absent, one or more clinical signs of a TBI are absent, and (a) GFAP level is less than about 100 pg / mL; (b) UCH-L1 level is less than about 700 pg / mL; or (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than about 700 pg / mL; or

[0429] (B) a trauma mechanism is present, one or more clinical signs of a TBI are present, and (a) GFAP level is greater than about 100 pg / mL; (b) UCH-L1 level is greater than about 700 pg / mL; or (c) GFAP level is greater than about 100 pg / mL and UCH-L1 level is greater than about 700 pg / mL.

[0430] In addition to the apparatus, device, or instrument, the system also comprises instructions. The instructions included in the system are for use in determining whether:

[0431] (A) the subject has not suffered from an actual acquired brain injury (AB I) if the subject’s (a) GFAP level is less than about 100 pg / mL; (b) UCH-L1 level is less than about 700 pg / mL; or (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than about 700 pg / mL; or

[0432] (B) the subject has sustained an actual ABI if the subject’s: subject’s (a) GFAP level is greater than about 100 pg / mL; (b) UCH-L1 level is greater than about 700 pg / mL; orDocket No. 15893; 43644.601(c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than about 700 pg / mL In some aspects, the instructions provide determining whether the subject’s:

[0433] (A) the subject has not suffered from an actual acquired brain injury (AB I) if the subject’s trauma mechanism is absent and(a) GFAP level is less than about 100 pg / mL; (b) UCH-L1 level is less than about 700 pg / mL; or (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than about 700 pg / mL; or

[0434] (B) the subject has sustained an actual ABI if the subject’s: subject’s trauma mechanism is present and (a) GFAP level is greater than about 100 pg / mL; (b) UCH-L1 level is greater than about 700 pg / mL; or (c) GFAP level is less than about 100 pg / mL and UCH- L1 level is less than about 700 pg / mL.

[0435] In still further aspects, the instructions provide determining whether the subject’s:

[0436] (A) the subject has not suffered from an actual acquired brain injury (ABI) if the subject’s trauma mechanism is absent, the one or more clinical signs of a TBI are absent, and (a) GFAP level is less than about 100 pg / mL; (b) UCH-L1 level is less than about 700 pg / mL; or (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than about 700 pg / mL; or

[0437] (B) the subject has sustained an actual ABI if the subject’s: subject’s trauma mechanism is present, the one or more clinical signs of a TBI are , and (a) GFAP level is greater than about 100 pg / mL; (b) UCH-L1 level is greater than about 700 pg / mL; or (c) GFAP level is less than about 100 pg / mL and UCH-L1 level is less than about 700 pg / mL The instructions that are included in the system can be provided as written instructions, such as in a product insert, product manual, excel document, etc., on a mobile device (e.g , a smart phone), on a website, in an e-mail, or any combination thereof.4. Treatment and Monitoring of Subjects Who Have Sustained an Injury to the Head

[0438] The subject identified in the methods described above may be treated or monitored. The subject (e.g., a human subject) identified or assessed in the methods described above as having an acquired brain injury, such as traumatic brain injury (e.g., a mild TBI, moderate TBI, severe TBI or moderate to severe TBI), may be treated or monitored. In some embodiments, the method further includes treating the subject (e.g., human subject (e.g., a human adult or human pediatric subject) determined as having a TBI with a traumatic brain injury treatment, such as any treatments known in the art. For example, treatment of traumatic brain injury can take a variety of forms depending on the severity of the injury toDocket No. 15893; 43644.601 the head. For example, for subjects suffering from mild TBI, the treatment may include one or more of: (1) rest (e.g., physical and / or mental rest) (2) abstaining from physical activities such as sports, work, school, play, or any combinations thereof; (3) avoiding light or wearing sunglasses when out in the light; (4) administering one or more types of medication, such as, for example, medication for relief of a headache or migraine (e.g., steroidal antiinflammatory drugs (e.g., corticosteroids (prednisone or dexamethasone)) or nonsteroidal anti-inflammatory drugs (NSAIDs, such as, aspirin, ibuprofen, naproxen sodium, etc.), medication to treat dizziness, anti-nausea medication (e.g., an anti-emetic), motion sickness medications (e.g., scopolamine, promethazine, dimenhydrinate, etc.), anti-depressant medication (e.g., a SSRI, such as fluoxetine (Prozac), paroxetine (Paxil), fluvoxamine (Luvox), citalopram (Celexa), escitalopram (Cipralex), sertraline (Zoloft), and / or psychedelic drugs, such as psilocybin and MDMA, etc.), anti-anxiety medication (e.g., a SSRI, such as fluoxetine (Prozac), paroxetine (Paxil), fluvoxamine (Luvox), citalopram (Celexa), escitalopram (Cipralex), sertraline (Zoloft), and / or psychedelic drugs, such as psilocybin, MDMA, etc.), sleep aid medication (e.g., melatonin, estazolam, flurazepam, quazepam, temazepam, triazolam, etc.), muscle relaxants (e.g., if there is injury or involvement of the neck or muscles around the neck in order to reduce inflammation that might be exacerbating a TBI), anti-inflammatory medications (e.g., steroidal anti-inflammatory drugs or NSAIDs ), medications to improve concentration and / or focus (e.g., psychostimulants such as methylphenidate, etc.) and / or one or more naturopathic medicines or treatments (e.g., non- hallucigenic mushrooms, herbal tea, acupuncture, medical marijuana, etc.); (5) providing one or more devices to treat nausea, such as acupressure wrist bands (e.g., a Psi band); (6) administering one or more nutrachemicals and / or nutritional compositions that contain one or more omega-3 fatty acids, one or more vitamins (e.g., vitamin B and / or vitamin D), one or more fatty acids and / or one or more antioxidants; (7) providing hyperbaric oxygen therapy;(8) light therapy (i.e., to help with mood swings, irritability and / or depression resulting from a TBI); (9) physical therapy for TBI (e.g., to treat dizziness, improve motor skills, etc.); (10) occupational therapy for TBI (e.g., to help improve memory, concentration, focus, etc); (11) individual, group counseling and / or psychotherapy to help treat depression, and / or anxiety resulting from a TBI; (12) sleep therapy to help treat sleep disorders (e.g., not sleeping or oversleeping) arising from a TBI; or (13) and any combinations of (1)-(12).

[0439] Treatment for patients suffering from moderate, severe or moderate-to-severe TBI might include administration of one or more appropriate medications (such as, for example, diuretics, anti-convulsant medications, medications to sedate and put an individual in a drug-Docket No. 15893; 43644.601 induced coma, or other pharmaceutical or biopharmaceutical medications (either known or developed in the future for treatment of TBI), one or more surgical procedures (such as, for example, removal of a hematoma, repairing a skull fracture, decompressive craniectomy, etc.), protecting the airway, and one or more therapies (such as, for example one or more rehabilitation, cognitive behavioral therapy, anger management, physical therapy, occupational therapy, a combination of physical and occupational therapy, counseling psychology, etc.). In some embodiments, the method further includes monitoring the subject, such as a human subject. In some embodiments, a subject may be monitored with CT scan or MRI procedure.5. Methods for Measuring the Level of UCH-L1

[0440] In the methods described above, UCH-L1 levels can be measured by any means, such as antibody dependent methods, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, protein immunostaining, electrophoresis analysis, a protein assay, a competitive binding assay, a functional protein assay, or chromatography or spectrometry methods, such as high- performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). Also, the assay can be employed in clinical chemistry format such as would be known by one skilled in the art.

[0441] In some embodiments, measuring the level of UCH-L1 includes contacting the sample with a first specific binding member and second specific binding member. In some embodiments the first specific binding member is a capture antibody and the second specific binding member is a detection antibody. In some embodiments, measuring the level of UCH- L1 includes contacting the sample, either simultaneously or sequentially, in any order: (1) a capture antibody (e.g, UCH-L1 -capture antibody), which binds to an epitope on UCH-L1 or UCH-L1 fragment to form a capture antib ody-UCH-Ll antigen complex (e.g, UCH-L1- capture antib ody-UCH-Ll antigen complex), and (2) a detection antibody (e.g., UCH-L1- detection antibody), which includes a detectable label and binds to an epitope on UCH-L1 that is not bound by the capture antibody, to form a UCH-L1 antigen-detection antibody complex (e.g., UCH-L1 antigen-UCH-Ll -detection antibody complex), such that a capture antibody -UCH-L1 antigen-detection antibody complex (e.g., UCH-L1 -capture antibody- UCH-L1 antigen-UCH-Ll -detection antibody complex) is formed, and measuring the amountDocket No. 15893; 43644.601 or concentration of UCH-L1 in the sample based on the signal generated by the detectable label in the capture antib ody-UCH-Ll antigen-detection antibody complex.

