Use of biomarkers to determine subacute traumatic brain injury (TBI) in subjects who have had a head computed tomography (CT) scan that is negative for TBI or who have not had a head CT scan

Abstract

Disclosed herein are methods to aid in determining whether a subject has a traumatic brain injury (TBI) by detecting the level of at least one biomarker, glial fibrillary acidic protein (GFAP), in a sample taken from a subject, such as a human subject, who has undergone a head CT scan that is negative for TBI.

Description

[Technical field]

[0001] This application claims priority to U.S. Patent Application No. 63 / 294,344, filed December 28, 2021, the contents of which are incorporated herein by reference.

[0002] The computer-readable nucleotide / amino acid sequence listing submitted herewith is incorporated by reference in its entirety into this specification and is identified as follows in a single 7,756-byte XML file named "40205_601_ST26.xml" created on December 28, 2022.

[0003] The present disclosure relates to a method for aiding in the diagnosis and assessment of a subject who has sustained, may have sustained, or is suspected of having sustained an injury, such as a traumatic brain injury (TBI), to the head. The method includes detecting a level of at least one biomarker, such as glial fibrillary acidic protein (GFAP), in one or more samples taken from the subject at one or more time points from about 24 hours to about three weeks after the subject sustains, may have sustained, or is suspected of having sustained a head injury, the subject also having a concurrent, prior, or subsequent (within a clinically relevant time frame) head computed tomography (CT) scan that is negative for TBI, and diagnosing the subject as more likely than not to have TBI if the level of the one or more biomarkers in the sample is higher than a reference level. [Background technology]

[0004] 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 and Glasgow Coma Scale (GCS) scores are not very comprehensive or sensitive in assessing mild TBI. Furthermore, head CT scans reveal nothing about mild TBI in most cases, are expensive, and expose patients to unnecessary radiation. In addition, a negative head CT does not mean that the patient is clearly not concussed, only that certain interventions, such as surgery, are not warranted. Physicians and patients need objective and reliable information to accurately assess this condition and facilitate appropriate triage and recovery. To date, there has been limited data available for the use of UCH-L1 and GFAP in acute care settings to aid in patient assessment and management. To date, there has been limited data available for the use of UCH-L1 and GFAP in the acute care setting to aid in patient assessment and management.

[0005] 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).

[0006] 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.

[0007] Furthermore, current brain injury trials rely on outcome measures, such as the Glasgow Outcome Scale-Extended, which capture a global phenomenon but cannot assess nuances in outcome. Summary of the Invention [Problem to be solved by the invention]

[0008] Sensitive endpoints are needed to determine how well patients have recovered from brain injury in order to test therapeutic and prophylactic agents. [Means for solving the problem]

[0009] In one aspect, the disclosure is directed to an improved method for aiding in the diagnosis and assessment of a subject, such as a human subject, who has sustained, may have sustained, or is suspected of having sustained a head injury. Such an improved method includes (1) an assay on a sample obtained from the subject about 24 hours to about 3 weeks after the actual or suspected head injury to measure or detect a level of a biomarker in the sample, the biomarker comprising glial fibrillary acidic protein (GFAP); and (2) simultaneously or sequentially performing a head computed tomography (CT) scan on the subject within a clinically relevant time frame, the improvement including diagnosing the subject as more likely than not to have a traumatic brain injury (TBI) if the level of the biomarker is higher than a reference level and the head CT scan is negative for TBI.

[0010] In yet another aspect, the reference level is correlated with a cutoff level related to: (a) a level in a subject suffering from a head injury; (b) the occurrence of a TBI in a subject; (c) a stage of a TBI in a subject, such as mild, moderate, severe, or moderate-severe; (d) a loss of consciousness in a subject; (e) an MRI that is positive rather than negative for TBI; (f) the occurrence of amnesia in a subject (i.e., the presence vs. absence of amnesia), or (g) a severity of a TBI in a subject.

[0011] In another embodiment, the improved method further comprises monitoring the subject for TBI if the level of the biomarker is higher than the reference level and the head CT scan is negative for TBI. In another embodiment, the improved method further comprises treating the subject for TBI if the level of the biomarker is higher than the reference level and the head CT scan is negative for TBI. In yet another embodiment, the improved method further comprises treating the subject for TBI if the level of the biomarker is higher than the reference level and the head CT scan is negative for TBI, and then monitoring the subject.

[0012] In yet another embodiment of the above improved method, the sample can be taken about 24.5 hours after the actual or suspected injury to the head. For example, the sample can be taken within about 25 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within about 26 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within about 27 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within about 28 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within about 29 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within 30 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within 31 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within 32 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within 33 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within 34 hours after the actual or suspected injury to the head. Alternatively, a sample can be taken within 35 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 36 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 37 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 38 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 39 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 40 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 41 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 42 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 43 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 44 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 45 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 46 hours of the actual or suspected injury to the head.Alternatively, the sample can be taken within 47 hours of the actual or suspected injury to the head. Alternatively, the sample can be taken within 48 hours of the actual or suspected injury to the head. Alternatively, the sample can be taken within 3 days of the actual or suspected injury to the head. Alternatively, the sample can be taken within 4 days of the actual or suspected injury to the head. Alternatively, the sample can be taken within 5 days of the actual or suspected injury to the head. Alternatively, the sample can be taken within 6 days after the actual or suspected injury to the head. Alternatively, the sample can be taken within 7 days (or 1 week) of the actual or suspected injury to the head. Alternatively, the sample can be taken within 2 weeks of the actual or suspected injury to the head. Alternatively, the sample can be taken within 3 weeks of the actual or suspected injury to the head.

[0013] In one embodiment, a subject is assessed or evaluated to have a TBI using the improved method described above. In another embodiment, a subject is assessed or evaluated to have a mild TBI using the improved method described above. In another embodiment, a subject is assessed or evaluated to have a moderate TBI using the improved method described above. In another embodiment, a subject is assessed or evaluated to have a severe TBI using the improved method described above. In another embodiment, a subject is assessed or evaluated to have a moderate to severe TBI using the improved method described above. In another embodiment, a subject is assessed or evaluated to have no TBI using the improved method described above.

