Method and system for diagnosing brain damage

Abstract

Disclosed herein are methods and systems for determining whether a subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated in a sample collected from a subject. The methods include determining whether a subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated in the sample and communicating the determination on or from a device. The methods may be used to aid in the diagnosis and assessment of a subject (e.g., a human subject) that has or may have suffered a head injury, such as determining whether the subject has suffered a mild, moderate, severe, or moderate-severe traumatic brain injury (TBI).

Description

[Technical field]

[0001] This application claims priority to U.S. Patent Application No. 63 / 250,966, filed September 30, 2021, U.S. Patent Application No. 63 / 254,285, filed October 11, 2021, and U.S. Patent Application No. 63 / 294,346, filed December 28, 2021, the contents of each of which are incorporated herein by reference.

[0002] The present disclosure relates to methods and systems to aid in the diagnosis and assessment of a subject (e.g., a human subject) who has suffered or may have suffered a head injury, such as mild, moderate, severe, or moderate-severe traumatic brain injury (TBI), by detecting levels of biomarkers such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or combinations thereof, in a sample taken from a subject (e.g., a human subject) who has suffered or is suspected of having suffered a head injury. [Background technology]

[0003] More than 5 million mild traumatic brain injuries (TBIs) occur annually in the United States alone. Currently, there are no simple, objective, and accurate measurements available to aid in patient evaluation. Indeed, much of TBI assessment and diagnosis is based on subjective data. Unfortunately, objective measurements such as head CT scans and Glasgow Coma Scale (GCS) scores are not very comprehensive or sensitive in assessing mild TBI. Furthermore, head CT scans often reveal nothing about mild TBI, are expensive, and expose patients to unnecessary radiation. In addition, a negative head CT does not mean that the patient is clearly not concussed, but only that certain interventions, such as surgery, are not warranted. Physicians and patients need objective and reliable information to accurately assess this condition and facilitate appropriate triage and recovery.

[0004] Mild TBI or concussion is much more difficult to detect objectively and is a daily challenge in emergency rooms worldwide. Concussion does not usually cause macroscopic pathology such as bleeding and abnormalities in conventional computed tomography scans of the brain, but often causes rapid onset neurological dysfunction that resolves spontaneously over days to weeks. Approximately 15% of mild TBI patients suffer from persistent cognitive dysfunction. There is an unmet need to detect and assess mild TBI victims in the field, in emergency rooms and clinics, on sports fields, and in military activities (e.g., combat).

[0005] Current algorithms for assessing the severity of brain injury include the Glasgow Coma Scale (GCS) score and other criteria. These criteria may be adequate to correlate acute severity, but are insufficiently sensitive to subtle pathology that may result in persistent deficits. GCS and other criteria also do not allow for differentiation between types of injury and may be inappropriate. Thus, patients classified into a single GCS level who enter clinical trials may have highly heterogeneous severity and type of injury. Inappropriate classification undermines the integrity of clinical trials, as outcomes vary accordingly. Improved classification of injury would allow for more accurate delineation of disease severity and type for TBI patients in clinical trials.

[0006] Furthermore, current brain injury trials rely on outcome measures, such as the Glasgow Outcome Scale-Extended, which capture global phenomena but cannot assess nuances in outcome. Thus, 30 consecutive trials of brain injury drugs have failed. Sensitive assessment criteria are needed to determine how well patients have recovered from brain injury in order to test therapeutic and preventative drugs. Summary of the Invention [Means for solving the problem]

[0007] In some aspects, the disclosure relates to a method for determining whether a subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated. In some embodiments, such a method is provided herein. In some embodiments, a method is provided herein that includes performing at least one assay for ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), at least one assay for glial fibrillary acidic protein (GFAP), or at least one assay for UCH-L1 and GFAP in at least one sample obtained from a human subject. The sample is obtained from the subject within about 48 hours after actual or suspected head injury. For example, in some embodiments, the sample is obtained within about 24 hours after actual or suspected head injury. In some embodiments, the sample is obtained within about 12 hours after actual or suspected head injury. In some embodiments, the method further includes determining whether the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated or not elevated, or that the assays for GFAP, UCH-L1, or GFAP and UCH-L1 should be repeated.

[0008] In some embodiments, the method includes determining that a subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated. A subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are determined to be elevated if: (i) only the level of GFAP in the sample is equal to or greater than about 35 pg / mL; (ii) the level of GFAP in the sample is equal to or greater than about 35 pg / mL and the level of UCH-L1 in the sample is less than, cannot be determined, or is not reported, about 400 pg / mL; (iii) the level of GFAP in the sample is equal to or greater than about 35 pg / mL and the level of UCH-L1 in the sample is equal to or greater than about 400 pg / mL; (iv) only the level of UCH-L1 in the sample is equal to or greater than about 400 pg / mL; or (v) the level of GFAP in the sample is not determinable or is not reported and the level of UCH-L1 in the sample is equal to or greater than about 400 pg / mL.

[0009] In some embodiments, the method includes determining that the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are not elevated. The subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are not elevated if only the subject level of GFAP in the sample is below about 35 pg / mL, only the level of UCH-L1 in the sample is below about 400 pg / mL, or if the subject level of GFAP in the sample is below about 35 pg / mL and the level of UCH-L1 in the sample is below about 400 pg / mL.

[0010] In some embodiments, the method includes determining that the assay for UCH-L1, GFAP, or UCH-L1 and GFAP should be repeated. If (i) only the level of UCH-L1 in the sample cannot be determined or is not reported; (ii) the level of GFAP in the sample is below about 35 pg / mL and the level of UCH-L1 in the sample is not determined or is not reported; (iii) only the level of GFAP in the sample is not determined or is not reported; (iv) the level of GFAP in the sample is not determined or is not reported and the level of UCH-L1 in the sample is below about 400 pg / mL; or (v) the level of GFAP in the sample is not determined or is not reported and the level of UCH-L1 in the sample is not determined or is not reported, the assay for UCH-L1, GFAP, or UCH-L1 and GFAP should be repeated. In some embodiments, the method includes transmitting the determination on or from at least one device.

[0011] In some embodiments, the method further comprises performing a head computed tomography (CT) scan, a magnetic resonance imaging (MRI) procedure, or both a CT scan or an MRI procedure on the subject if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated. In some embodiments, the method further comprises determining not to perform a head computed tomography (CT) scan, a magnetic resonance imaging (MRI) procedure, or both a head CT scan and an MRI procedure on the subject if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are not elevated.

[0012] In some embodiments, the method further includes diagnosing the subject as having traumatic brain injury (TBI) if the level of GFAP alone is equal to or greater than about 35 pg / mL, the level of UCH-L1 alone is equal to or greater than about 400 pg / mL, or the level of GFAP is equal to or greater than about 35 pg / mL and / or the level of UCH-L1 is equal to or greater than about 400 pg / mL, regardless of whether a head CT scan is negative for TBI or whether any head CT scan is performed.

[0013] In some embodiments, the method further comprises treating the subject for mild, moderate, moderate-severe, or severe TBI if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated. In some embodiments, the method further comprises monitoring the subject if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated.

[0014] In some embodiments, the sample is administered within about 5 minutes, about 10 minutes, about 12 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, or within about 24 hours of an actual or suspected head injury. The antibody is harvested within about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, or within about 48 hours.

[0015] In another embodiment, the sample is collected within about 8 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 9 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 10 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 11 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 12 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 13 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 14 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 15 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 16 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 17 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 18 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 19 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 20 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 21 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 22 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 23 hours to about 48 hours after the actual or suspected injury to the head. In yet other embodiments, the sample is collected within about 24 hours to about 48 hours after actual or suspected head injury, and in yet other embodiments, the sample is collected within about 25 hours to about 48 hours after actual or suspected head injury.In yet another embodiment, the sample is collected within about 26 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 27 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 28 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 29 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 30 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 31 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 32 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 33 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 34 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 35 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 36 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 37 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 38 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 39 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 40 hours to about 48 hours after the actual or suspected injury to the head.

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

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

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

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

[0020] In some embodiments, the subject has suffered an orthopedic injury in addition to the actual or suspected injury to the head, hi some embodiments, the orthopedic injury and the injury to the head may occur simultaneously.

[0021] In some embodiments, the sample is selected from the group consisting of a whole blood sample, a capillary blood sample, a serum sample, a cerebrospinal fluid sample, a mixed venous and capillary blood sample, a mixed capillary blood and interstitial fluid sample, a tissue sample, a body fluid, and a plasma sample.

[0022] In another embodiment, the present disclosure relates to a system. Specifically, the system of the present disclosure comprises: An assay for ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), an assay for glial fibrillary acidic protein (GFAP), or an assay for UCH-L1 and GFAP; and NON-POINT-OF-CARE DEVICES FOR PERFORMING ASSAYS FOR UCH-L1, ASSAYS FOR GFAP, OR ASSAYS FOR UCH-L1 AND GFAP Including, The device determines the amount of UCH-L1, GFAP, or UCH-L1 and GFAP in a sample obtained from the subject; The amount of UCH-L1, GFAP or UCH-L1 and GFAP determined in the sample is: a. Elevated if (i) only the level of GFAP in the sample is equal to or exceeds about 35 pg / mL; (ii) the level of GFAP in the sample is equal to or exceeds about 35 pg / mL and the level of UCH-L1 in the sample is lower than, cannot be determined, or is not reported to be about 400 pg / mL; (iii) the level of GFAP in the sample is equal to or exceeds about 35 pg / mL and the level of UCH-L1 in the sample is equal to or exceeds about 400 pg / mL; (iv) only the level of UCH-L1 in the sample is equal to or exceeds about 400 pg / mL; or (v) the level of GFAP in the sample is not determinable or is not reported and the level of UCH-L1 in the sample is equal to or exceeds about 400 pg / mL; b. (i) only the level of GFAP in the sample is below about 35 pg / mL; (ii) only the level of UCH-L1 in the sample is below about 400 pg / mL; or (iii) not elevated if the level of GFAP in the sample is below about 35 pg / mL and the level of UCH-L1 in the sample is below about 400 pg / mL; c. (i) the level of UCH-L1 in the sample alone cannot be determined or is not reported; (ii) the level of GFAP in the sample is below about 35 pg / mL and the level of UCH-L1 in the sample is not determined or is not reported; (iii) the level of GFAP in the sample alone cannot be determined or is not reported; (iv) the level of GFAP in the sample is not determined or is not reported and the level of UCH-L1 in the sample is below about 400 pg / mL; or (v) the level of GFAP in the sample is not determined or is not reported and the level of UCH-L1 in the sample is not determined or is not reported, requiring that the assays for UCH-L1 and GFAP be repeated. is communicated by or from the device as

[0023] In some embodiments, the sample is administered within about 5 minutes, within about 10 minutes, within about 12 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 60 minutes, within about 90 minutes, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 24 hours, within about 28 hours, within about 29 hours, within about 30 hours, within about 31 hours, within about 32 hours, within about 33 hours, within about 34 hours, within about 35 hours, within about 36 hours, within about 37 hours, within about 38 hours, within about 39 hours, within about 40 hours, within about 42 hours, within about 44 hours, within about 46 hours, within about 48 hours, within about 49 hours, within about 50 hours, within about 50 hours, within about 51 hours, within about 52 hours, within about 53 hours, within about 54 hours, within about 55 hours, within about 56 hours, within about 57 hours, within about 58 hours, within about 59 hours, within about 60 hours, within about 61 hours, within about 62 hours, within about 63 hours, within about 64 hours, within about 65 hours, within about 66 hours, within about 67 hours, within about 68 hours, within about 69 hours, within about 70 hours, within about 71 hours, within about 72 hours, within about 73 hours, within about 74 hours, within about 75 hours, within The antibody is harvested within about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, or within about 48 hours.

[0024] In another embodiment, the sample is collected within about 8 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 9 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 10 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 11 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 12 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 13 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 14 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 15 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 16 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 17 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 18 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 19 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 20 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 21 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 22 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 23 hours to about 48 hours after the actual or suspected injury to the head. In yet other embodiments, the sample is collected within about 24 hours to about 48 hours after actual or suspected head injury, and in yet other embodiments, the sample is collected within about 25 hours to about 48 hours after actual or suspected head injury.In yet another embodiment, the sample is collected within about 26 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 27 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 28 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 29 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 30 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 31 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 32 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 33 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 34 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 35 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 36 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 37 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 38 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 39 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 40 hours to about 48 hours after the actual or suspected injury to the head.

[0025] The assay for UCH-L1 and / or the assay for GFAP can be performed simultaneously or sequentially, in any order.

[0026] In some embodiments, the sample is obtained after the subject has been subjected to a physical shake, a blunt impact from an external mechanical or other force resulting in a closed or open head injury, one or more falls, an injury to the head caused by an explosion or blast, or other type of blunt force trauma. In some embodiments, the sample is obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin. In some embodiments, the chemical or toxin is fire, mold, asbestos, a pesticide, a insecticide, an organic solvent, a paint, a glue, a gas, an organometallic, a drug of abuse, or a combination of one or more thereof. In some embodiments, the sample is obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection (e.g., SARS-CoV-2), a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof.

[0027] In some embodiments, the assay (e.g., an assay for GFAP and / or an assay for UCH-L1) is an immunoassay or a clinical chemistry assay. In some embodiments, the assay is a single molecule detection assay or a non-point-of-care assay. In some embodiments, the volume of the at least one sample is about 10 μL to about 30 μL. For example, in some embodiments, the volume of the at least one sample is about 20 μL.

[0028] In some embodiments, the assay for UCH-L1, the assay for GFAP, or the assay for UCH-L1 and at least one of the assays for GFAP are performed about 10 minutes to about 20 minutes later. For example, in some embodiments, the assay for UCH-L1, the assay for GFAP, or the assay for UCH-L1 and the assay for GFAP are performed about 15 minutes later.

[0029] In some embodiments, the subject has suffered an orthopedic injury in addition to the actual or suspected injury to the head, hi some embodiments, the orthopedic injury and the injury to the head may occur simultaneously.

[0030] In some embodiments, the sample is selected from the group consisting of a whole blood sample, a capillary blood sample, a serum sample, a cerebrospinal fluid sample, a mixed venous and capillary blood sample, a mixed capillary blood and interstitial fluid sample, a tissue sample, a body fluid, and a plasma sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present disclosure relates to methods and systems that use one or more biomarkers, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, to aid in the diagnosis and assessment of a subject (e.g., a human subject) that has sustained or may have sustained a head injury, such as mild, moderate, severe, or moderate-severe traumatic brain injury (TBI). These methods include detecting one or more biomarker levels in one or more samples taken from the subject (e.g., a human subject) within 48 hours of the actual or suspected head injury. In some aspects, the subject has sustained an orthopedic injury in addition to the actual or suspected head injury. In some embodiments, the orthopedic injury is sustained simultaneously with the head injury.

[0032] In some aspects, the disclosure relates to methods and systems for determining whether a subject has elevated levels of GFAP, UCH-L1, or GFAP and UCH-L1. The method includes determining whether a subject has elevated levels of GFAP, UCH-L1, or GFAP and UCH-L1, and communicating the determination on or from at least one device. The method includes detecting levels of GFAP, UCH-L1, or GFAP and UCH-L1 in a sample obtained within about 48 hours after actual or suspected head injury. For example, the method may include detecting levels of GFAP, UCH-L1, or GFAP and UCH-L1 in a sample obtained within about 48 hours, within about 24 hours, or within about 12 hours after actual or suspected head injury. In some embodiments, the method includes detecting levels of GFAP, UCH-L1, or GFAP and UCH-L1 in a sample obtained within about 12 hours to about 48 hours, or within about 24 hours to about 48 hours. In still other embodiments, the method includes detecting levels of GFAP, UCH-L1, or GFAP and UCH-L1 within about 12 hours (e.g., within about 12 hours, within about 11 hours, within about 10 hours, within about 9 hours, within about 8 hours, within about 7 hours, within about 6 hours, within about 5 hours, within about 4 hours, within about 3 hours, within about 2 hours, within about 1 hour, or within about 30 minutes) after an actual or suspected injury to the head.

[0033] The present disclosure relates to methods and systems that aid in determining whether a subject (e.g., a human subject) who has or may have suffered such a head injury would benefit from, and therefore undergo, a head computed tomography (CT) scan, a magnetic resonance imaging (MRI) procedure, or both a head CT scan and an MRI procedure, based on the levels of one or more biomarkers, such as UCH-L1, GFAP, or a combination thereof. These methods include detecting the levels of at least one biomarker, such as UCH-L1, GFAP, or a combination thereof, in one or more samples taken from the subject (e.g., a human subject) at a time point within about 48 hours after the head injury (e.g., actual injury) or suspected head injury. For example, the methods can include detecting the levels of UCH-L1, GFAP, or UCH-L1 and GFAP within about 48 hours, within about 24 hours, or within about 12 hours after the actual or suspected head injury. After injury (e.g., actual injury) or suspected injury to the head, detection of a biomarker such as UCH-L1, GFAP, or a combination thereof, higher than the reference level of the biomarker, aids in determining whether the subject should undergo a head CT scan and / or MRI procedure. For example, a subject (e.g., a human subject) having a level of a biomarker such as UCH-L1, GFAP, or a combination thereof, higher than the reference level of the biomarker, such as UCH-L1, GFAP, or a combination thereof, may also be identified as likely to have a positive head CT scan and / or MRI, and thus may benefit from having a CT scan and / or MRI procedure. Alternatively, a subject (e.g., a human subject) having a level of a biomarker such as UCH-L1, GFAP, or a combination thereof, lower than the reference level of the biomarker, such as UCH-L1, GFAP, or a combination thereof, may be identified as likely to have a negative head CT scan and / or MRI, and thus may not benefit from a CT scan and / or MRI procedure.

[0034] The section headings used in this section and throughout the disclosure herein are for organizational purposes only and are not intended to be limiting.

[0035] 1.Definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of discrepancy, the present document, including definitions, will prevail. In the practice or testing of this disclosure, methods and materials similar or equivalent to those described herein may be used, but the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0036] As used herein, the terms "comprise(s)," "include(s)," "having," "having," "may be," and "containing" are open-ended transitional phrases, transitional terms, or transitional words that do not exclude additional acts or structures. The singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise," "consist of," or "consist essentially of" the embodiments or elements presented herein, whether expressly stated or not.

[0037] In this specification, to recite numerical ranges, each intervening number with the same precision is expressly contemplated. For example, for the range of 6 to 9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated, and for the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0038] As used herein, an "affinity matured antibody" refers to an antibody that has been subjected to a process to reduce the affinity (i.e., K D , k d or k aThe term affinity matured antibody is used to refer to an antibody with one or more alterations in one or more CDRs that result in an improvement over a parent antibody without the alterations. Exemplary affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Various procedures for generating affinity matured antibodies are known in the art, including screening combinatorial antibody libraries prepared using BioDisplay technology. For example, Marks et al., BioTechnology, 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues has been described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-3319 (1995) and Hawkins et al., J. Mol. Biol., 226:889-896 (1992). Selective mutagenesis positions and selective mutations at contact or hypermutation positions with activity enhancing amino acid residues are described in U.S. Patent No. 6,914,128 B1.

[0039] As used herein, "antibody" and "antibodies" include mammalian antibodies, including monoclonal antibodies, polyspecific antibodies, human antibodies, humanized antibodies (fully humanized or partially humanized), avian (e.g., duck or goose) antibodies, shark antibodies, whale antibodies, and non-primate (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, etc.) antibodies or non-human primate (e.g., monkey, chimpanzee, etc.) antibodies. It refers to, but is not limited to, animal antibodies, recombinant antibodies, chimeric antibodies, single chain Fvs ("scFvs"), single chain antibodies, single domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs ("sdFvs") and anti-idiotypic ("anti-Id") antibodies, dual domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual variable domain immunoglobulins and methods for producing them are described in Wu, C. et al., Nature Biotechnology, 25(11):1290-1297 (2007) and PCT International Application No. 2001 / 058956, the contents of each of which are incorporated herein by reference), and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of molecule. For simplicity, antibodies to an analyte are often referred to herein as "anti-analyte antibodies" or simply "analyte antibodies" (e.g., anti-UCH-L1 antibodies or UCH-L1 antibodies).

[0040] As used herein, an "antibody fragment" refers to a portion of an intact antibody that contains the antigen-binding site or variable region. The portion does not contain the heavy chain constant domain of the Fc region of the intact antibody (i.e., CH2, CH3 or CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing three CDRs of a light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing three CDRs of a heavy chain variable region.

[0041] "Area under the curve" or "AUC" refers to the area under the ROC curve. The AUC under the ROC curve is a measure of accuracy. An AUC of 1 represents a perfect test, whereas an AUC of 0.5 represents a non-significant test. A preferred AUC may be at least about 0.700, at least about 0.750, at least about 0.800, at least about 0.850, at least about 0.900, at least about 0.910, at least about 0.920, at least about 0.930, at least about 0.940, at least about 0.950, at least about 0.960, at least about 0.970, at least about 0.980, at least about 0.990, or at least about 0.995.

[0042] As used herein, "beads" and "particles" are used interchangeably and refer to a substantially spherical solid support. One example of a bead or particle is a microparticle. Microparticles that may be used herein may be of any type known in the art. For example, the bead or particle may be a magnetic bead or particle. The magnetic beads / particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or magnetic fluid. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO2, MnAs, MnBi, EuO, and NiO / Fe. Examples of ferrimagnetic materials include NiFe2O4, CoFe2O4, Fe3O4 (or FeO.Fe2O3). The beads may be magnetic and have a solid core portion surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion may be a layer around the non-magnetic core. Microparticles can be of any size that works in the methods described herein, for example, from about 0.75 to about 5 nm, or from about 1 to about 5 nm, or from about 1 to about 3 nm.

[0043] As used herein, "binding protein" is used to refer to a monomeric or multimeric protein that binds to and forms a complex with a binding partner, such as, for example, a polypeptide, an antigen, a compound or other molecule, or a substrate of any kind. A binding protein specifically binds to a binding partner. Binding proteins include antibodies, as well as their antigen-binding fragments and various other forms and derivatives thereof known in the art and described herein below, and other molecules that contain one or more antigen-binding domains that bind to an antigen molecule or a specific site (epitope) on an antigen molecule. Thus, binding proteins include, but are not limited to, antibodies that are tetrameric immunoglobulins, IgG molecules, IgG1 molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, affinity matured antibodies, and any such antibody fragments that retain the ability to bind to an antigen.

[0044] As used herein, "bispecific antibodies" are used to refer to full-length antibodies generated by quadroma technology (see Milstein et al., Nature, 305(5934):537-540 (1983)), chemical conjugation of two different monoclonal antibodies (see Staerz et al., Nature, 314(6012):628-631 (1985)), or the knob-into-hole (KIH) method or similar techniques that introduce mutations within the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14):6444-6448 (1993)), resulting in a number of different immunoglobulin species, only one of which is a functional bispecific antibody. A bispecific antibody binds one antigen (or epitope) in one of its two binding arms (one pair of HC / LC) and binds a different antigen (or epitope) in its second arm (a different pair of HC / LC). By this definition, a bispecific antibody has two significantly different antigen-binding arms (both specificity and CDR sequences) and is monovalent for each antigen to which it binds.

[0045] As used herein, "CDR" is used to refer to the "complementarity determining region" in the variable sequence of an antibody. There are three CDRs in each of the heavy and light chain variable regions. From the N-terminus of the heavy or light chain, these regions are designated as "CDR1", "CDR2" and "CDR3" for each variable region. As used herein, the term "CDR set" refers to a group of three CDRs occurring in a single variable region that bind to an antigen. Thus, an antigen-binding site may contain six CDRs, including a CDR set from each of the heavy and light chain variable regions. A polypeptide containing a single CDR (e.g., CDR1, CDR2 or CDR3) may be referred to as a "molecular recognition unit". Crystal structure analysis of antigen-antibody complexes supports that the amino acid residues of the CDRs make extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR3. Thus, the molecular recognition units may be primarily responsible for the specificity of an antigen-binding site. In general, the CDR residues are directly and most substantially involved in influencing binding to antigen.

[0046] The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system that is applicable to any variable region of an antibody, but also provides the exact residue boundaries that define the three CDRs. These CDRs may be referred to as "Kabat CDRs." Chothia and colleagues (Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); and Chothia et al., Nature, 342:877-883 (1989)) have described the Kabat CDRs in a numbered system that is applicable to any variable region of an antibody. It was found that certain sub-portions within the CDRs adopt nearly identical peptide backbone conformations despite great diversity at the amino acid sequence level. These sub-portions were designated "L1", "L2" and "L3", or "H1", "H2" and "H3", with "L" and "H" designating the light and heavy chain regions, respectively. These regions are sometimes referred to as "Chothia CDRs", which have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are described in Padlan, FASEB, and others. J., 9:133-139 (1995); and MacCallum, J. Mol. Biol., 262(5):732-745 (1996). Still other CDR boundary definitions may not strictly follow the system defined herein and may be shorter or longer in light of predicted or experimental findings that a particular residue or group of residues or even the entire CDR does not significantly affect binding to the antigen, but still overlap with the Kabat CDRs. The methods used herein may use CDRs defined according to any of these systems, although certain embodiments use the Kabat-defined CDRs or the Chothia-defined CDRs.

[0047] As used herein, "communicated" or "communicating" refers to conveying, sending, and / or reporting a piece of information. In some embodiments, the communicated information is a piece of information (e.g., a result) obtained by performing an assay, such as the amount or presence of a biomarker in a sample. The information obtained by performing an assay can be communicated by computer, in a document and / or spreadsheet, on a mobile device (e.g., a smartphone), on a website, by email, or any combination thereof. In some other embodiments, the information is communicated on or from an instrument or device. In other embodiments, the information is communicated by being displayed, such as on an instrument or device.

[0048] "Component", "components" or "at least one component" generally refers to capture antibodies, detection reagents or detection conjugates, calibrators, controls, sensitivity panels, containers, buffers, diluents, salts, enzymes, cofactors for enzymes, detection reagents, pretreatment reagents / solutions, substrates (e.g., in solution), stop solutions, etc. that may be included in a kit for assaying a test sample, such as a patient urine sample, whole blood sample, serum sample or plasma sample, according to the methods described herein and other methods known in the art. Some components may be in solution or may be lyophilized for reconstitution for use in the assay.

[0049] As used herein, "correlates to" means compared to.