[0442] In some embodiments, the first specific binding member is immobilized on a solid support. In some embodiments, the second specific binding member is immobilized on a solid support. In some embodiments, the first specific binding member is a UCH-L1 antibody as described below.

[0443] In some embodiments, the sample is diluted or undiluted. The sample can be from about 1 to about 25 microliters, about 1 to about 24 microliters, about 1 to about 23 microliters, about 1 to about 22 microliters, about 1 to about 21 microliters, about 1 to about 20 microliters, about 1 to about 18 microliters, about 1 to about 17 microliters, about 1 to about 16 microliters, about 15 microliters or about 1 microliter, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, about 10 microliters, about 11 microliters, about 12 microliters, about 13 microliters, about 14 microliters, about 15 microliters, about 16 microliters, about 17 microliters, about 18 microliters, about 19 microliters, about 20 microliters, about 21 microliters, about 22 microliters, about 23 microliters, about 24 microliters or about 25 microliters. In some embodiments, the sample is from about 1 to about 150 microliters or less or from about 1 to about 25 microliters or less.

[0444] Some instruments (such as, for example the Abbott Laboratories instrument ARCHITECT®, and other core laboratory instruments) other than a point-of-care device may be capable of measuring levels of UCH-L1 in a sample higher or greater than 25,000 pg / mL.

[0445] Other methods of detection include the use of or can be adapted for use on a nanopore device or nanowell device. Examples of nanopore devices are described in International Patent Publication No. WO 2016 / 161402, which is hereby incorporated by reference in its entirety. Examples of nanowell device are described in International Patent Publication No. WO 2016 / 161400, which is hereby incorporated by reference in its entirety6. UCH-L1 Antibodies

[0446] The methods described herein may use an isolated antibody that specifically binds to ubiquitin carboxy -terminal hydrolase LI (“UCH-L1”) (or fragments thereof), referred to as “UCH-L1 antibody.” The UCH-L1 antibodies can be used to assess the UCH-L1 status as a measure of traumatic brain injury, detect the presence of UCH-L1 in a sample, quantify the amount of UCH-L1 present in a sample, or detect the presence of and quantify the amount of UCH-L1 in a sample.Docket No. 15893; 43644.601 a. Ubiquitin Carboxy-Terminal Hydrolase LI (UCH-L1)

[0447] Ubiquitin carboxy-terminal hydrolase LI (“UCH-L1”), which is also known as “ubiquitin C-terminal hydrolase,” is a deubiquitinating enzyme. UCH-L1 is a member of a gene family whose products hydrolyze small C-terminal adducts of ubiquitin to generate the ubiquitin monomer. Expression of UCH-L1 is highly specific to neurons and to cells of the diffuse neuroendocrine system and their tumors. It is abundantly present in all neurons (accounts for 1-2% of total brain protein), expressed specifically in neurons and testis / ovary. The catalytic triad of UCH-L1 contains a cysteine at position 90, an aspartate at position 176, and a histidine at position 161 that are responsible for its hydrolase activity.

[0448] Human UCH-L1 may have the following amino acid sequence:

[0449] MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLL LFPLTAQHENFRKKQIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGF EDGSVLKQFLSETEKMSPEDRAKCFEKNEAIQAAHDAVAQEGQCRVDDKVNFHFIL FNN VDGHLYELDGRM PF P VNHG A S SEDTLLKD AAKVCREFTEREQGEVRF S AVALC KAA (SEQ ID NO: 1).

[0450] The human UCH-L1 may be a fragment or variant of SEQ ID NO: 1. The fragment of UCH-L1 may be between 5 and 225 amino acids, between 10 and 225 amino acids, between 50 and 225 amino acids, between 60 and 225 amino acids, between 65 and 225 amino acids, between 100 and 225 amino acids, between 150 and 225 amino acids, between 100 and 175 amino acids, or between 175 and 225 amino acids in length. The fragment may comprise a contiguous number of amino acids from SEQ ID NO: 1. b. UCH-Ll-Recognizing Antibody

[0451] The antibody is an antibody that binds to UCH-L1, a fragment thereof, an epitope of UCH-L1, or a variant thereof. The antibody may be a fragment of the anti-UCH-Ll antibody or a variant or a derivative thereof. The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, a fully human antibody or an antibody fragment, such as a Fab fragment, or a mixture thereof. Antibody fragments or derivatives may comprise F(ab’)2, Fv or scFv fragments. The antibody derivatives can be produced by peptidomimetics. Further, techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies.

[0452] The anti-UCH-Ll antibodies may be a chimeric anti-UCH-Ll or humanized anti- UCH-Ll antibody. In one embodiment, both the humanized antibody and chimeric antibodyDocket No. 15893; 43644.601 are monovalent. In one embodiment, both the humanized antibody and chimeric antibody comprise a single Fab region linked to an Fc region.

[0453] Human antibodies may be derived from phage-display technology or from transgenic mice that express human immunoglobulin genes. The human antibody may be generated as a result of a human in vivo immune response and isolated. See, for example, Funaro et al., BMC Biotechnology, 2008(8):85. Therefore, the antibody may be a product of the human and not animal repertoire. Because it is of human origin, the risks of reactivity against self-antigens may be minimized. Alternatively, standard yeast display libraries and display technologies may be used to select and isolate human anti-UCH-Ll antibodies. For example, libraries of naive human single chain variable fragments (scFv) may be used to select human anti-UCH-Ll antibodies. Transgenic animals may be used to express human antibodies.

[0454] Humanized antibodies may be antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.

[0455] The antibody is distinguishable from known antibodies in that it possesses different biological function(s) than those known in the art.(1) Epitope

[0456] The antibody may immunospecifically bind to UCH-L1 (SEQ ID NO: 1), a fragment thereof, or a variant thereof. The antibody may immunospecifically recognize and bind at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, or at least ten amino acids within an epitope region. The antibody may immunospecifically recognize and bind to an epitope that has at least three contiguous amino acids, at least four contiguous amino acids, at least five contiguous amino acids, at least six contiguous amino acids, at least seven contiguous amino acids, at least eight contiguous amino acids, at least nine contiguous amino acids, or at least ten contiguous amino acids of an epitope region. c. Antibody Preparation / Production

[0457] Antibodies may be prepared by any of a variety of techniques, including those well known to those skilled in the art. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies via conventional techniques, or via transfection of antibody genes, heavy chains, and / or light chains into suitable bacterialDocket No. 15893; 43644.601 or mammalian cell hosts, to allow for the production of antibodies, wherein the antibodies may be recombinant. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium -phosphate precipitation, DEAE-dextran transfection and the like. Although it is possible to express the antibodies in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells is preferable, and most preferable in mammalian host cells, because such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.

[0458] Exemplary mammalian host cells for expressing the recombinant antibodies include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Set. USA, 77: 4216-4220 (1980)), used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982), NSO myeloma cells, COS cells, and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0459] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure may be performed. For example, it may be desirable to transfect a host cell with DNA encoding functional fragments of either the light chain and / or the heavy chain of an antibody. Recombinant DNA technology may also be used to remove some, or all, of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigens of interest. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies. In addition, bifunctional antibodies may be produced in which one heavy and one light chain are an antibody (i.e., binds human UCH-L1) and the other heavy and light chain are specific for an antigen other than human UCH-L1 by crosslinking an antibody to a second antibody by standard chemical crosslinking methods.

[0460] In a preferred system for recombinant expression of an antibody, or antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chainDocket No. 15893; 43644.601 genes are each operatively linked to CMV enhancer / AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection / amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. Still further, the method of synthesizing a recombinant antibody may be by culturing a host cell in a suitable culture medium until a recombinant antibody is synthesized. The method can further comprise isolating the recombinant antibody from the culture medium.

[0461] Methods of preparing monoclonal antibodies involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity. Such cell lines may be produced from spleen cells obtained from an immunized animal. The animal may be immunized with UCH-L1 or a fragment and / or variant thereof. The peptide used to immunize the animal may comprise amino acids encoding human Fc, for example the fragment crystallizable region or tail region of human antibody. The spleen cells may then be immortalized by, for example, fusion with a myeloma cell fusion partner. A variety of fusion techniques may be employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports that growth of hybrid cells, but not myeloma cells. One such technique uses hypoxanthine, aminopterin, thymidine (HAT) selection. Another technique includes electrofusion. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity may be used.

[0462] Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. Affinity chromatography is an example of a method that can be used in a process to purify the antibodies.Docket No. 15893; 43644.601

[0463] The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen -binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab’)2 fragment, which comprises both antigenbinding sites.