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

[0015] In the improved method, the reference level for GFAP is about 5 pg / mL to about 115 pg / mL. In the improved method, the reference level for GFAP is about 5 pg / mL to about 110 pg / mL. In the improved method, the reference level for GFAP is about 5 pg / mL to about 105 pg / mL. In some embodiments, the reference level for GFAP is about 5 pg / mL to about 100 pg / mL. In some embodiments, the reference level for GFAP is about 5 pg / mL to about 90 pg / mL. In still other embodiments, the reference level for GFAP is about 5 pg / mL to about 80 pg / mL. In still other embodiments, the reference level for GFAP is about 5 pg / mL to about 70 pg / mL. In still other embodiments, the reference level for GFAP is about 5 pg / mL to about 60 pg / mL. In yet further embodiments, the reference level for GFAP is about 5 pg / mL to about 50 pg / mL. In yet further embodiments, the reference level for GFAP is about 5 pg / mL to about 40 pg / mL. In yet other embodiments, the reference level for GFAP is about 5 pg / mL to about 30 pg / mL. In yet other embodiments, the reference level for GFAP is about 5 pg / mL to about 25 pg / mL. In yet other embodiments, the reference level for GFAP is about 5 pg / mL to about 20 pg / mL.

[0016] In other aspects, the reference level for GFAP is about 10 pg / mL to about 115 pg / mL. In the improved method, the reference level for GFAP is about 10 pg / mL to about 110 pg / mL. In the improved method, the reference level for GFAP is about 10 pg / mL to about 105 pg / mL. In some embodiments, the reference level for GFAP is about 10 pg / mL to about 100 pg / mL. In some embodiments, the reference level for GFAP is about 10 pg / mL to about 90 pg / mL. In still other embodiments, the reference level for GFAP is about 10 pg / mL to about 80 pg / mL. In still other embodiments, the reference level for GFAP is about 10 pg / mL to about 70 pg / mL. In still other embodiments, the reference level for GFAP is about 10 pg / mL to about 60 pg / mL. In yet further embodiments, the reference level for GFAP is about 10 pg / mL to about 50 pg / mL. In yet further embodiments, the reference level for GFAP is about 10 pg / mL to about 40 pg / mL. In yet other embodiments, the reference level for GFAP is about 10 pg / mL to about 30 pg / mL. In yet other embodiments, the reference level for GFAP is about 10 pg / mL to about 25 pg / mL. In yet other embodiments, the reference level for GFAP is about 10 pg / mL to about 20 pg / mL.

[0017] In yet another aspect, the reference level for GFAP is about 15 pg / mL to about 115 pg / mL. In the improved method, the reference level for GFAP is about 15 pg / mL to about 110 pg / mL. In the improved method, the reference level for GFAP is about 15 pg / mL to about 105 pg / mL. In some embodiments, the reference level for GFAP is about 15 pg / mL to about 100 pg / mL. In some embodiments, the reference level for GFAP is about 15 pg / mL to about 90 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 80 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 70 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 60 pg / mL. In yet further embodiments, the reference level for GFAP is about 15 pg / mL to about 50 pg / mL. In yet further embodiments, the reference level for GFAP is about 15 pg / mL to about 40 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 30 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 25 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 20 pg / mL.

[0018] In the improved method, the sample can be selected from the group consisting of a blood sample, a urine sample, a cerebrospinal fluid sample, a tissue sample, a body fluid sample, a saliva sample, an oropharyngeal sample, and a nasopharyngeal sample. The blood sample can include a whole blood sample, a serum sample, or a plasma sample. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a plasma sample. In still other embodiments, the sample is a serum sample. In still other embodiments, the sample is a saliva sample. In still other embodiments, the sample is an oropharyngeal sample. In still other embodiments, the sample is a nasopharyngeal sample. Such samples can be obtained in a variety of ways. For example, the sample is obtained after the subject has suffered physical shaking, blunt impact from an external mechanical or other force resulting in closed or open head trauma, injury to the head caused by one or more falls, explosions or blasts, or other types of blunt trauma. Alternatively, the sample is obtained after the subject has ingested or been exposed to fire, chemicals, toxins, or a combination of chemicals and toxins. Examples of chemicals or toxins are mold, asbestos, pesticides, insecticides, organic solvents, paints, adhesives, gases, organometallics, addictive drugs, or a combination of one or more thereof. Additionally, 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.

[0019] The above improved methods can be performed on any subject, such as a human subject, regardless of factors selected from the group consisting of the subject's clinical condition, the subject's clinical laboratory values, the subject's classification as suffering from mild, moderate, severe, or moderate-severe TBI, the subject's presentation of low, moderate, or high levels of GFAP, and the timing of any events in which the subject sustained or may have sustained a head injury.

[0020] In the improved method, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In still other embodiments, the assay is a clinical chemistry assay. In still other embodiments, the assay is a single molecule detection assay. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a clinical chemistry assay, and the sample is whole blood. In yet further embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum.

[0021] In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a clinical chemistry assay, the subject is a human, and the sample is serum. In still further embodiments, the assay is a single molecule detection assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a clinical chemistry assay, and the sample is plasma. In still further embodiments, the assay is a single molecule detection assay, and the sample is plasma. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is saliva. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is saliva. In still other embodiments, the assay is a clinical chemistry assay, and the sample is saliva. In still further embodiments, the assay is a single molecule detection assay, and the sample is saliva. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is an oropharyngeal or nasopharyngeal specimen. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is an oropharyngeal or nasopharyngeal specimen. In still other embodiments, the assay is a clinical chemistry assay, and the sample is an oropharyngeal or nasopharyngeal specimen. In yet further embodiments, the assay is a single molecule detection assay, and the sample is an oropharyngeal or nasopharyngeal specimen.

[0022] In another aspect, the disclosure is directed to a method for aiding in the diagnosis and assessment of a subject, such as a human subject, who has sustained, may have sustained, or is suspected of having sustained a head injury, the method comprising: a.(1) an assay on a sample obtained from a subject from about 24 hours to about 3 weeks after an actual or suspected head injury to measure or detect levels of a biomarker in the sample, the biomarker including glial fibrillary acidic protein (GFAP), and (2) a simultaneous or sequential head computed tomography (CT) scan within a clinically relevant time frame; and b. diagnosing the subject as more likely than not to have a traumatic brain injury (TBI) if the level of the biomarker is higher than the reference level and the head CT scan is negative for TBI; Includes.