[0050] As used herein, "CT scan" refers to a computed tomography (CT) scan. A CT scan combines a series of X-ray images taken from different angles and uses computer processing to create cross-sectional images or slices of bones, blood vessels, and soft tissues within the body. A CT scan may use X-ray CT, positron emission tomography (PET), single photon emission computed tomography (SPECT), computer axial tomography (CAT scan), or computer-assisted tomography. A CT scan may be a conventional CT scan or a spiral / spiral CT scan. In a conventional CT scan, the scan is acquired slice by slice, stopping and moving to the next slice after each slice scan, e.g., from above the abdomen to the pelvis. A conventional CT scan requires the patient to hold their breath to avoid motion artifacts. A spiral / spiral CT scan is a continuous scan, acquired in a spiral, and the scanned images are continuous, making the process much quicker.

[0051] As used herein, a "derivative" of an antibody may refer to an antibody that has one or more modifications to its amino acid sequence when compared to the original or parent antibody, and may exhibit a modified domain structure. A derivative may not only adopt an amino acid sequence capable of specifically binding to a target (antigen), but may further adopt the typical domain configuration found in a natural antibody. Typical examples of antibody derivatives are antibodies coupled to other polypeptides, rearranged antibody domains or antibody fragments. A derivative may also comprise at least one further compound, e.g. a protein domain, which is linked by covalent or non-covalent bonds. Linking may be based on gene fusion according to methods known in the art. The further domain present in a fusion protein comprising an antibody may preferably be linked by a flexible linker, advantageously a peptide linker, which comprises multiple, hydrophilic, peptide-linked amino acids of sufficient length to span the distance between the C-terminus of the further protein domain and the N-terminus of the antibody or vice versa. The antibody may have a conformation suitable for biological activity or may be linked to an effector molecule that selectively binds, for example, a solid support, a biologically active substance (e.g., a cytokine or growth hormone), a chemical agent, a peptide, a protein, or a drug.

[0052] "Determined by an assay" is used herein to refer to the determination of a reference level by any suitable assay. In some embodiments, the determination of the reference level can be achieved by the same type of assay as that applied to the sample from the subject (e.g., immunoassay, clinical chemistry assay, single molecule detection assay, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis or protein immunostaining, electrophoretic analysis, protein assay, competitive binding assay, functional protein assay, or chromatographic or spectroscopic methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS)). In some embodiments, the determination of the reference level can be achieved by the same type of assay as that applied to the sample from the subject under the same assay conditions. As mentioned herein, the present disclosure presents exemplary reference levels (e.g., calculated by comparing reference levels at different time points). It is well within the skill of the art to adapt the present disclosure herein to other assays and obtain assay-specific reference levels for these other assays based on the description presented by the present disclosure. For example, assay-specific reference levels may be obtained using a set of training samples that includes samples obtained from subjects known to have sustained a head injury (e.g., samples obtained from human subjects known to have sustained (i) mild TBI and / or (ii) moderate, severe, or moderate-severe TBI) and samples obtained from subjects (e.g., human subjects) known to have not sustained a head injury. It is understood that a reference level "determined by an assay" and having a recited level of "sensitivity" and / or "specificity" is used herein to refer to a reference level that has been determined to result in the method of the recited sensitivity and / or specificity when employed in the methods of the present disclosure.It is well within the skill of one in the art to determine the sensitivity and specificity associated with a given reference level in the methods of the present disclosure, for example by repeating statistical analyses on the assay data using several different possible reference levels.

[0053] In practice, when distinguishing subjects as having or not having traumatic brain injury, or as having mild versus moderate, severe, or moderate-severe traumatic brain injury, one skilled in the art will balance the effect of raising the cutoff on sensitivity and specificity. Raising or lowering the cutoff has well-defined and predictable effects on sensitivity and specificity, as well as other standard statistical measures. It is well known that raising the cutoff will improve specificity but likely worsen sensitivity (the proportion of people who have the disease and test positive). In contrast, lowering the cutoff will improve sensitivity but worsen specificity (the proportion of people who do not have the disease and test negative). The ramifications of detecting traumatic brain injury or determining mild versus moderate, severe, or moderate-severe traumatic brain injury will be readily apparent to one skilled in the art. In discriminating whether a subject has or does not have traumatic brain injury or mild versus moderate, severe, or moderate-severe traumatic brain injury, the higher the cutoff, the better the specificity as more true negatives (i.e., subjects with no traumatic brain injury, no mild traumatic brain injury, no moderate traumatic brain injury, no severe traumatic brain injury, or no moderate-severe traumatic brain injury) are distinguished from subjects with traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury. However, at the same time, raising the cutoff necessarily reduces sensitivity, as it reduces the number of cases identified as positive overall, as well as the number of true positives. Conversely, a lower cutoff improves sensitivity as more true positives (i.e., subjects with traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury) are distinguished from subjects without traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury. At the same time, however, specificity necessarily decreases, as lowering the cutoff increases the number of cases identified as overall positives, as well as the number of false positives.

[0054] In general, high sensitivity values ​​help the skilled artisan to exclude a disease or condition (such as traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury), and high specificity values ​​help the skilled artisan to include a disease or condition. Whether the skilled artisan wants to exclude or include a disease depends on what the consequences are for the patient for each type of error. Therefore, without fully disclosing the underlying information on how the values ​​were selected, it is not possible to know or predict the exact balancing used to derive the test cutoff. Balancing specificity and sensitivity against other factors will vary depending on the individual case. For this reason, it may be preferable to provide alternative cutoff (e.g., reference) values ​​for the physician or medical practitioner to choose from.

[0055] As used herein, "bispecific antibody" is used to refer to a full-length antibody (see PCT Publication No. 02 / 02773) that can bind to two different antigens (or epitopes) in each of its two binding arms (HC / LC pair). Thus, a bispecific binding protein has two identical antigen-binding arms with identical specificity and identical CDR sequences, and is bivalent for each antigen to which it binds.

[0056] As used herein, "dual variable domain" is used to refer to two or more antigen-binding sites on a binding protein, which may be a bivalent binding protein (two antigen-binding sites), a tetravalent binding protein (four antigen-binding sites), or a multivalent binding protein. A DVD may be monospecific, i.e., capable of binding one antigen (or one specific epitope), or multispecific, i.e., capable of binding two or more antigens (i.e., two or more epitopes of the same target antigen molecule or two or more epitopes of different target antigens). A preferred DVD-binding protein comprises two heavy chain DVD polypeptides and two light chain DVD polypeptides and is referred to as a "DVD immunoglobulin" or "DVD-Ig". Such DVD-Ig binding proteins are thus tetrameric and similar to IgG molecules, but provide more antigen-binding sites than IgG molecules. Thus, each half of a tetrameric DVD-Ig molecule is similar to half of an IgG molecule and contains a heavy chain DVD polypeptide and a light chain DVD polypeptide, but unlike the heavy and light chain pair of an IgG molecule, which results in a single antigen-binding domain, the heavy and light chain pair of a DVD-Ig results in two or more antigen-binding sites.

[0057] Each antigen-binding site of a DVD-Ig binding protein may be derived from a donor ("parent") monoclonal antibody and may comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) with the CDRs involved in binding to the antigen, a total of six CDRs per antigen-binding site. Thus, a DVD-Ig binding protein that binds to two different epitopes (i.e., two different epitopes on two different antigen molecules or two different epitopes on the same antigen molecule) comprises an antigen-binding site derived from a first parent monoclonal antibody and an antigen-binding site of a second parent monoclonal antibody.

[0058] The design, expression and characterization of DVD-Ig binding molecules are described in PCT Publication No. 2007 / 024715, U.S. Patent No. 7,612,181 and Wu et al., Nature Biotech., 25:1290-1297 (2007). A preferred example of such a DVD-Ig molecule comprises a heavy chain comprising the structural formula: VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker, provided that it is not CH1, X2 is an Fc region, and n is 0 or 1, preferably 1; and a light chain comprising the structural formula: VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker, provided that it is not CH1, X2 does not comprise an Fc region, and n is 0 or 1, preferably 1. Such a DVD-Ig can include two such heavy chains and two such light chains, where each chain includes a variable domain linked in tandem with no intervening constant region between the variable domains, where the heavy and light chains can associate to form a functional antigen binding site in tandem, and where a pair of heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein with four functional antigen binding sites. In another example, a DVD-Ig molecule can include heavy and light chains including three variable domains (VD1, VD2, VD3), each linked in tandem with no intervening constant region between the variable domains, where the pair of heavy and light chains can associate to form three antigen binding sites, and where a pair of heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein with six antigen binding sites.

[0059] In a preferred embodiment, a DVD-Ig binding protein not only binds to the same target molecule that its parent monoclonal antibodies bind, but also retains one or more desired properties of one or more of its parent monoclonal antibodies. Preferably, such additional properties are one or more antibody parameters of the parent monoclonal antibodies. Antibody parameters that can be contributed to a DVD-Ig binding protein derived from one or more of its parent monoclonal antibodies include, but are not limited to, antigen specificity, antigen affinity, potency, biological function, epitope recognition, protein stability, protein solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross-reactivity, and binding to orthologous antigens.

[0060] DVD-Ig binding proteins bind to at least one epitope of UCH-L1, GFAP, or UCH-L1 and GFAP. Non-limiting examples of DVD-Ig binding proteins include (1) DVD-Ig binding proteins that bind to one or more epitopes of UCH-L1, DVD-Ig binding proteins that bind to an epitope of human UCH-L1 and an epitope of UCH-L1 of another species (e.g., mouse), and DVD-Ig binding proteins that bind to an epitope of human UCH-L1 and an epitope of another target molecule; (2) DVD-Ig binding proteins that bind to one or more epitopes of GFAP, DVD-Ig binding proteins that bind to an epitope of human GFAP and an epitope of GFAP of another species (e.g., mouse). or (3) a DVD-Ig binding protein that binds to one or more epitopes of UCH-L1 and GFAP, a DVD-Ig binding protein that binds to an epitope of human UCH-L1, human GFAP and an epitope of UCH-L1 of another species (e.g., mouse), and a DVD-Ig binding protein that binds to an epitope of human UCH-L1, human GFAP and an epitope of another target molecule.

[0061] As used herein, "dynamic range" refers to the range over which the readout of an assay is proportional to the amount of target molecule or analyte in the sample being analyzed.

[0062] "Epitope" or "epitopes" or "epitope of interest" refers to a site on any molecule that can be recognized and bind to a complementary site on its specific binding partner. The molecule and specific binding partner are part of a specific binding pair. For example, an epitope can be on a polypeptide, a protein, a hapten, a carbohydrate antigen (such as, but not limited to, a glycolipid, glycoprotein, or lipopolysaccharide), or a polysaccharide. The specific binding partner can be, but is not limited to, an antibody.

[0063] As used herein, "Fab (fragment antigen-binding) fragment" or "Fab fragment" refers to a fragment of an antibody that binds to an antigen and contains one complete light chain and part of one heavy chain, which is one antigen-binding site. Fab is a monovalent fragment consisting of the VL, VH, CL and CH1 domains. Fab is composed of one constant domain and one variable domain of each heavy and light chain. The variable domain contains a paratope (antigen-binding site) that includes a set of complementarity determining regions at the amino terminus of the monomer. Thus, each arm of the Y binds to an epitope on the antigen. Fab fragments can be produced as described in the art, for example, using papain, an enzyme that can be used to cleave an immunoglobulin monomer into two Fab fragments and an Fc fragment, or can be produced by recombinant means.

[0064] As used herein, "F(ab')2 fragment" refers to an antibody generated by pepsin digestion of a whole IgG antibody, removing most of the Fc region while leaving part of the hinge region intact. The F(ab')2 fragment is a bivalent fragment with two antigen-binding F(ab) portions linked together by disulfide bonds, resulting in a molecular weight of approximately 110 kDa. Bivalent antibody fragments (F(ab')2 fragments) are smaller than whole IgG molecules, allowing better tissue penetration and thus facilitating better antigen recognition in immunohistochemistry. The use of F(ab')2 fragments also avoids non-specific binding to Fc receptors or protein A / G on live cells. The F(ab')2 fragments can bind and precipitate antigens.

[0065] As used herein, "framework" (FR) or "framework sequence" may refer to the remaining sequence of the variable region, excluding the CDRs. The exact definition of the CDR sequence may be determined by different systems (see, for example, above), and the meaning of the framework sequence is subject to different interpretations accordingly. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) also divide the framework regions on the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. As mentioned by others, if a particular subregion is not specified as FR1, FR2, FR3, or FR4, the framework region represents the combination of FRs in the variable region of a single, naturally occurring immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FR refers to two or more of the four subregions that make up a framework region.

[0066] Human heavy and light chain FR sequences are known in the art that can be used as heavy and light chain "acceptor" framework sequences (or simply, "acceptor" sequences) to humanize non-human antibodies using techniques known in the art. In one embodiment, the human heavy and light chain acceptor sequences are selected from framework sequences listed in publicly available databases, such as V-base (hypertext transferprotocol: / / vbase.mrc-cpe.cam.ac.uk / ) or the international ImMunoGeneTics® (IMGT®) information system (hypertext transferprotocol: / / imgt.cines.fr / texts / IMGTrepertoire / LocusGenes / ).

[0067] As used herein, a "functional antigen-binding site" can refer to a site on a binding protein (e.g., an antibody) that can bind to a target antigen. The antigen-binding affinity of an antigen-binding site may not be as strong as that of the parent binding protein, e.g., the parent antibody, from which the antigen-binding site is derived, but the ability to bind to an antigen must be measurable using any one of a variety of known methods for assessing antigen-binding proteins, e.g., antibodies. Furthermore, the antigen-binding affinity of each of the antigen-binding sites of a multivalent protein, e.g., a multivalent antibody herein, does not need to be quantitatively the same.

[0068] "GFAP" is used herein to describe glial fibrillary acidic protein, a protein that is encoded by the GFAP gene in humans and by GFAP gene counterparts in other species, and that can be produced (e.g., by recombinant means in other species).

[0069] "GFAP status" can mean the level or amount of GFAP at a point in time (such as a point in time with a single measurement of GFAP), the level or amount of GFAP associated with monitoring (such as monitoring with repeated testing to identify increases or decreases in the amount of GFAP in a subject), the level or amount of GFAP associated with a treatment for traumatic brain injury (whether primary brain injury and / or secondary brain injury), or a combination thereof.

[0070] As used herein, "Glasgow Coma Scale" or "GCS" refers to a 15-point scale (described, for example, in 1974 by Graham Teasdale and Bryan Jennett, Lancet 1974;2:81-4) that provides a practical method for assessing impairment in level of consciousness in patients who have suffered brain injury. The test measures best motor response, verbal response and eye opening response with these values: I. best motor response (6: follows two-part request; 5: brings hand to head / neck stimulus above clavicle; 4: arm bent sharply at elbow but not primarily abnormal in character; 3: arm bent at elbow, clearly primarily abnormal in character; 2: arm extended at elbow; 1: no arm / leg movement, no interfering factors; NT: paralyzed or other limiting factors); II. verbal response (5: gives name, place and date correctly; 4: disoriented but communicates coherently; 3: understandable words; 2: moans / growls only; 1: no audible response, no interfering factors; NT: factors impeding communication) and III. eyes open (4: open before stimulus; 3: after verbal or shouted request; 2: after fingertip stimulus; 1: eyes not open at any time, no interfering factors; NT: closed due to local factors). The final score is determined by adding the values ​​of I + II + III. If the GCS score is 13-15, the subject is considered to have mild TBI. If the GCS score is 9-12, the subject is considered to have moderate TBI. If the GCS score is 8 or less, typically 3-8, the subject is considered to have severe TBI.

[0071] As used herein, the term "Glasgow Outcome Scale (GOS)" refers to a global scale for functional outcome that assesses a patient's condition into one of five categories: death, vegetative state, severe disability, moderate disability, or good recovery. The term "Extended Glasgow Outcome Scale (GOSE)" or "GOSE", used interchangeably herein, provides a more detailed categorization into eight categories by subdividing the severe disability, moderate disability, and good recovery categories into upper and lower categories as shown in Table 1.

[0072] [Table 1]

[0073] As used herein, "humanized antibody" is used to describe an antibody that contains heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but where at least a portion of the VH and / or VL sequences have been altered to be more "human-like", i.e., more similar to human germline variable sequences. A "humanized antibody" is an antibody or variant, derivative, analog or fragment thereof that immunospecifically binds to an antigen of interest and contains a framework (FR) region that has substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) that has substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR that has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of the non-human antibody CDR. A humanized antibody comprises substantially all of at least one, but typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In certain embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically the Fc region of a human immunoglobulin. In some embodiments, the humanized antibody contains at least the variable domain of the heavy chain as well as the light chain. The antibody may also contain the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only a humanized light chain. In some embodiments, the humanized antibody contains only a humanized heavy chain. In specific embodiments, the humanized antibody contains only humanized variable domains of the light chain and / or humanized heavy chain.

[0074] The humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including, without limitation, IgG1, IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions, using techniques well known in the art.

[0075] The framework regions and CDRs of a humanized antibody need not correspond exactly to the parental sequences, for example, the donor antibody CDR or consensus framework may be mutagenized by substitution, insertion and / or deletion of at least one amino acid residue such that the CDR or framework residue at this site does not correspond to the donor antibody or consensus framework. However, in a preferred embodiment, such mutations are not extensive mutations. Typically, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues correspond to the residues of the parental FR and CDR sequences. The term "consensus framework" as used herein refers to a framework region within a consensus immunoglobulin sequence. The term "consensus immunoglobulin sequence" as used herein refers to a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, for example, Winnaker, "From Genes to Clones" (Verlagsgesellschaft, Weinheim, 1987)). Thus, a "consensus immunoglobulin sequence" may comprise a "consensus framework region" and / or a "consensus CDR." Within a family of immunoglobulins, each position within a consensus sequence is occupied by the amino acid that occurs most frequently at that position within the family. If two amino acids occur equally frequently, either may be included within the consensus sequence.

[0076] "Identical" or "identity" as used herein in the context of two or more polypeptide or polynucleotide sequences may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions in both sequences where identical residues occur to determine the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to determine the percentage of sequence identity. If the two sequences are of different length or the alignment results in one or more sticky ends and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator of the calculation, but not in the numerator.

[0077] "Head injury" or "head injury", used interchangeably herein, refers to any trauma to the scalp, skull, or brain. Such injury may include only a minor bump on the head, or may be a severe brain injury. Such injury includes primary injury to the brain and / or secondary injury to the brain. Primary brain injury occurs during the initial insult and results from the displacement of the brain's physical structures. More specifically, primary brain injury is physical damage to the parenchyma (tissue, blood vessels) that occurs during the traumatic event, resulting in shearing and compression of the surrounding brain tissue. Secondary brain injury occurs subsequent to the primary injury and may involve a series of cellular processes. More specifically, secondary brain injury refers to changes that develop over a period of time (hours to days) following the primary brain injury. Secondary brain injury includes a whole cascade of cellular, chemical, tissue, or vascular changes in the brain that contribute to further destruction of brain tissue.

[0078] Injuries to the head can be closed or open (penetrating). A closed head injury is a trauma to the scalp, skull, or brain without the penetration of the skull by an impacting object. An open head injury is a trauma to the scalp, skull, or brain with the penetration of the skull by an impacting object. Head injuries can be caused by physical shaking of a person, by external mechanical or other forces resulting in closed or open head injuries (vehicle accidents such as with a car, plane, train, etc.; blows to the head such as with a baseball bat or from a firearm), cerebrovascular accidents (e.g., strokes), one or more falls (e.g., during sports or other activities), blunt impact from an explosion or blast (collectively, "blast injuries"), and by other types of blunt force trauma. Alternatively, head injuries can be caused by ingestion of and / or exposure to chemicals, toxins, or combinations of chemicals and toxins. Examples of such chemicals and / or toxins include fire, mold, asbestos, pesticides and insecticides, organic solvents, paints, adhesives, gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organometallics (such as methylmercury, tetraethyl lead, and organotin), and / or one or more addictive drugs. Alternatively, head injury may be caused as a result of the subject suffering from an autoimmune disease, metabolic disorder, brain tumor, hypoxia, viral infection (e.g., SARS-CoV-2), fungal infection, bacterial infection, meningitis, hydrocephalus, or any combination thereof. In some cases, it is not possible to ascertain whether any such event or injury has occurred or occurred. For example, there may be no medical history for the patient or subject, the subject may be unable to speak, the subject may be aware of what event the subject has been exposed to, etc. Such circumstances are described herein as the subject "possibly suffering from head injury" or as "suspected injury." In certain embodiments herein, closed head injury does not include, and specifically excludes, cerebrovascular accidents, such as stroke.

[0079] As used herein, an "isolated polynucleotide" can mean a polynucleotide that, by its origin, is not associated with all or a portion of a polynucleotide with which the "isolated polynucleotide" is found in nature; that is operably linked to a polynucleotide with which it is not linked in nature; or that does not exist in nature as part of a larger sequence (e.g., by genomic, cDNA, or synthetic origin, or a combination of portions thereof).

[0080] As used herein, "label" and "detectable label" refer to a moiety attached to an antibody or analyte such that the reaction of the antibody with the analyte is detectable, and the antibody or analyte so labeled is said to be "detectably labeled." The label may provide a signal detectable by visual or instrumental means. Various labels include signal generators such as chromogens, fluorescent compounds, chemiluminescent compounds, radioactive compounds, and the like. Representative examples of labels include moieties that provide light, e.g., acridinium compounds, and moieties that provide fluorescence, e.g., fluorescein. Other labels are also described herein. In this regard, the moiety itself may not be detectable, but may become detectable upon reaction with yet another moiety. The term "detectably labeled" is intended to encompass such labels.

[0081] Any suitable detectable label known in the art may be used. For example, detectable labels include radioactive labels (such as 3H, 14C, 32P, 33P, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm), enzyme labels (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase), chemiluminescent labels (acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, etc.), fluorescent labels (fluorescein, ... The label may be a fluorescent label (e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, etc.), rhodamine, phycobiliprotein, R-phycoerythrin, quantum dots (e.g., cadmium selenide capped with zinc sulfide), calorimetric labels, or immunopolymerase chain reaction labels. An introduction to labels, labeling procedures, and detection of labels can be found in Polak and Van Noorden, "Introduction to Immunocytochemistry," 2nd ed., Springer Verlag, NY (1997), and Haugland, "Handbook of Fluorescent Probes and Research Chemicals" (1996), a combination handbook and catalogue published by Molecular Probes, Inc., Eugene, Oregon. Fluorescent labels can be used in FPIA (see, e.g., U.S. Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are incorporated by reference herein in their entireties).Acridinium compounds can be used as detectable labels in homogeneous chemiluminescent assays (see, e.g., Adamczyk et al., Bioorg. Med. Chem. Lett., 16:1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett., 4:2313-2317 (2004); Adamczyk et al., Bioorg. Med. Chem. Lett., 14:3917-3921 (2004) and Adamczyk et al., Org. Lett., 5:3779-3782 (2003)).

[0082] In one embodiment, the acridinium compound is acridinium-9-carboxamide. Methods for preparing acridinium 9-carboxamide are described in Mattingly, J. Biolumin. Chemilumin., 6:107-114 (1991); Adamczyk et al., J. Org. Chem., 63:5636-5639 (1998); Adamczyk et al., Tetrahedron, 55:10899-10914 (1999); Adamczyk et al., Org. Lett., 1:779-781 (1999); Adamczyk et al., Bioconjugate Chem., 11:714-724 (2000); Mattingly et al., Luminescence Biotechnology: Instruments and Applications, edited by Dyke, KV, CRC Press: Boca Raton, 77-105 (2002); Adamczyk et al., Org. Lett., 5:3779-3782 (2003); and U.S. Pat. Nos. 5,468,646, 5,543,524, and 5,783,699, each of which is incorporated by reference in its entirety for its teachings herein.

[0083] Another example of an acridinium compound is acridinium-9-carboxylate aryl ester. An example of the acridinium-9-carboxylate aryl ester of formula II is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available from Cayman Chemical, Ann Arbor, MI). Methods for preparing acridinium-9-carboxylate aryl ester are described in McCapra et al., Photochem. Photobiol., 4:1111-21 (1965); Razavi et al., Luminescence, 15:245-249 (2000); Razavi et al., Luminescence, 15:239-244 (2000) and U.S. Patent No. 5,241,070 (each of which is incorporated herein by reference in its entirety for its teachings therein). Such acridinium-9-carboxylate aryl esters are effective chemiluminescent indicators for hydrogen peroxide generated in the oxidation of an analyte by at least one oxidase in terms of signal intensity and / or signal rapidity. The chemiluminescence process of acridinium-9-carboxylate aryl esters is completed quickly, i.e., in less than one second, whereas the chemiluminescence of acridinium-9-carboxamides lasts for more than two seconds. However, acridinium-9-carboxylate aryl esters lose their chemiluminescent properties in the presence of proteins. Therefore, their use requires the absence of proteins at the time of signal generation and detection. Methods for separating or removing proteins in a sample are well known to those skilled in the art and include, but are not limited to, ultrafiltration, extraction, precipitation, dialysis, chromatography and / or digestion (see, for example, Wells, "High Throughput Bioanalytical Sample Preparation. Methods and Automation Strategies", Elsevier (2003)).The amount of protein removed or separated from the test sample can be about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95%. Further details regarding acridinium-9-carboxylate aryl esters and their uses are set forth in U.S. Patent Application No. 11 / 697,835, filed April 9, 2007. Acridinium-9-carboxylate aryl esters can be dissolved in any suitable solvent, such as degassed anhydrous N,N-dimethylformamide (DMF) or aqueous sodium cholate.

[0084] A "linking sequence" or "linking peptide sequence" refers to a naturally occurring or artificial polypeptide sequence connected to one or more polypeptide sequences of interest (e.g., full length sequences, sequence fragments, etc.). The term "connected" refers to the joining of the linking sequence to the polypeptide sequence of interest. Such polypeptide sequences are preferably joined by one or more peptide bonds. The linking sequence may have a length of about 4 to about 50 amino acids. Preferably, the length of the linking sequence is about 6 to about 30 amino acids. A naturally occurring linking sequence may be modified by amino acid substitution, addition, or deletion to create an artificial linking sequence. Linking sequences may be used for many purposes, including use in recombinant Fab. Exemplary linking sequences include, but are not limited to: (i) histidine (His) tags, such as a 6×His tag, having an amino acid sequence of HHHHHH (SEQ ID NO: 3), are useful as linking sequences to facilitate isolation and purification of polypeptides and antibodies of interest; (ii) enterokinase cleavage sites, such as a His tag, are used in the isolation and purification of proteins and antibodies of interest. Enterokinase cleavage sites are often used in conjunction with His tags in the isolation and purification of proteins and antibodies of interest. A variety of enterokinase cleavage sites are known in the art. Examples of enterokinase cleavage sites include, but are not limited to, the amino acid sequence of DDDDK (SEQ ID NO: 4) and its derivatives (e.g., ADDDDK (SEQ ID NO: 5) and the like); (iii) other sequences can also be used to link or connect the light chain variable region and / or heavy chain variable region of the single chain variable region fragment. Examples of other linking sequences can be found in Bird et al., Science, 242:423-426 (1988); Huston et al., PNAS USA 85:5879-5883 (1988) and McCafferty et al., Nature, 348:552-554 (1990). Linking sequences can also be modified for additional functions, such as conjugation of drugs or conjugation to solid supports. In the context of the present disclosure, the monoclonal antibody may contain linking sequences such as, for example, a His tag, an enterokinase cleavage site, or both.