[0464] The Fv fragment can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions IgG or IgA immunoglobulin molecules. The Fv fragment may be derived using recombinant techniques. The Fv fragment includes a non-covalent VH: : VL heterodimer including an antigen-binding site that retains much of the antigen recognition and binding capabilities of the native antibody molecule.

[0465] The antibody, antibody fragment, or derivative may comprise a heavy chain and a light chain complementarity determining region (“CDR”) set, respectively interposed between a heavy chain and a light chain framework (“FR”) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other. The CDR set may contain three hypervariable regions of a heavy or light chain V region.

[0466] Other suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, but not limited to, methods that select recombinant antibody from a peptide or protein library (e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, RNA, cDNA, yeast or the like, display library); e.g., as available from various commercial vendors such as Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid / Planegg, Del.), Biovation (Aberdeen, Scotland, UK) BioInvent (Lund, Sweden), using methods known in the art. See U.S. Patent Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862. Alternative methods rely upon immunization of transgenic animals (e.g., SCID mice, Nguyen et al. (1997) Microbiol. Immunol. 41 :901-907; Sandhu et al. (1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol. 93: 154-161) that are capable of producing a repertoire of human antibodies, as known in the art and / or as described herein. Such techniques, include, but are not limited to, ribosome display (Hanes et al. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998) Proc. Natl. Acad. Sci. USA, 95: 14130-14135); single cell antibody producing technologies (e.g., selected lymphocyte antibody method ("SLAM") (U.S. Patent No. 5,627,052, Wen et al. (1987) J. Immunol. 17:887-892; Babcook et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848); gel microdroplet and flow cytometry (Powell et al. (1990) Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass).; Gray et al. (1995) J. Imm.Docket No. 15893; 43644.601Meth. 182: 155-163; Kenny et al. (1995) Bio / Technol. 13:787-790); B-cell selection (Steenbakkers et al. (1994) Afo / ec. Biol. Reports 19: 125-134 (1994)).

[0467] An affinity matured antibody may be produced by any one of a number of procedures that are known in the art. For example, see Marks et al., BioTechnology, 10: 779- 783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91 : 3809-3813 (1994); Schier c / a / „ Gene, 169: 147-155 (1995); Yelton et al., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol., 154(7): 3310-3319 (1995); Hawkins et al, J. Mol. Biol., 226: 889-896 (1992). Selective mutation at selective mutagenesis positions and at contact or hypermutation positions with an activity enhancing amino acid residue is described in U.S. Patent No. 6,914,128 Bl.

[0468] Antibody variants can also be prepared using delivering a polynucleotide encoding an antibody to a suitable host such as to provide transgenic animals or mammals, such as goats, cows, horses, sheep, and the like, that produce such antibodies in their milk. These methods are known in the art and are described for example in U.S. Patent Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.

[0469] Antibody variants also can be prepared by delivering a polynucleotide to provide transgenic plants and cultured plant cells (e.g., but not limited to tobacco, maize, and duckweed) that produce such antibodies, specified portions or variants in the plant parts or in cells cultured therefrom. For example, Cramer et al. (1999) Curr. Top. Microbiol. Immunol. 240:95-118 and references cited therein, describe the production of transgenic tobacco leaves expressing large amounts of recombinant proteins, e.g., using an inducible promoter.Transgenic maize have been used to express mammalian proteins at commercial production levels, with biological activities equivalent to those produced in other recombinant systems or purified from natural sources. See, e.g., Hood et al., Adv. Exp. Med. Biol. (1999) 464: 127- 147 and references cited therein. Antibody variants have also been produced in large amounts from transgenic plant seeds including antibody fragments, such as single chain antibodies (scFvs), including tobacco seeds and potato tubers. See, e.g., Conrad et al. (1998) Plant Mol. Biol. 38: 101-109 and reference cited therein. Thus, antibodies can also be produced using transgenic plants, according to known methods.

[0470] Antibody derivatives can be produced, for example, by adding exogenous sequences to modify immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic. Generally, part or all of theDocket No. 15893; 43644.601 non-human or human CDR sequences are maintained while the non-human sequences of the variable and constant regions are replaced with human or other amino acids.

[0471] Small antibody fragments may be diabodies having two antigen-binding sites, wherein fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH VL). See for example, EP 404,097; WO 93 / 11161; and Hollinger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. See also, U.S. Patent No. 6,632,926 to Chen et al. which is hereby incorporated by reference in its entirety and discloses antibody variants that have one or more amino acids inserted into a hypervariable region of the parent antibody and a binding affinity for a target antigen which is at least about two fold stronger than the binding affinity of the parent antibody for the antigen.

[0472] The antibody may be a linear antibody. The procedure for making a linear antibody is known in the art and described in Zapata et al., (1995) Protein Eng. 8(10): 1057-1062. Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[0473] The antibodies may be recovered and purified from recombinant cell cultures by known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification.

[0474] It may be useful to detectably label the antibody. Methods for conjugating antibodies to these agents are known in the art. For the purpose of illustration only, antibodies can be labeled with a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, or the like. Such labeled antibodies can be used for diagnostic techniques, either in vivo, or in an isolated test sample. They can be linked to a cytokine, to a ligand, to another antibody. Suitable agents for coupling to antibodies to achieve an antitumor effect include cytokines, such as interleukin 2 (IL-2) and Tumor Necrosis Factor (TNF); photosensitizers, for use in photodynamic therapy, including aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine; radionuclides, such as iodine-131 (1311), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), technetium- 99m (99mTc), rhenium-186 (186Re), and rhenium-188 (188Re); antibiotics, such asDocket No. 15893; 43644.601 doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacterial, plant, and other toxins, such as diphtheria toxin, pseudomonas exotoxin A, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-alpha toxin, cytotoxin from Chinese cobra (naja atra), and gelonin (a plant toxin); ribosome inactivating proteins from plants, bacteria and fungi, such as restrictocin (a ribosome inactivating protein produced by Aspergillus restrictus), saporin (a ribosome inactivating protein from Saponaria officinalis), and RNase; tyrosine kinase inhibitors; ly207702 (a difluorinated purine nucleoside); liposomes containing anti cystic agents (e.g., antisense oligonucleotides, plasmids which encode for toxins, methotrexate, etc.); and other antibodies or antibody fragments, such as F(ab).

[0475] Antibody production via the use of hybridoma technology, the selected lymphocyte antibody method (SLAM), transgenic animals, and recombinant antibody libraries is described in more detail below.(1) Anti-UCH-Ll Monoclonal Antibodies Using Hybridoma Technology

[0476] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, second edition, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988); Hammerling, et al., In Monoclonal Antibodies and T-Cell Hybridomas, (Elsevier, N.Y., 1981). It is also noted that the term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0477] Methods of generating monoclonal antibodies as well as antibodies produced by the method may comprise culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from an animal, e.g., a rat or a mouse, immunized with UCH-L1 with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention. Briefly, rats can be immunized with a UCH-L1 antigen. In a preferred embodiment, the UCH-L1 antigen is administered with an adjuvant to stimulate the immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes). Such adjuvants may protect the polypeptide from rapid dispersal by sequestering it in a localDocket No. 15893; 43644.601 deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. Preferably, if a polypeptide is being administered, the immunization schedule will involve two or more administrations of the polypeptide, spread out over several weeks; however, a single administration of the polypeptide may also be used.

[0478] After immunization of an animal with a UCH-L1 antigen, antibodies and / or antibody-producing cells may be obtained from the animal. An anti-UCH-Ll antibodycontaining serum is obtained from the animal by bleeding or sacrificing the animal. The serum may be used as it is obtained from the animal, an immunoglobulin fraction may be obtained from the serum, or the anti-UCH-Ll antibodies may be purified from the serum. Serum or immunoglobulins obtained in this manner are polyclonal, thus having a heterogeneous array of properties.

[0479] Once an immune response is detected, e.g., antibodies specific for the antigen UCH- L1 are detected in the rat serum, the rat spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example, cells from cell line SP20 available from the American Type Culture Collection (ATCC, Manassas, Va., US). Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding UCH-L1. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing rats with positive hybridoma clones.

[0480] In another embodiment, antibody -producing immortalized hybridomas may be prepared from the immunized animal. After immunization, the animal is sacrificed, and the splenic B cells are fused to immortalized myeloma cells as is well known in the art. See, e.g., Harlow and Lane, supra. In a preferred embodiment, the myeloma cells do not secrete immunoglobulin polypeptides (a non-secretory cell line). After fusion and antibiotic selection, the hybridomas are screened using UCH-L1, or a portion thereof, or a cell expressing UCH-L1. In a preferred embodiment, the initial screening is performed using an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA), preferably an ELISA. An example of ELISA screening is provided in PCT Publication No. WO 00 / 37504.