[0023] In yet another aspect, the reference level is correlated with a cutoff level related to: (a) a level in a subject suffering from a head injury; (b) the occurrence of a TBI in a subject; (c) a stage of a TBI in a subject, such as mild, moderate, severe, or moderate-severe; (d) a loss of consciousness in a subject; (e) an MRI that is positive rather than negative for TBI; (f) the occurrence of amnesia in a subject (i.e., the presence vs. absence of amnesia), or (g) a severity of a TBI in a subject.

[0024] In another embodiment, the above method further comprises monitoring the subject for TBI if the level of the biomarker is higher than the reference level and the head CT scan is negative for TBI. In another embodiment, the method further comprises treating the subject for TBI if the level of the biomarker is higher than the reference level and the head CT scan is negative for TBI. In yet another embodiment, the method further comprises monitoring the subject following treating the subject for TBI if the level of the biomarker is higher than the reference level and the head CT scan is negative for TBI.

[0025] In yet another aspect of the above method, the sample can be taken about 24.5 hours after the actual or suspected injury to the head. For example, the sample can be taken within about 25 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within about 26 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within about 27 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within about 28 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within about 29 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within 30 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within 31 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within 32 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within 33 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within 34 hours after the actual or suspected injury to the head. Alternatively, a sample can be taken within 35 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 36 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 37 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 38 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 39 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 40 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 41 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 42 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 43 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 44 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 45 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 46 hours of the actual or suspected injury to the head.Alternatively, the sample can be taken within 47 hours of the actual or suspected injury to the head. Alternatively, the sample can be taken within 48 hours of the actual or suspected injury to the head. Alternatively, the sample can be taken within 3 days of the actual or suspected injury to the head. Alternatively, the sample can be taken within 4 days of the actual or suspected injury to the head. Alternatively, the sample can be taken within 5 days of the actual or suspected injury to the head. Alternatively, the sample can be taken within 6 days after the actual or suspected injury to the head. Alternatively, the sample can be taken within 7 days (or 1 week) of the actual or suspected injury to the head. Alternatively, the sample can be taken within 2 weeks of the actual or suspected injury to the head. Alternatively, the sample can be taken within 3 weeks of the actual or suspected injury to the head.

[0026] In the improved method, the reference level for GFAP is about 5 pg / mL to about 115 pg / mL. In the improved method, the reference level for GFAP is about 5 pg / mL to about 110 pg / mL. In the improved method, the reference level for GFAP is about 5 pg / mL to about 105 pg / mL. In some embodiments, the reference level for GFAP is about 5 pg / mL to about 100 pg / mL. In some embodiments, the reference level for GFAP is about 5 pg / mL to about 90 pg / mL. In still other embodiments, the reference level for GFAP is about 5 pg / mL to about 80 pg / mL. In still other embodiments, the reference level for GFAP is about 5 pg / mL to about 70 pg / mL. In still other embodiments, the reference level for GFAP is about 5 pg / mL to about 60 pg / mL. In yet further embodiments, the reference level for GFAP is about 5 pg / mL to about 50 pg / mL. In yet further embodiments, the reference level for GFAP is about 5 pg / mL to about 40 pg / mL. In yet other embodiments, the reference level for GFAP is about 5 pg / mL to about 30 pg / mL. In yet other embodiments, the reference level for GFAP is about 5 pg / mL to about 25 pg / mL. In yet other embodiments, the reference level for GFAP is about 5 pg / mL to about 20 pg / mL.

[0027] In other aspects, the reference level for GFAP is about 10 pg / mL to about 115 pg / mL. In the improved method, the reference level for GFAP is about 10 pg / mL to about 110 pg / mL. In the improved method, the reference level for GFAP is about 10 pg / mL to about 105 pg / mL. In some embodiments, the reference level for GFAP is about 10 pg / mL to about 100 pg / mL. In some embodiments, the reference level for GFAP is about 10 pg / mL to about 90 pg / mL. In still other embodiments, the reference level for GFAP is about 10 pg / mL to about 80 pg / mL. In still other embodiments, the reference level for GFAP is about 10 pg / mL to about 70 pg / mL. In still other embodiments, the reference level for GFAP is about 10 pg / mL to about 60 pg / mL. In yet further embodiments, the reference level for GFAP is about 10 pg / mL to about 50 pg / mL. In yet further embodiments, the reference level for GFAP is about 10 pg / mL to about 40 pg / mL. In yet other embodiments, the reference level for GFAP is about 10 pg / mL to about 30 pg / mL. In yet other embodiments, the reference level for GFAP is about 10 pg / mL to about 25 pg / mL. In yet other embodiments, the reference level for GFAP is about 10 pg / mL to about 20 pg / mL.

[0028] In yet another aspect, the reference level for GFAP is about 15 pg / mL to about 115 pg / mL. In the improved method, the reference level for GFAP is about 15 pg / mL to about 110 pg / mL. In the improved method, the reference level for GFAP is about 15 pg / mL to about 105 pg / mL. In some embodiments, the reference level for GFAP is about 15 pg / mL to about 100 pg / mL. In some embodiments, the reference level for GFAP is about 15 pg / mL to about 90 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 80 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 70 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 60 pg / mL. In yet further embodiments, the reference level for GFAP is about 15 pg / mL to about 50 pg / mL. In yet further embodiments, the reference level for GFAP is about 15 pg / mL to about 40 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 30 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 25 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 20 pg / mL.

[0029] In one embodiment, a subject is assessed or evaluated to have a TBI using the above method. In another embodiment, a subject is assessed or evaluated to have a mild TBI using the above method. In another embodiment, a subject is assessed or evaluated to have a moderate TBI using the above method. In another embodiment, a subject is assessed or evaluated to have a severe TBI using the above method. In another embodiment, a subject is assessed or evaluated to have a moderate to severe TBI using the above method. In yet a further embodiment, a subject is assessed or evaluated to have no TBI using the above method.

[0030] The above method can further include treating a subject, such as a human subject assessed or evaluated as having mild, moderate, severe, or moderate-severe TBI, with a treatment for TBI (e.g., a surgical treatment, a therapeutic treatment, or a combination thereof). Any such treatment known in the art and further described herein can be used. Furthermore, in further embodiments, any subject being treated for TBI can also be optionally monitored during and after any course of treatment. Alternatively, the method can further include monitoring a subject assessed as having mild, moderate, severe, or moderate-severe TBI (such as a subject who may not yet have received any treatment).