[0085] As used herein, a "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical, except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigen (although, for example, cross-reactivity or shared reactivity may occur). Furthermore, in contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies herein include, inter alia, "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological properties.

[0086] "Magnetic Resonance Imaging" or "MRI," as used interchangeably herein, refers to a medical imaging technique (e.g., referred to interchangeably herein as "MRI," "MRI procedure," or "MRI exam") used in radiology to produce images of the anatomy and physiological processes of the body, both in health and disease. MRI is a form of medical imaging that measures the response of atomic nuclei in body tissues to radio frequency radio waves when placed in a strong magnetic field, producing images of internal organs. MRI scanners are based on the science of nuclear magnetic resonance (NMR) and use strong magnetic fields, radio waves, and field gradients to produce images of the inside of the body.

[0087] As used herein, "multivalent binding protein" is used to refer to a binding protein that contains two or more antigen-binding sites (also referred to herein as "antigen-binding domains"). Multivalent binding proteins are preferably engineered to have three or more antigen-binding sites and are generally not naturally occurring antibodies. The term "multispecific binding protein" refers to binding proteins that can bind to two or more related or unrelated targets, including binding proteins capable of binding to two or more different epitopes of the same target molecule.

[0088] "Negative predictive value" or "NPV," used interchangeably herein, refers to the probability that a subject will have a negative outcome given that they have a negative test result.

[0089] "Orthopedic injury" refers to one or more injuries to one or more parts of the skeletal musculoskeletal system, including injuries to the bones, muscles, cartilage, tendons, ligaments, joints, and other connective tissues that collectively support and connect the tissues and organs. In one embodiment, an orthopedic injury is the result of a sudden accident and may require medical attention. Examples of orthopedic injuries include dislocations (e.g., dislocations to a joint), fractures (e.g., stress fractures or compression fractures) or fractures (e.g., fractures to one or more bones), sprains (e.g., sprains to the ankle, wrist, knee, shoulder, etc.), tears (e.g., ligament tears such as ACL tears or meniscus tears, cartilage tears such as lip tears, or tendon tears and / or muscle tears such as rotator cuff tears), or overuse injuries (e.g., plantar fasciitis, tennis elbow, carpal tunnel syndrome, etc.). In one embodiment, the orthopedic injury is a fracture. In another embodiment, the orthopedic injury is a fracture. In another embodiment, the orthopedic injury is a sprain. In yet another embodiment, the orthopedic injury is a tear. In yet another embodiment, the orthopedic injury is one or more of a fracture, break, sprain, or tear.

[0090] A "non-point-of-care device" is a device that is neither a point-of-care device nor a single-use device. A "point-of-care" device is a device that is used to provide medical diagnostic testing at or near the point-of-care (i.e., outside of a laboratory) at the time and place of patient care (such as in a hospital, clinic, emergency or other medical facility, the patient's home, a nursing home, and / or a long-term care and / or hospice facility). A point-of-care instrument does not simultaneously perform assays on more than one clinical sample. Examples of point-of-care devices include point-of-care devices made by Abbott Laboratories (Abbott Park, IL) (e.g., i-STAT and i-STAT Alinity, Universal Biosensors (Rowville, Australia)) (see U.S. Patent Publication No. 2006 / 0134713), Axis-Shield PoC AS (Oslo, Norway), and Clinical Lab Products (Los Angeles, USA). In some embodiments, the point-of-care device is a single-use device. The term "single-use device" or "single-use device" refers to a clinical diagnostic device that processes and performs clinical diagnostic assays on a unit-use basis (such as a single-use cartridge) on a single patient sample. A non-point-of-care device refers to any device that does not meet any of the above limitations of a point-of-care or single-use device. In some embodiments, a non-point-of-care device may be a relatively large device, such as a benchtop device. Thus, in some embodiments, a non-point-of-care device is not a handheld device. In some embodiments, a non-point-of-care device can perform assays on more than one clinical sample simultaneously. Suitable non-point-of-care devices include, for example, the Architect or Alinity platforms manufactured by Abbott Core Laboratories.

[0091] "Positive predictive value" or "PPV," used interchangeably herein, refers to the probability that a subject will have a positive outcome given a positive test result.

[0092] "Quality control reagents" in the context of the immunoassays and kits described herein include, but are not limited to, calibrators, controls, and sensitivity panels. "Calibrators" or "standards" (e.g., a plurality, one or more) are typically used to establish a calibration curve for interpolating the concentration of an analyte, such as an antibody or analyte. Alternatively, a single calibrator that is near a reference or control level (e.g., a "low", "medium", or "high" level) may be used. Multiple calibrators (i.e., more than one calibrator or varying amounts of calibrators) may be used to comprise a "sensitivity panel".

[0093] A "receiver operating characteristic" curve or "ROC" curve refers to a graphical plot illustrating the operation of a binary classifier system as its discrimination threshold is varied. For example, an ROC curve can be a plot of true positive rate against false positive rate for different possible cutoff points of a diagnostic test. An ROC curve is created by plotting the proportion of true positives among positives (TPR=true positive rate) against the proportion of false positives among negatives (FPR=false positive rate) at various threshold situations. TPR is also known as sensitivity, and FPR is [1-specificity or true negative rate]. ROC curves demonstrate a trade-off between sensitivity and specificity (any increase in sensitivity is accompanied by a decrease in specificity); the closer the curve is to the left and then upper boundaries of the ROC space, the more accurate the test; the closer the curve is to the 45 degree diagonal of the ROC space, the less accurate the test; the slope of the tangent at the cutoff point gives the likelihood ratio (LR) for this value of the test and the area under the curve is a measure of the accuracy of the test.

[0094] "Recombinant antibody" and "recombinant antibodies" refer to antibodies prepared by recombinant methods, by one or more steps including cloning a nucleic acid sequence encoding all or a portion of one or more monoclonal antibodies into a suitable expression vector, and then expressing the antibody in a suitable host cell. The terms include, but are not limited to, recombinantly produced monoclonal antibodies, chimeric antibodies, humanized antibodies (fully or partially humanized antibodies), multispecific or multivalent structures formed from antibody fragments, bifunctional antibodies, heteroconjugate Abs, DVD-Ig®, and other antibodies described in (i) herein (dual variable domain immunoglobulins and methods for making them are described in Wu, C. et al., Nature Biotechnology, 25:1290-1297 (2007)). The term "bifunctional antibody" as used herein refers to an antibody that contains a first arm that has specificity for one antigenic site and a second arm that has specificity for a different antigenic site, i.e., a bifunctional antibody has dual specificity.

[0095] "Reference level" as used herein refers to an assay cut-off value used to evaluate diagnostic, prognostic or therapeutic efficacy, and is herein linked or associated with various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression, non-progression or improvement of disease, etc.). The present disclosure provides exemplary reference levels. However, it is well known that reference levels may vary depending on the nature of the immunoassay (e.g., antibody utilized, reaction conditions, purity of sample, etc.), and assays may be compared and standardized. Moreover, it is well within the skill of the artisan to adapt the present disclosure herein to other immunoassays and obtain immunoassay-specific reference levels for these other immunoassays based on the description provided by the present disclosure. Although the exact values ​​of the reference levels may vary between assays, it is intended that the findings described herein are generally applicable and can be extrapolated to other assays.

[0096] In certain aspects described herein, the reference level is said to be determined by any assay having a certain specificity and sensitivity.

[0097] As used herein, "risk assessment," "risk classification," "risk identification," or "risk stratification" of a subject (e.g., patient) refers to the assessment of factors, including biomarkers, to predict the risk of developing a future event, including disease onset or disease progression, so that treatment decisions for the subject can be made on a more informed, condition-based basis.

[0098] As used herein, "sample," "test sample," "specimen," "sample from a subject," and "patient sample" may be used interchangeably and may be a sample of blood, such as whole blood (including, for example, capillary blood, venous blood, mixed samples of venous and capillary blood, mixed samples of capillary blood and interstitial fluid, dried blood spots, etc.), tissue, urine, serum, plasma, amniotic fluid, saliva, lower respiratory tract specimens, such as, but not limited to, endotracheal aspirate or bronchoalveolar lavage, nasal mucus, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample may be used directly as obtained from the patient or may be pretreated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., as discussed herein or otherwise known in the art to alter the characteristics of the sample.

[0099] A variety of cell types, tissues or bodily fluids may be utilized to obtain samples. Such cell types, tissues and fluids may include biopsy and autopsy samples, oropharyngeal specimens, nasopharyngeal specimens, nasal mucus specimens, sections of tissue such as frozen sections taken for histological purposes, blood (such as whole blood, capillary blood, venous blood, mixed venous and capillary blood samples, mixed capillary and interstitial fluid samples, dried blood spots, etc.), plasma, serum, red blood cells, platelets, anal samples (such as anal swab samples), interstitial fluid, cerebrospinal fluid, etc. Cell types and tissues may also include lymphatic fluid, cerebrospinal fluid, or any fluid collected by aspiration. Tissues or cell types may be prepared by removing cell samples from humans and non-human animals, but may also be arrived at by using previously isolated cells (e.g., isolated by another person, at another time, and / or for another purpose). Archival tissues, such as tissues with treatment history or outcome history, may also be used. Isolation and / or purification of proteins or nucleotides may not be necessary. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a capillary blood sample. In some embodiments, the sample is a dried blood spot. In some embodiments, the sample is a serum sample. In still other embodiments, the sample is a plasma sample. In some embodiments, the sample is an oropharyngeal specimen. In other embodiments, the sample is a nasopharyngeal specimen. In other embodiments, the sample is sputum. In other embodiments, the sample is an endotracheal aspirate. In still other embodiments, the sample is bronchoalveolar lavage fluid. In still other embodiments, the sample is nasal mucus.

[0100] "Sensitivity" refers to the proportion of subjects whose outcome is positive who are correctly identified as positive (e.g., the proportion of subjects who correctly identify those with the disease or medical condition for which they are being tested). For example, this can include correctly identifying subjects with TBI from subjects without TBI, correctly identifying subjects with moderate, severe, or moderate-severe TBI from subjects with mild TBI, correctly identifying subjects with mild TBI from subjects with moderate, severe, or moderate-severe TBI, correctly identifying subjects with moderate, severe, or moderate-severe TBI from subjects without TBI, or correctly identifying subjects with mild TBI from subjects without TBI, etc.).

[0101] As used herein, the "specificity" of an assay refers to the proportion of subjects whose outcome is negative who are correctly identified as negative (e.g., the proportion of subjects who correctly identify that they do not have the disease or medical condition for which they are being tested). For example, this can include correctly identifying subjects with TBI from subjects without TBI, correctly identifying subjects without moderate, severe, or moderate-severe TBI from subjects with mild TBI, correctly identifying subjects without mild TBI from subjects with moderate, severe, or moderate-severe TBI, or correctly identifying subjects without any TBI, or correctly identifying subjects with mild TBI from subjects without TBI, etc.

[0102] A "series of calibration compositions" refers to a plurality of compositions comprising a known concentration of (1) UCH-L1, each of which differs from the other compositions in the series in its concentration of UCH-L1; and / or a known concentration of (2) GFAP, each of which differs from the other compositions in the series in its concentration of GFAP.

[0103] As used interchangeably herein, "solid phase" or "solid support" refers to any material that can be attached to and / or attract and immobilize (1) one or more capture agents or specific binding partners for capture, or (2) one or more detection agents or specific binding partners for detection. The solid phase can be selected for its intrinsic ability to attract and immobilize the capture agent. Alternatively, the solid phase has a linking agent attached thereto that has the ability to attract and immobilize (1) the capture agent or specific binding partner for capture, or (2) the detection agent or specific binding partner for detection. For example, the linking agent can include a charged substance that is oppositely charged to the capture agent (e.g., specific binding partner for capture) or detection agent (e.g., specific binding partner for detection) itself, or a charged substance conjugated to (1) the capture agent or specific binding partner for capture, or (2) the detection agent or specific binding partner for detection. In general, the linking agent can be any binding partner (preferably a specific binding partner) that is capable of being immobilized (attached to) a solid phase and immobilizing (1) a capture agent or a specific binding partner for capture, or (2) a detection agent or a specific binding partner for detection, via a binding reaction. The linking agent allows the indirect binding of the capture agent to the solid phase material before the performance of the assay or during the performance of the assay. For example, the solid phase can be plastic, derivatized plastic, magnetic or non-magnetic metal, glass, or silicon, including, for example, test tubes, microtiter wells, sheets, beads, microparticles, chips, and other configurations known to those skilled in the art.

[0104] As used herein, "specific binding" or "specifically binding to" can refer to the interaction of an antibody, protein, or peptide with a second chemical species, where the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure, rather than proteins in general. If an antibody is specific for epitope "A", then the presence of a molecule containing epitope A (or free A, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[0105] A "specific binding partner" is a member of a specific binding pair. A specific binding pair includes two different molecules that specifically bind to each other through chemical or physical means. Thus, in addition to the typical immunoassay specific binding pair of antigen and antibody, other specific binding pairs can include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzymes and enzyme inhibitors, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding member, e.g., analyte analogs. Immunoreactive specific binding members include antigens, antigen fragments, and monoclonal and polyclonal antibodies, as well as antibodies, including complexes and fragments thereof, whether isolated or recombinantly produced.

[0106] "Statistically significant" as used herein refers to the likelihood that a relationship between two or more variables is caused by something other than random chance. Statistical hypothesis testing is used to determine whether the results of a data set are statistically significant. In statistical hypothesis testing, a statistically significant result is reached whenever the observed p-value for the test statistic is less than the significance level defined for the study. The p-value is the probability of obtaining a result at least as extreme as the result observed if the null hypothesis is true. Examples of statistical hypothesis analysis include Wilcoxon's matched rank test, t-test, chi-square test, or Fisher's exact test. "Significant" as used herein refers to a change that has not been determined to be statistically significant (e.g., may not have been subjected to statistical hypothesis testing).

[0107] As used interchangeably herein, "subject" and "patient" refer to any vertebrate, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates (e.g., monkeys such as cynomolgus or rhesus monkeys, chimpanzees) and humans). In some embodiments, the subject can be human or non-human. In some embodiments, the subject is human. The subject or patient may be undergoing other forms of treatment. In some embodiments, the subject is a human who may be undergoing other forms of treatment. In some embodiments, the subject is a human helper subject, such as a horse, dog, or other species that helps humans perform their daily tasks (e.g., companion animals) or occupations (e.g., service animals).

[0108] As used herein, "treating", "treating" or "treatment" are each used interchangeably to describe preventing, alleviating or inhibiting the progression of a disease and / or injury or one or more symptoms of such disease to which such term applies. Depending on the subject's condition, the term also refers to preventing disease, including preventing the onset of a disease or preventing symptoms associated with a disease. Treatment can be performed acutely or chronically. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to contraction of the disease. Such prevention or reduction of the severity of a disease prior to contraction refers to administration of a pharmaceutical composition to a subject who is not affected by the disease at the time of administration. "Preventing" also refers to preventing the recurrence of a disease or one or more symptoms associated with such disease. "Treatment" and "therapeutically" refer to the act of treating, where "treating" is as defined above.

[0109] "Traumatic brain injury" or "TBI", used interchangeably herein, refers to a complex injury with a wide range of symptoms and disability. TBI is most often an acute event that resembles other injuries. TBI can be classified as "mild", "moderate", "moderate-severe" or "severe". Causes of TBI are diverse and include, for example, physical shaking by a person, automobile accidents, injuries from weapons, cerebrovascular accidents (e.g., strokes), falls, explosions or blasts, and other types of blunt force trauma. Other causes of TBI include ingestion of and / or exposure to one or more chemicals or toxins, such as fire, mold, asbestos, pesticides and insecticides, organic solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide, and cyanides), organometallics (such as methylmercury, tetraethyl lead, and organotins), one or more addictive drugs, or combinations thereof. Alternatively, TBI may occur in subjects suffering from autoimmune disease, metabolic disorders, brain tumors, hypoxia, viral infections (e.g., SARS-CoV-2), fungal infections, bacterial infections, meningitis, hydrocephalus, or any combination 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, cerebrovascular accidents such as stroke.

[0110] As used herein, "mild TBI" refers to head injury in which the subject may or may not experience loss of consciousness. In subjects who experience loss of consciousness, the loss of consciousness is typically brief, usually lasting only a few seconds or minutes. Mild TBI is also referred to as concussion, mild head trauma, mild TBI, mild brain injury, and mild head injury. MRI and CT scans are often normal, but individuals with mild TBI may have cognitive problems such as headaches, difficulty thinking, memory problems, attention deficits, mood swings, and frustration.

[0111] Mild TBI is the most common type of TBI and is often overlooked at the time of initial injury. Typically, subjects have a Glasgow Coma Scale (GCS) score between 13-15 (such as 13-15 or 14-15). Of people with mild TBI, fifteen percent (15%) have symptoms that persist for three months or more. Common symptoms of mild TBI include fatigue, headache, visual impairment, memory loss, decreased attention / concentration, sleep disorders, vertigo / loss of balance, irritability (emotional disturbance), feelings of depression, and seizures. Other symptoms associated with mild TBI include nausea, loss of smell, sensitivity to light and sound, mood changes, confusion or confusion, and / or slowed thinking.

[0112] "Moderate TBI" as used herein refers to a brain injury in which loss of consciousness and / or confusion and disorientation occurs for 1-24 hours and the subject has a Glasgow Coma Scale score between 9-13 (e.g., 9-12 or 9-13). Individuals with moderate TBI may have abnormal brain imaging results. "Severe TBI" as used herein refers to a brain injury in which loss of consciousness occurs for more than 24 hours, memory loss after injury or penetrating skull injury occurs for more than 24 hours, and the subject has a Glasgow Coma Scale score between 3-8. Deficits range from high level cognitive impairment to coma. Survivors may have limited arm or leg function, speech or language abnormalities, loss of thinking ability, or emotional problems. Individuals with severe injuries may remain in a prolonged state of refractory status. For many people with severe TBI, prolonged rehabilitation is often required to maximize function and independence.

[0113] As used herein, "moderate-severe" TBI includes changes over time from moderate to severe TBI, and thus refers to a range of brain damage (e.g., over time) including moderate TBI alone, severe TBI alone, and combined moderate-severe TBI. For example, in some clinical situations, a subject may be initially diagnosed with a moderate TBI, but over time (minutes, hours, or days), the subject will have a severe TBI (e.g., in the setting of a cerebral hemorrhage). Alternatively, in some clinical situations, a subject may be initially diagnosed with a severe TBI, but over time (minutes, hours, or days), the subject will have a moderate TBI. Such subjects are considered examples of patients that can be classified as "moderate-severe." Common symptoms of moderate to severe TBI include problems with attention, concentration, distractibility, memory, processing speed, confusion, perseveration, impulsivity, language processing and / or "executive function", not understanding spoken words (receptive aphasia), difficulty speaking and being understood (expressive aphasia), slurred speech, very fast or very slow speech, problems reading, problems writing, interpreting touch, temperature, movement, limb position and fine discrimination, difficulties integrating or patterning sensory impressions into psychologically meaningful data, partial or total loss of vision, weak eye muscles and double vision (diplopia), blurred vision, difficulty judging distances. Problems with hearing, involuntary eye movements (nystagmus), light intolerance (photophobia), hearing problems such as reduced or lost hearing, ringing in the ears (tinnitus), hypersensitivity to sound, loss or diminished sense of smell (anosmia), loss or diminished sense of taste, convulsions associated with epilepsy, which can be of several types and may involve problems with consciousness, sensory perception or movement, bowel and bladder control, sleep disorders, loss of energy, changes in appetite, regulation of internal temperature, menstrual difficulties, addictive behaviors, problems with emotional capacity or stability, lack of motivation, irritability, aggression, depression, disinhibition, or cognitive deficits including denial / lack of consciousness. Subjects with moderate to severe TBI can have a Glasgow Coma Scale (GCS) score of 3 to 12 (including a range of 9 to 12 for moderate TBI and 3 to 8 for severe TBI).

[0114] "Ubiquitin carboxy-terminal hydrolase L1" or "UCH-L1," as used interchangeably herein, refers to the deubiquitinating enzyme encoded by the UCH-L1 gene in humans and by UCH-L1 gene counterparts in other species. UCH-L1, also known as ubiquitin carboxyl-terminal esterase L1 and ubiquitin thiolesterase, is a member of a gene family whose products hydrolyze small C-terminal appendages of ubiquitin to generate ubiquitin monomers.

[0115] "UCH-L1 status" can mean the level or amount of UCH-L1 at a point in time (such as a point in time involving a single measurement of UCH-L1), the level or amount of UCH-L1 associated with monitoring (such as monitoring involving repeated testing to identify increases or decreases in the amount of UCH-L1 in a subject), the level or amount of UCH-L1 associated with treatment for traumatic brain injury (whether primary brain injury and / or secondary brain injury), or a combination thereof.

[0116] As used herein, "variant" is used to describe a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or the ability to stimulate an immune response. As used herein, variant is also used to describe a protein with an amino acid sequence that is substantially identical to a reference protein with an amino acid sequence that retains at least one biological activity. It is recognized in the art that conservative substitutions of amino acids, i.e., replacing an amino acid with a different amino acid that has similar properties (e.g., hydrophilicity, degree of charge, and distribution of charged regions), typically involve small changes. As understood in the art, these small changes can be identified, in part, by considering the hydrophobicity index of an amino acid (Kyte et al., J. Mol. Biol. 157:105-132 (1982)). The hydrophobicity index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophilicity indexes may be substituted and still retain protein function. In one embodiment, amino acids with hydrophobicity indexes of ±2 are substituted. The hydrophilicity of amino acids may also be used to identify substitutions that result in proteins that retain biological function. Consideration of the hydrophilicity of amino acids in the context of a peptide allows for the calculation of the maximum local average hydrophilicity of the peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity (U.S. Pat. No. 4,554,101, incorporated herein by reference). Substitution with amino acids with similar hydrophilicity values ​​may result in peptides that retain biological activity, such as immunogenicity as understood in the art. Substitutions may be made with amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the particular side chain of the amino acid.Consistent with this observation is the understanding that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size and other properties, and in particular on the side chains of those amino acids. "Variants" may also be used to refer to antigenically reactive fragments of anti-UCH-L1 antibodies that differ in amino acid sequence from the corresponding fragments of anti-UCH-L1 antibodies, but that are still antigenically reactive and can compete with the corresponding fragments of anti-UCH-L1 antibodies for binding to UCH-L1. "Variants" may also be used to describe polypeptides or fragments thereof that have been processed differently, such as by proteolysis, phosphorylation, or other post-translational modifications, but that retain their antigenic reactivity.

[0117] As used herein, "vector" is used to describe a nucleic acid molecule that can carry another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., mammalian non-episomal vectors) can be integrated into the genome of the host cell upon introduction into the host cell, and thereby replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Since plasmids are the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions, may also be used. In this regard, RNA forms of vectors, including viral RNA vectors, may also be used in the context of the present disclosure.

[0118] Unless otherwise specified, technical and scientific terms used herein shall have the same meaning as commonly understood by those skilled in the art. For example, the terminology used in connection with and techniques for cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well-known and commonly used in the art. The meaning and scope of the terms shall be clear, but in the unlikely event of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0119] 2. Methods and systems for determining whether a subject has elevated levels of GFAP, UCH-L1, or GFAP and UCH-L1 In some aspects, the present disclosure relates to methods and systems for determining whether a subject has elevated levels of GFAP, UCH-L1, or GFAP and UCH-L1. In some embodiments, the methods and systems for determining whether a subject has elevated levels of GFAP, UCH-L1, or GFAP and UCH-L1 aid in the diagnosis and assessment of whether a subject has suffered a head injury. In some embodiments, the methods and systems for determining whether a subject has elevated levels of GFAP, UCH-L1, or GFAP and UCH-L1 can aid in determining whether a subject requires further assessment, such as by a head computed tomography (CT) scan and / or magnetic resonance imaging (MRI) procedure.

[0120] In some embodiments, the method includes performing at least one assay for UCH-L1, at least one assay for GFAP, or at least one assay for UCH-L1 and at least one assay for GFAP in at least one sample obtained from a subject (e.g., from a human subject). In some embodiments, the sample is obtained within about 48 hours after actual or suspected head injury. In other embodiments, the sample is obtained within about 24 hours after actual or suspected head injury. In yet other embodiments, the sample is obtained within about 12 hours after actual or suspected head injury. The method includes determining whether the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated based on a comparison of the level of GFAP in the sample to a baseline level of GFAP, the level of UCH-L1 in the sample to a baseline level of UCH-L1, or the level of GFAP in the sample to a baseline level of GFAP and the level of UCH-L1 in the sample to a baseline level of UCH-L1.

[0121] In some embodiments, the method includes obtaining a sample within about 48 hours (e.g., within about 48 hours, within about 24 hours, or within about 12 hours) of actual or suspected damage to the subject, and contacting the sample with an antibody to ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and / or an antibody to glial fibrillary acidic protein (GFAP) to form an antibody-biomarker complex. The method also includes detecting the resulting antibody-biomarker complex(es).

[0122] In some embodiments, the sample is taken from a subject (e.g., a human subject) within about 48 hours of injury, an actual or suspected injury to the head. For example, the sample may be taken within about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 60, 90 minutes, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours after the actual or suspected injury to the head. within about 21 hours, within about 22 hours, within about 23 hours, within about 24 hours, within about 25 hours, within about 26 hours, within about 27 hours, within about 28 hours, within about 29 hours, within about 30 hours, within about 31 hours, within about 32 hours, within about 33 hours, within about 34 hours, within about 35 hours, within about 36 hours, within about 37 hours, within about 38 hours, within about 39 hours, within about 40 hours, within about 41 hours, within about 42 hours, within about 43 hours, within about 44 hours, within about 45 hours, within about 46 hours, within about 47 hours, or within about 48 hours from the subject (e.g., a human subject).