[0481] Anti-UCH-Ll antibody-producing hybridomas are selected, cloned, and further screened for desirable characteristics, including robust hybridoma growth, high antibody production, and desirable antibody characteristics. Hybridomas may be cultured and expanded in vivo in syngeneic animals, in animals that lack an immune system, e.g., nudeDocket No. 15893; 43644.601 mice, or in cell culture in vitro. Methods of selecting, cloning and expanding hybridomas are well known to those of ordinary skill in the art.

[0482] In a preferred embodiment, hybridomas are rat hybridomas. In another embodiment, hybridomas are produced in a non-human, non-rat species such as mice, sheep, pigs, goats, cattle, or horses. In yet another preferred embodiment, the hybridomas are human hybridomas, in which a human non-secretory myeloma is fused with a human cell expressing an anti-UCH-Ll antibody.

[0483] Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce two identical Fab fragments) or pepsin (to produce an F(ab')2 fragment). A F(ab')2 fragment of an IgG molecule retains the two antigen-binding sites of the larger ("parent") IgG molecule, including both light chains (containing the variable light chain and constant light chain regions), the CHI domains of the heavy chains, and a disulfide-forming hinge region of the parent IgG molecule. Accordingly, an F(ab')2 fragment is still capable of crosslinking antigen molecules like the parent IgG molecule.(2) Anti-UCH-Ll Monoclonal Antibodies Using SLAM

[0484] In another aspect of the invention, recombinant antibodies are generated from single, isolated lymphocytes using a procedure referred to in the art as the selected lymphocyte antibody method (SLAM), as described in U.S. Patent No. 5,627,052; PCT Publication No. WO 92 / 02551; and Babcook et al., Proc. Natl. Acad. Sci. USA, 93: 7843-7848 (1996). In this method, single cells secreting antibodies of interest, e.g., lymphocytes derived from any one of the immunized animals are screened using an antigen-specific hemolytic plaque assay, wherein the antigen UCH-L1, a subunit of UCH-L1, or a fragment thereof, is coupled to sheep red blood cells using a linker, such as biotin, and used to identify single cells that secrete antibodies with specificity for UCH-L1. Following identification of antibodysecreting cells of interest, heavy- and light-chain variable region cDNAs are rescued from the cells by reverse transcriptase-PCR (RT-PCR) and these variable regions can then be expressed, in the context of appropriate immunoglobulin constant regions (e.g., human constant regions), in mammalian host cells, such as COS or CHO cells. The host cells transfected with the amplified immunoglobulin sequences, derived from in vivo selected lymphocytes, can then undergo further analysis and selection in vitro, for example, by panning the transfected cells to isolate cells expressing antibodies to UCH-L1. The amplified immunoglobulin sequences further can be manipulated in vitro, such as by in vitro affinityDocket No. 15893; 43644.601 maturation method. See, for example, PCT Publication No. WO 97 / 29131 and PCT Publication No. WO 00 / 56772.(3) Anti-UCH-Ll Monoclonal Antibodies Using Transgenic Animals

[0485] In another embodiment of the invention, antibodies are produced by immunizing a non-human animal comprising some, or all, of the human immunoglobulin locus with a UCH-L1 antigen. In an embodiment, the non-human animal is a XENOMOUSE® transgenic mouse, an engineered mouse strain that comprises large fragments of the human immunoglobulin loci and is deficient in mouse antibody production. See, e.g., Green et al., Nature Genetics, 7: 13-21 (1994) and U.S. Patent Nos. 5,916,771; 5,939,598; 5,985,615;5,998,209; 6,075,181; 6,091,001; 6,114,598; and 6,130,364. See also PCT Publication Nos. WO 91 / 10741; WO 94 / 02602; WO 96 / 34096; WO 96 / 33735; WO 98 / 16654; WO 98 / 24893; WO 98 / 50433; WO 99 / 45031; WO 99 / 53049; WO 00 / 09560; and WO 00 / 37504. The XENOMOUSE® transgenic mouse produces an adult-like human repertoire of fully human antibodies and generates antigen-specific human monoclonal antibodies. The XENOMOUSE® transgenic mouse contains approximately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and x light chain loci. See Mendez et al., Nature Genetics, 15: 146-156 (1997), Green and Jakobovits, J. Exp. Med., 188: 483-495 (1998), the disclosures of which are hereby incorporated by reference.(4) Anti-UCH-Ll Monoclonal Antibodies Using Recombinant Antibody Libraries

[0486] In vitro methods also can be used to make the antibodies of the invention, wherein an antibody library is screened to identify an antibody having the desired UCH-L1 -binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include methods described in, for example, U.S. Patent No. 5,223,409 (Ladner et al.); PCT Publication No. WO 92 / 18619 (Kang et al.); PCT Publication No. WO 91 / 17271 (Dower et al.); PCT Publication No. WO 92 / 20791 (Winter et al.); PCT Publication No. WO 92 / 15679 (Markland et al ); PCT Publication No. WO 93 / 01288 (Breitling et al ); PCT Publication No. WO 92 / 01047 (McCafferty et al ); PCT Publication No. WO 92 / 09690 (Garrard et al.); Fuchs et al., Bio / Technology, 9: 1369-1372 (1991); Hay et al., Hum.Antibod. Hybridomas, 3: 81-85 (1992); Huse et al., Science, 246: 1275-1281 (1989); McCafferty et al., Nature, 348: 552-554 (1990); Griffiths et al., EMBO J., 12: 725-734 (1993); Hawkins et al., J. Mol. Biol., 226: 889-896 (1992); Clackson et al., Nature, 352: 624- 628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA, 89: 3576-3580 (1992); Garrard et al., Bio / Technology, 9: 1373-1377 (1991); Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137Docket No. 15893; 43644.601(1991); Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991); U.S. Patent Application Publication No. 2003 / 0186374; and PCT Publication No. WO 97 / 29131, the contents of each of which are incorporated herein by reference.

[0487] The recombinant antibody library may be from a subject immunized with UCH-L1, or a portion of UCH-L1. Alternatively, the recombinant antibody library may be from a naive subject, i.e., one who has not been immunized with UCH-L1, such as a human antibody library from a human subject who has not been immunized with human UCH-L1. Antibodies of the invention are selected by screening the recombinant antibody library with the peptide comprising human UCH-L1 to thereby select those antibodies that recognize UCH-L1. Methods for conducting such screening and selection are well known in the art, such as described in the references in the preceding paragraph. To select antibodies of the invention having particular binding affinities for UCH-L1, such as those that dissociate from human UCH-L1 with a particular Koff rate constant, the art-known method of surface plasmon resonance can be used to select antibodies having the desired Koff rate constant. To select antibodies of the invention having a particular neutralizing activity for hUCH-Ll, such as those with a particular IC50, standard methods known in the art for assessing the inhibition of UCH-L1 activity may be used.

[0488] In one aspect, the invention pertains to an isolated antibody, or an antigen-binding portion thereof, that binds human UCH-L1. Preferably, the antibody is a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody.

[0489] For example, antibodies can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. Such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv, or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies include those disclosed in Brinkmann et al., J. Immunol. Methods, 182: 41-50 (1995); Ames et al., J. Immunol. Methods, 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24: 952-958 (1994); Persic et al., Gene, 187: 9-18Docket No. 15893; 43644.601(1997); Burton et al., Advances in Immunology, 57: 191-280 (1994); PCT Publication No. WO 92 / 01047; PCT Publication Nos. WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743; and 5,969,108.

[0490] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies including human antibodies or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab', and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication No. WO 92 / 22324; Mullinax et al., BioTechniques, 12(6): 864- 869 (1992); Sawai et al., Am. J. Reprod. Immunol., 34: 26-34 (1995); and Better et al., Science, 240: 1041-1043 (1988). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203: 46-88 (1991); Shu et al., Proc. Natl. Acad. Sci. USA, 90: 7995-7999 (1993); and Skerra et a / ., Science, 240: 1038-1041 (1988).