[0031] In the above method, the sample can be selected from the group consisting of a blood sample, a urine sample, a cerebrospinal fluid sample, a tissue sample, a body fluid sample, a saliva sample, an oropharyngeal sample, and a nasopharyngeal sample. In some embodiments, the sample is a whole blood sample. The blood sample can be a whole blood sample, a serum sample, or a plasma sample. In some embodiments, the sample is a plasma sample. In still other embodiments, the sample is a serum sample. In still other embodiments, the sample is a saliva sample. In still other embodiments, the sample is an oropharyngeal sample. In still other embodiments, the sample is a nasopharyngeal sample. Such samples can be obtained in a variety of ways. For example, the sample is obtained after the subject has suffered physical shaking, blunt impact from an external mechanical or other force resulting in a closed or open head injury, head injury caused by one or more falls, an explosion or blast, or other type of blunt trauma. Alternatively, the sample is obtained after the subject has ingested or been exposed to fire, a chemical, a toxin, or a combination of a chemical and a toxin. Examples of chemicals or toxins are mold, asbestos, pesticides, insecticides, organic solvents, paints, adhesives, gases, organometallics, addictive drugs, or a combination of one or more thereof. Additionally, 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.

[0032] The above methods can be performed on any subject, such as a human subject, regardless of factors selected from the group consisting of the subject's clinical condition, the subject's clinical laboratory values, the subject's classification as suffering from mild, moderate, severe, or moderate-severe TBI, the subject's presentation of low, moderate, or high levels of GFAP, and the timing of any events in which the subject sustained or may have sustained a head injury.

[0033] In the above methods, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In still other embodiments, the assay is a clinical chemistry assay. In still other embodiments, the assay is a single molecule detection assay. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a clinical chemistry assay, and the sample is whole blood. In still further embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a clinical chemistry assay, and the sample is serum. In still further embodiments, the assay is a single molecule detection assay, and the sample is serum. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In yet other embodiments, the assay is a clinical chemistry assay and the sample is plasma. In yet further embodiments, the assay is a single molecule detection assay and the sample is plasma. In yet other embodiments, the assay is an immunoassay, the subject is a human and the sample is saliva. In yet other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is saliva. In yet other embodiments, the assay is a clinical chemistry assay and the sample is saliva. In yet further embodiments, the assay is a single molecule detection assay and the sample is saliva. In yet other embodiments, the assay is an immunoassay, the subject is a human and the sample is an oropharyngeal or nasopharyngeal specimen. In yet other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is an oropharyngeal or nasopharyngeal specimen. In yet other embodiments, the assay is a clinical chemistry assay and the sample is an oropharyngeal or nasopharyngeal specimen.In yet a further embodiment, the assay is a single molecule detection assay and the sample is an oropharyngeal or nasopharyngeal specimen.

[0034] In yet another embodiment, the disclosure relates to an improvement in a method for aiding in the diagnosis and assessment of a subject, such as a human subject, who has sustained or may have sustained a head injury. The improvement includes performing an assay on a sample obtained from the subject from about 24 hours to about 3 weeks after an actual or suspected head injury to measure or detect a level of a biomarker in the sample, the biomarker comprising glial fibrillary acidic protein (GFAP); and diagnosing the subject as more likely than not to have traumatic brain injury (TBI) if the level of the biomarker is higher than a reference level and the subject has a head computed tomography (CT) scan negative for TBI within a clinically relevant time frame, or if no head CT scan is performed on the subject.

[0035] In yet another aspect, the reference level is correlated with a cutoff level related to: (a) a level in a subject suffering from a head injury; (b) the occurrence of a TBI in a subject; (c) a stage of a TBI in a subject, such as mild, moderate, severe, or moderate-severe; (d) a loss of consciousness in a subject; (e) an MRI that is positive rather than negative for TBI; (f) the occurrence of amnesia in a subject (i.e., the presence vs. absence of amnesia), or (g) a severity of a TBI in a subject.

[0036] In another embodiment, the improved method further comprises monitoring the subject for TBI if the level of the biomarker is higher than the reference level and, optionally, if performed, a head CT scan is negative for TBI. In another embodiment, the improved method further comprises treating the subject for TBI if the level of the biomarker is higher than the reference level and, optionally, if performed, a head CT scan is negative for TBI. In yet another embodiment, the improved method further comprises treating the subject for TBI if the level of the biomarker is higher than the reference level and, optionally, if performed, a head CT scan is negative for TBI, followed by monitoring the subject.

[0037] In yet another aspect of the improved method, the sample can be taken about 24.5 hours after the actual or suspected injury to the head. For example, the sample can be taken within about 25 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within about 26 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within about 27 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within about 28 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within about 29 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within 30 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within 31 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within 32 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within 33 hours after the actual or suspected injury to the head. Alternatively, the sample can be taken within 34 hours after the actual or suspected injury to the head. Alternatively, a sample can be taken within 35 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 36 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 37 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 38 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 39 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 40 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 41 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 42 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 43 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 44 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 45 hours of the actual or suspected injury to the head. Alternatively, a sample can be taken within 46 hours of the actual or suspected injury to the head.Alternatively, the sample can be taken within 47 hours of the actual or suspected injury to the head. Alternatively, the sample can be taken within 48 hours of the actual or suspected injury to the head. Alternatively, the sample can be taken within 3 days of the actual or suspected injury to the head. Alternatively, the sample can be taken within 4 days of the actual or suspected injury to the head. Alternatively, the sample can be taken within 5 days of the actual or suspected injury to the head. Alternatively, the sample can be taken within 6 days after the actual or suspected injury to the head. Alternatively, the sample can be taken within 7 days (1 week) of the actual or suspected injury to the head. Alternatively, the sample can be taken within 2 weeks of the actual or suspected injury to the head. Alternatively, the sample can be taken within 3 weeks of the actual or suspected injury to the head.