[0123] In yet another embodiment, the sample is collected within about 8 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 9 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 10 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 11 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 12 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 13 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 14 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 15 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 16 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 17 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 18 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 19 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 20 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 21 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 22 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 23 hours to about 48 hours after the actual or suspected injury to the head. In yet other embodiments, the sample is collected within about 24 hours to about 48 hours after actual or suspected head injury, and in yet other embodiments, the sample is collected within about 25 hours to about 48 hours after actual or suspected head injury.In yet another embodiment, the sample is collected within about 26 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 27 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 28 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 29 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 30 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 31 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 32 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 33 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 34 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 35 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 36 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 37 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 38 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 39 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 40 hours to about 48 hours after the actual or suspected injury to the head.

[0124] In some embodiments, the onset of presence of a biomarker such as UCH-L1, GFAP, or a combination thereof occurs within about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours after actual or suspected head injury. within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, within about 24 hours, within about 25 hours, within about 26 hours, within about 27 hours, within about 28 hours, within about 29 hours, within about 30 hours, within about 31 hours, within about 32 hours, within about 33 hours, within about 34 hours, within about 35 hours, within about 36 hours, within about 37 hours, within about 38 hours, within about 39 hours, within about 40 hours, within about 41 hours, within about 42 hours, within about 43 hours, within about 44 hours, within about 45 hours, within about 46 hours, within about 47 hours, or within about 48 hours.

[0125] In other aspects, the onset of presence of a biomarker such as UCH-L1, GFAP, or a combination thereof, occurs within about 8 hours to about 48 hours, within about 9 hours to about 48 hours, within about 10 hours to about 48 hours, within about 11 hours to about 48 hours, within about 12 hours to about 48 hours, within about 13 hours to about 48 hours, within about 14 hours to about 48 hours, within about 15 hours to about 48 hours, within about 16 hours to about 48 hours, within about 17 hours to about 48 hours, within about 18 hours to about 48 hours, within about 19 hours to about 48 hours, within about 20 hours to about 48 hours, within about 21 hours to about 48 hours, within about 22 hours appears within about 48 hours, within about 23 hours to about 48 hours, within about 24 hours to about 48 hours, within about 25 hours to about 48 hours, within about 26 hours to about 48 hours, within about 27 hours to about 48 hours, within about 29 hours to about 48 hours, within about 30 hours to about 48 hours, within about 31 hours to about 48 hours, within about 32 hours to about 48 hours, within about 33 hours to about 48 hours, within about 34 hours to about 48 hours, within about 35 hours to about 48 hours, within about 36 hours to about 48 hours, within about 37 hours to about 48 hours, within about 38 hours to about 48 hours, within about 39 hours to about 48 hours, or within about 40 hours to about 48 hours.

[0126] In still further embodiments, the method comprises performing at least one assay for UCH-L1, at least one assay for GFAP, or at least one assay for UCH-L1 and at least one assay for GFAP on at least one sample obtained from the subject, and determining whether the subject's levels of UCH-L1, GFAP, or GFAP and UCH-L1 are elevated based on the results of the assays. In some embodiments, the method comprises determining that the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated. In some embodiments, the methods include determining that the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated if the level of only GFAP in the sample is equal to or greater than about 35 pg / mL, the level of only UCH-L1 in the sample is equal to or greater than about 400 pg / mL, the level of GFAP in the sample is equal to or greater than about 35 pg / mL and the level of UCH-L1 is less than about 400 pg / mL, or the level of GFAP in the sample is equal to or greater than about 35 pg / mL and the level of UCH-L1 is less than about 400 pg / mL, cannot be determined by an assay for UCH-L1, or is not reported by an assay for UCH-L1. In some embodiments, the method includes determining that the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated if the level of GFAP alone is equal to or greater than about 35 pg / mL, the level of UCH-L1 alone is equal to or greater than about 400 pg / mL, or the level of GFAP is equal to or greater than about 35 pg / mL and the level of UCH-L1 is equal to or greater than about 400 pg / mL. In some embodiments, the method includes determining that the subject's levels of GFAP and UCH-L1 are elevated if the level of GFAP cannot be determined by an assay for GFAP or is not reported by an assay for GFAP and the level of UCH-L1 is equal to or greater than about 400 pg / mL.

[0127] In some embodiments, the method includes determining that the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are not elevated. In some embodiments, the method includes determining that the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are not elevated if only the level of GFAP in the sample is below about 35 pg / mL, only the level of UCH-L1 in the sample is below about 40 pg / mL, or the level of GFAP in the sample is below about 35 pg / mL and the level of UCH-L1 in the sample is below about 400 pg / mL.

[0128] In some embodiments, the method includes determining that assays for UCH-L1, GFAP, or UCH-L1 and GFAP should be repeated. In some embodiments, the method includes determining that assays for UCH-L1, GFAP, or UCH-L1 and GFAP should be repeated if the level of UCH-L1 alone in the sample cannot be determined or reported, the level of GFAP is below about 35 pg / mL and the level of UCH-L1 cannot be determined or reported by an assay for UCH-L1, or the level of GFAP alone in the sample cannot be determined or reported. In some embodiments, the method includes determining that assays for UCH-L1 and GFAP should be repeated if the level of GFAP cannot be determined or reported by an assay for GFAP and the level of UCH-L1 is below about 400 pg / mL. In some embodiments, the method includes determining that if the level of GFAP cannot be determined by or is not reported by an assay for GFAP and the level of UCH-L1 cannot be determined by or is not reported by an assay for UCH-L1, then the assays for UCH-L1 and GFAP should be repeated.

[0129] In some embodiments, the method includes communicating a decision on or from at least one device (e.g., a decision that the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated, a decision that the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are not elevated, or a decision that the assay for GFAP, UCH-L1, or GFAP and UCH-L1 should be repeated). Suitable devices are described herein, including non-point-of-care devices (e.g., the Architect platform marketed by Abbott Core Laboratories), which may include a user interface that displays and thereby communicates the decision.

[0130] In some embodiments, the device includes software that performs one or more tasks. In some embodiments, the device includes software that automatically determines the next appropriate step in the methods described herein. For example, the device may include software that determines whether the levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated, whether the levels are not elevated, and / or whether the assay needs to be repeated. The software may display this determination, such as on a graphical user interface.

[0131] In some embodiments, the device stores software that directs the processor to perform a given task. In some embodiments, the software stores machine-readable instructions that direct the processor to perform a given task. The machine-readable instructions may be one or more executable programs or part(s) of an executable program for execution by a computer. The program 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 processor. Alternatively, the entire program and / or parts thereof may be instead executed by a device other than the processor and / or embodied in firmware or dedicated hardware. Additionally or alternatively, the process may be implemented by one or more hardware circuits (e.g., discrete and / or integrated analog and / or digital circuits, FPGAs, ASICs, comparators, operational amplifiers (op-amps), logic circuits, etc.) that are structured to perform the corresponding operations without executing software or firmware.

[0132] 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, and the like. The machine-readable instructions described herein may be stored as data (e.g., portions of instructions, code, representations of code, and the like) that can be utilized to generate, manufacture, and / or create 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 placing, modifying, adapting, updating, combining, adding, configuring, decrypting, unpacking, distributing, rearranging, editing, and the like to make the instructions directly readable, interpretable, and / or executable by the computing device and / or other machines. For example, the machine-readable instructions may be stored in multiple portions that are individually compressed, encrypted, and stored on separate computing devices, and the portions when decrypted, unpacked, and combined form a set of executable instructions that implement a program, such as the program described herein.

[0133] In another example, the machine-readable instructions may be stored in a state in which they can be read by a computer, but require the addition of a library (e.g., a dynamic link library (DLL)), a software development kit (SDK), an application programming interface (API), etc., to execute the instructions on a particular computing device or other device. In another example, the machine-readable instructions may require configuration (e.g., saving settings, inputting data, recording network addresses, etc.) before the machine-readable instructions and / or 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 corresponding program(s) regardless of the particular format or state of the machine-readable instructions and / or corresponding program(s) when stored or otherwise at rest or in transmission.

[0134] The machine-readable instructions described herein may be expressed in any past, present, or future command language, scripting language, programming language, etc. For example, the machine-readable instructions may be expressed using any of the following languages: C, C++, Java, C#, Perl, Python, JavaScript, HyperText Markup Language (HTML), Structured Query Language (SQL), Swift, etc.

[0135] The machine-readable instructions may be stored on a non-transitory computer and / or machine-readable medium, such as a hard disk drive, flash memory, read-only memory, compact disk, digital versatile disk, cache, random access memory, and / or any other storage device or storage disk on which information is stored for any period of time (e.g., long term, permanently, for a short period of time, for temporary buffering, and / or for caching of information). As used herein, the term non-transitory computer-readable medium is expressly defined to include any type of computer-readable storage device and / or storage disk, and to exclude propagating signals and to exclude transmission media.

[0136] In some embodiments, the method further comprises performing a head computed tomography (CT) scan, a magnetic resonance imaging (MRI) procedure, or both a CT scan or an MRI procedure on the subject if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated. For example, in some embodiments, the method further comprises performing a head CT scan on the subject if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated. In another example, in some embodiments, the method further comprises performing an MRI procedure on the subject if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated. In some embodiments, the method further comprises performing a head CT scan and an MRI procedure on the subject if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated.

[0137] In some embodiments, the method further comprises not performing a head computed tomography (CT) scan, a magnetic resonance imaging (MRI) procedure, or both a head CT scan or an MRI procedure on the subject if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are not elevated. In other words, the method comprises "precluding" the need for a head CT scan, an MRI procedure, or both, if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are not elevated.

[0138] In some embodiments, the method further includes diagnosing the subject as having traumatic brain injury (TBI) if the level of GFAP alone is equal to or greater than about 35 pg / mL, the level of UCH-L1 alone is equal to or greater than about 400 pg / mL, or the level of GFAP is equal to or greater than about 35 pg / mL and the level of UCH-L1 is equal to or greater than about 400 pg / mL, regardless of whether a head CT scan is negative for TBI or whether any head CT scan is performed.

[0139] In some embodiments, the method further comprises treating the subject for mild, moderate, moderate-severe, or severe TBI if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are determined to be elevated. For example, in some embodiments, the method further comprises treating the subject for mild TBI if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are determined to be elevated. In some embodiments, the method further comprises treating the subject for moderate-severe TBI if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are determined to be elevated. In some embodiments, the method further comprises treating the subject for severe TBI if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are determined to be elevated. In some embodiments, the selection of an appropriate treatment may be facilitated by results from a head CT scan, an MRI procedure, or both, if performed on the subject. For example, results from a head CT scan and / or MRI procedure can help further distinguish between mild, moderate-severe, or severe TBI in a subject. Such a distinction can aid in selecting an appropriate treatment for the subject. In some embodiments, the method further includes monitoring the subject for elevated levels of GFAP, UCH-L1, or GFAP and UCH-L1.

[0140] In some embodiments, the method further comprises treating a subject (e.g., a human subject) assessed to have mild, moderate, severe, or moderate-severe traumatic brain injury with a traumatic brain injury treatment, as described below. In still other embodiments, the method further comprises treating a subject (e.g., a human subject) assessed to have mild traumatic brain injury with a traumatic brain injury treatment, as described below. In still other embodiments, the method further comprises treating a subject (e.g., a human subject) assessed to have moderate traumatic brain injury with a traumatic brain injury treatment, as described below. In still other embodiments, the method further comprises treating a subject (e.g., a human subject) assessed to have severe traumatic brain injury with a traumatic brain injury treatment. In some embodiments, the method further comprises monitoring a subject (e.g., a human subject) assessed to have mild traumatic brain injury, as described below. In other embodiments, the method further comprises monitoring a subject (e.g., a human subject) assessed to have moderate traumatic brain injury, as described below. In yet other embodiments, the method further comprises monitoring the subject (e.g., a human subject) assessed as having a severe traumatic brain injury, as described below. In yet other embodiments, the method further comprises monitoring the subject (e.g., a human subject) assessed as having a moderate to severe traumatic brain injury.

[0141] At least one assay for GFAP and at least one assay for UCH-L1 may be performed simultaneously. Alternatively, an assay for GFAP and an assay for UCH-L1 may be performed sequentially. The assays may be performed sequentially in any order. For example, an assay for GFAP may be performed first, followed by an assay for UCH-L1. In another example, an assay for UCH-L1 may be performed first, followed by an assay for GFAP.

[0142] In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 are performed about 10 minutes to about 20 minutes, respectively. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 are performed about 10 minutes, respectively. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 are performed about 11 minutes, respectively. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 are performed about 12 minutes, respectively. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 are performed about 13 minutes, respectively. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 are performed about 14 minutes, respectively. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 are performed about 15 minutes, respectively. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 are performed about 16 minutes after each. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 are performed about 17 minutes after each. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 are performed about 18 minutes after each. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 are performed about 19 minutes after each. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 are performed about 20 minutes after each.The nature of the assays used in the methods described herein is not determinative, and the tests can be any assay known in the art, such as, for example, immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, or protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, or chromatographic or spectroscopic methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). Nevertheless, any test or assay capable of performing the claimed methods can be used, such as assays with various sensitivities and those described herein. Furthermore, the assays used in the methods described herein can be used in clinical chemistry formats as would be known to one of skill in the art. Such assays are described in further detail in Sections 5-9 of the present specification. It is known in the art that values ​​(e.g., reference levels, cutoffs, thresholds, specificity, sensitivity, calibrator and / or control concentrations, etc.) used in an assay using a particular sample type (e.g., an immunoassay using serum or a point-of-care device using whole blood) can be extrapolated to other assay formats using techniques known in the art, such as assay standardization. For example, one way that assay standardization can be performed is by applying a factor to the calibrator used in the assay to increase or decrease the sample concentration lead to obtain a slope that matches the comparator method. Other methods of standardizing the results obtained in one assay to another are well known and described in the literature (see, for example, David Wild, Immunoassay Handbook, 4th Edition, Chapter 3.5, pages 315-322, the contents of which are incorporated herein by reference).

[0143] 3. Using Reference Levels to Help Diagnose and Assess Whether a Subject Has Suspected of Having Suffered or Having Suspected of Having Suffered a Head Injury The present disclosure relates, among other methods, to methods of assessing or aiding in the diagnosis and assessment of whether a subject (e.g., a human subject) has suffered or may have suffered a head injury. In some embodiments, the method for determining whether a subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated can aid in determining whether a subject has suffered a traumatic brain injury. In some embodiments, the method can aid in determining the extent of traumatic brain injury in a subject (e.g., a human subject) with actual or suspected head injury, for example, determining whether a subject (e.g., a human subject) has mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate to severe traumatic brain injury. As used herein, "determining whether a subject (e.g., a human subject) has mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury" refers to the fact that the above-described methods, for example, when used in conjunction with other information (e.g., clinical evaluation data), can determine that a subject is more likely than not to have mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury. The method can include performing an assay on a sample obtained from a subject (e.g., a human subject) within about 48 hours after an actual or suspected head injury to measure or detect levels of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and / or glial fibrillary acidic protein (GFAP) in the sample, and determining whether the subject (e.g., a human subject) has suffered a mild, moderate, severe, or moderate-severe traumatic brain injury (TBI) based on the levels of GFAP, and / or UCH-L1.In some embodiments, the method can include performing an assay on a sample obtained from a subject (e.g., a human subject) within about 24 hours after an actual or suspected head injury to measure or detect a level of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and / or glial fibrillary acidic protein (GFAP) in the sample, and determining whether the subject (e.g., a human subject) has suffered a mild, moderate, severe, or moderate-severe traumatic brain injury (TBI) based on the level of GFAP, and / or UCH-L1. In another embodiment, the method can include performing an assay on a sample obtained from a subject (e.g., a human subject) within about 12 hours after an actual or suspected head injury to measure or detect a level of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and / or glial fibrillary acidic protein (GFAP) in the sample, and determining whether the subject (e.g., a human subject) has suffered a mild, moderate, severe, or moderate-severe traumatic brain injury (TBI) based on the level of GFAP, and / or UCH-L1. In some embodiments, the subject is determined to have mild, moderate, severe, or moderate-severe TBI based on determining whether the levels of GFAP and / or UCH-L1 are elevated in a sample obtained from the subject. In some embodiments, the subject is determined to have mild, moderate, severe, or moderate-severe TBI if the levels of GFAP and / or UCH-L1 are determined to be elevated. In some embodiments, determining whether the levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated relies on comparing the level of GFAP in the sample to a reference level for GFAP, the level of UCH-L1 in the sample to a reference level for UCH-L1, or the level of GFAP in the sample to a reference level for GFAP, and comparing the level of UCH-L1 in the sample to a reference level for UCH-L1. The sample can be a biological sample.

[0144] In some embodiments, the method can include obtaining a sample within about 48 hours of actual or suspected injury to the subject and contacting the sample with an antibody to a biomarker for TBI, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, to form an antibody-biomarker complex. In another aspect, the method can include obtaining a sample within about 24 hours of actual or suspected injury to the subject and contacting the sample with an antibody to a biomarker for TBI, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, to form an antibody-biomarker complex. In yet a further aspect, the method can include obtaining a sample within about 12 hours of actual or suspected injury to the subject and contacting the sample with an antibody to a biomarker for TBI, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, to form an antibody-biomarker complex. The method also includes detecting the resulting antibody-biomarker complex.

[0145] In some embodiments, the sample is taken from a subject (e.g., a human subject) within about 48 hours of actual or suspected head injury. For example, the sample may be taken within about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 60, 90 minutes, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours after actual or suspected head injury. within about 21 hours, within about 22 hours, within about 23 hours, within about 24 hours, within about 25 hours, within about 26 hours, within about 27 hours, within about 28 hours, within about 29 hours, within about 30 hours, within about 31 hours, within about 32 hours, within about 33 hours, within about 34 hours, within about 35 hours, within about 36 hours, within about 37 hours, within about 38 hours, within about 39 hours, within about 40 hours, within about 41 hours, within about 42 hours, within about 43 hours, within about 44 hours, within about 45 hours, within about 46 hours, within about 47 hours, or within about 48 hours from the subject (e.g., a human subject).

[0146] In another embodiment, the sample is collected within about 8 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 9 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 10 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 11 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 12 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 13 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 14 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 15 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 16 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 17 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 18 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 19 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 20 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 21 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 22 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 23 hours to about 48 hours after the actual or suspected injury to the head. In yet other embodiments, the sample is collected within about 24 hours to about 48 hours after actual or suspected head injury, and in yet other embodiments, the sample is collected within about 25 hours to about 48 hours after actual or suspected head injury.In yet another embodiment, the sample is collected within about 26 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 27 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 28 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 29 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 30 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 31 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 32 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 33 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 34 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 35 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 36 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 37 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 38 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 39 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 40 hours to about 48 hours after the actual or suspected injury to the head.

[0147] In some embodiments, the onset of presence of a biomarker, such as UCH-L1, GFAP, or a combination thereof, occurs within about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours after actual or suspected head injury. within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, within about 24 hours, within about 25 hours, within about 26 hours, within about 27 hours, within about 28 hours, within about 29 hours, within about 30 hours, within about 31 hours, within about 32 hours, within about 33 hours, within about 34 hours, within about 35 hours, within about 36 hours, within about 37 hours, within about 38 hours, within about 39 hours, within about 40 hours, within about 41 hours, within about 42 hours, within about 43 hours, within about 44 hours, within about 45 hours, within about 46 hours, within about 47 hours, or within about 48 hours.

[0148] In other aspects, the onset of presence of a biomarker such as UCH-L1, GFAP, or a combination thereof, occurs within about 8 hours to about 48 hours, within about 9 hours to about 48 hours, within about 10 hours to about 48 hours, within about 11 hours to about 48 hours, within about 12 hours to about 48 hours, within about 13 hours to about 48 hours, within about 14 hours to about 48 hours, within about 15 hours to about 48 hours, within about 16 hours to about 48 hours, within about 17 hours to about 48 hours, within about 18 hours to about 48 hours, within about 19 hours to about 48 hours, within about 20 hours to about 48 hours, within about 21 hours to about 48 hours, within about 22 hours appears within about 48 hours, within about 23 hours to about 48 hours, within about 24 hours to about 48 hours, within about 25 hours to about 48 hours, within about 26 hours to about 48 hours, within about 27 hours to about 48 hours, within about 29 hours to about 48 hours, within about 30 hours to about 48 hours, within about 31 hours to about 48 hours, within about 32 hours to about 48 hours, within about 33 hours to about 48 hours, within about 34 hours to about 48 hours, within about 35 hours to about 48 hours, within about 36 hours to about 48 hours, within about 37 hours to about 48 hours, within about 38 hours to about 48 hours, within about 39 hours to about 48 hours, or within about 40 hours to about 48 hours.

[0149] In some embodiments, the subject receives a GCS score before or after the assay is performed. In some embodiments, the subject (e.g., a human subject) is suspected to have moderate, severe, or moderate-severe traumatic brain injury based on the GCS score. In some embodiments, the baseline level of a biomarker, such as UCH-L1, GFAP, or a combination thereof, correlates with the subject having moderate, severe, or moderate-severe traumatic brain injury. In some embodiments, the baseline level of a biomarker, such as UCH-L1, GFAP, or a combination thereof, correlates with a GCS score of 9-13 (moderate TBI). In some embodiments, the baseline level of a biomarker, such as UCH-L1, GFAP, or a combination thereof, correlates with a GCS score of 3-8 (severe TBI). In some embodiments, the baseline levels of biomarkers such as UCH-L1, GFAP, or a combination thereof are correlated with a GCS score of 3-12 (moderate, severe, or moderate-severe TBI). In some embodiments, the subject is suspected to have mild traumatic brain injury based on the GCS score. In some embodiments, the baseline levels of biomarkers such as UCH-L1, GFAP, or a combination thereof are correlated with subjects having mild traumatic brain injury. In some embodiments, the baseline levels of biomarkers such as UCH-L1, GFAP, or a combination thereof are correlated with a GCS score of 13-15 (mild TBI).

[0150] Generally, the reference level of a biomarker, such as UCH-L1, GFAP, or a combination thereof, can also be used as a benchmark against which results obtained when assaying a test sample for a biomarker, such as UCH-L1, GFAP, or a combination thereof, can be assessed. Generally, in making such a comparison, the reference level of a biomarker, such as UCH-L1, GFAP, or a combination thereof, is obtained by performing or executing a particular assay a sufficient number of times and under appropriate conditions so as to link or correlate the presence, amount, or concentration of the analyte with a particular stage or endpoint of TBI or with a particular symptom. Typically, the reference level of a biomarker, such as UCH-L1, GFAP, or a combination thereof, is obtained using an assay of a reference subject (or population of subjects). The biomarker, such as UCH-L1, GFAP, or a combination thereof, that is measured can include fragments thereof, degradation products thereof, and / or enzymatic cleavage products thereof.

[0151] In certain embodiments, the baseline level may be correlated to a control subject (eg, a human subject) who has not suffered a head injury.

[0152] In some embodiments, the method includes determining that the subject has traumatic brain injury if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated. For example, in some embodiments, the method includes determining that the subject has mild, moderate, severe, or moderate-severe traumatic brain injury if the level of GFAP alone in a sample obtained from the subject is equal to or exceeds a threshold of 35 pg / mL, the level of GFAP in a sample obtained from the subject is equal to or exceeds a threshold of 35 pg / mL and the level of UCH-L1 is below a threshold of about 400 pg / mL, cannot be determined, or is not reported. In some embodiments, the method includes determining that the subject has mild, moderate, severe, or moderate-severe traumatic brain injury if the level of UCH-L1 alone in the sample is equal to or exceeds a threshold of about 400 pg / mL, or if the level of GFAP in the sample from the subject is equal to or exceeds a threshold of 35 pg / mL and the level of UCH-L1 in the sample is equal to or exceeds a threshold of about 400 pg / mL. In some embodiments, the method includes determining that the subject has mild, moderate, severe, or moderate-severe traumatic brain injury if the level of GFAP in the sample from the subject cannot be determined or is not reported and the level of UCH-L1 in the sample is equal to or exceeds a threshold of about 400 pg / mL.

[0153] In still further embodiments, the method includes determining that the subject is likely free of traumatic brain injury if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are not elevated. For example, in some embodiments, the method includes determining that the subject is likely free of traumatic brain injury if only the level of GFAP in the sample is below a threshold of about 35 pg / mL, only the level of UCH-L1 in the sample is below a threshold of about 400 pg / mL, or if the level of GFAP in a sample obtained from the subject is below a threshold of about 35 pg / mL and the level of UCH-L1 in the sample is below a threshold of about 400 pg / mL.

[0154] In some embodiments, the method further comprises treating a subject (e.g., a human subject) assessed to have mild, moderate, severe, or moderate-severe traumatic brain injury with a traumatic brain injury treatment, as described below. In still other embodiments, the method further comprises treating a subject (e.g., a human subject) assessed to have mild traumatic brain injury with a traumatic brain injury treatment, as described below. In still other embodiments, the method further comprises treating a subject (e.g., a human subject) assessed to have moderate traumatic brain injury with a traumatic brain injury treatment, as described below. In still other embodiments, the method further comprises treating a subject (e.g., a human subject) assessed to have severe traumatic brain injury with a traumatic brain injury treatment. In some embodiments, the method further comprises monitoring a subject (e.g., a human subject) assessed to have mild traumatic brain injury, as described below. In other embodiments, the method further comprises monitoring a subject (e.g., a human subject) assessed to have moderate traumatic brain injury, as described below. In yet other embodiments, the method further comprises monitoring the subject (e.g., a human subject) assessed as having a severe traumatic brain injury, as described below. In yet other embodiments, the method further comprises monitoring the subject (e.g., a human subject) assessed as having a moderate to severe traumatic brain injury.

[0155] The nature of the assay utilized in the methods described herein is not critical, and the test can be any assay known in the art, such as, for example, immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis or protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, or chromatographic or spectroscopic methods, such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). Regardless, any test or assay capable of performing the claimed methods, such as, for example, assays with various sensitivities and sensitivity described herein, may be used. Furthermore, the assays used in the methods described herein may be used in clinical chemistry formats that would be known to one of skill in the art. Such assays are described in further detail in Sections 5-9 of this specification. It is known in the art that values ​​(e.g., reference levels, cut-offs, thresholds, specificity, sensitivity, calibrator and / or control concentrations, etc.) used in an assay using a specific sample type (e.g., immunoassays using serum or non-point-of-care devices using whole blood) can be extrapolated to other assay formats using techniques known in the art, such as assay standardization. For example, one way in which assay standardization can be performed is by applying a factor to the calibrator used in the assay to make the sample concentration reading higher or lower to obtain a slope that matches the comparison method. Other methods of standardizing the results obtained in one assay to another are also known and described in the literature (see, for example, David Wild, "Immunoassay Handbook", 4th edition, chapter 3.5, pages 315-322, the contents of which are incorporated herein by reference).