[0491] Alternative to screening of recombinant antibody libraries by phage display, other methodologies known in the art for screening large combinatorial libraries can be applied to the identification of antibodies of the invention. One type of alternative expression system is one in which the recombinant antibody library is expressed as RNA-protein fusions, as described in PCT Publication No. WO 98 / 31700 (Szostak and Roberts), and in Roberts and Szostak, Proc. Natl. Acad. Sci. USA, 94: 12297-12302 (1997). In this system, a covalent fusion is created between an mRNA and the peptide or protein that it encodes by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3' end. Thus, a specific mRNA can be enriched from a complex mixture of mRNAs (e.g., a combinatorial library) based on the properties of the encoded peptide or protein, e.g., antibody, or portion thereof, such as binding of the antibody, or portion thereof, to the dual specificity antigen. Nucleic acid sequences encoding antibodies, or portions thereof, recovered from screening of such libraries can be expressed by recombinant means as described above (e.g., in mammalian host cells) and, moreover, can be subjected to further affinity maturation by either additional rounds of screening of mRNA-peptide fusions in which mutations have been introduced into the originally selected sequence(s), or by otherDocket No. 15893; 43644.601 methods for affinity maturation in vitro of recombinant antibodies, as described above. A preferred example of this methodology is PROfusion display technology.

[0492] In another approach, the antibodies can also be generated using yeast display methods known in the art. In yeast display methods, genetic methods are used to tether antibody domains to the yeast cell wall and display them on the surface of yeast. Such yeast can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Examples of yeast display methods that can be used to make the antibodies include those disclosed in U.S. Patent No. 6,699,658 (Wittrup et al.) incorporated herein by reference. d. Production of Recombinant UCH-L1 Antibodies

[0493] Antibodies may be produced by any of a number of techniques known in the art. For example, expression from host cells, wherein expression vector(s) encoding the heavy and light chains is (are) transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection, and the like. Although it is possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells is preferable, and most preferable in mammalian host cells, because such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.

[0494] Exemplary mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Set. USA, 77: 4216-4220 (1980), used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982), NS0 myeloma cells, COS cells, and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0495] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedureDocket No. 15893; 43644.601 may be performed. For example, it may be desirable to transfect a host cell with DNA encoding functional fragments of either the light chain and / or the heavy chain of an antibody of this invention. Recombinant DNA technology may also be used to remove some, or all, of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigens of interest. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention. In addition, bifunctional antibodies may be produced in which one heavy and one light chain are an antibody of the invention ( / .< ., binds human UCH-L1) and the other heavy and light chain are specific for an antigen other than human UCH-L1 by crosslinking an antibody of the invention to a second antibody by standard chemical crosslinking methods.

[0496] In a preferred system for recombinant expression of an antibody, or antigen-binding portion thereof, of the invention, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to CMV enhancer / AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection / amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. Still further, the invention provides a method of synthesizing a recombinant antibody of the invention by culturing a host cell of the invention in a suitable culture medium until a recombinant antibody of the invention is synthesized. The method can further comprise isolating the recombinant antibody from the culture medium.(1) Humanized Antibody

[0497] The humanized antibody may be an antibody or a variant, derivative, analog or portion thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. The humanized antibody may be from a non-human species antibody that binds the desired antigen having one or more complementarityDocket No. 15893; 43644.601 determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.

[0498] As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and 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 ( / .<?., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. According to one aspect, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In some embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and / or of a heavy chain.

[0499] The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including without limitation IgG 1, 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.

[0500] The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework may be mutagenized by substitution, insertion and / or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. In one embodiment, such mutations, however, will not be extensive. Usually, at least 90%, at least 95%, at least 98%, or at least 99% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in the consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In aDocket No. 15893; 43644.601 family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.[0501 J The humanized antibody may be designed to minimize unwanted immunological response toward rodent anti-human antibodies, which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients. The humanized antibody may have one or more amino acid residues introduced into it from a source that is non-human. These non-human residues are often referred to as “import” residues, which are typically taken from a variable domain. Humanization may be performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. For example, see U.S. Patent No. 4,816,567, the contents of which are herein incorporated by reference. The humanized antibody may be a human antibody in which some hypervariable region residues, and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanization or engineering of antibodies of the present invention can be performed using any known method, such as but not limited to those described in U.S. Patent Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.

[0502] The humanized antibody may retain high affinity for UCH-L1 and other favorable biological properties. The humanized antibody may be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristics, such as increased affinity for UCH-L1, is achieved. In general, the hypervariable region residues may be directly and most substantially involved in influencing antigen binding.Docket No. 15893; 43644.601

[0503] As an alternative to humanization, human antibodies (also referred to herein as “fully human antibodies”) can be generated. For example, it is possible to isolate human antibodies from libraries via PROfusion and / or yeast related technologies. It is also possible to produce transgenic animals (e.g., mice that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. The humanized or fully human antibodies may be prepared according to the methods described in U.S. Patent Nos. 5,770,429; 5,833,985; 5,837,243; 5,922,845; 6,017,517; 6,096,311; 6,111,166; 6,270,765; 6,303,755; 6,365,116; 6,410,690; 6,682,928; and 6,984,720, the contents each of which are herein incorporated by reference. e. Anti-UCH-Ll antibodies

[0504] Anti-UCH-Ll antibodies may be generated using the techniques described above as well as using routine techniques known in the art. In some embodiments, the anti-UCH-Ll antibody may be an unconjugated UCH-L1 antibody, such as UCH-L1 antibodies available from United State Biological (Catalog Number: 031320), Cell Signaling Technology (Catalog Number: 3524), Sigma-Aldrich (Catalog Number: HPA005993), Santa Cruz Biotechnology, Inc. (Catalog Numbers: sc-58593 or sc-58594), R&D Systems (Catalog Number: MAB6007), Novus Biologicals (Catalog Number: NB600-1160), Biorbyt (Catalog Number: orb33715), Enzo Life Sciences, Inc. (Catalog Number: ADI-905-520-1), Bio-Rad (Catalog Number: VMA00004), BioVision (Catalog Number: 6130-50), Abeam (Catalog Numbers: ab75275 or abl04938), Invitrogen Antibodies (Catalog Numbers: 480012), ThermoFisher Scientific (Catalog Numbers: MAI-46079, MA5-17235, MAI-90008, or MAI -83428), EMD Millipore (Catalog Number: MABN48), or Sino Biological Inc. (Catalog Number: 50690-R011). The anti-UCH-Ll antibody may be conjugated to a fluorophore, such as conjugated UCH-L1 antibodies available from BioVision (Catalog Number: 6960-25) or Aviva Systems Biology (Cat. Nos. OAAF01904-FITC).

[0505] Alternatively, the antibodies described in WO 2018 / 081649, U.S. Patent No.11,078,298, WO 2018 / 067468 and / or Bazarian et al., “Accuracy of a rapid GFAP / UCH-L1 test for the prediction of intracranial injuries on head CT after mild traumatic brain injury”,Docket No. 15893; 43644.601Acad. Emerg. e , (August 6, 2021), the contents of which are herein incorporated by reference, can also be used.7. Methods for Measuring the Level of GFAP

[0506] In the methods described above, GFAP levels can be measured by any means, such as antibody dependent methods, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, or protein immunostaining, electrophoresis analysis, a protein assay, a competitive binding assay, a functional protein assay, or chromatography or spectrometry methods, such as high- performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). Also, the assay can be employed in clinical chemistry format such as would be known by one skilled in the art.

[0507] In some embodiments, measuring the level of GFAP includes contacting the sample with a first specific binding member and second specific binding member. In some embodiments the first specific binding member is a capture antibody and the second specific binding member is a detection antibody. In some embodiments, measuring the level of GFAP includes contacting the sample, either simultaneously or sequentially, in any order: (1) a capture antibody (e.g., GFAP-capture antibody), which binds to an epitope on GFAP or GFAP fragment to form a capture antibody-GFAP antigen complex (e.g., GFAP-capture antibody-GFAP antigen complex), and (2) a detection antibody e.g., GFAP-detection antibody), which includes a detectable label and binds to an epitope on GFAP that is not bound by the capture antibody, to form a GFAP antigen-detection antibody complex (e.g., GFAP antigen-GFAP-detection antibody complex), such that a capture antibody-GFAP antigen-detection antibody complex (e.g., GFAP-capture antibody-GFAP antigen-GFAP- detection antibody complex) is formed, and measuring the amount or concentration of GFAP in the sample based on the signal generated by the detectable label in the capture antibody- GFAP antigen-detection antibody complex.

[0508] In some embodiments, the first specific binding member is immobilized on a solid support. In some embodiments, the second specific binding member is immobilized on a solid support. In some embodiments, the first specific binding member is a GFAP antibody as described below.