[0038] In the improved method, the reference level for GFAP is about 5 pg / mL to about 115 pg / mL. In the improved method, the reference level for GFAP is about 5 pg / mL to about 110 pg / mL. In the improved method, the reference level for GFAP is about 5 pg / mL to about 105 pg / mL. In some embodiments, the reference level for GFAP is about 5 pg / mL to about 100 pg / mL. In some embodiments, the reference level for GFAP is about 5 pg / mL to about 90 pg / mL. In still other embodiments, the reference level for GFAP is about 5 pg / mL to about 80 pg / mL. In still other embodiments, the reference level for GFAP is about 5 pg / mL to about 70 pg / mL. In still other embodiments, the reference level for GFAP is about 5 pg / mL to about 60 pg / mL. In yet further embodiments, the reference level for GFAP is about 5 pg / mL to about 50 pg / mL. In yet further embodiments, the reference level for GFAP is about 5 pg / mL to about 40 pg / mL. In yet other embodiments, the reference level for GFAP is about 5 pg / mL to about 30 pg / mL. In yet other embodiments, the reference level for GFAP is about 5 pg / mL to about 25 pg / mL. In yet other embodiments, the reference level for GFAP is about 5 pg / mL to about 20 pg / mL.

[0039] In other aspects, the reference level for GFAP is about 10 pg / mL to about 115 pg / mL. In the improved method, the reference level for GFAP is about 10 pg / mL to about 110 pg / mL. In the improved method, the reference level for GFAP is about 10 pg / mL to about 105 pg / mL. In some embodiments, the reference level for GFAP is about 10 pg / mL to about 100 pg / mL. In some embodiments, the reference level for GFAP is about 10 pg / mL to about 90 pg / mL. In still other embodiments, the reference level for GFAP is about 10 pg / mL to about 80 pg / mL. In still other embodiments, the reference level for GFAP is about 10 pg / mL to about 70 pg / mL. In still other embodiments, the reference level for GFAP is about 10 pg / mL to about 60 pg / mL. In yet further embodiments, the reference level for GFAP is about 10 pg / mL to about 50 pg / mL. In yet further embodiments, the reference level for GFAP is about 10 pg / mL to about 40 pg / mL. In yet other embodiments, the reference level for GFAP is about 10 pg / mL to about 30 pg / mL. In yet other embodiments, the reference level for GFAP is about 10 pg / mL to about 25 pg / mL. In yet other embodiments, the reference level for GFAP is about 10 pg / mL to about 20 pg / mL.

[0040] In yet another aspect, the reference level for GFAP is about 15 pg / mL to about 115 pg / mL. In the improved method, the reference level for GFAP is about 15 pg / mL to about 110 pg / mL. In the improved method, the reference level for GFAP is about 15 pg / mL to about 105 pg / mL. In some embodiments, the reference level for GFAP is about 15 pg / mL to about 100 pg / mL. In some embodiments, the reference level for GFAP is about 15 pg / mL to about 90 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 80 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 70 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 60 pg / mL. In yet further embodiments, the reference level for GFAP is about 15 pg / mL to about 50 pg / mL. In yet further embodiments, the reference level for GFAP is about 15 pg / mL to about 40 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 30 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 25 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 20 pg / mL.

[0041] In one embodiment, a subject is assessed or evaluated to have a TBI using the improved method described above. In another embodiment, a subject is assessed or evaluated to have a mild TBI using the improved method described above. In another embodiment, a subject is assessed or evaluated to have a moderate TBI using the improved method described above. In another embodiment, a subject is assessed or evaluated to have a severe TBI using the improved method described above. In another embodiment, a subject is assessed or evaluated to have a moderate to severe TBI using the improved method described above. In yet a further embodiment, a subject is assessed or evaluated to have no TBI using the improved method described above.

[0042] The improved method can further include treating a subject, such as a human subject assessed or evaluated as having mild, moderate, severe, or moderate-severe TBI, with a treatment for TBI (e.g., a surgical treatment, a therapeutic treatment, or a combination thereof). Any such treatment known in the art and further described herein can be used. Furthermore, in further embodiments, any subject being treated for TBI can also be optionally monitored during and after any course of treatment. Alternatively, the method can further include monitoring a subject assessed as having mild, moderate, severe, or moderate-severe TBI (such as a subject who may not yet have received any treatment).

[0043] In the improved method, the sample can be selected from the group consisting of a blood sample, a urine sample, a cerebrospinal fluid sample, a tissue sample, a body fluid sample, a saliva sample, an oropharyngeal sample, and a nasopharyngeal sample. The blood sample can be a whole blood sample, a serum sample, or a plasma sample. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a plasma sample. In still other embodiments, the sample is a serum sample. In still other embodiments, the sample is a saliva sample. In still other embodiments, the sample is an oropharyngeal sample. In still other embodiments, the sample is a nasopharyngeal sample. Such samples can be obtained in a variety of ways. For example, the sample is obtained after the subject has suffered physical shaking, blunt impact from an external mechanical or other force resulting in a closed or open head injury, head injury caused by one or more falls, an explosion or blast, or other types of blunt trauma. Alternatively, the sample is obtained after the subject has ingested or been exposed to fire, a chemical, a toxin, or a combination of a chemical and a toxin. Examples of chemicals or toxins are mold, asbestos, pesticides, insecticides, organic solvents, paints, adhesives, gases, organometallics, addictive drugs, or a combination of one or more thereof. Additionally, 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.

[0044] The above improved methods can be performed on any subject, such as a human subject, regardless of factors selected from the group consisting of the subject's clinical condition, the subject's clinical laboratory values, the subject's classification as suffering from mild, moderate, severe, or moderate-severe TBI, the subject's presentation of low, moderate, or high levels of GFAP, and the timing of any events in which the subject sustained or may have sustained a head injury.

[0045] In the improved method, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In still other embodiments, the assay is a clinical chemistry assay. In still other embodiments, the assay is a single molecule detection assay. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a clinical chemistry assay, and the sample is whole blood. In still further embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a clinical chemistry assay, and the sample is serum. In still further embodiments, the assay is a single molecule detection assay, and the sample is serum. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is plasma. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In yet other embodiments, the assay is a clinical chemistry assay and the sample is plasma. In yet further embodiments, the assay is a single molecule detection assay and the sample is plasma. In yet other embodiments, the assay is an immunoassay, the subject is a human and the sample is saliva.