[0156] 4. Using Reference Levels to Help Decide Whether to Perform a CT Scan and / or MRI in Subjects Who Have Suffered or May Have Suffered Head Injury The present disclosure relates, among other methods, to a method for aiding in the decision of whether to perform a computed tomography (CT) scan and / or magnetic resonance imaging on a subject (e.g., a human subject) who has or may have suffered actual or suspected injury to the head. In some embodiments, a method for determining whether a subject has elevated levels of GFAP, UCH-L1, or GFAP and UCH-L1 can aid in the decision of whether to perform a CT scan or MRI on the subject. As used herein, "determining whether to perform a CT scan on a subject" refers to, for example, the fact that the aforementioned method can be used with other information (e.g., clinical evaluation data) to determine that a subject (e.g., a human subject) is more likely than not to have a positive head CT scan. As used herein, "determining whether to perform an MRI on a subject" refers to, for example, the fact that the aforementioned method can be used with other information (e.g., clinical evaluation data) to determine that a subject (e.g., a human subject) is more likely than not to have a positive head MRI scan. Specifically, such methods can include the steps of: (a) performing an assay on a sample obtained from the subject within about 48 hours after actual or suspected head injury to determine whether the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated; and (b) determining whether to perform a CT scan and / or MRI on the subject (e.g., human subject) based on whether the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated. In some aspects, the assay is performed on a sample obtained from the subject within about 24 hours after actual or suspected head injury. In still further aspects, the assay is performed on a sample obtained from the subject within about 12 hours after actual or suspected head injury. In some embodiments, the method includes performing a head CT scan or MRI procedure on the subject if the levels of GFAP, UCH-L1, or GFAP and UCH-L1 are determined to be elevated. In some aspects, a CT scan is performed on the subject. In other aspects, an MRI procedure is performed on the subject.In yet further aspects, a CT scan and an MRI are performed on the subject (the order in which the CT scan and MRI are performed is not critical). In some embodiments, the method includes not performing a head CT scan or MRI procedure on the subject if the levels of GFAP, UCH-L1, or GFAP and UCH-L1 are not determined to be elevated. In other words, the method includes "ruling out" the need for a head CT scan, MRI procedure, or both, if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are not elevated. The sample can be a biological sample.

[0157] In some embodiments, the method can include obtaining a sample (e.g., a human subject) within about 48 hours of actual or suspected injury to the subject and contacting the sample with an antibody to a biomarker for TBI, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, to form an antibody-biomarker complex. In some embodiments, the method can include obtaining a sample (e.g., a human subject) within about 24 hours of actual or suspected injury to the subject and contacting the sample with an antibody to a biomarker for TBI, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, to form an antibody-biomarker complex. In some embodiments, the method includes obtaining a sample (e.g., from a human subject) within about 12 hours of actual or suspected injury to the subject and contacting the sample with an antibody to a biomarker for TBI, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, to form an antibody-biomarker complex. The method also includes detecting the antibody-biomarker complex thus obtained.

[0158] In some embodiments, the sample is taken from a subject (e.g., a human subject) within about 2 hours of actual or suspected injury to the head. For example, the sample can be taken from the subject within about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about 2 hours after actual or suspected injury to the head. In some embodiments, the onset of presence of a biomarker such as UCH-L1, GFAP, or a combination thereof occurs within about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours after actual or suspected head injury. within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, within about 24 hours, within about 25 hours, within about 26 hours, within about 27 hours, within about 28 hours, within about 29 hours, within about 30 hours, within about 31 hours, within about 32 hours, within about 33 hours, within about 34 hours, within about 35 hours, within about 36 hours, within about 37 hours, within about 38 hours, within about 39 hours, within about 40 hours, within about 41 hours, within about 42 hours, within about 43 hours, within about 44 hours, within about 45 hours, within about 46 hours, within about 47 hours, or within about 48 hours.

[0159] In another embodiment, the sample is collected within about 8 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 9 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 10 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 11 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 12 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 13 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 14 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 15 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 16 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 17 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 18 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 19 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 20 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 21 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 22 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 23 hours to about 48 hours after the actual or suspected injury to the head. In yet other embodiments, the sample is collected within about 24 hours to about 48 hours after actual or suspected head injury, and in yet other embodiments, the sample is collected within about 25 hours to about 48 hours after actual or suspected head injury.In yet another embodiment, the sample is collected within about 26 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 27 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 28 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 29 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 30 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 31 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 32 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 33 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 34 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 35 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 36 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 37 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 38 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 39 hours to about 48 hours after the actual or suspected injury to the head. In yet another embodiment, the sample is collected within about 40 hours to about 48 hours after the actual or suspected injury to the head.

[0160] In some embodiments, the subject undergoes a CT scan before or after the assay is performed. In some embodiments, the subject is suspected of having traumatic brain injury based on a CT scan. In some embodiments, the baseline level of a biomarker such as UCH-L1, GFAP, or a combination thereof is correlated with a positive head CT scan.

[0161] Generally, a reference level of a biomarker, such as UCH-L1, GFAP, or a combination thereof, can be used as a reference benchmark for assessing the results obtained when assaying a test sample for UCH-L1, GFAP, or a combination thereof. Generally, in making such a comparison, a reference level of a biomarker, such as UCH-L1, GFAP, or a combination thereof, is obtained by performing a specific assay a sufficient number of times and under appropriate conditions so as to link or correlate the presence, amount, or concentration of the analyte with a particular stage or endpoint of TBI or with a particular symptom. Typically, a reference level of a biomarker, such as UCH-L1, GFAP, or a combination thereof, is obtained using an assay of a reference subject (or population of subjects). The biomarker, such as UCH-L1, GFAP, or a combination thereof, that is measured can include fragments thereof, degradation products thereof, and / or enzymatic cleavage products thereof.

[0162] In yet further embodiments, the method includes performing a head CT scan or MRI on the subject if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated. For example, in some embodiments, the method includes performing a head CT scan or MRI procedure on the subject if the level of only GFAP in a sample obtained from the subject is equal to or exceeds a threshold value of 35 pg / mL, or the level of GFAP and the level of UCH-L1 are lower than, indeterminable, or not reported at a threshold value of about 400 pg / mL. In some embodiments, the method includes performing a head CT scan or MRI procedure on the subject if the level of only UCH-L1 in the sample is equal to or exceeds a threshold value of about 400 pg / mL, or the level of GFAP in a sample obtained from the subject is equal to or exceeds a threshold value of 35 pg / mL and the level of UCH-L1 in the sample is equal to or exceeds a threshold value of about 400 pg / mL. In some embodiments, the methods include performing a head CT scan or MRI procedure on the subject if the level of GFAP in a sample obtained from the subject cannot be determined or is not reported and the level of UCH-L1 in the sample is equal to or exceeds a threshold value of about 400 pg / mL.

[0163] In some embodiments, the method includes determining that the subject does not require a head CT scan or MRI if the subject's levels of GFAP and UCH-L1 are not elevated. For example, in some embodiments, the method includes determining that the subject does not require a head CT scan or MRI procedure if only the level of GFAP in the sample is below about 35 pg / mL, only the level of UCH-L1 in the sample is below about 400 pg / mL, or if the level of GFAP in the sample obtained from the subject is below a threshold of about 35 pg / mL and the level of UCH-L1 in the sample is below a threshold of about 400 pg / mL.

[0164] In some embodiments, the methods further include treating the subject (e.g., a human subject) with a traumatic brain injury treatment and / or monitoring the subject, as described below.

[0165] The nature of the assay used in the methods described herein is not critical and the test can be any assay known in the art, such as, for example, immunoassays, protein immunoprecipitation, immunoelectrophoresis, Western blots, or protein immunostaining, or spectroscopy such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). Moreover, the assays can be used in clinical chemistry formats that would be known to one of skill in the art. Such assays are described in further detail in Sections 6-10 herein.

[0166] 5. Treatment and Monitoring of Subjects Suffering from Traumatic Brain Injury In the above methods, subjects (e.g., human subjects) identified or assessed as having elevated levels of GFAP, UCH-L1, or GFAP and UCH-L1 that may indicate traumatic brain injury may be treated or monitored. In some embodiments, the method further includes treating the subjects (e.g., human subjects) determined to have elevated levels of GFAP, UCH-L1, or GFAP and UCH-L1 with a traumatic brain injury treatment, such as any treatment known in the art. For example, traumatic brain injury treatment may take a variety of forms depending on the severity of the injury to the head. For example, in subjects suffering from mild TBI, treatment may include one or more of rest, refraining from physical activity such as sports, avoiding light or wearing sunglasses when outside in bright light, medication to relieve headaches or migraines, antiemetic medication, etc. Treatment for patients suffering from moderate, severe, or moderate-severe TBI may include administration of one or more appropriate medications (such as, for example, diuretics, anticonvulsants, medications to sedate the individual and put them into a drug-induced coma, or other pharmaceutical or biopharmaceutical medications (known or future developed for the treatment of TBI), one or more surgical procedures (such as, for example, removal of a hematoma, repair of a skull fracture, decompressive craniectomy, etc.), and one or more therapies (such as, for example, one or more rehabilitation, cognitive behavioral therapy, anger management, counseling psychology, etc.). In some embodiments, the method further includes monitoring the subject (e.g., a human subject) assessed as having elevated levels of GFAP, UCH-L1, or GFAP and UCH-L1 (e.g., which may be indicative of, or may be, mild, moderate, severe, or moderate-severe traumatic brain injury). For example, monitoring a subject assessed to have elevated levels of GFAP, UCH-L1, or GFAP and UCH-L1 may include monitoring using CT scans and / or MRI procedures.In some embodiments, subjects identified as having mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury, or traumatic brain injury such as mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury, may be monitored using CT scans and / or MRIs.

[0167] 6. Methods for Measuring UCH-L1 Levels In the methods described above, UCH-L1 levels can be measured by any means, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, antibody-dependent methods such as SDS-PAGE and Western blot analysis, protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, or chromatographic methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS), such as those described in WO2018 / 067468, WO2018 / 191531, WO2018 / 218169 and WO2019 / 112860, or spectroscopic methods. The contents of each of the above patent documents are incorporated herein by reference. The assay can also be used in clinical chemistry formats that would be known to those skilled in the art.

[0168] In some embodiments, measuring the level of UCH-L1 comprises contacting the sample with a first specific binding member and a second specific binding member. In some embodiments, the first specific binding member is a capture antibody and the second specific binding member is a detection antibody. In some embodiments, measuring the level of UCH-L1 comprises contacting the sample with, simultaneously or sequentially, in any order, (1) a capture antibody (e.g., a UCH-L1 capture antibody) that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form a capture antibody-UCH-L1 antigen complex (e.g., a UCH-L1 capture antibody-UCH-L1 antigen complex), and (2) a detectable label that binds to an epitope on UCH-L1 not bound by the capture antibody to form a UCH-L1 antigen complex. - contacting the sample with a detection antibody (e.g., a UCH-L1 detection antibody) which forms a detection antibody complex (e.g., a UCH-L1 antigen-UCH-L1 detection antibody complex) to form a capture antibody-UCH-L1 antigen-detection antibody complex (e.g., a UCH-L1 capture antibody-UCH-L1 antigen-UCH-L1 detection antibody complex), and measuring the amount or concentration of UCH-L1 in the sample based on a signal generated by a detectable label in the capture antibody-UCH-L1 antigen-detection antibody complex.

[0169] In some embodiments, the first specific binding member is immobilized on a solid support. In some embodiments, the second specific binding member is immobilized on a solid support. In some embodiments, the first specific binding member is a UCH-L1 antibody described below.

[0170] In some embodiments, the sample is diluted or undiluted. In some embodiments, the sample is about 1 to about 30 microliters. In some embodiments, the sample is about 10 to about 30 microliters. In some embodiments, the sample is about 20 microliters. In some embodiments, the sample is 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, or about 15 microliters. In some embodiments, the sample is about 1 microliter, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, about 10 microliters, about 11 microliters, about 12 microliters, about 13 microliters, about 14 microliters, about 15 microliters, about 16 microliters, about 17 microliters, about 18 microliters, about 19 microliters, about 20 microliters, about 21 microliters, about 22 microliters, about 23 microliters, about 24 microliters, about 25 microliters, about 26 microliters, about 27 microliters, about 28 microliters, about 29 microliters, or about 30 microliters. In some embodiments, the sample is about 1 to about 150 microliters or less, or about 1 to about 30 microliters or less.

[0171] Some non-point-of-care meters (e.g., Abbott Laboratories meters ARCHITECT®, Alinity, and other core testing meters) may be capable of measuring UCH-L1 levels in samples of 25,000 pg / mL or greater.

[0172] Other detection methods may include or be adapted for use in nanopore or nanowell devices. Examples of nanopore devices are described in International Patent Publication No. WO 2016 / 161402, which is incorporated herein by reference in its entirety. Examples of nanowell devices are described in International Patent Publication No. WO 2016 / 161400, which is incorporated herein by reference in its entirety.

[0173] 7.UCH-L1 antibody The methods described herein may use isolated antibodies that specifically bind to ubiquitin carboxy-terminal hydrolase L1 ("UCH-L1") (or fragments thereof), referred to as "UCH-L1 antibodies." UCH-L1 antibodies may be used to assess UCH-L1 status as a measure of traumatic brain injury, or to detect the presence of UCH-L1 in a sample, to quantitate the amount of UCH-L1 present in a sample, or to detect the presence of UCH-L1 in a sample and quantitate the amount of UCH-L1 in a sample.

[0174] Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) ("UCH-L1"), 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 ubiquitin monomers. Expression of UCH-L1 is highly specific to cells of the neurons and diffuse neuroendocrine system and their tumors. UCH-L1 is abundant in all neurons (accounting for 1-2% of total brain protein) and is specifically expressed in neurons and testis / ovary. The catalytic triad of UCH-L1 contains a cysteine ​​at position 90, an aspartic acid at position 176, and a histidine at position 161, which contribute to its hydrolase activity.

[0175] Human UCH-L1 has the following amino acid sequence:

[0176] [ka] may have.

[0177] Human UCH-L1 can be a fragment or variant of SEQ ID NO: 1. Fragments of UCH-L1 can be between 5 and 225 amino acids, between 10 and 225 amino acids, between 50 and 225 amino acids, between 60 and 225 amino acids, between 65 and 225 amino acids, between 100 and 225 amino acids, between 150 and 225 amino acids, between 100 and 225 amino acids, or between 175 and 225 amino acids in length. Fragments can include consecutively numbered amino acids derived from SEQ ID NO: 1.

[0178] b.UCH-L1 recognition antibody The antibody is an antibody that binds to UCH-L1, a fragment thereof, an epitope of UCH-L1 or a variant thereof. The antibody may be a fragment of an anti-UCH-L1 antibody or a variant or derivative thereof. The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, a fully human antibody or an antibody fragment such as a Fab fragment or a mixture thereof. The antibody fragment or derivative may include a F(ab')2 fragment, an Fv fragment or a scFv fragment. The antibody derivative may be produced by peptidomimetics. Furthermore, techniques described for producing single chain antibodies may be adapted to produce single chain antibodies.

[0179] The anti-UCH-L1 antibody may be a chimeric or humanized anti-UCH-L1 antibody. In one embodiment, both the humanized and chimeric antibodies are monovalent. In one embodiment, both the humanized and chimeric antibodies comprise a single Fab region linked to an Fc region.

[0180] Human antibodies can be derived by phage display technology or transgenic mice expressing human immunoglobulin genes. Human antibodies can be generated and isolated as a result of a human immune response in vivo. See, for example, Funaro et al., BMC Biotechnology, 2008(8):85. Thus, the antibody can be a product of a human and may not be an animal repertoire. Because the antibody is of human origin, the risk of reactivity against self-antigens can be minimized. Alternatively, standard yeast display libraries and yeast display technology can be used to select and isolate human anti-UCH-L1 antibodies. For example, a library of naive human single chain variable fragments (scFv) can be used to select human anti-UCH-L1 antibodies. Transgenic animals can be used to express human antibodies.

[0181] A humanized antibody can be an antibody molecule derived from a non-human species antibody that binds to a desired antigen and has one or more complementarity determining regions (CDRs) from the non-human species and a framework region derived from a human immunoglobulin molecule.

[0182] The antibody is distinguishable from known antibodies in that it possesses a different biological function than antibodies known in the art.

[0183] (1) Epitope The antibody may immunospecifically bind to UCH-L1 (SEQ ID NO: 1), a fragment thereof, or a variant thereof. The antibody may immunospecifically recognize and bind to at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, or at least 10 amino acids within the epitope region. The antibody may immunospecifically recognize and bind to an epitope having at least 3 consecutive amino acids, at least 4 consecutive amino acids, at least 5 consecutive amino acids, at least 6 consecutive amino acids, at least 7 consecutive amino acids, at least 8 consecutive amino acids, at least 9 consecutive amino acids, or at least 10 consecutive amino acids within the epitope region.

[0184] c. Preparation / production of antibodies Antibodies may be prepared by any of a variety of techniques, including techniques well known to those of skill in the art. In general, antibodies may be produced by cell culture methods, including the production of monoclonal antibodies, via conventional techniques or by transfection of the antibody genes, heavy and / or light chains into a suitable bacterial or mammalian cell host to allow for the production of antibodies, which may be recombinant. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although antibodies can be expressed in prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells is preferred, and most preferred in mammalian host cells, since such eukaryotic cells (and particularly mammalian cells) are more likely than prokaryotic cells to assemble, properly fold, and secrete immunologically active antibodies.

[0185] Exemplary mammalian host cells for expressing recombinant antibodies include Chinese hamster ovary (CHO) cells (including dhfr-CHO cells described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)) used with a DHFR selectable marker as described, for example, in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982), NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a sufficient time to allow for expression of the antibody within the host cell or, more preferably, secretion of the antibody into the culture medium in which the host cell is grown. The antibody may be recovered from the culture medium using standard protein purification methods.

[0186] The host cells may also be used to generate functional antibody fragments, such as Fab fragments or scFv molecules. It is understood that variations on the above procedures may be implemented. For example, it may be desirable to transfect the host cells with DNA encoding functional fragments of the antibody light and / or heavy chains. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also encompassed by antibodies. In addition, bifunctional antibodies, in which one heavy and one light chain is an antibody (i.e., binds human UCH-L1) and the other heavy and light chains are specific for an antigen other than human UCH-L1, may also be generated by crosslinking the antibody to a second antibody via standard chemical crosslinking methods.

[0187] In a preferred system for recombinantly expressing an antibody or antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, each of the antibody heavy chain gene and the antibody light chain gene is operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene that allows for selection of CHO cells transfected with the vector using methotrexate selection / amplification. The host cells of the selected transformants are cultured to allow expression of the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology methods are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. Still further, a method for synthesizing a recombinant antibody is by culturing the host cells in an appropriate culture medium until the recombinant antibody is synthesized. The method may further comprise the step of isolating the recombinant antibody from the culture medium.

[0188] The method of preparing monoclonal antibodies involves the preparation of immortal cell lines capable of producing antibodies with the desired specificity. Such cell lines can be generated from spleen cells obtained from immunized animals. The animals can be immunized with UCH-L1 or fragments and / or variants thereof. The peptide used to immunize the animals can include amino acids encoding human Fc, e.g., the Fc (fragment crystallizable) region or tail region of a human antibody. The spleen cells are then immortalized, e.g., by fusion with a myeloma cell fusion partner. Various fusion techniques can be utilized. For example, spleen cells and myeloma cells are combined with a non-ionic detergent for several minutes and then plated at low density on a selective medium that supports the growth of hybrid cells but not myeloma cells. One such technique uses hypoxanthine, aminopterin, thymidine (HAT) selection. Another technique involves electrofusion. After a sufficient time, usually about 1-2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants are tested for binding activity to the polypeptide. Hybridomas with high reactivity and specificity can be used.

[0189] Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. In addition, various techniques can be utilized to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies can then be harvested from the ascites fluid or blood. Contaminants can be removed from the antibody by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. Affinity chromatography is an example of a method that can be used in the process of purifying the antibody.

[0190] The proteolytic enzyme papain preferentially cleaves IgG molecules into several fragments, two of which (F(ab) fragments) contain a covalent heterodimer, each of which contains an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules into several fragments, including the F(ab')2 fragment, which contains both antigen-binding sites.

[0191] Fv fragments can be generated by preferential proteolytic cleavage of IgM, and in rare cases, IgG or IgA immunoglobulin molecules. Fv fragments can be derived using recombinant methods. Fv fragments contain a non-covalent VH::VL heterodimer that contains an antigen-binding site that retains much of the antigen recognition and binding capabilities of a native antibody molecule.

[0192] Each antibody, antibody fragment or derivative may comprise a set of heavy and light chain complementarity determining regions ("CDRs") interposed between a set of heavy and light chain frameworks ("FRs") that provide support for the CDRs and define the spatial relationship of the CDRs to each other. The set of CDRs may contain three hypervariable regions, consisting of heavy chain V regions or light chain V regions.

[0193] Other suitable methods of making or isolating antibodies with the requisite specificity may be used, including, but not limited to, selection of recombinant antibodies from peptide or protein libraries (e.g., but not limited to, display libraries such as bacteriophage libraries, ribosomal libraries, oligonucleotide libraries, RNA libraries, cDNA libraries, yeast libraries, etc.) commercially available from a variety of commercial sources, such as Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid / Planegg, Del.), Biovation (Aberdeen, Scotland, UK), BioInvent (Lund, Sweden), using methods known in the art. See U.S. Patent Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; and 5,976,862. An alternative method relies on immunization of transgenic animals capable of generating a repertoire of human antibodies (e.g., SCID mice, Nguyen et al. (1997), Microbiol. Immunol., 41:901-907; Sandhu et al. (1996), Crit. Rev. Biotechnol., 16:95-118; Eren et al. (1998), Immunol., 93:154-161), as known in the art and / or described herein.Such techniques include ribosome display (Hanes et al. (1997), Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998), Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibody generation techniques (e.g., the selected lymphocyte antibody assay ("SLAM") (U.S. Patent No. 5,627,052; Wen et al. (1987) J. Immunol., 17:887-892; Babcook et al. (1996), Proc. Natl. Acad. Sci. USA, 93:7843-7848); gel microdroplets and flow cytometry (Powell et al. (1990), Biotechnol., 8:333-337; One Cell Systems (Cambridge, Mass.); Gray et al. (1995), J. Imm. Meth., 182:155-163; Kenny et al. (1995), Bio / Technol., 13:787-790); B cell selection (Steenbakkers et al. (1994), Molec. Biol. Reports, 19:125-134 (1994)).

[0194] Affinity matured antibodies can be produced by any one of a number of procedures known in the art, for example, Marks et al., BioTechnology, 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues has been described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-3319 (1995); Hawkins et al., J. Mol. Biol., 226:889-896 (1992). Selective mutagenesis positions and selective mutations at contact or hypermutation positions with activity enhancing amino acid residues have been described in U.S. Patent No. 6,914,128 B1.

[0195] Antibody variants can also be prepared using delivery of a polynucleotide encoding the antibody into a suitable host to produce a transgenic animal or mammal, such as a goat, cow, horse, sheep, etc., which produces such antibodies in their milk. These methods are known in the art and described, for example, in U.S. Patent Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362 and 5,304,489.

[0196] Antibody variants can also be prepared by delivering polynucleotides to produce transgenic plants and cultured plant cells (e.g., but not limited to, tobacco, corn, and duckweed) that produce such antibodies, specified portions, or variants in plant parts or cells cultured therefrom. For example, Cramer et al. (1999), Curr. Top. Microbiol. Immunol., 240:95-118, and references cited therein, describe the production of transgenic tobacco leaves that express large amounts of recombinant proteins, e.g., using inducible promoters. Transgenic corn has been used to express mammalian proteins at commercial production levels with biological activity equivalent to mammalian proteins produced in other recombinant systems or purified from natural sources. See, 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 quantities from transgenic plant seeds that contain antibody fragments, such as single chain antibodies (scFv), including tobacco seeds and potato tubers. See, e.g., Conrad et al. (1998), Plant Mol. Biol., 38:101-109 and references cited therein. Thus, antibodies can also be produced using transgenic plants according to known methods.

[0197] Derivatives of antibodies can be made, for example, by adding exogenous sequences to modify immunogenicity or to reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life or any other suitable characteristic. Generally, the non-human sequences of the variable and constant regions are replaced with human or other amino acids while maintaining some or all of the non-human or human CDR sequences.

[0198] A small antibody fragment may be a diabody having two antigen-binding sites, where the fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) (VH VL) on the same polypeptide chain. See, 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 complementary domains on another chain and create two antigen-binding sites. See also U.S. Pat. No. 6,632,926 by Chen et al., which is incorporated by reference in its entirety and which also discloses antibody mutants in which one or more amino acids are inserted into a hypervariable region of a parent antibody and which have a binding affinity for a target antigen that is at least about two-fold stronger than the binding affinity of the parent antibody for that antigen.

[0199] The antibody may be a linear antibody. Procedures for making linear antibodies are known in the art and are described in Zapata et al. (1995), Protein Eng., 8(10):1057-1062. Briefly, these antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific.

[0200] Antibodies can be recovered and purified from recombinant cell culture by known methods, including, but not limited to, Protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification.

[0201] It may be useful to detectably label the antibody. Methods for conjugating the antibody to these agents are known in the art. By way of example only, the antibody may be labeled with a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, or the like. Such labeled antibodies may be used for diagnostic methods in vivo or in an isolated test sample. Such labeled antibodies may be linked to a cytokine, to a ligand, to another antibody.Agents suitable for coupling to antibodies to achieve an antitumor effect include cytokines such as interleukin 2 (IL-2) and tumor necrosis factor (TNF); photosensitizers for use in photodynamic therapy, including aluminum (III) phthalocyanine tetrasulfonic acid, hematoporphyrin and phthalocyanine; iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), technetium-99m (99mTc), rhenium-186 (186R), and iodine-131 (131I). e) and rhenium-188 (188Re); antibiotics such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin; diphtheria toxin, pseudomonas exotoxin A, staphylococcal enterotoxin A, abrin A toxin, ricin A (deglycosylated ricin A and native ricin A), transforming growth factor-alpha toxin, cytotoxin derived from the Taiwan cobra (naja naja atra), and cytotoxin derived from the Taiwan cobra (naja naja atra). ribosome-inactivating proteins derived from plants, bacteria and fungi, such as restrictocin (a ribosome-inactivating protein produced by Aspergillus restrictus), saporin (a ribosome-inactivating protein derived from Saponaria officinalis) and RNase; tyrosine kinase inhibitors; ly207702 (a difluorinated purine nucleoside); liposomes containing anticystants (e.g., antisense oligonucleotides, plasmids encoding toxins, methotrexate, etc.) and other antibodies or antibody fragments such as F(ab).