[0509] In some embodiments, the sample is diluted or undiluted. The sample can be from about 1 to about 25 microliters, about 1 to about 24 microliters, about 1 to about 23Docket No. 15893; 43644.601 microliters, about 1 to about 22 microliters, about 1 to about 21 microliters, about 1 to about 20 microliters, about 1 to about 18 microliters, about 1 to about 17 microliters, about 1 to about 16 microliters, about 15 microliters or about 1 microliter, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, about 10 microliters, about 11 microliters, about 12 microliters, about 13 microliters, about 14 microliters, about 15 microliters, about 16 microliters, about 17 microliters, about 18 microliters, about 19 microliters, about 20 microliters, about 21 microliters, about 22 microliters, about 23 microliters, about 24 microliters or about 25 microliters. In some embodiments, the sample is from about 1 to about 150 microliters or less or from about 1 to about 25 microliters or less.

[0510] Some instruments (such as, for example the Abbott Laboratories instrument ARCHITECT®, and other core laboratory instruments) other than a point-of-care device may be capable of measuring levels of GFAP in a sample higher or greater than 25,000 pg / mL.

[0511] Other methods of detection include the use of or can be adapted for use on a nanopore device or nanowell device. Examples of nanopore devices are described in International Patent Publication No. WO 2016 / 161402, which is hereby incorporated by reference in its entirety. Examples of nanowell device are described in International Patent Publication No. WO 2016 / 161400, which is hereby incorporated by reference in its entirety8. GFAP Antibodies

[0512] The methods described herein may use an isolated antibody that specifically binds to Glial fibrillary acidic protein (“GFAP”) (or fragments thereof), referred to as “GFAP antibody.” The GFAP antibodies can be used to assess the GFAP status as a measure of traumatic brain injury, detect the presence of GFAP in a sample, quantify the amount of GFAP present in a sample, or detect the presence of and quantify the amount of GFAP in a sample. a. Glial fibrillary acidic protein (GFAP)

[0513] Glial fibrillary acidic protein (GFAP) is a 50 kDa intracytoplasmic filamentous protein that constitutes a portion of the cytoskeleton in astrocytes, and it has proved to be the most specific marker for cells of astrocytic origin. GFAP protein is encoded by the GFAP gene in humans. GFAP is the principal intermediate filament of mature astrocytes. In the central rod domain of the molecule, GFAP shares considerable structural homology with the other intermediate filaments. GFAP is involved in astrocyte motility and shape by providingDocket No. 15893; 43644.601 structural stability to astrocytic processes. Glial fibrillary acidic protein and its breakdown products (GFAP-BDP) are brain-specific proteins released into the blood as part of the pathophysiological response after traumatic brain injury (TBI). Following injury to the human CNS caused by trauma, genetic disorders, or chemicals, astrocytes proliferate and show extensive hypertrophy of the cell body and processes, and GFAP is markedly upregulated. In contrast, with increasing astrocyte malignancy, there is a progressive loss of GFAP production. GFAP can also be detected in Schwann cells, enteric glia cells, salivary gland neoplasms, metastasizing renal carcinomas, epiglottic cartilage, pituicytes, immature oligodendrocytes, papillary meningiomas, and myoepithelial cells of the breast.

[0514] Human GFAP may have the following amino acid sequence:

[0515] MERRRITSAARRSYVSSGEMMVGGLAPGRRLGPGTRLSLARMPPPLPTRVD FSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAK EPTKLADVYQAELRELRLRLDQLTANSARLEVERDNLAQDLATVRQKLQDETNLRL EAENNLAAYRQEADEATLARLDLERKIESLEEEIRFLRKIHEEEVRELQEQLARQQVH VELDVAI<PDLTAALI<EIRTQYEAMASSNMHEAEEWYRSI<FADLTDAAARNAELLR QAKHEANDYRRQLQSLTCDLESLRGTNESLERQMREQEERHVREAASYQEALARLE EEGQSLKDEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRE TSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKESKQEHKDVM (SEQ ID NO: 2).

[0516] The human GFAP may be a fragment or variant of SEQ ID NO: 2. The fragment of GFAP may be between 5 and 400 amino acids, between 10 and 400 amino acids, between 50 and 400 amino acids, between 60 and 400 amino acids, between 65 and 400 amino acids, between 100 and 400 amino acids, between 150 and 400 amino acids, between 100 and 300 amino acids, or between 200 and 300 amino acids in length. The fragment may comprise a contiguous number of amino acids from SEQ ID NO: 2. The human GFAP fragment or variant of SEQ ID NO: 2 may be a GFAP breakdown product (BDP). The GFAP BDP may be 38 kDa, 42 kDa (fainter 41 kDa), 47 kDa (fainter 45 kDa); 25 kDa (fainter 23 kDa); 19 kDa, or 20 kDa. b. GFAP-Recognizing Antibody

[0517] The antibody is an antibody that binds to GFAP, a fragment thereof, an epitope of GFAP, or a variant thereof. The antibody may be a fragment of the anti-GFAP antibody or a variant or a derivative thereof. The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, a fully human antibody or an antibodyDocket No. 15893; 43644.601 fragment, such as a Fab fragment, or a mixture thereof. Antibody fragments or derivatives may comprise F(ab’)2, Fv or scFv fragments. The antibody derivatives can be produced by peptidomimetics. Further, techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies.

[0518] The anti-GFAP antibodies may be a chimeric anti-GFAP or humanized anti-GFAP antibody. In one embodiment, both the humanized antibody and chimeric antibody are monovalent. In one embodiment, both the humanized antibody and chimeric antibody comprise a single Fab region linked to an Fc region.

[0519] Human antibodies may be derived from phage-display technology or from transgenic mice that express human immunoglobulin genes. The human antibody may be generated as a result of a human in vivo immune response and isolated. See, for example, Funaro et al., BMC Biotechnology, 2008(8):85. Therefore, the antibody may be a product of the human and not animal repertoire. Because it is of human origin, the risks of reactivity against self-antigens may be minimized. Alternatively, standard yeast display libraries and display technologies may be used to select and isolate human anti-GFAP antibodies. For example, libraries of naive human single chain variable fragments (scFv) may be used to select human anti-GFAP antibodies. Transgenic animals may be used to express human antibodies.

[0520] Humanized antibodies may be antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.

[0521] The antibody is distinguishable from known antibodies in that it possesses different biological function(s) than those known in the art.(1) Epitope

[0522] The antibody may immunospecifically bind to GFAP (SEQ ID NO: 2), a fragment thereof, or a variant thereof. The antibody may immunospecifically recognize and bind at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, or a...

Claims

Docket No. 15893; 43644.601CLAIMSWhat is claimed is:

1. A method of adjudicating a traumatic brain injury (TBI) diagnosis, the method comprising:(a) receiving diagnostic criteria of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head, wherein the diagnostic criteria comprises:(i) the presence or absence of a trauma mechanism;(ii) the presence or absence of a neuroimaging intracranial abnormality;(iii) the presence or absence of one or more clinical signs of a TBI; and(iv) the presence or absence of one or more confounding factors;(b) executing a TBI adjudication algorithm by compiling the diagnostic criteria; and(c) adjudicating a TBI diagnosis based on the diagnostic criteria compiled in step (b), wherein a subject is diagnosed as having a TBI when:(i) a trauma mechanism and a neuroimaging intracranial abnormality are present; or(ii) a trauma mechanism is present, a neuroimaging intracranial abnormality is absent, one or more clinical signs indicative of TBI is present, and one or more confounding factors is absent.

2. The method of claim 1, wherein the subject is diagnosed as not having a TBI when a trauma mechanism is absent.

3. The method of claim 1 or 2, wherein the subject is diagnosed as not having a TBI when:(i) a trauma mechanism is present;(ii) a neuroimaging intracranial abnormality is absent; and(iii) one more clinical signs of a TBI is absent.

4. The method of any one of claims 1-3, wherein the subject is diagnosed as not having a TBI diagnosis when:(i) a trauma mechanism is present;(ii) a neuroimaging intracranial abnormality is absent;(iii) one more clinical signs of a TBI is present; andDocket No. 15893; 43644.601(iv) the one or more clinical signs of TBI is better accounted for by the presence of the one or more confounding factors.

5. The method of any one of claims 1-4, wherein the trauma mechanism is selected from the group consisting of an injury involving:(i) impact of an object on the subject’s head;(ii) the subject’s head striking a hard object or surface;(iii) acceleration or deceleration of the subject’s head;(iv) a force generated from a blast or explosion; and(v) combinations of (i)-(iv).

6. The method of any one of claims 1-5, wherein the neuroimaging is selected from the group consisting of a computed tomography (CT) scan of the subject’s head and a magnetic resonance imaging of the subject’s head.

7. The method of any one of claims 1-6, wherein the one or more clinical signs is selected from the group consisting of:(i) loss of consciousness;(ii) post-traumatic or peri-traumatic amnesia;(iii) alteration of mental status; and(iv) combinations of (i)-(iii).