[0046] In yet other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is saliva. In yet other embodiments, the assay is a clinical chemistry assay, and the sample is saliva. In yet further embodiments, the assay is a single molecule detection assay, and the sample is saliva. In yet other embodiments, the assay is an immunoassay, the subject is a human, and the sample is an oropharyngeal or nasopharyngeal specimen. In yet other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is an oropharyngeal or nasopharyngeal specimen. In yet other embodiments, the assay is a clinical chemistry assay, and the sample is an oropharyngeal or nasopharyngeal specimen. In yet further embodiments, the assay is a single molecule detection assay, and the sample is an oropharyngeal or nasopharyngeal specimen. [Brief description of the drawings]

[0047] [Figure 1] Figure 1 shows a ROC analysis of GFAP levels correlating with Glasgow Coma Scale (GCS) score severity (i.e., GCS mild vs. moderate / severe) for subjects with CT scans negative for TBI. Samples were assessed within 2 weeks of injury. [Diagram 2] Figure 2 shows the ROC analysis of GFAP levels correlating with the Glasgow Outcome Scale Extended (GOSE) for subjects with CT scans negative for TBI. Samples were assessed within 2 weeks of injury. [Diagram 3] Figure 3 shows a ROC analysis of GFAP levels correlating with MRI results (i.e., positive vs. negative) for subjects with CT scans that were negative for TBI. Samples were assessed within 2 weeks of injury. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] The present disclosure relates to improved methods to aid in the diagnosis and assessment of a subject, such as a human subject, who has sustained, may have sustained, or is suspected of having sustained a head injury, such as a traumatic brain injury (e.g., mild, moderate, severe, or moderate-severe TBI), using biomarkers such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, optionally where the subject has undergone at least one head computed tomography (CT) scan that is negative for TBI, within a clinically relevant time frame.

[0049] The methods described herein include detecting one or more biomarker levels in one or more samples taken from a subject, such as a human subject, at a time point from about 24 hours to about 3 weeks after an actual or suspected head injury. Optionally, if performed, a head CT scan can be performed in any order, simultaneously or sequentially, at the same time, prior to, or after one or more biomarker levels are detected in one or more samples taken from the subject. Detection of a level of one or more biomarkers, such as UCH-L1, GFAP, or a combination thereof, higher than a reference level helps accurately assess or diagnose a subject, such as a human subject, as more likely than not to have a TBI, if the subject optionally undergoes a head CT scan that is negative for TBI. In other words, in some aspects, the improved methods described herein allow for the identification of subjects who have suffered a TBI but may otherwise be inaccurately diagnosed as not having a TBI if the diagnosis was based solely on the results of one or more head CT scans.

[0050] The section headings used in this section and throughout the disclosure herein are for organizational purposes only and are not intended to be limiting.

[0051] 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 discrepancies, 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.

[0052] As used herein, the terms "comprise(s)," "include(s)," "having," "having," "may be," and "containing" are open-ended transitional phrases, transitional terms, or transitional words that do not exclude additional acts or structures. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise," "consist of," or "consist essentially of" the embodiments or elements presented herein, whether expressly stated or not.

[0053] 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.

[0054] 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.

[0055] As used herein, an "analog assay" refers to an assay in which the presence and / or concentration of an analyte in a test sample is determined by measuring the total signal (e.g., fluorescence, color, etc.) generated by the analyte in the entire reaction mixture (e.g., a single reaction vessel). In an analog assay, noise is indistinguishable from signal. An example of an analog assay is an assay in which the presence and / or concentration of an analyte is determined by measuring the total signal generated from multiple beads or microparticles contained in a single reaction vessel.

[0056] 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. 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).

[0057] 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.

[0058] "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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] A "clinically relevant time frame" is a time frame (e.g., seconds, minutes, or hours) within which a prudent and prudent medical practitioner (e.g., physician, nurse, paramedic, or other) would reasonably review the results of one or more biomarker tests associated with an imaging procedure, such as a head CT scan, or that conforms to guidelines established by an oversight entity (e.g., a standard setting body such as the World Health Organization (WHO), a Medical Review Board, a regulatory approval authority such as the FDA, EMEA, or other, etc.).

[0065] "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.

[0066] As used herein, "correlates to" means compared to.

[0067] 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.

[0068] A head CT scan is "negative" for TBI if no intracranial lesion(s) are observed in the image taken from a subject who has sustained, may have sustained, or is suspected to have sustained a head injury. To further clarify, a subject's head CT scan is "negative" for TBI if no lesions are found or identified, although in some embodiments, a subject may still experience symptoms (e.g., of TBI) even if the head CT is negative. Given that not all injuries or lesions can be visualized by head CT, the majority of subjects will have a negative head CT for TBI. Thus, the methods and assays described herein can be used to provide an assessment or determination of subjects who have a negative head CT but may still have a TBI.

[0069] "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, a set of training samples including samples obtained from human subjects known to have sustained a head injury, more particularly, samples obtained from human subjects known to have sustained (i) mild TBI, and / or (ii) moderate, severe, or moderate-severe TBI, as well as samples obtained from human subjects known not to have sustained a head injury, may be used to obtain assay-specific reference levels. It is understood that a reference level "determined by an assay" and having a recited level of "sensitivity" and / or "specificity" is used herein to refer to a reference level that has been determined to result in the method of the present disclosure having the recited sensitivity and / or specificity.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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] As used herein, a "digital assay" refers to an assay in which an analyte is captured and molecules of the analyte are separated and interrogated (e.g., to detect the presence and / or concentration of the analyte in a sample). In a digital assay, noise is separated from signal. In a digital assay, results are assigned a value of 1 or 0. Examples of digital assays include one or more of the following (which may overlap, but are not mutually exclusive): single molecule detection assays, nanowell assays, single molecule enzyme-linked immunosorbent assays, direct capture counting assays, etc.

[0074] "Drug of abuse" is used herein to refer to one or more addictive substances (such as narcotics) taken for non-medical (e.g., recreational and / or mind-altering effects) reasons. Excessive indulgence in, use of, or dependence on such drugs of abuse is often referred to as "drug abuse." Examples of drugs of abuse include alcohol, barbiturates, benzodiazepines, cannabis, cocaine, hallucinogens (such as ketamine, mescaline (peyote), PCP, psilocybin, DMT, and / or LSD), methaqualone, opioids, amphetamines (including methamphetamine), anabolic steroids, inhalants (i.e., substances that contain volatile substances that contain psychoactive properties, such as, for example, nitrites, spray paints, cleaning fluids, markers, glues, etc.), and combinations thereof.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] "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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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 / ).

[0087] 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.

[0088] "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).