[0202] The production of antibodies through hybridoma technology, selected lymphocyte antibody technique (SLAM), transgenic animals and the use of recombinant antibody libraries are described in more detail below.

[0203] (1) Anti-UCH-L1 monoclonal antibody using hybridoma technology Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma methods known in the art, including those taught in, for example, Harlow et al., Antibodies: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, NY, 1981). It is also noted that the term "monoclonal antibody" as used herein is not limited to antibodies produced via hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0204] The monoclonal antibodies produced by the method as well as the method of producing the antibodies may include a step of culturing hybridoma cells secreting the antibodies of the present disclosure, where the hybridomas are preferably produced by fusing spleen cells isolated from an animal, e.g., a rat or mouse, immunized with UCH-L1 with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones secreting antibodies capable of binding to the polypeptides of the present disclosure. Briefly, rats may be immunized with UCH-L1 antigen. In a preferred embodiment, the UCH-L1 antigen is administered with an adjuvant to stimulate the immune response. Such adjuvants include Freund's complete or incomplete adjuvant, RIBI (muramyl dipeptide) or ISCOM (immunostimulating complex). Such adjuvants may protect the polypeptide from rapid dispersion by encapsulating it in a local deposit, or they may contain substances which stimulate the host to secrete factors which are chemotactic for macrophages and other components of the immune system. If a polypeptide is being administered, the immunization schedule preferably involves two or more administrations of the polypeptide, spread out over a period of weeks, although a single administration of the polypeptide may also be used.

[0205] After immunization of an animal with a UCH-L1 antigen, antibodies and / or antibody-producing cells can be obtained from the animal. Anti-UCH-L1 antibody-containing serum can be obtained from the animal by bleeding or sacrificing the animal. The serum can be used as obtained from the animal, an immunoglobulin fraction can be obtained from the serum, or anti-UCH-L1 antibodies can be purified from the serum. The serum or immunoglobulins thus obtained are polyclonal and therefore have a range of heterogeneous characteristics.

[0206] Once an immune response is detected, for example, antibodies specific to the antigen UCH-L1 are detected in rat serum, the rat spleen is removed and spleen cells are isolated. The spleen cells are then fused to any suitable myeloma cells, for example, cells derived from SP20, a cell line available from American Type Culture Collection (ATCC, Manassas, Va., US), by well-known techniques. Hybridomas are selected and cloned by limiting dilution. Hybridoma clones are then assayed for cells secreting antibodies capable of binding to UCH-L1 by methods known in the art. Rats can be immunized with positive hybridoma clones to produce ascites fluid that generally contains high levels of antibodies.

[0207] In another embodiment, immortalized hybridomas producing antibodies can be prepared from the immunized animals. After immunization, the animals are sacrificed and 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 (non-secretory cell lines). After fusion and antibiotic selection, the hybridomas are screened using UCH-L1 or a portion thereof or cells expressing UCH-L1. In a preferred embodiment, initial screening is performed using an enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), preferably an ELISA. An example of ELISA screening is provided in PCT Publication No. 00 / 37504.

[0208] Hybridomas producing anti-UCH-L1 antibodies are selected, cloned, and further screened for desired characteristics, including robust hybridoma growth, high antibody production, and desired antibody characteristics. Hybridomas can be cultured and expanded in vivo in syngeneic animals, animals lacking an immune system, such as nude mice, or in in vitro cell cultures. Methods for selecting, cloning, and expanding hybridomas are well known to those skilled in the art.

[0209] In a preferred embodiment, the hybridomas are rat hybridomas. In another embodiment, the hybridomas are made in a non-human, non-rat species, such as mouse, sheep, pig, goat, cow or horse. In yet another preferred embodiment, the hybridomas are human hybridomas, in which a human non-secretory myeloma is fused with a human cell expressing an anti-UCH-L1 antibody.

[0210] Antibody fragments that recognize specific epitopes can be produced by known techniques. For example, the Fab and F(ab')2 fragments of the present disclosure can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (resulting in two identical Fab fragments) or pepsin (resulting in an F(ab')2 fragment). The F(ab')2 fragment of an IgG molecule retains the two antigen-binding sites of the larger ("parent") IgG molecule, including both light chains (containing the light chain variable and constant regions), the CH1 domain of the heavy chain, and the disulfide-forming hinge region of the parent IgG molecule. Thus, the F(ab')2 fragment is capable of cross-linking antigen molecules in the same way as the parent IgG molecule.

[0211] (2) Anti-UCH-L1 monoclonal antibody using SLAM In another aspect of the disclosure, recombinant antibodies are produced 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. 92 / 02551 and Babcook et al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996). In this method, 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 a single cell, e.g., a lymphocyte from any one of the immunized animals, secreting the antibody of interest is screened using an antigen-specific hemolytic plaque assay, which is used to identify a single cell that secretes an antibody with specificity for UCH-L1. After identifying the antibody-secreting cells of interest, cDNAs for the heavy and light chain variable regions are rescued from the cells by reverse transcriptase PCR (RT-PCR), and these variable regions are then expressed in the context of an appropriate immunoglobulin constant region (e.g., a human constant region) in a mammalian host cell, such as a COS or CHO cell. Host cells transfected with the amplified immunoglobulin sequences derived from in vivo selected lymphocytes may then undergo further analysis and selection in vitro, for example, by panning the transfected cells to isolate cells expressing antibodies against UCH-L1. The amplified immunoglobulin sequences may be further manipulated in vitro, such as by in vitro affinity maturation methods. See, e.g., PCT Publication Nos. 97 / 29131 and 00 / 56772.

[0212] (3) Anti-UCH-L1 monoclonal antibody using transgenic animals In another embodiment of the disclosure, the antibodies are produced by immunizing a non-human animal that contains a portion or all of the human immunoglobulin loci with the UCH-L1 antigen. In one embodiment, the non-human animal is a XENOMOUSE® transgenic mouse, an engineered mouse strain that contains large fragments of the human immunoglobulin loci and is deficient in the production of mouse antibodies. See, e.g., Green et al., Nature Genetics, 7:13-21 (1994), and U.S. Patent Nos. 5,916,771; 5,939,598; 5,985,615; 5,998,209; 6,075,181; 6,091,001; 6,114,598, and 6,130,364. See also PCT Publication Nos. 91 / 10741; PCT Publication No. 94 / 02602; PCT Publication No. 96 / 34096; PCT Publication No. 96 / 33735; PCT Publication No. 98 / 16654; PCT Publication No. 98 / 24893; PCT Publication No. 98 / 50433; PCT Publication No. 99 / 45031; PCT Publication No. 99 / 53049; PCT Publication No. 00 / 09560, and PCT Publication No. 00 / 37504. XENOMOUSE® transgenic mice produce an adult-like human repertoire of fully human antibodies and develop antigen-specific human monoclonal antibodies. The XENOMOUSE® transgenic mice contain approximately 80% of the human antibody repertoire by introducing megabase-sized, germline-configured YAC fragments of human heavy chain and x light chain loci. See Mendez et al., Nature Genetics, 15:146-156 (1997); Green and Jakobovits, J. Exp. Med., 188:483-495 (1998), the disclosures of which are incorporated by reference.

[0213] (4) Anti-UCH-L1 monoclonal antibody using a recombinant antibody library In vitro methods may also be used to generate the antibodies of the present disclosure, where antibody libraries are screened to identify antibodies with the desired UCH-L1 binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art, and are described, for example, in U.S. Pat. No. 5,223,409 (Ladner et al.); PCT Publication No. 92 / 18619 (Kang et al.); PCT Publication No. 91 / 17271 (Dower et al.); PCT Publication No. 92 / 20791 (Winter et al.); PCT Publication No. 92 / 15679 (Markland et al.); PCT Publication No. 93 / 01288 (Breitling et al.); PCT Publication No. 92 / 01047 (McCafferty et al.); PCT Publication No. 92 / 09690 (Garrard et al.); Fuchs et al., Bio / Technology, 9:1369-1372 (1991); Hay et al., Bio / Technology, 9:1369-1372 (1992 ... et al., Hum. Antibod. Hybrodomas, 3:81-85 (1992); Huse et al., Science, 246:1275-1281 (1989); McCafferty et al., Nature, 348:552-554 (1990); Griffiths et al., EMBOJ., 12:725-734 (1993); Hawkins et al., J. Mol. Biol. , 226:889-896 (1992); Clackson et al., Nature, 352:624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992); Garrard et al., Bio / Technology, 9:1373-1377 (1991); Hoogenboom et al., Nucl. Acids Res., 19:4133-4137 (1991); Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991); U.S. Patent Application Publication No. 2003 / 0186374 and PCT Publication No. 97 / 29131.

[0214] The recombinant antibody library may be derived from a subject immunized with UCH-L1 or a portion of UCH-L1. Alternatively, the recombinant antibody library may be derived from a naive subject, i.e., a subject not immunized with UCH-L1, such as a human antibody library derived from a human subject not immunized with human UCH-L1. The antibodies of the present disclosure are selected by screening the recombinant antibody library with peptides comprising human UCH-L1, thereby selecting antibodies that recognize UCH-L1. Methods for performing such screening and selection are well known in the art, such as those described in the references in the preceding paragraph. The antibodies of the present disclosure having a specific binding affinity for UCH-L1, and which are isolated from human UCH-L1 by a specific K off To select an antibody of the present disclosure, such as an antibody that dissociates with a desired K off Surface plasmon resonance techniques known in the art can be used to select antibodies with specific rate constants. 50 To select an antibody of the present disclosure, such as an antibody with, standard methods known in the art for assessing inhibition of UCH-L1 activity can be used.

[0215] In one aspect, the present disclosure relates to an isolated antibody or an antigen-binding portion thereof that binds to human UCH-L1. Preferably, the antibody is a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody.

[0216] For example, antibodies can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. Such phage can be used to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen-binding domain that binds to the antigen of interest can be selected or identified with the antigen, for example, using labeled antigen or antigen bound or captured to a solid surface or bead. The phage used in these methods are typically filamentous phage that contain phage-expressed fd and M13 binding domains, together with Fab, Fv or disulfide-stabilized Fv antibody domains recombinantly fused to phage gene III or phage gene VIII proteins.Examples of phage display methods which can be used to make antibodies include those described in Brinkmann et al., J. Immunol. Methods, 182:41-50 (1995); Ames et al., J. Immunol. Methods, 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic et al., Gene, 187:9-18 (1997); Burton et al., Advances in Immunology, 57:191-280 (1994); PCT Publication No. 92 / 01047; PCT Publication No. 90 / 02809; PCT Publication No. 91 / 10737; PCT Publication No. 92 / 01047; PCT Publication No. 92 / 18619; PCT Publication No. 93 / 11236; PCT Publication No. 95 / 15982; PCT Publication No. 95 / 20401 and U.S. Pat. Nos. 5,698,426 and 5,223,409. Nos. 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

[0217] After phage selection, as described in the above references, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, which can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described in detail below. Techniques can also be utilized to recombinantly generate, for example, Fab fragments, Fab' fragments, and F(ab')2 fragments, using methods known in the art, such as those disclosed in PCT Publication No. 92 / 22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); Sawai et al., Am. J. Reprod. Immunol., 34:26-34 (1995), and Better et al., Science, 240:1041-1043 (1988). Examples of techniques that can be used to make single chain Fv antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88 (1991); Shu et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999 (1993), and Skerra et al., Science, 240:1038-1041 (1988).

[0218] Other methods known in the art for screening large combinatorial libraries, which are alternatives to screening recombinant antibody libraries by phage display, can also be applied to identify the antibodies of the present disclosure. One type of alternative expression system is the expression system in which recombinant antibody libraries are expressed as RNA-protein fusions, as described in PCT Publication No. 98 / 31700 (Szostak and Roberts) and Roberts and Szostak, Proc. Natl. Acad. Sci. USA, 94:12297-12302 (1997). In this system, a covalent fusion is created between the mRNA and the peptide or protein it encodes, by in vitro translation of a synthetic mRNA that carries the peptidyl acceptor antibiotic puromycin at its 3' end. Thus, specific mRNAs can be enriched from a complex mixture of mRNAs (e.g., combinatorial libraries) based on the properties of the encoded peptides or proteins, e.g., antibodies or portions thereof, such as the binding of the antibodies or portions thereof to bispecific antigens. Nucleic acid sequences encoding antibodies or portions thereof recovered from screening of such libraries can be expressed by recombinant means as described above (e.g., in mammalian host cells) and subjected to further affinity maturation by further rounds of screening of mRNA-peptide fusions in which mutations are introduced into the originally selected sequences, or by other methods for affinity maturation of recombinant antibodies in vitro, as described above. A preferred example of this methodology is the PROfusion display technology.

[0219] In another approach, antibodies can also be made using yeast display methods known in the art. In yeast display methods, genetic methods are used to tether antibody domains to the yeast cell wall and display them on the surface of the yeast. In particular, such yeast can be used to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Examples of yeast display methods that can be used to make antibodies include the yeast display method disclosed in U.S. Patent No. 6,699,658 (Wittrup et al.), which is incorporated herein by reference.

[0220] d. Preparation of recombinant UCH-L1 antibody Antibodies may be produced by any of a number of techniques known in the art, such as expression from a host cell in which expression vectors encoding the heavy and light chains are transfected into the host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is possible to express the antibodies of the present disclosure in prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, and most preferably in mammalian host cells, is preferred, since such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells to assemble, properly fold, and secrete immunologically active antibodies.

[0221] Exemplary mammalian host cells for expressing recombinant antibodies of the disclosure include Chinese hamster ovary (CHO) cells (including dhfr-CHO cells, as described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), used with a DHFR selectable marker as described, for example, in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982), NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a sufficient time to allow for expression of the antibody within the host cell or, more preferably, secretion of the antibody into the culture medium in which the host cell is grown. The antibody may be recovered from the culture medium using standard protein purification methods.

[0222] The host cells may also be used to generate functional antibody fragments, such as Fab fragments or scFv molecules. It is understood that variations on the above procedures may be performed. For example, it may be desirable to transfect the host cells with DNA encoding functional fragments of the light and / or heavy chains of the antibodies of the present disclosure. 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 are not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the present disclosure. In addition, bifunctional antibodies, in which one heavy chain and one light chain is an antibody of the present disclosure (i.e., an antibody that binds human UCH-L1) and the other heavy and light chains are specific for an antigen other than human UCH-L1, may also be generated by crosslinking an antibody of the present disclosure to a second antibody via standard chemical crosslinking methods.

[0223] In a preferred system for recombinantly expressing an antibody of the present disclosure or an antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, each of the antibody heavy chain gene and the antibody light chain gene is operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene to allow for selection of CHO cells transfected with the vector using methotrexate selection / amplification. The host cells of the selected transformants are cultured to allow expression of the antibody heavy and light chains and for intact antibody to be recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. Still further, the present invention provides a method of synthesizing a recombinant antibody of the present disclosure by culturing the host cells of the present disclosure in an appropriate culture medium until the recombinant antibody of the present disclosure is synthesized. The method may further comprise the step of isolating the recombinant antibody from the culture medium.

[0224] (1) Humanized antibodies A humanized antibody may be an antibody, or a variant, derivative, analog or portion thereof, that immunospecifically binds to an antigen of interest and comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody. A humanized antibody may be derived from a non-human species antibody that binds to a desired antigen and has one or more complementarity determining regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule.

[0225] As used herein, the term "substantially" in the context of a CDR refers to a CDR that has an amino acid sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, but typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. According to one aspect, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically the Fc region of a human immunoglobulin. In some embodiments, the humanized antibody contains both a light chain and at least the variable domains of a heavy chain. The antibody may also comprise the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, a humanized antibody contains only a humanized light chain. In some embodiments, a humanized antibody contains only a humanized heavy chain. In specific embodiments, a humanized antibody contains only humanized variable domains of the light and / or heavy chain.

[0226] The humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including, without limitation, IgG1, IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions, using techniques well known in the art.

[0227] The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequences, for example, the donor antibody CDR or consensus framework may be mutagenized by substitution, insertion and / or deletion of at least one amino acid residue such that the CDR or framework residue at this site does not correspond to the donor antibody or consensus framework. However, in one embodiment, such mutations are not extensive mutations. Typically, at least 90%, at least 95%, at least 98% or at least 99% of the humanized antibody residues correspond to the residues of the parental FR and CDR sequences. The term "consensus framework" as used herein refers to a framework region in a consensus immunoglobulin sequence. The term "consensus immunoglobulin sequence" as used herein refers to a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, for example, Winnaker, "From Genes to Clones" (Verlagsgesellschaft, Weinheim, Germany, 1987)). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

[0228] Humanized antibodies can be designed to minimize undesired immunological responses to anti-human rodent antibodies that limit the duration and effectiveness of therapeutic applications of these moieties in human recipients. A humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human residues are often referred to as "import" residues, typically taken from a variable domain. Humanization can be performed by substituting hypervariable region sequences with the corresponding sequences of a human antibody. Such "humanized" antibodies are thus chimeric antibodies in which substantially less than intact human variable domains are substituted by the corresponding sequences from a non-human species. See, for example, U.S. Pat. No. 4,816,567, the contents of which are incorporated herein by reference. Humanized antibodies can be human antibodies in which some hypervariable region residues, and possibly some FR residues, are substituted by residues from analogous sites in rodent antibodies. Humanization or engineering of the antibodies of the present disclosure may be carried out using any known method, such as, but not limited to, those described in U.S. Pat. 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.

[0229] Humanized antibodies may retain high affinity for UCH-L1 and other favorable biological properties. Humanized antibodies may be prepared by analyzing parental sequences and various conceptual humanized products using three-dimensional models for the parental and humanized sequences. Three-dimensional immunoglobulin models are publicly available. Computer programs are available which illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these representations allows analysis of the residues for their key role in the functioning of the candidate immunoglobulin sequence, i.e., for residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues may be selected and combined from the recipient and import sequences such that the desired antibody characteristic, such as increased affinity for UCH-L1, is achieved. In general, hypervariable region residues may be directly and most substantially involved in influencing antigen binding.

[0230] As an alternative to humanization, human antibodies (also referred to herein as "fully human antibodies") can be produced. For example, human antibodies can be isolated from libraries via PROfusion and / or yeast-related technologies. It is also possible to produce transgenic animals (e.g., mice that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production). For example, the expression of the antibody heavy chain joining region (JC) in chimeric and germline mutant mice can be expressed in a variety of ways. HHomozygous deletion of the .) gene results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. Humanized or fully human antibodies may be prepared according to the methods described in U.S. Pat. Nos. 5,770,429; 5,833,985; 5,837,243; 5,922,845; 6,017,517; 6,096,311; 6,111,166; 6,270,765; 6,303,755; 6,365,116; 6,410,690; 6,682,928 and 6,984,720, the contents of each of which are incorporated herein by reference.

[0231] e. Anti-UCH-L1 antibody Anti-UCH-L1 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-L1 antibody is available from United State Biological (product number: 031320), Cell Signaling Technology (product number: 3524), Sigma-Aldrich (product number: HPA005993), Santa Cruz Biotechnology, Inc. (product number: sc-58593 or sc-58594), R&D Systems (product number: MAB6007), Novus Biologicals (product number: NB600-1160), Biobyt (product number: orb33715), Enzo Life Sciences, Inc. (product number: ADI-905-520-1), Bio-Rad (product number: VMA00004), BioVision (product number: 6130-50), Abcam (product number: ab75275 or ab104938), Invitrogen The anti-UCH-L1 antibody may be an unconjugated UCH-L1 antibody, such as the UCH-L1 antibody commercially available from ThermoFisher Scientific (Model No.: MA1-46079, MA5-17235, MA1-90008, or MA1-83428), EMD Millipore (Model No.: MABN48) or Sino Biological Inc. (Model No.: 50690-R011). The anti-UCH-L1 antibody may be conjugated to a fluorophore, such as the conjugated UCH-L1 antibody commercially available from BioVision (Model No.: 6960-25) or Aviva Systems Biology (Model No.: OAAF01904-FITC).

[0232] Alternatively, antibodies described in WO2018 / 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,” Acad. Emerg. Med. (August 6, 2021), the contents of which are incorporated herein by reference, may be used.

[0233] 8. Methods for Measuring GFAP Levels In the methods described above, GFAP levels can be measured by any means, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, antibody-dependent methods such as SDS-PAGE and Western blot analysis, or protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, or chromatographic methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS), such as those described in WO2018 / 067474, WO2018 / 191531, WO2018 / 218169, and WO2019 / 112860, the contents of each of which are incorporated herein by reference. The assay can also be used in clinical chemistry formats that would be known to those skilled in the art.

[0234] In some embodiments, measuring the level of GFAP comprises contacting the sample with a first specific binding member and a second specific binding member, hi some embodiments, the first specific binding member is a capture antibody and the second specific binding member is a detection antibody. In some embodiments, the step of measuring the level of GFAP includes contacting the sample simultaneously or sequentially, in any order, with (1) a capture antibody (e.g., a GFAP capture antibody) that binds to an epitope on GFAP or a GFAP fragment to form a capture antibody-GFAP antigen complex (e.g., a GFAP capture antibody-GFAP antigen complex), and (2) a detection antibody (e.g., a GFAP detection antibody) that contains a detectable label and binds to an epitope on GFAP to which the capture antibody is not bound to form a GFAP antigen-detection antibody complex (e.g., a GFAP antigen-GFAP detection antibody complex) to form a capture antibody-GFAP antigen-detection antibody complex (e.g., a GFAP capture antibody-GFAP antigen-GFAP detection antibody complex), and measuring the amount or concentration of GFAP in the sample based on a signal generated by the detectable label in the capture antibody-GFAP antigen-detection antibody complex.

[0235] 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.

[0236] In some embodiments, the sample is diluted or undiluted. In some embodiments, the sample is about 1 to about 30 microliters. In some embodiments, the sample is about 10 to about 30 microliters. In some embodiments, the sample is about 20 microliters. In some embodiments, the sample is 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, or about 15 microliters. In some embodiments, the sample is about 1 microliter, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, about 10 microliters, about 11 microliters, about 12 microliters, about 13 microliters, about 14 microliters, about 15 microliters, about 16 microliters, about 17 microliters, about 18 microliters, about 19 microliters, about 20 microliters, about 21 microliters, about 22 microliters, about 23 microliters, about 24 microliters, about 25 microliters, about 26 microliters, about 27 microliters, about 28 microliters, about 29 microliters, or about 30 microliters. In some embodiments, the sample is about 1 to about 150 microliters or less, or about 1 to about 30 microliters or less.

[0237] Some non-point-of-care meters (e.g., Abbott Laboratories meters ARCHITECT®, Alinity, and other core testing meters) may be capable of measuring GFAP levels in samples greater than 25,000 pg / mL.

[0238] Other detection methods can include or be adapted for use in nanopore or nanowell devices. Examples of nanopore devices are described in International Patent Publication No. WO 2016 / 161402, which is incorporated herein by reference in its entirety. Examples of nanowell devices are described in International Patent Publication No. WO 2016 / 161400, which is incorporated herein by reference in its entirety.

[0239] 9.GFAP antibody The methods described herein may use isolated antibodies that specifically bind to glial fibrillary acidic protein ("GFAP") (or fragments thereof), referred to as "GFAP antibodies." GFAP antibodies may be used to assess GFAP status as a measure of traumatic brain injury, to detect the presence of GFAP in a sample, to quantitate the amount of GFAP present in a sample, or to detect the presence of GFAP in a sample and quantitate the amount of GFAP in a sample.

[0240] Glial fibrillary acidic protein (GFAP) Glial fibrillary acidic protein (GFAP) is a 50 kDa cytoplasmic fibrillary protein that constitutes part of the cytoskeleton in astrocytes and has been demonstrated to be the most specific marker for cells of astrocyte origin. GFAP protein is encoded by the GFAP gene in humans. GFAP is the major intermediate fiber of mature astrocytes. In the central rod domain of the molecule, GFAP shares a large structural homology with other intermediate fibers. GFAP is involved in astrocyte motility and shape by providing structural stability to astrocyte processes. Glial fibrillary acidic protein and its degradation products (GFAP-BDP) are brain-specific proteins released into the blood as part of the pathophysiological response after traumatic brain injury (TBI). Following injury to the human CNS caused by trauma, genetic injury or chemicals, astrocytes proliferate and show extensive hypertrophy of cell bodies and cell processes, and GFAP is significantly upregulated. In contrast, there is a progressive loss of GFAP production in growing astrocytic malignancies.GFAP can also be detected in Schwann cells, gastrointestinal glial cells, salivary gland neoplasms, metastatic renal carcinomas, epiglottic cartilage, pituitary cells, immature oligodendrocytes, papillary meningiomas, and myoepithelial cells of the mammary gland.

[0241] Human GFAP may have the following amino acid sequence:

[0242] [ka]

[0243] The human GFAP may be a fragment or variant of SEQ ID NO: 2. The fragment of GFAP may be between 5 and 400 amino acids, between 10 and 400 amino acids, between 50 and 400 amino acids, between 60 and 400 amino acids, between 65 and 400 amino acids, between 100 and 400 amino acids, between 150 and 400 amino acids, between 100 and 300 amino acids or between 200 and 300 amino acids in length. The fragment may comprise consecutive amino acids from SEQ ID NO: 2. The fragment or variant of human GFAP of SEQ ID NO: 2 may be a GFAP degradation product (BDP). The GFAP BDP may be 38 kDa, 42 kDa (faint band: 41 kDa), 47 kDa (faint band: 45 kDa); 25 kDa (faint band: 23 kDa); 19 kDa or 20 kDa.

[0244] b.GFAP recognition antibody The antibody is an antibody that binds to GFAP, a fragment thereof, an epitope of GFAP or a variant thereof. The antibody may be a fragment of an anti-GFAP antibody or a variant or derivative thereof. The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, a fully human antibody or an antibody fragment such as a Fab fragment or a mixture thereof. The antibody fragment or derivative may include a F(ab')2 fragment, an Fv fragment or a scFv fragment. The antibody derivative may be produced by peptidomimetics. Furthermore, techniques described for producing single chain antibodies may be adapted to produce single chain antibodies.