8. The method of claim 7, wherein executing the TBI adjudication algorithm further comprises compiling an injury severity indicator based on the one or more clinical signs, wherein the one or more clinical signs are selected from the group consisting of:(i) a duration of the subject’s loss of consciousness;(ii) a duration of the subject’s post-traumatic or peri -traumatic amnesia; and(iii) a Glasgow Coma Scale (GCS) score, a Ranchos Los Amigos Scale score, and / or a Rivermead Post-Concussion Symptoms Questionnaire score of the subject after a duration of time.

9. The method of any one of claims 1-8, wherein the one or more confounding factors is selected from the group consisting of:(i) acute musculoskeletal pain;Docket No. 15893; 43644.601(ii) psychological stress;(iii) use of a drug of abuse;(iv) pulmonary / circulatory disruption;(v) syncopy prior to a fall; and(vi) combinations of (i)-(vi).

10. The method of any one of claims 1-9, wherein the diagnostic criteria further comprises the presence of an acute symptom selected from the group consisting of:(i) a subjective alteration in mental status selected from the group consisting of feeling confused, feeling disoriented, feeling dazed, and combinations thereof;(ii) a physical symptom selected from the group consisting of a headache, nausea, dizziness, balance problems, vision problems, light sensitivity, noise sensitivity, and combinations thereof;(iii) a cognitive symptom selected from the group consisting of feeling slowed down, mental fog, difficulty concentrating, memory problems, and combinations thereof; and(iv) an emotional symptom selected from the group consisting of unusual emotional lability, emotional irritability, and combinations thereof.

11. The method of claim 10, wherein the diagnostic criteria further comprises the presence of an impairment on acute clinical examination or the presence of an elevated biomarker indicative of an intracranial injury.

12. The method of claim 11, wherein the impairment on acute clinical examination is selected from the group consisting of:(i) a cognitive impairment on acute clinical examination;(ii) a balance impairment on acute clinical examination; and(iii) an oculomotor impairment or symptom provocation in response to vestibular-oculomotor challenge on acute clinical examination.

13. The method of claim 11, wherein the elevated biomarker indicative of an intracranial injury is selected from the group consisting of APOA1, ADAMI 0, brain derived nerve growth factor (BDNF), calcium binding protein (SI 00b), C-reactive protein (CRP), glial fibrillary acidic protein (GFAP), glial fibrillary acidic protein breakdown productsDocket No. 15893; 43644.601(GFAP-BDP), neuron specific enolase (NSE), NF-L, peroxidredoxin 6 (PRDX6), Tau, p-Tau, ubiquitin carboxy -terminal hydrolase LI (UCH-L1), and combinations thereof.

14. The method of any one of claims 10-13, wherein executing the TBI adjudication algorithm further comprises compiling the presence of the acute symptom, the presence of the impairment on acute clinical examination, and the presence of the elevated biomarker indicative of an intracranial injury.

15. The method of claim 14, wherein a subject is diagnosed as having a TBI when:(i) a mechanism of trauma is present;(ii) at least two acute symptoms are present;(iii) either at least one impairment on acute clinical examination is present or at least one elevated biomarker indicative of an intracranial injury is present; and(iv) the at least two acute symptoms and either the at least one impairment on acute clinical examination or at least one elevated biomarker indicative of the intracranial injury are not better accounted for by the one or more confounding factors.

16. The method of any of claims 1-15, wherein the diagnosis is a mild TBI.

17. The method of any of claims 1-15, wherein a moderate, moderate-to-severe, or severe TBI is diagnosed when:(i) a duration of the subject’s loss of consciousness exceeds 30 minutes;(ii) a duration of the subject’s post-traumatic or peri -traumatic amnesia exceeds 24 hours; or(iii) a GCS score of the subject after 30 minutes is less than 13.

18. The method of any one of claims 1-17, which is a computer-implemented method.

19. The method of any one of claims 1-18, wherein the diagnostic criteria is received from a database comprising electronic medical records of the subject.Docket No. 15893; 43644.60120. The method of any one of claims 1-19, further comprising storing the adjudicated TBI diagnosis in a database comprising an electronic medical record of the subject.

21. The method of any one of claims 1-20, further comprising recommending a treatment based on the adjudicated TBI diagnosis.

22. The method of claim 21, wherein the treatment comprises a prescription for a medication.

23. The method of claim 22, wherein the prescription is automatically sent to a preferred pharmacy of the subject.

24. A method of diagnosing or aiding in a diagnosis of a traumatic brain injury (TBI) diagnosis, the method comprising:(a) receiving diagnostic criteria of a subject that has sustained, may have sustained, or is suspected of sustaining an injury to the head, wherein the diagnostic criteria comprises:(i) the presence or absence of a trauma mechanism; and(ii) a level of glial fibrillary acidic protein (GFAP) and / or a level of ubiquitin carboxy -terminal hydrolase LI (UCH-L1) measured in a sample obtained from the subject; and(b) diagnosing the subject as having a TBI or aiding in the diagnosis that the subject is more likely than not to have a TBI when:(i) a trauma mechanism is present; and(ii) the level of GFAP is greater than or equal to about 80 pg / mL, the level of UCH-L1 is greater than or equal to about 500 pg / mL, or the level of GFAP is greater than or equal to about 80 pg / mL and the level of UCH-L1 is greater than or equal to about 500 pg / mL.

25. The method of claim 24, wherein a TBI is diagnosed in the subject or the subject is determined to more likely than not to have a TBI when:(i) a trauma mechanism is present; andDocket No. 15893; 43644.601(ii) the level of GFAP is greater than or equal to about 80 pg / mL, the level of UCH-L1 is greater than or equal to about 1000 pg / mL, or the level of GFAP is greater than or equal to about 80 pg / mL and the level of UCH-L1 is greater than or equal to about 1000 pg / mL.

26. The method of claim 24, wherein a TBI is diagnosed in the subject or the subject is determined to more likely than not to have a TBI when:(i) a trauma mechanism is present; and(ii) the level of GFAP is greater than or equal to about 100 pg / mL, the level of UCH-L1 is greater than or equal to about 500 pg / mL, or the level of GFAP is greater than or equal to about 100 pg / mL and the level of UCH-L1 is greater than or equal to about 500 pg / mL.

27. The method of claim 24, wherein a TBI is diagnosed in the subject or the subject is determined to more likely than not to have a TBI when:(i) a trauma mechanism is present; and(ii) the level of GFAP is greater than or equal to about 100 pg / mL, the level of UCH-L1 is greater than or equal to about 1000 pg / mL, or the level of GFAP is greater than or equal to about 100 pg / mL and the level of UCH-L1 is greater than or equal to about 1000 pg / mL.

28. The method of claim 24, wherein a TBI is diagnosed in the subject or the subject is determined to more likely than not to have a TBI when:(i) a trauma mechanism is present; and(ii) the level of GFAP is greater than or equal to about 120 pg / mL, the level of UCH-L1 is greater than or equal to about 500 pg / mL, or the level of GFAP is greater than or equal to about 120 pg / mL and the level of UCH-L1 is greater than or equal to about 500 pg / mL.

29. The method of claim 24, wherein TBI is diagnosed in the subject or the subject is determined to more likely than not to have a TBI when:(i) a trauma mechanism is present; and(ii) the level of GFAP is greater than or equal to about 120 pg / mL, the level of UCH-L1 is greater than or equal to about 1000 pg / mL, or the level of GFAP is greaterDocket No. 15893; 43644.601 than or equal to about 120 pg / mL and the level of UCH-L1 is greater than or equal to about 1000 pg / mL.

30. The method of any one of claims 24-29, wherein TBI is diagnosed in the subject or the subject is determined to more likely than not to have a TBI when:(i) a trauma mechanism is present; and(ii) the level of GFAP is greater than between about 80 pg / mL to about 120 pg / mL, the level of UCH-L1 is greater than between about 500 pg / mL to about 1000 pg / mL, or the level of GFAP is greater than between about 80 pg / mL to about 120 pg / mL and the level of UCH-L1 is greater than between about 500 pg / mL to about 1000 pg / mL.

31. The method of any one of claims 24-30, wherein the subject is diagnosed as not having or likely not to have a TBI when a trauma mechanism is absent.