[0089] "GFAP status" can mean the level or amount of GFAP at a point in time (such as a point in time involving a single measurement of GFAP), the level or amount of GFAP associated with monitoring (such as monitoring involving 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.

[0090] 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.

[0091] 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.

[0092] [Table 1]

[0093] 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.

[0094] 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.

[0095] 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.

[0096] "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.

[0097] "Head injury" or "head injury", used interchangeably herein, refers to any trauma to the scalp, skull, or brain. Such injury may include only a minor bump on the skull, or may be a severe brain injury. Such injury includes primary injury to the brain and / or secondary injury to the brain. Primary brain injury occurs during the initial insult and results from the displacement of the brain's physical structures. More specifically, primary brain injury is physical damage to the parenchyma (tissue, blood vessels) that occurs during the traumatic event, resulting in shearing and compression of the surrounding brain tissue. Secondary brain injury occurs subsequent to the primary injury and may involve a series of cellular processes. More specifically, secondary brain injury refers to changes that develop over a period of time (hours to days) following the primary brain injury. Secondary brain injury includes a whole cascade of cellular, chemical, tissue, or vascular changes in the brain that contribute to further destruction of brain tissue.

[0098] 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 concussion 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), by cerebrovascular accidents (e.g., strokes), one or more falls (e.g., during sports or other activities), by blunt impact from an explosion or blast (collectively, "blast injuries"), and by other types of blunt trauma. Alternatively, head injuries can be caused by ingestion and / or exposure to fire, chemicals, toxins, or combinations of chemicals and toxins. Examples of such chemicals and / or toxins include 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 having suffered a head injury." In certain embodiments herein, closed head injury does not include, and specifically excludes, cerebrovascular accidents such as strokes.

[0099] As used herein, an "intracranial lesion" refers to an area of ​​damage within the brain. An intracranial lesion can be an abnormality visible on a brain imaging test, such as a CT scan or magnetic resonance imaging (MRI). On a CT or MRI scan, a brain lesion may appear as a dark or pale spot that does not look like normal brain tissue.

[0100] 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).

[0101] 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.

[0102] Any suitable detectable label known in the art may be used. For example, detectable labels include radioactive labels (such as 3H, 14C, 32P, 33P, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm), enzyme labels (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase), chemiluminescent labels (acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, etc.), fluorescent labels (fluorescein, ... The label may be a fluorescent label (e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, etc.), rhodamine, phycobiliprotein, R-phycoerythrin, quantum dots (e.g., cadmium selenide capped with zinc sulfide), a calorimetric label, or an immunopolymerase chain reaction label. An introduction to labels, labeling procedures, and detection of labels can be found in Polak and Van Noorden, "Introduction to Immunocytochemistry," 2nd ed., Springer Verlag, NY (1997), and Haugland, "Handbook of Fluorescent Probes and Research Chemicals" (1996), a combination handbook and catalogue published by Molecular Probes, Inc., Eugene, Oregon. Fluorescent labels can be used in FPIA (see, e.g., U.S. Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are incorporated by reference herein in their entireties).Acridinium compounds can be used as detectable labels in homogeneous chemiluminescent assays (see, e.g., Adamczyk et al., Bioorg. Med. Chem. Lett., 16:1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett., 4:2313-2317 (2004); Adamczyk et al., Bioorg. Med. Chem. Lett., 14:3917-3921 (2004) and Adamczyk et al., Org. Lett., 5:3779-3782 (2003)).

[0103] 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.

[0104] 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.

[0105] 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.

[0106] "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 high 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.

[0107] 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.

[0108] 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.

[0109] "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.

[0110] As used herein, a "reference level" refers to an assay cutoff value used to assess 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, etc.). As used herein, an "absolute amount" refers to the absolute value of the change or difference between at least two assay results taken or sampled at different time points, which, similar to a reference level, 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, etc.). As used herein, an "absolute value" refers to the magnitude of a real number (e.g., the difference between two compared levels (e.g., a level taken at a first time point and a level taken at a second time point)) regardless of its sign, i.e., whether its sign is positive or negative.

[0111] The present disclosure provides exemplary reference levels and absolute amounts (e.g., calculated by comparing reference levels at different time points). However, it is well known that reference levels and absolute amounts may vary depending on the nature of the immunoassay (e.g., the antibody used, reaction conditions, sample purity, etc.), and that assays can be compared and standardized. Furthermore, it is within the ordinary skill of a person skilled in the art to adapt the disclosure herein to other immunoassays and obtain immunoassay-specific reference levels and absolute amounts for those other immunoassays based on the explanation provided by the present disclosure. Although the exact values ​​of reference levels and absolute amounts may vary between assays, the findings described herein are generally applicable and should be able to be extrapolated to other assays.

[0112] "Point-of-care device" refers to a device used to provide medical diagnostic testing at or near the point of care (i.e., outside a laboratory), at the time and place of patient care (such as a hospital, clinic, urgent care facility or other medical care facility, the patient's home, a nursing home and / or long-term care facility and / or hospice facility). Examples of point-of-care devices include point-of-care devices made by Abbott Laboratories (Abbott Park, IL) (e.g., i-STAT and i-STAT Alinity, Universal Biosensors (Rowville, Australia)) (see U.S. Patent Application Publication No. 2006 / 0134713), Axis-Shield PoC AS (Oslo, Norway) and Clinical Lab Products (Los Angeles, USA). In some embodiments, the point-of-care device is a single-use device. The term "single-use device" or "single-use instrument" refers to a clinical diagnostic instrument that processes and performs clinical diagnostic assays on a unit-use basis (such as a single-use cartridge) on a single patient sample. Point-of-care instruments do not simultaneously perform assays on more than one clinical sample. However, point-of-care instruments may have the capability to measure more than one parameter (e.g., more than one analyte) in an individual clinical sample per unit-use basis.

[0113] "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.

[0114] "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".

[0115] 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.

[0116] "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.

[0117] 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.

[0118] A "sample," "test sample," "specimen," "sample from a subject," "biological sample," and "patient sample," as used interchangeably herein, may be a blood sample such as whole blood (including, e.g., capillary blood, venous blood, dried blood spots, etc.), serum or plasma, tissue, saliva, urine, amniotic fluid, an oropharyngeal specimen, a nasopharyngeal specimen, sputum, a lower respiratory tract specimen such as an endotracheal aspirate or bronchoalveolar lavage, cerebrospinal fluid, placental cells or tissue, vascular endothelial cells, white blood cells, or mononuclear cells. The sample may be used directly as obtained from the patient, or may be pretreated to alter the characteristics of the sample, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., as discussed herein or otherwise known in the art. Additionally, the sample can be a nasopharyngeal or oropharyngeal sample obtained using one or more swabs, which once obtained are placed into a sterile tube containing viral transport medium (VTM) or universal transport medium (UTM) and either retained there or transferred to another medium for testing.