[0245] The anti-GFAP antibody may be a chimeric or humanized anti-GFAP antibody. In one embodiment, both the humanized and chimeric antibodies are monovalent. In one embodiment, both the humanized and chimeric antibodies comprise a single Fab region linked to an Fc region.

[0246] Human antibodies can be derived from phage display technology or transgenic mice expressing human immunoglobulin genes. Human antibodies can be generated and isolated as a result of a human in vivo immune response. See, for example, Funaro et al., BMC Biotechnology, 2008(8):85. Thus, the antibody can be a product of a human and may not be an animal repertoire. Because the antibody is of human origin, the risk of reactivity against self-antigens can be minimized. Alternatively, standard yeast display libraries and yeast display technology can be used to select and isolate human anti-GFAP antibodies. For example, a library of naive human single chain variable fragments (scFv) can be used to select human anti-GFAP antibodies. Transgenic animals can be used to express human antibodies.

[0247] A humanized antibody can be an antibody molecule derived from a non-human species antibody that binds to a desired antigen and has one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.

[0248] The antibody is distinguishable from known antibodies in that it possesses a different biological function than antibodies known in the art.

[0249] (1) Epitope The antibody may immunospecifically bind to GFAP (SEQ ID NO: 2), a fragment or variant thereof. The antibody may immunospecifically recognize and bind to at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acids within the epitope region. The antibody may immunospecifically recognize and bind to an epitope having at least 3 consecutive amino acids, at least 4 consecutive amino acids, at least 5 consecutive amino acids, at least 6 consecutive amino acids, at least 7 consecutive amino acids, at least 8 consecutive amino acids, at least 9 consecutive amino acids or at least 10 consecutive amino acids within the epitope region.

[0250] c. Preparation / production of antibodies Antibodies may be prepared by any of a variety of techniques, including techniques well known to those of skill in the art. In general, antibodies may be produced by cell culture methods, including the production of monoclonal antibodies, via conventional techniques or by transfection of the antibody genes, heavy and / or light chains into a suitable bacterial or mammalian cell host to allow for the production of antibodies, which may be recombinant. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although antibodies can be expressed in prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells is preferred, and most preferred in mammalian host cells, since such eukaryotic cells (and, in particular, mammalian cells) are more likely to assemble, properly fold, and secrete immunologically active antibodies than prokaryotic cells.

[0251] Exemplary mammalian host cells for expressing recombinant antibodies include Chinese hamster ovary (CHO cells) used with a DHFR selectable marker as described, for example, in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982) (including dhfr-CHO cells described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a sufficient time to allow for expression of the antibody within the host cell or, more preferably, secretion of the antibody into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0252] The host cells may also be used to generate functional antibody fragments, such as Fab fragments or scFv molecules. It is understood that variations on the above procedures may be performed. For example, it may be desirable to transfect the host cells with DNA encoding functional fragments of the light and / or heavy chains of the antibody. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also encompassed by antibody. In addition, bifunctional antibodies, in which one heavy and one light chain is an antibody (i.e., binds human GFAP) and the other heavy and light chains are specific for an antigen other than human GFAP, may be generated by crosslinking the antibody to a second antibody via standard chemical crosslinking methods.

[0253] In a preferred system for recombinantly expressing an antibody or antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, each of the antibody heavy chain gene and the antibody light chain gene is operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene that allows for selection of CHO cells transfected with the vector using methotrexate selection / amplification. The host cells of the selected transformants are cultured to allow expression of the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology methods are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. Still further, a method for synthesizing a recombinant antibody is by culturing the host cells in an appropriate culture medium until the recombinant antibody is synthesized. The method may further include isolating the recombinant antibody from the culture medium.

[0254] Methods for preparing monoclonal antibodies include the preparation of immortal cell lines capable of producing antibodies with the desired specificity. Such cell lines can be made from spleen cells obtained from immunized animals. Animals can be immunized with GFAP or fragments and / or variants thereof. The peptide used to immunize the animals can include amino acids encoding human Fc, e.g., the fragment crystallizable region or tail region of a human antibody. The spleen cells can then be immortalized, e.g., by fusion with a myeloma cell fusion partner. Various fusion methods can be utilized. For example, spleen cells and myeloma cells can be combined with a non-ionic detergent for a few minutes and then plated at low density on a selection medium that supports the growth of hybrid cells but not myeloma cells. One such technique uses hypoxanthine, aminopterin, thymidine (HAT) selection. Another technique involves electrofusion. After a sufficient time, usually about 1-2 weeks, colonies of hybrids can be observed. Single colonies are selected and their culture supernatants are tested for binding activity to the polypeptide. Hybridomas with high reactivity and specificity can be used.

[0255] Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. In addition, various techniques can be utilized to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies can then be harvested from the ascites fluid or blood. Contaminants can be removed from the antibody by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. Affinity chromatography is an example of a method that can be used in the process of purifying the antibody.

[0256] The proteolytic enzyme papain preferentially cleaves IgG molecules into several fragments, two of which (F(ab) fragments) contain covalent heterodimers each containing an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules into several fragments, including the F(ab')2 fragment, which contains both antigen-binding sites.

[0257] Fv fragments can be generated by preferential proteolytic cleavage of IgM, and in rare cases, IgG or IgA immunoglobulin molecules. Fv fragments can be derived using recombinant methods. Fv fragments contain a non-covalent VH::VL heterodimer that contains an antigen-binding site that retains much of the antigen recognition and binding capabilities of a native antibody molecule.

[0258] Each antibody, antibody fragment or derivative may comprise a set of heavy and light chain complementarity determining regions ("CDRs") interposed between a set of heavy and light chain frameworks ("FRs") that provide support for the CDRs and define the spatial relationship of the CDRs to each other. The set of CDRs may contain three hypervariable regions, consisting of heavy chain V regions or light chain V regions.

[0259] Other suitable methods for making or isolating antibodies with the requisite specificity can be used, including, but not limited to, selection of recombinant antibodies from peptide or protein libraries (e.g., but not limited to, display libraries such as bacteriophage libraries, ribosomal libraries, oligonucleotide libraries, RNA libraries, cDNA libraries, yeast libraries, etc.) commercially available from a variety of commercial sources, such as Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid / Planegg, Del.), Biovation (Aberdeen, Scotland, UK), BioInvent (Lund, Sweden), using methods known in the art. See U.S. Patent Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; and 5,976,862. An alternative method relies on immunization of transgenic animals capable of generating a repertoire of human antibodies (e.g., SCID mice, Nguyen et al. (1997), Microbiol. Immunol., 41:901-907; Sandhu et al. (1996), Crit. Rev. Biotechnol., 16:95-118; Eren et al. (1998), Immunol., 93:154-161), as known in the art and / or described herein.Such techniques include ribosome display (Hanes et al. (1997), Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998), Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibody generation techniques (e.g., the selected lymphocyte antibody assay ("SLAM") (U.S. Patent No. 5,627,052; Wen et al. (1987) J. Immunol., 17:887-892; Babcook et al. (1996), Proc. Natl. Acad. Sci. USA, 93:7843-7848); gel microdroplets and flow cytometry (Powell et al. (1990), Biotechnol., 8:333-337; One Cell Systems (Cambridge, Mass); Gray et al. (1995), J. Imm. Meth., 182:155-163; Kenny et al. (1995), Bio / Technol., 13:787-790); B cell selection (Steenbakkers et al. (1994), Molec. Biol. Reports, 19:125-134 (1994)).

[0260] Affinity matured antibodies can be produced by any one of a number of procedures known in the art, for example, Marks et al., BioTechnology, 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues has been described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-3319 (1995); Hawkins et al., J. Mol. Biol., 226:889-896 (1992). Selective mutagenesis positions and selective mutations at contact or hypermutation positions with activity enhancing amino acid residues are described in U.S. Pat. No. 6,914,128.

[0261] Antibody variants can also be prepared using delivery of a polynucleotide encoding the antibody into a suitable host to produce a transgenic animal or mammal, such as a goat, cow, horse, sheep, etc., which produces such antibodies in their milk. These methods are known in the art and described, for example, in U.S. Patent Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362 and 5,304,489.

[0262] Antibody variants may also be prepared by delivering polynucleotides to produce transgenic plants and cultured plant cells (such as, but not limited to, tobacco, corn, and duckweed) that produce such antibodies, specified portions, or variants in plant parts or cells cultured therefrom. For example, Cramer et al. (1999), Curr. Top. Microbiol. Immunol., 240:95-118 and references cited therein, describe the production of transgenic tobacco leaves that express large amounts of recombinant proteins, for example, using inducible promoters. Transgenic corn has been used to express mammalian proteins at commercial production levels with biological activity equivalent to mammalian proteins produced in other recombinant systems or purified from natural sources. See, for example, Hood et al., Adv. Exp. Med. Biol. (1999), 464:127-147 and references cited therein. Antibody variants have also been produced in large quantities from transgenic plant seeds that contain antibody fragments, such as single chain antibodies (scFv), including tobacco seeds and potato tubers. See, e.g., Conrad et al. (1998), Plant Mol. Biol., 38:101-109 and references cited therein. Thus, antibodies can also be produced using transgenic plants according to known methods.

[0263] Derivatives of antibodies can be made, for example, by adding exogenous sequences to modify immunogenicity or to reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life or any other suitable characteristic. Generally, the non-human sequences of the variable and constant regions are replaced with human or other amino acids while maintaining some or all of the non-human or human CDR sequences.

[0264] A small antibody fragment may be a diabody having two antigen-binding sites, where the fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) (VH VL) on the same polypeptide chain. See, e.g., EP 404,097; WO 93 / 1161 and Hollinger et al. (1993), Proc. Natl. Acad. Sci. USA, 90:6444-6448. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. See also U.S. Pat. No. 6,632,926 by Chen et al., which is incorporated by reference in its entirety and which also discloses antibody mutants in which one or more amino acids are inserted into a hypervariable region of a parent antibody and which have a binding affinity for a target antigen that is at least about two-fold stronger than the binding affinity of the parent antibody for that antigen.

[0265] The antibody may be a linear antibody. Procedures for making linear antibodies are known in the art and are described in Zapata et al. (1995), Protein Eng., 8(10):1057-1062. Briefly, these antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0266] Antibodies can be recovered and purified from recombinant cell cultures by known methods, including, but not limited to, Protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification.

[0267] It may be useful to detectably label the antibody. Methods for conjugating the antibody to these agents are known in the art. By way of example only, the antibody may be labeled with a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, etc. Such labeled antibodies may be used for diagnostic methods in vivo or in an isolated test sample. Such labeled antibodies may be linked to a cytokine, a ligand, another antibody. Drugs suitable for coupling to antibodies to achieve an antitumor effect include cytokines such as interleukin 2 (IL-2) and tumor necrosis factor (TNF); photosensitizers for use in photodynamic therapy, including aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrin and phthalocyanine; radionuclides such as iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), technetium-99m (99mTc), rhenium-186 (186Re) and rhenium-188 (188Re); antibiotics such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin and carboplatin; diphtheria toxin, Pseudomonas aeruginosa exotoxin A, Staphylococcus aureus exotoxin A, Staphylococcus aureus exotoxin B, Staphylococcus aureus exotoxin C, Staphylococcus aureus exotoxin D, Staphylococcus aureus exotoxin E, Staphylococcus aureus exotoxin F ... bacterial, plant and other toxins such as ricin A, abrin A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-alpha toxin, cytotoxin derived from Taiwan cobra (Naja naja atra) and gelonin (a plant toxin); ribosome-inactivating proteins derived from plants, bacteria and fungi such as restrictocin (a ribosome-inactivating protein produced by Aspergillus restrictus), saporin (a ribosome-inactivating protein derived from Saponaria officinalis) and RNase; tyrosine kinase inhibitors; ly207702 (a difluorinated purine nucleoside); liposomes containing anticystants (e.g., antisense oligonucleotides, plasmids encoding toxins, methotrexate, etc.) and other antibodies or antibody fragments such as F(ab).

[0268] The production of antibodies through hybridoma technology, selected lymphocyte antibody technique (SLAM), transgenic animals and the use of recombinant antibody libraries are described in more detail below.

[0269] (1) Anti-GFAP monoclonal antibody using hybridoma technology Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma methods known in the art, including those taught in, for example, Harlow et al., Antibodies: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, NY, 1981). It is also noted that the term "monoclonal antibody" as used herein is not limited to antibodies produced via hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0270] The monoclonal antibodies produced by the method as well as the method of producing the antibodies may include a step of culturing hybridoma cells secreting the antibodies of the present disclosure, where the hybridomas are preferably produced by fusing spleen cells isolated from an animal, e.g., a rat or mouse, immunized with GFAP with myeloma cells, and then screening the hybridomas resulting from the fusion for hybridoma clones secreting antibodies capable of binding to the polypeptides of the present disclosure. Briefly, rats can be immunized with GFAP antigen. In a preferred embodiment, the GFAP antigen is administered with an adjuvant to stimulate the immune response. Such adjuvants include Freund's complete or incomplete adjuvant, RIBI (muramyl dipeptide) or ISCOM (immunostimulating complex). Such adjuvants may protect the polypeptide from rapid dispersion by encapsulating it in a localized deposit, or such adjuvants may contain substances which stimulate the host to secrete factors which are chemotactic for macrophages and other components of the immune system. If a polypeptide is being administered, the immunization schedule preferably involves two or more administrations of the polypeptide spread out over a period of weeks, although a single administration of the polypeptide may also be used.

[0271] After immunization of an animal with a GFAP antigen, antibodies and / or antibody-producing cells can be obtained from the animal. Anti-GFAP antibody-containing serum can be obtained from the animal by bleeding the animal or sacrificing the animal. The serum can be used as obtained from the animal, an immunoglobulin fraction can be obtained from the serum, or the anti-GFAP antibodies can be purified from the serum. The serum or immunoglobulins thus obtained are polyclonal and therefore have a heterogeneous set of characteristics.

[0272] Once an immune response is detected, for example, in rat serum, antibodies specific to the antigen GFAP are detected, the rat spleen is removed and spleen cells are isolated.The spleen cells are then fused to any suitable myeloma cells, for example, cells derived from SP20, a cell line available from American Type Culture Collection (ATCC, Manassas, Va., US), by well-known techniques.Hybridomas are selected and cloned by limiting dilution.The hybridoma clones are then assayed for cells secreting antibodies capable of binding to GFAP by methods known in the art.By immunizing rats with positive hybridoma clones, ascites fluid that generally contains high levels of antibodies can be produced.

[0273] In another embodiment, antibody-producing immortalized hybridomas can be prepared from the immunized animals. After immunization, the animals are sacrificed and 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 (non-secretory cell lines). After fusion and antibiotic selection, the hybridomas are screened using GFAP or a portion thereof or cells expressing GFAP. In a preferred embodiment, initial screening is performed using an enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), preferably an ELISA. An example of ELISA screening is provided in PCT Publication No. 00 / 37504.

[0274] Hybridomas producing anti-GFAP antibodies are selected, cloned, and further screened for desired characteristics, including robust hybridoma growth, high antibody production, and desired antibody characteristics. Hybridomas can be cultured and expanded in vivo in syngeneic animals, animals lacking an immune system, such as nude mice, or in in vitro cell culture. Methods for selecting, cloning, and expanding hybridomas are well known to those of skill in the art.

[0275] In a preferred embodiment, the hybridomas are rat hybridomas. In another embodiment, the hybridomas are made in a non-human, non-rat species, such as mouse, sheep, pig, goat, cow or horse. In yet another preferred embodiment, the hybridomas are human hybridomas, in which a human non-secretory myeloma has been fused with a human cell expressing an anti-GFAP antibody.

[0276] Antibody fragments that recognize specific epitopes can be produced by known techniques. For example, the Fab and F(ab')2 fragments of the present disclosure can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (resulting in two identical Fab fragments) or pepsin (resulting in an F(ab')2 fragment). The F(ab')2 fragment of an IgG molecule retains the two antigen-binding sites of the larger ("parent") IgG molecule, including both light chains (containing the light chain variable region and the light chain constant region), the CH1 domain of the heavy chain, and the disulfide-forming hinge region of the parent IgG molecule. Thus, the F(ab')2 fragment is still capable of cross-linking antigen molecules, just like the parent IgG molecule.

[0277] (2) Anti-GFAP monoclonal antibody using SLAM In another aspect of the present disclosure, recombinant antibodies are produced 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. 92 / 02551 and Babcook et al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996). In this method, single cells secreting the antibody of interest, e.g., lymphocytes from any one of the immunized animals, are screened using an antigen-specific hemolytic plaque assay in which the antigen, GFAP, a subunit of GFAP or a fragment thereof, is coupled to sheep red blood cells using a linker such as biotin and used to identify single cells secreting antibodies with specificity for GFAP. After identifying the antibody-secreting cells of interest, cDNAs for the heavy and light chain variable regions are rescued from the cells by reverse transcriptase PCR (RT-PCR), and these variable regions can then be expressed in the context of appropriate immunoglobulin constant regions (e.g., human constant regions) in mammalian host cells, such as COS or CHO cells. Host cells transfected with the amplified immunoglobulin sequences derived from in vivo selected lymphocytes can then be subjected to further analysis and selection in vitro, for example, by panning the transfected cells to isolate cells expressing antibodies against GFAP. The amplified immunoglobulin sequences can be further manipulated in vitro, such as by in vitro affinity maturation methods. See, e.g., PCT Publication Nos. 97 / 29131 and 00 / 56772.

[0278] (3) Anti-GFAP monoclonal antibody using transgenic animals In another embodiment of the disclosure, the antibodies are produced by immunizing a non-human animal that contains a portion or all of the human immunoglobulin loci with the GFAP antigen. In one embodiment, the non-human animal is a XENOMOUSE® transgenic mouse, an engineered mouse strain that contains large fragments of the human immunoglobulin loci and is deficient in the production of mouse antibodies. See, e.g., Green et al., Nature Genetics, 7:13-21 (1994), and U.S. Patent Nos. 5,916,771; 5,939,598; 5,985,615; 5,998,209; 6,075,181; 6,091,001; 6,114,598, and 6,130,364. See also PCT Publication Nos. 91 / 10741; PCT Publication No. 94 / 02602; PCT Publication No. 96 / 34096; PCT Publication No. 96 / 33735; PCT Publication No. 98 / 16654; PCT Publication No. 98 / 24893; PCT Publication No. 98 / 50433; PCT Publication No. 99 / 45031; PCT Publication No. 99 / 53049; PCT Publication No. 00 / 09560, and PCT Publication No. 00 / 37504. XENOMOUSE® transgenic mice produce an adult-like human repertoire of fully human antibodies and develop antigen-specific human monoclonal antibodies. The XENOMOUSE® transgenic mice contain approximately 80% of the human antibody repertoire by introducing megabase-sized, germline-configured YAC fragments of human heavy-chain and x-light-chain loci. See Mendez et al., Nature Genetics, 15:146-156 (1997); Green and Jakobovits, J. Exp. Med., 188:483-495 (1998), the disclosures of which are incorporated herein by reference.

[0279] (4) Anti-GFAP monoclonal antibody using a recombinant antibody library In vitro methods can also be used to generate the antibodies of the present disclosure, where antibody libraries are screened to identify antibodies with the desired GFAP binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art, and are described, for example, in U.S. Patent No. 5,223,409 (Ladner et al.); PCT Publication No. 92 / 18619 (Kang et al.); PCT Publication No. 91 / 17271 (Dower et al.); PCT Publication No. 92 / 20791 (Winter et al.); PCT Publication No. 92 / 15679 (Markland et al.); PCT Publication No. 93 / 01288 (Breitling et al.); PCT Publication No. 92 / 01047 (McCafferty et al.); PCT Publication No. 92 / 09690 (Garrard et al.); Fuchs et al., Bio / Technology, 9:1369-1372 (1991); Hay et al., Hum. Antibod. H. ybrodomas, 3:81-85 (1992); Huse et al., Science, 246:1275-1281 (1989); McCafferty et al., Nature, 348:552-554 (1990); Griffiths et al., EMBOJ., 12:725-734 (1993); Hawkins et al., J. Mol. Biol., 226:889-896 (1992); Clackson et al., Nature, 352:624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992); Garrard et al., Bio / Technology, 9:1373-1377 (1991); Hoogenboom et al., Nucl. Acids Res., 19:4133-4137 (1991); Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991); U.S. Patent Application Publication No. 2003 / 0186374 and PCT Publication No. 97 / 29131, the contents of each of which are incorporated herein by reference.

[0280] The recombinant antibody library may be derived from a subject immunized with GFAP or a portion of GFAP. Alternatively, the recombinant antibody library may be derived from a naive subject, i.e., a subject not immunized with GFAP, such as a human antibody library derived from a human subject not immunized with human GFAP. The antibodies of the present disclosure are selected by screening the recombinant antibody library with peptides comprising GFAP, thereby selecting antibodies that recognize GFAP. Methods for such screening and selection are well known in the art, such as those described in the references in the preceding paragraph. The antibodies of the present disclosure having a specific binding affinity for GFAP, and which are isolated from human GFAP by a specific K off Surface plasmon resonance techniques known in the art can be used to select antibodies, such as antibodies that dissociate with a desired K off Antibodies having a specific IC 50 To select antibodies, such as those having the following structure, standard methods known in the art for assessing inhibition of GFAP activity can be used.

[0281] In one aspect, the disclosure relates to an isolated antibody or antigen-binding portion thereof that binds to human GFAP. Preferably, the antibody is a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody.

[0282] For example, antibodies can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. Such phage can be used to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen-binding domain that binds to the antigen of interest can be selected or identified with the antigen, for example, using labeled antigen or antigen bound or captured to a solid surface or bead. The phage used in these methods are typically filamentous phage that contain phage-expressed fd and M13 binding domains, together with Fab, Fv or disulfide-stabilized Fv antibody domains recombinantly fused to phage gene III or phage gene VIII proteins.Examples of phage display methods that can be used to generate antibodies include those described in Brinkmann et al., J. Immunol. Methods, 182:41-50 (1995); Ames et al., J. Immunol. Methods, 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic et al., Gene, 187:9-18 (1997); Burton et al., Advances in Immunology, 57:191-280 (1994); PCT Publication No. 92 / 01047; PCT Publication No. 90 / 02809; PCT Publication No. 91 / 10737; PCT Publication No. 92 / 01047; PCT Publication No. 92 / 18619; PCT Publication No. 93 / 11236; PCT Publication No. 95 / 15982; PCT Publication No. 95 / 20401 and U.S. Pat. Nos. 5,698,426 and 5,223,409. Nos. 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

[0283] After phage selection, as described in the above references, the antibody coding regions from the phage are isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, which can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described in detail below. Techniques can also be used to recombinantly generate, for example, Fab fragments, Fab' fragments, and F(ab')2 fragments, using methods known in the art, such as those disclosed in PCT Publication No. 92 / 22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); Sawai et al., Am. J. Reprod. Immunol., 34:26-34 (1995), and Better et al., Science, 240:1041-1043 (1988). Examples of techniques that can be used to make single chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88 (1991); Shu et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999 (1993), and Skerra et al., Science, 240:1038-1041 (1988).

[0284] Other methods known in the art for screening large combinatorial libraries, which are alternatives to screening recombinant antibody libraries by phage display, can also be applied to identify the antibodies of the present disclosure. One type of alternative expression system is the expression system in which recombinant antibody libraries are expressed as RNA-protein fusions, as described in PCT Publication No. 98 / 31700 (Szostak and Roberts) and Roberts and Szostak, Proc. Natl. Acad. Sci. USA, 94:12297-12302 (1997). In this system, covalent fusions are created between the mRNA and the peptide or protein it encodes by in vitro translation of synthetic mRNA carrying the peptidyl acceptor antibiotic puromycin at its 3' end. Thus, specific mRNAs can be enriched from a complex mixture of mRNAs (e.g., combinatorial libraries) based on the properties of the encoded peptides or proteins, e.g., antibodies or portions thereof, such as the binding of the antibodies or portions thereof to bispecific antigens. Nucleic acid sequences encoding antibodies or portions thereof recovered from screening of such libraries can be expressed by recombinant means (e.g., in mammalian host cells) as described above and subjected to further affinity maturation by further rounds of screening of mRNA-peptide fusions in which mutations are introduced into the originally selected sequences, or by other methods for affinity maturation of recombinant antibodies in vitro as described above. A preferred example of this methodology is the PROfusion display technology.

[0285] In another approach, antibodies can also be made using yeast display methods known in the art. In yeast display methods, genetic techniques are used to tether antibody domains to the yeast cell wall and display them on the surface of the yeast. In particular, such yeast can be used to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Examples of yeast display methods that can be used to make antibodies include the yeast display method disclosed in U.S. Patent No. 6,699,658 (Wittrup et al.), which is incorporated herein by reference.

[0286] d. Generation of recombinant GFAP antibodies Antibodies may be produced by any of a number of techniques known in the art, such as expression from a host cell in which expression vectors encoding the heavy and light chains have been transfected into the host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is possible to express the antibodies of the present disclosure in prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, and most preferably in mammalian host cells, is preferred, since such eukaryotic cells (and particularly mammalian cells) are more likely than prokaryotic cells to assemble, properly fold, and secrete immunologically active antibodies.

[0287] Exemplary mammalian host cells for expressing recombinant antibodies of the disclosure include Chinese hamster ovary (CHO cells) used with a DHFR selectable marker as described, for example, in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a sufficient time to allow for expression of the antibody within the host cell or, more preferably, secretion of the antibody into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0288] The host cells can also be used to generate functional antibody fragments, such as Fab fragments or scFv molecules. It is understood that variations on the above procedures can be performed. For example, it may be desirable to transfect the host cells with DNA encoding functional fragments of the light and / or heavy chains of the antibodies of the present disclosure. Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the present disclosure. In addition, bifunctional antibodies in which one heavy and one light chain is an antibody of the present disclosure (i.e., binds human GFAP) and the other heavy and light chains are specific for an antigen other than human GFAP can be generated by crosslinking the antibody of the present disclosure to a second antibody via standard chemical crosslinking methods.