32. The method of any one of claims 24-31, wherein the subject is diagnosed as not having or likely not to have a TBI when:(i) a trauma mechanism is present and the level of GFAP is less than about 80 pg / mL, the level of UCH-L1 is less than about 500 pg / mL, the level of GFAP is less than about 80 pg / mL, the level of UCH-L1 is less than about 500 pg / mL;(ii) a trauma mechanism is present and the level of GFAP is less than about 80 pg / mL, the level of UCH-L1 is less than about 1000 pg / mL, or the level of GFAP is less than about 80 pg / mL and the level of UCH-L1 is less than about 1000 pg / mL;(iii) a trauma mechanism is present and the level of GFAP is less than about 100 pg / mL, the level of UCH-L1 is less than about 500 pg / mL, or the level of GFAP is less than about 100 pg / mL and the level of UCH-L1 is less than about 500 pg / mL;(iv) a trauma mechanism is present and the level of GFAP is less than about 100 pg / mL, the level of UCH-L1 is less than about 1000 pg / mL, or the level of GFAP is less than about 100 pg / mL and the level of UCH-L1 is less than about 1000 pg / mL;(v) a trauma mechanism is present and the level of GFAP is less than about 120 pg / mL, the level of UCH-L1 is less than about 500 pg / mL, or the level of GFAP is less than about 120 pg / mL and the level of UCH-L1 is less than about 500 pg / mL; orDocket No. 15893; 43644.601(vi) a trauma mechanism is present and the level of GFAP is less than about 120 pg / mL, the level of UCH-L1 is less than about 1000 pg / mL, or the level of GFAP is less than about 120 pg / mL and the level of UCH-L1 is less than about 1000 pg / mL .

33. The method of any one of claims 24-32, wherein the subject is diagnosed as not having or likely not to have a TBI when:(i) a trauma mechanism is present; and(ii) the level of GFAP is less than between about 80 pg / mL to about 120 pg / mL, the level of UCH-L1 is less than between about 500 pg / mL to about 1000 pg / mL, or level of GFAP is less than between about 80 pg / mL to about 120 pg / mL and the level of UCH-L1 is less than between about 500 pg / mL to about 1000 pg / mL.

34. The method of any one of claims 24-33, wherein the diagnostic criteria further comprises:(iii) the presence or absence of one or more clinical signs of a TBI.

35. The method of claim 34, wherein the subject is diagnosed as having a TBI or more likely than not to have a TBI when:(i) a trauma mechanism is present, the level of GFAP is greater than about 80 pg / mL and / or the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is absent;(ii) a trauma mechanism is present, the level of GFAP is greater than about 80 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is absent;(iii) a trauma mechanism is present, the level of GFAP is greater than about 100 pg / mL and / or the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is absent;(iv) a trauma mechanism is present, the level of GFAP is greater than about 100 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is absent;(v) a trauma mechanism is present and the level of GFAP is greater than about 120 pg / mL and the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is absent; orDocket No. 15893; 43644.601(vi) a trauma mechanism is present and the level of GFAP is greater than about 120 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is absent.

36. The method of claim 34, wherein the subject is diagnosed as having a TBI or more likely than not to have a TBI when:(i) a trauma mechanism is present, the level of GFAP is greater than about 80 pg / mL and / or the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is present;(ii) a trauma mechanism is present, the level of GFAP is greater than about 80 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is present;(iii) a trauma mechanism is present, the level of GFAP is greater than about 100 pg / mL and / or the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is present;(iv) a trauma mechanism is present, the level of GFAP is greater than about 100 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is present;(v) a trauma mechanism is present and the level of GFAP is greater than about 120 pg / mL and / or the level of UCH-L1 is greater than about 500 pg / mL, and the one or more clinical signs of a TBI is present;(vi) a trauma mechanism is present and the level of GFAP is greater than about 120 pg / mL and / or the level of UCH-L1 is greater than about 1000 pg / mL, and the one or more clinical signs of a TBI is present; or(vii) a trauma mechanism is present and the level of GFAP is greater than about 100 pg / mL and / or the level of UCH-L1 is greater than about 700 pg / mL, and the one or more clinical signs of a TBI is present.

37. The method of claim 34, wherein the subject is diagnosed as having a TBI or more likely than not to have a TBI when:(i) a trauma mechanism is present;(ii) the level of GFAP is greater than between about 80 pg / mL to about 120 pg / mL and / or the level of UCH-L1 is greater than between about 500 pg / mL to about 1000 pg / mL; andDocket No. 15893; 43644.601(iii) the one or more clinical signs of a TBI is absent.

38. The method of claim 34, wherein the subject is diagnosed as having a TBI or more likely than not to have a TBI when:(i) a trauma mechanism is present;(ii) the level of GFAP is greater than between about 80 pg / mL to about 120 pg / mL and / or the level of UCH-L1 is greater than between about 500 pg / mL to about 1000 pg / mL; and(iii) the one or more clinical signs of a TBI is present.

39. The method of any one of claims 24-38 wherein the diagnostic criteria further comprises:(iv) the presence or absence of one or more confounding factors.

40. The method of claim 38, wherein the subject is diagnosed as not having or likely not to have a TBI when:(i) a trauma mechanism is present;(ii) one more clinical signs of a TBI is present; and(iv) the one or more clinical signs of TBI is better accounted for by the presence of the one or more confounding factors.

41. The method of any one of claims 24-40, wherein the trauma mechanism is selected from the group consisting of an injury involving:(i) impact of an object on the subject’s head;(ii) the subject’s head striking a hard object or surface;(iii) acceleration or deceleration of the subject’s head;(iv) a force generated from a blast or explosion; and(v) combinations of (i)-(i v).

42. The method of any one of claims 24-41, wherein the one or more clinical signs is selected from the group consisting of:(i) loss of consciousness;(ii) post-traumatic or peri-traumatic amnesia;(iii) alteration of mental status; andDocket No. 15893; 43644.601(iv) combinations of (i)-(iii).

43. The method of claim 42, wherein the diagnostic criteria further comprises an injury severity indicator based on the one or more clinical signs, wherein the one or more clinical signs are selected from the group consisting of:(i) a duration of the subject’s loss of consciousness;(ii) a duration of the subject’s post-traumatic or peri -traumatic amnesia; and(iii) a Glasgow Coma Scale (GCS) score, a Ranchos Los Amigos Scale score, and / or a Rivermead Post-Concussion Symptoms Questionnaire score of the subject after a duration of time.

44. The method of any one of claims 24-43, wherein the one or more confounding factors is selected from the group consisting of:(i) acute musculoskeletal pain;(ii) psychological stress;(iii) use of a drug of abuse;(iv) pulmonary / circulatory disruption;(v) syncopy prior to a fall; and(vi) combinations of (i)-(vi).

45. The method of any one of claims 24-44, wherein the diagnostic criteria further comprises the presence of an acute symptom selected from the group consisting of:(i) a subjective alteration in mental status selected from the group consisting of feeling confused, feeling disoriented, feeling dazed, and combinations thereof;(ii) a physical symptom selected from the group consisting of a headache, nausea, dizziness, balance problems, vision problems, light sensitivity, noise sensitivity, and combinations thereof;(iii) a cognitive symptom selected from the group consisting of feeling slowed down, mental fog, difficulty concentrating, memory problems, and combinations thereof; and(iv) an emotional symptom selected from the group consisting of unusual emotional lability, emotional irritability, and combinations thereof.Docket No. 15893; 43644.60146. The method of any one of claims 24-45, wherein the diagnostic criteria further comprises the presence of an impairment on acute clinical examination.

47. The method of claim 46, wherein the impairment on acute clinical examination is selected from the group consisting of:(i) a cognitive impairment on acute clinical examination;(ii) a balance impairment on acute clinical examination; and(iii) an oculomotor impairment or symptom provocation in response to vestibular-oculomotor challenge on acute clinical examination.

48. The method of any one of claims 45-47, wherein a subject is diagnosed as having a TBI or more likely than not to have a TBI when:(i) a mechanism of trauma is present;(ii) at least two acute symptoms are present;(iii) either at least one impairment on acute clinical examination is present or at least one elevated biomarker indicative of an intracranial injury is present; and(iv) the at least two acute symptoms and either the at least one impairment on acute clinical examination or at least one elevated biomarker indicative of the intracranial injury are not better accounted for by the one or more confounding factors.

49. The method of any of claims 24-48, wherein the diagnosis is a mild TBI.

50. The method of any of claims 24-49, wherein a moderate, moderate-to-severe, or severe TBI is diagnosed when:(i) a duration of the subject’s loss of consciousness exceeds 30 minutes;(ii) a duration of the subject’s post-traumatic or peri -traumatic amnesia exceeds 24 hours; or(iii) a GCS score of the subject after 30 minutes is less than 13.

51. The method of any one of claims 24-50, wherein the method is a computer- implemented method.Docket No. 15893; 43644.60152. The method of any one of claims 24-51, wherein neuroimaging (e.g., brainimaging test, such as head CT or magnetic resonance imaging (MRI)) is done following a diagnosis of or to further aid in a diagnosis of TBI.

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