[0289] In a preferred system for recombinantly expressing an antibody of the present disclosure or an antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, each of the antibody heavy chain gene and the antibody light chain gene is operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene to allow for selection of CHO cells transfected with the vector using methotrexate selection / amplification. The host cells of the selected transformants are cultured to allow expression of the antibody heavy and light chains and for intact antibody to be recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. Still further, the present invention provides a method of synthesizing a recombinant antibody of the present disclosure by culturing the host cells of the present disclosure in an appropriate culture medium until the recombinant antibody of the present disclosure is synthesized. The method may further comprise the step of isolating the recombinant antibody from the culture medium.

[0290] (1) Humanized antibodies A humanized antibody may be an antibody, or a variant, derivative, analog or portion thereof, that immunospecifically binds to an antigen of interest and comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody. A humanized antibody may be derived from a non-human species antibody that binds to a desired antigen and has one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.

[0291] As used herein, the term "substantially" in the context of CDRs refers to a CDR having an amino acid sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, but typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are framework regions of a human immunoglobulin consensus sequence. According to one aspect, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically the Fc region of a human immunoglobulin. In some embodiments, the humanized antibody contains both a light chain and at least the variable domains of a heavy chain. The antibody may also comprise the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, a humanized antibody contains only a humanized light chain. In some embodiments, a humanized antibody contains only a humanized heavy chain. In certain embodiments, a humanized antibody contains only humanized variable domains of the light chain and / or the heavy chain.

[0292] The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including, without limitation, IgG1, IgG2, IgG3, and IgG4. The humanized antibody can comprise sequences from more than one class or isotype, and particular constant domains can be selected to optimize desired effector functions, using techniques well known in the art.

[0293] The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequences, for example, the donor antibody CDR or consensus framework may be mutagenized by substitution, insertion and / or deletion of at least one amino acid residue such that the CDR or framework residue at this site does not correspond to the donor antibody or consensus framework. However, in one embodiment, such mutations are not extensive mutations. Typically, at least 90%, at least 95%, at least 98% or at least 99% of the humanized antibody residues correspond to the residues of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to a framework region in a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, for example, Winnaker, "From Genes to Clones" (Verlagsgesellschaft, Weinheim, Germany, 1987)). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

[0294] Humanized antibodies can be designed to minimize undesired immunological responses to anti-human rodent antibodies that limit the duration and effectiveness of therapeutic applications of these moieties in human recipients. Humanized antibodies can have one or more amino acid residues introduced into them from a source that is non-human. These non-human residues are often referred to as "import" residues, typically taken from the variable domain. Humanization can be performed by substituting hypervariable region sequences with the corresponding sequences of a human antibody. Such "humanized" antibodies are thus chimeric antibodies in which substantially less than intact human variable domains are substituted by the corresponding sequences from a non-human species. See, e.g., U.S. Pat. No. 4,816,567, the contents of which are incorporated herein by reference. Humanized antibodies can be human antibodies in which some hypervariable region residues, and possibly some FR residues, are substituted by residues from analogous sites in rodent antibodies. Humanization or engineering of the antibodies of the present disclosure can be carried out using any known method, such as, but not limited to, those methods described in U.S. Pat. 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.

[0295] The humanized antibody may retain high affinity for GFAP and other favorable biological properties. The humanized antibody may be prepared by analyzing the parental sequences and various conceptual humanized products using three-dimensional models for the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available. Computer programs are available which illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for GFAP, is achieved. In general, the hypervariable region residues may be directly and most substantially involved in influencing antigen binding.

[0296] As an alternative to humanization, human antibodies (also referred to herein as "fully human antibodies") can be produced. For example, human antibodies can be isolated from libraries via PROfusion and / or yeast-related technologies. It is also possible to produce transgenic animals (e.g., mice that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production). For example, the expression of the antibody heavy chain joining region (JC) in chimeric and germline mutant mice can be expressed in a variety of ways. HHomozygous deletion of the .ALPHA.-Asp.I.) gene results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies upon antigen challenge. Humanized or fully human antibodies can be prepared according to the methods described in U.S. Patent Nos. 5,770,429; 5,833,985; 5,837,243; 5,922,845; 6,017,517; 6,096,311; 6,111,166; 6,270,765; 6,303,755; 6,365,116; 6,410,690; 6,682,928 and 6,984,720. The contents of each of the above patents are incorporated herein by reference.

[0297] e. Anti-GFAP antibody Anti-GFAP antibodies may be made using the techniques described above as well as using routine techniques known in the art. In some embodiments, the anti-GFAP antibody is from Dako (product no. M0761), ThermoFisher Scientific (product no. MA5-12023, A-21282, 13-0300, MA1-19170, MA1-19395, MA5-15086, MA5-16367, MA1-35377, MA1-06701, or MA1-20035), AbCam (product no. ab10062, ab4648, ab68428, ab33922, ab207165, ab190288, ab115898, or ab21837), EMD Millipore (product no. FCMAB257P, MAB360, MAB3402, 04-1031, 04-1062, MAB5628), Santa The antibody may be an unconjugated GFAP antibody, such as a GFAP antibody commercially available from Cruz (Model No.: sc-166481, sc-166458, sc-58766, sc-56395, sc-51908, sc-135921, sc-71143, sc-65343 or sc-33673), Sigma-Aldrich (Model No.: G3893 or G6171) or Sino Biological Inc. (Model No.: 100140-R012-50). The anti-GFAP antibody may be conjugated to a fluorophore, such as conjugated GFAP antibodies commercially available from ThermoFisher Scientific (cat. no.: A-21295 or A-21294), EMD Millipore (cat. no.: MAB3402X, MAB3402B, MAB3402B or MAB3402C3) or AbCam (cat. no.: ab49874 or ab194325).

[0298] Alternatively, antibodies described in WO2018 / 067474 and / or Bazarian et al., “Accuracy of a rapid GFAP / UCH-L1 test for the prediction of intracranial injuries on head CT after mild traumatic brain injury,” Acad. Emerg. Med. (August 6, 2021), the contents of which are incorporated herein by reference, may be used.

[0299] 10. Method Variations The disclosed method of determining the presence or amount of an analyte of interest (UCH-L1 and / or GFAP) present in a sample may be as described above. The method may also be adapted with other methods for analyzing the analyte in mind. Examples of well-known variations include, but are not limited to, sandwich immunoassays (e.g., monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonal sandwich immunoassays), competitive inhibition immunoassays (e.g., forward competitive inhibition immunoassays and reverse competitive inhibition immunoassays), enzyme multiplexed immunoassay method (EMIT), competitive binding assays, bioluminescence energy transfer (BRET), one-step antibody detection assays, homogeneous assays, heterogeneous assays, capture-on-the-fly assays, and the like, including enzyme detection (enzyme immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISA)).

[0300] Immunoassay Analytes and / or peptides or fragments thereof of interest (e.g., UCH-L1 and / or GFAP and / or peptides or fragments thereof, i.e., UCH-L1 and / or GFAP fragments) can be analyzed using UCH...

Claims

1. a. performing at least one assay for ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and at least one assay for glial fibrillary acidic protein (GFAP) in at least one sample obtained from a human subject, wherein the sample is obtained from the subject within about 48 hours after an actual or suspected head injury; b.

1. A subject's levels of GFAP and UCH-L1 are elevated if (i) the level of GFAP in the sample is equal to or greater than about 35 pg / mL and the level of UCH-L1 in the sample is less than, cannot be determined, or is not reported; (ii) the level of GFAP in the sample is equal to or greater than about 35 pg / mL and the level of UCH-L1 in the sample is equal to or greater than about 400 pg / mL; or (iii) the level of GFAP in the sample is not determinable or is not reported and the level of UCH-L1 in the sample is equal to or greater than about 400 pg / mL; 2. If the level of GFAP in the sample is below about 35 pg / mL and the level of UCH-L1 in the sample is below about 400 pg / mL, the subject's levels of GFAP and UCH-L1 are not elevated; or 3. If (i) the level of GFAP in the sample is below about 35 pg / mL and the level of UCH-L1 in the sample cannot be determined or is not reported; (ii) the level of GFAP in the sample is not determined or is not reported and the level of UCH-L1 in the sample is below about 400 pg / mL; or (iii) the level of GFAP in the sample is not determined or is not reported and the level of UCH-L1 in the sample cannot be determined or is not reported, the assays for UCH-L1 and GFAP should be repeated. determining c. Communicating the determination from steps b(1)-(3) on or from at least one device, wherein the device is a non-point-of-care device. A method comprising:

2. If the subject's levels of GFAP and UCH-L1 are elevated, the subject should undergo a head computed tomography (CT) scan, a magnetic resonance imaging (MRI) procedure, or both a CT scan and an MRI procedure; or that if the subject's levels of GFAP and UCH-L1 are not elevated, then the subject should not undergo a head CT scan or MRI procedure; The method of claim 1 , wherein:

3. 2. The method of claim 1, wherein a level of GFAP equal to or greater than about 35 pg / mL and a level of UCH-L1 equal to or greater than about 400 pg / mL indicates that the subject should be diagnosed with traumatic brain injury (TBI), regardless of whether a head CT scan is negative for TBI or whether any head CT scan is performed.

4. The method of claim 1, wherein elevated levels of GFAP and UCH-L1 in a subject indicate that the subject should be treated for mild, moderate, moderate to severe, or severe TBI.

5. The method of claim 1, wherein if the subject's levels of GFAP and UCH-L1 are elevated, it is indicated that the subject should be monitored.

6. The sample is collected within about 5 minutes, within about 10 minutes, within about 12 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 60 minutes, within about 90 minutes, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 24 hours, within about 25 minutes, within about 26 hours, within about 27 hours, within about 28 hours, within about 29 hours, within about 30 minutes, within about 31 minutes, within about 32 minutes, within about 33 minutes, within about 34 hours, within about 35 minutes, within about 36 hours, within about 37 hours, within about 38 hours, within about 39 hours, within about 40 minutes, within about 41 minutes, within about 42 hours, within about 43 hours, within about 44 hours, within about 45 minutes, within about 46 hours, within about 47 hours, within about 48 hours, within about 49 hours, within about 50 minutes, within about 51 hours, within about 52 hours, within about 53 hours, within about 54 hours, within about 55 hours, within about 56 hours, within about 57 hours, within about 58 hours, within about 59 hours, within about 60 minutes, within about 61 hours, within about 62 hours, within about 63 hours, within about 64 hours, within about 65 hours, within about 66 hours, within about 67 hours, within about 68 hours, within about 69 2. The method of claim 1, wherein the sample is collected within 2 hours, within about 23 hours, within about 24 hours, within about 25 hours, within about 26 hours, within about 27 hours, within about 28 hours, within about 29 hours, within about 30 hours, within about 31 hours, within about 32 hours, within about 33 hours, within about 34 hours, within about 35 hours, within about 36 hours, within about 37 hours, within about 38 hours, within about 39 hours, within about 40 hours, within about 41 hours, within about 42 hours, within about 43 hours, within about 44 hours, within about 45 hours, within about 46 hours, within about 47 hours, or within about 48 hours.

7. 2. The method of claim 1, wherein at least one assay for UCH-L1 and at least one assay for GFAP are performed simultaneously or sequentially, in any order.

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

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

10. 10. The method of claim 9, wherein the chemical or toxin is fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, adhesives, gases, organometallics, addictive drugs, or a combination of one or more thereof.

11. 10. The method of claim 1, wherein the sample is obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection, a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof.

12. 10. The method of claim 1, wherein the assay is an immunoassay or a clinical chemistry assay.

13. The method of claim 1 , wherein the assay is a single molecule detection assay.

14. 10. The method of claim 1, wherein the volume of at least one sample is from about 10 μL to about 30 μL.

15. 15. The method of claim 14, wherein the volume of at least one sample is about 20 μL.

16. 2. The method of claim 1, wherein at least one assay for UCH-L1, at least one assay for GFAP, or at least one assay for UCH-L1 and at least one assay for GFAP is performed in about 10 minutes to about 20 minutes.

17. 17. The method of claim 16, wherein at least one assay for UCH-L1, at least one assay for GFAP, or at least one assay for UCH-L1 and at least one assay for GFAP is performed in about 15 minutes.

18. 10. The method of claim 1, wherein the subject has suffered an orthopedic injury and an actual or suspected injury to the head.

19. 10. The method of claim 1, wherein the sample is selected from the group consisting of a whole blood sample, a serum sample, and a plasma sample.

20. Assays for ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and assays for glial fibrillary acidic protein (GFAP); and Non-point-of-care device for performing an assay for UCH-L1 and an assay for GFAP - Patent application Including, the device determines the amount of UCH-L1 and GFAP in a sample obtained from the subject; The amounts of UCH-L1 and GFAP determined in the samples were: a. Elevated if (i) the level of GFAP in the sample is equal to or greater than about 35 pg / mL and the level of UCH-L1 in the sample is less than, cannot be determined, or is not reported as being about 400 pg / mL; (ii) the level of GFAP in the sample is equal to or greater than about 35 pg / mL and the level of UCH-L1 in the sample is equal to or greater than about 400 pg / mL; or (iii) the level of GFAP in the sample is not determinable or is not reported as being about 400 pg / mL and the level of UCH-L1 in the sample is equal to or greater than about 400 pg / mL; b. Not elevated if the level of GFAP in the sample is below about 35 pg / mL and the level of UCH-L1 in the sample is below about 400 pg / mL; or c. (i) the level of GFAP in the sample is below about 35 pg / mL and the level of UCH-L1 in the sample cannot be determined or is not reported; (ii) the level of GFAP in the sample is not determined or is not reported and the level of UCH-L1 in the sample is below about 400 pg / mL; or (iii) the level of GFAP in the sample is not determined or is not reported and the level of UCH-L1 in the sample cannot be determined or is not reported, the assays for UCH-L1 and GFAP need to be repeated. transmitted on or from the device as system.

21. The sample is taken within about 5 minutes, within about 10 minutes, within about 12 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 60 minutes, within about 90 minutes, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, 21. The system of claim 20, wherein the sample is collected within about 23 hours, within about 24 hours, within about 25 hours, within about 26 hours, within about 27 hours, within about 28 hours, within about 29 hours, within about 30 hours, within about 31 hours, within about 32 hours, within about 33 hours, within about 34 hours, within about 35 hours, within about 36 hours, within about 37 hours, within about 38 hours, within about 39 hours, within about 40 hours, within about 41 hours, within about 42 hours, within about 43 hours, within about 44 hours, within about 45 hours, within about 46 hours, within about 47 hours, or within about 48 hours.

22. The system of claim 20, wherein the assay for UCH-L1 and the assay for GFAP are performed simultaneously or sequentially in any order.

23. 21. The system of claim 20, wherein the sample is obtained after the subject has experienced physical shaking, blunt impact from an external mechanical or other force resulting in closed or open head injury, one or more falls, head injury caused by an explosion or blast, or other type of blunt force trauma.

24. 21. The system of claim 20, wherein the sample is obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin.

25. 25. The system of claim 24, wherein the chemical or toxin is fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, adhesives, gases, organometallics, addictive drugs, or a combination of one or more thereof.

26. 21. The system of claim 20, wherein the sample is obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection, a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof.

27. 21. The system of claim 20, wherein the assay is an immunoassay or a clinical chemistry assay.

28. 21. The system of claim 20, wherein the assay is a single molecule detection assay.

29. 21. The system of claim 20, wherein the volume of at least one sample is between about 10 μL and about 30 μL.

30. 30. The system of claim 29, wherein the volume of at least one sample is about 20 μL.

31. 21. The system of claim 20, wherein at least one assay for UCH-L1, at least one assay for GFAP, or at least one assay for UCH-L1 and at least one assay for GFAP is performed in about 10 minutes to about 20 minutes.

32. 32. The system of claim 31, wherein the assay for UCH-L1, the assay for GFAP, or at least one assay for UCH-L1 and GFAP is performed in about 15 minutes.

33. 21. The system of claim 20, wherein the subject has suffered an orthopedic injury and an actual or suspected injury to the head.

34. 21. The system of claim 20, wherein the sample is selected from the group consisting of a whole blood sample, a serum sample, and a plasma sample.

35. a. performing at least one assay for ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), at least one assay for glial fibrillary acidic protein (GFAP), or at least one assay for UCH-L1 and at least one assay for GFAP in at least one sample obtained from a human subject, wherein the sample is obtained from the subject within about 48 hours after an actual or suspected head injury; b.

1. A subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated if (i) the level of GFAP alone in the sample is equal to or greater than about 35 pg / mL; (ii) the level of GFAP alone in the sample is equal to or greater than about 35 pg / mL and the level of UCH-L1 alone in the sample is less than, cannot be determined, or is not reported as being about 400 pg / mL; (iii) the level of GFAP alone in the sample is equal to or greater than about 400 pg / mL and the level of UCH-L1 alone in the sample is equal to or greater than about 400 pg / mL; (iv) the level of GFAP alone in the sample is equal to or greater than about 400 pg / mL; or (v) the level of GFAP alone in the sample is not determinable or not reported as being about 400 pg / mL and the level of UCH-L1 alone in the sample is equal to or greater than about 400 pg / mL; 2. (i) the subject's level of GFAP is not elevated if only GFAP in the sample is below about 35 pg / mL; (ii) UCH-L1 is not elevated if only UCH-L1 in the sample is below about 400 pg / mL; or (iii) GFAP and UCH-L1 are not elevated if the level of GFAP in the sample is below about 35 pg / mL and the level of UCH-L1 in the sample is below about 400 pg / mL; or 3. If (i) only the level of UCH-L1 in the sample cannot be determined or is not reported; (ii) the level of GFAP in the sample is below about 35 pg / mL and the level of UCH-L1 in the sample is not determined or is not reported; (iii) only the level of GFAP in the sample is not determined or is not reported; (iv) the level of GFAP in the sample is not determined or is not reported and the level of UCH-L1 in the sample is below about 400 pg / mL; or (v) the level of GFAP in the sample is not determined or is not reported and the level of UCH-L1 in the sample is not determined or is not reported, the assays for UCH-L1 and GFAP should be repeated. determining that: c. Communicating the determination from steps b(1)-(3) on or from at least one device, where the device is a non-point-of-care device. A method comprising:

36. If the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated, then the subject should undergo a head computed tomography (CT) scan, a magnetic resonance imaging (MRI) procedure, or both a CT scan and an MRI procedure; or If the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are not elevated, then neither a head CT scan nor an MRI procedure should be performed on the subject. The method of claim 35, wherein:

37. 36. The method of claim 35, wherein a level of GFAP equal to or greater than about 35 pg / mL, a level of UCH-L1 equal to or greater than about 400 pg / mL, or a level of GFAP equal to or greater than about 35 pg / mL and a level of UCH-L1 equal to or greater than about 400 pg / mL indicates that the subject should be diagnosed with traumatic brain injury (TBI), regardless of whether a head CT scan is negative for TBI or whether any head CT scan is performed.

38. The method of claim 35, wherein elevated levels of GFAP, UCH-L1, or GFAP and UCH-L1 in a subject indicate that the subject should be treated for mild, moderate, moderate to severe, or severe TBI.

39. The method of claim 35, wherein if the subject's levels of GFAP, UCH-L1, or GFAP and UCH-L1 are elevated, it is indicated that the subject should be monitored.

40. The sample is taken within about 5 minutes, within about 10 minutes, within about 12 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 60 minutes, within about 90 minutes, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 24 hours, within about 26 hours, within about 28 hours, within about 30 minutes, within about 31 minutes, within about 32 minutes, within about 33 minutes, within about 34 hours, within about 35 minutes, within about 36 hours, within about 37 hours, within about 38 hours, within about 39 hours, within about 40 minutes, within about 41 minutes, within about 42 minutes, within about 44 hours, within about 45 minutes, within about 46 hours, within about 47 hours, within about 48 hours, within about 49 hours, within about 50 minutes, within about 51 minutes, within about 52 hours, within about 53 minutes, within about 54 hours, within about 55 minutes, within about 56 hours, within about 57 hours, within about 58 hours, within about 59 hours, within about 60 minutes, within about 61 minutes, within about 62 hours, within about 63 hours, within about 64 hours, within about 65 minutes, within about 66 hours, within about 67 hours, within about 68 hours, within about 69 hours, within about 70 minutes, within about 71 hours, within about 72 36. The method of claim 35, wherein the sample is collected within about 23 hours, within about 24 hours, within about 25 hours, within about 26 hours, within about 27 hours, within about 28 hours, within about 29 hours, within about 30 hours, within about 31 hours, within about 32 hours, within about 33 hours, within about 34 hours, within about 35 hours, within about 36 hours, within about 37 hours, within about 38 hours, within about 39 hours, within about 40 hours, within about 41 hours, within about 42 hours, within about 43 hours, within about 44 hours, within about 45 hours, within about 46 hours, within about 47 hours, or within about 48 hours.

41. 36. The method of claim 35, wherein at least one assay for UCH-L1 and at least one assay for GFAP are performed simultaneously or sequentially, in any order.

42. 36. The method of claim 35, wherein the sample is obtained after the subject has been subjected to physical shaking, blunt impact from an external mechanical or other force resulting in closed or open head injury, head injury caused by one or more falls, explosion or blast, or other type of blunt force trauma.

43. 36. The method of claim 35, wherein the sample is obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin.

44. 44. The method of claim 43, wherein the chemical or toxin is fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, adhesives, gases, organometallics, addictive drugs, or a combination of one or more thereof.

45. 36. The method of claim 35, wherein the sample is obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection, a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof.

46. 36. The method of claim 35, wherein the assay is an immunoassay or a clinical chemistry assay.

47. 36. The method of claim 35, wherein the assay is a single molecule detection assay.

48. 36. The method of claim 35, wherein the volume of at least one sample is from about 10 μL to about 30 μL.

49. 49. The method of claim 48, wherein the volume of at least one sample is about 20 μL.

50. 36. The method of claim 35, wherein the at least one assay for UCH-L1, the at least one assay for GFAP, or the at least one assay for UCH-L1 and the at least one assay for GFAP are performed in about 10 minutes to about 20 minutes.

51. 51. The method of claim 50, wherein at least one assay for UCH-L1, at least one assay for GFAP, or at least one assay for UCH-L1 and at least one assay for GFAP is performed in about 15 minutes.

52. 36. The method of claim 35, wherein the subject has suffered an orthopedic injury and an actual or suspected injury to the head.

53. 36. The method of claim 35, wherein the sample is selected from the group consisting of a whole blood sample, a serum sample, and a plasma sample.

54. an assay for ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), an assay for glial fibrillary acidic protein (GFAP), or an assay for UCH-L1 and an assay for GFAP; and Non-point-of-care device for performing an assay for UCH-L1, an assay for GFAP, or an assay for UCH-L1 and an assay for GFAP Including, The device determines the amount of UCH-L1, GFAP, or UCH-L1 and GFAP in a sample obtained from the subject; and The amount of UCH-L1, GFAP, or UCH-L1 and GFAP determined in the sample is a. Elevated if (i) the level of GFAP alone in the sample is equal to or greater than about 35 pg / mL; (ii) the level of GFAP alone in the sample is equal to or greater than about 35 pg / mL and the level of UCH-L1 alone in the sample is less than, cannot be determined, or is not reported as being about 400 pg / mL; (iii) the level of GFAP alone in the sample is equal to or greater than about 35 pg / mL and the level of UCH-L1 alone in the sample is equal to or greater than about 400 pg / mL; (iv) the level of UCH-L1 alone in the sample is equal to or greater than about 400 pg / mL; or (v) the level of GFAP alone in the sample is not determinable or is not reported as being about 400 pg / mL and the level of UCH-L1 alone in the sample is equal to or greater than about 400 pg / mL; b. (i) the level of only GFAP in the sample is below about 35 pg / mL; (ii) the level of only UCH-L1 in the sample is below about 400 pg / mL; or (iii) not elevated if the level of GFAP in the sample is below about 35 pg / mL and the level of UCH-L1 in the sample is below about 400 pg / mL; or c. (i) the level of UCH-L1 alone cannot be determined or is not reported in the sample; (ii) the level of GFAP in the sample is below about 35 pg / mL and the level of UCH-L1 in the sample is below about 35 pg / mL; (iii) the level of GFAP alone cannot be determined or is not reported in the sample; (iv) the level of GFAP in the sample is below about 400 pg / mL and the level of UCH-L1 in the sample is below about 400 pg / mL; or (v ... and the assays for UCH-L1 and GFAP need to be repeated. transmitted on or from the device as system.

55. The sample is taken within about 5 minutes, within about 10 minutes, within about 12 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 60 minutes, within about 90 minutes, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, 55. The system of claim 54, wherein the sample is collected within about 23 hours, within about 24 hours, within about 25 hours, within about 26 hours, within about 27 hours, within about 28 hours, within about 29 hours, within about 30 hours, within about 31 hours, within about 32 hours, within about 33 hours, within about 34 hours, within about 35 hours, within about 36 hours, within about 37 hours, within about 38 hours, within about 39 hours, within about 40 hours, within about 41 hours, within about 42 hours, within about 43 hours, within about 44 hours, within about 45 hours, within about 46 hours, within about 47 hours, or within about 48 hours.

56. 55. The system of claim 54, wherein the assay for UCH-L1 and the assay for GFAP are performed simultaneously or sequentially, in any order.

57. 55. The system of claim 54, wherein the sample is obtained after the subject has been subjected to physical shaking, blunt impact from an external mechanical or other force resulting in closed or open head injury, head injury caused by one or more falls, explosion or blast, or other type of blunt force trauma.

58. 55. The system of claim 54, wherein the sample is obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin.

59. 59. The system of claim 58, wherein the chemical or toxin is fire, mold, asbestos, pesticides, insecticides, organic solvents, paints, adhesives, gases, organometallics, addictive drugs, or a combination of one or more thereof.

60. 55. The system of claim 54, wherein the sample is obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection, a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof.

61. 55. The system of claim 54, wherein the assay is an immunoassay or a clinical chemistry assay.

62. 55. The system of claim 54, wherein the assay is a single molecule detection assay.

63. 55. The system of claim 54, wherein the volume of at least one sample is from about 10 μL to about 30 μL.

64. 64. The system of claim 63, wherein the volume of at least one sample is about 20 μL.

65. 55. The system of claim 54, wherein the at least one assay for UCH-L1, the at least one assay for GFAP, or the at least one assay for UCH-L1 and the at least one assay for GFAP are performed in about 10 minutes to about 20 minutes.

66. 66. The system of claim 65, wherein the assay for UCH-L1, the assay for GFAP, or at least one of the assays for UCH-L1 and GFAP is performed in about 15 minutes.

67. 55. The system of claim 54, wherein the subject has suffered an orthopedic injury and an actual or suspected injury to the head.

68. 55. The system of claim 54, wherein the sample is selected from the group consisting of a whole blood sample, a serum sample, and a plasma sample.