[0119] 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, tissue sections such as frozen sections taken for histological purposes, blood (such as whole blood, dried blood spots, etc.), plasma, serum, saliva, red blood cells, platelets, 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 blood sample (e.g., a whole blood sample, a serum sample, or a plasma sample). In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a capillary blood sample. In some embodiments, the sample is a dried blood spot. In some embodiments, the sample is a serum sample. In 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. In still other embodiments, the sample is a saliva sample.

[0120] As used herein, the "sensitivity" of an assay refers to the proportion of subjects with a positive outcome that are correctly identified as positive (e.g., correctly identify subjects who have the disease or medical condition for which they are being tested). For example, this can include correctly identifying subjects with a TBI that is significantly different from subjects with no TBI, correctly identifying subjects with a moderate, severe, or moderate-severe TBI that is significantly different from subjects with a mild TBI, correctly identifying subjects with a mild TBI that is significantly different from subjects with a moderate, severe, or moderate-severe TBI, correctly identifying subjects with a moderate, severe, or moderate-severe TBI that is significantly different from subjects with no TBI, or correctly identifying subjects with a mild TBI that is significantly different from subjects with no TBI, etc.

[0121] 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 who do not have a TBI that is significantly different from subjects who have a TBI, correctly identifying subjects who do not have a moderate, severe, or moderate-severe TBI that is significantly different from subjects who have a mild TBI, correctly identifying subjects who do not have a mild TBI that is significantly different from subjects who have a moderate, severe, or moderate-severe TBI, etc.).

[0122] A "series of calibration compositions" refers to a plurality of compositions comprising UCH-L1 of known concentrations, each of which differs from the other compositions in the series in its concentration of UCH-L11; and / or (2) GFAP of known concentrations, each of which differs from the other compositions in the series in its concentration of GFAP.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] "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).

[0127] As used herein, "subacute" refers to an injury that is between the acute and chronic stages (such as, for example, traumatic brain injury). In some embodiments, subacute refers to an injury from about 24 hours to about 3 weeks after an actual or suspected injury to the head.

[0128] 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.

[0129] As used herein, "treating", "treating" or "treatment" are each used interchangeably to describe preventing, alleviating or inhibiting the progression of a disease and / or injury or one or more symptoms of such disease to which such term applies. Depending on the subject's condition, the term also refers to preventing disease, including preventing the onset of a disease or preventing symptoms associated with a disease. Treatment can be performed acutely or chronically. The term also refers to reducing the severity of a disease or symptoms associated with such disease before contraction of the disease. Such prevention or reduction of the severity of a disease before contraction refers to administration of a pharmaceutical composition to a subject who is not affected by the disease at the time of administration. "Preventing" also refers to preventing the recurrence of a disease or one or more symptoms associated with such disease. "Treatment" and "therapeutically" refer to the act of treating, where "treating" is as defined above.

[0130] "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 concussion by a person, automobile accidents, firearm injuries, cerebrovascular accidents (e.g., strokes), falls, explosions or blasts, and other types of blunt trauma. Other causes of TBI include ingestion of and / or exposure to one or more fires, chemicals or toxins, such as 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 an autoimmune disease, metabolic disorder, brain tumor, hypoxia, viral infection (e.g., SARS-CoV-2, meningitis, etc.), fungal infection (e.g., meningitis), bacterial infection (e.g., meningitis), 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.

[0131] 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.

[0132] 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.

[0133] "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.

[0134] 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).

[0135] "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.

[0136] "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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 2. A method for aiding in the determination of traumatic brain injury (TBI) in subjects with head computed tomography (CT) scans that are negative for TBI Among other methods, the present disclosure relates to a method for aiding in the determination of whether a subject, such as a human subject who has sustained, may have sustained, or is suspected of having sustained a head injury, and who has undergone one or more head CT scans that are negative for TBI, is more likely than not to have sustained a traumatic brain injury (TBI). Specifically, such a method can include the steps of (a) (1) performing at least one assay on a sample obtained from the subject from about 24 hours to about 3 weeks after an actual or suspected head injury to measure or detect the level of a biomarker in the sample, the biomarker including ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, and (2) simultaneously or sequentially (in any order) performing at least one head CT scan on the subject within a clinically relevant time frame, and (b) diagnosing the subject as more likely than not to have a TBI if the level of the biomarker is higher than a reference level and the head CT scan is negative for TBI. The sample can be a biological sample.

[0141] In yet another aspect, the disclosure relates to a method for aiding in the determination of whether a subject, such as a human subject, has sustained, may sustain, or is suspected to have sustained a head injury. Specifically, the method includes performing an assay on a sample obtained from the subject from about 24 hours to about 3 weeks after the actual or suspected head injury to measure or detect a level of a biomarker in the sample, the biomarker comprising ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, and the method includes diagnosing that the level of the biomarker is higher than a reference level and that the subject has a negative head computed tomography (CT) scan for TBI within a clinically relevant time frame, or that the subject has not had a head CT scan, is more likely than not to have subacute traumatic brain injury. The sample can be a biological sample. In some aspects, a head CT scan is performed on the subject. In other aspects, a head CT scan is not performed on the subject.

[0142] As referred to herein, the at least one assay and, optionally, if performed, the at least one head CT scan can be performed simultaneously or sequentially, in any order. If performed sequentially, the assay and head CT scan can be performed within a clinically relevant time frame, such as, for example, within about 1 minute of each other, within about 2 minutes of each other, within about 3 minutes of each other, within about 4 minutes of each other, within about 5 minutes of each other, within about 10 minutes of each other, within about 15 minutes of each other, within about 20 minutes of each other, within about 25 minutes of each other, within about 30 minutes of each other, within about 45 minutes of each other, within about 50 minutes of each other, within about 60 minutes of each other, within about 1 hour of each other, within about 1.5 hours of each other, within about 2 hours of each other, within about 3 hours of each other, within about 4 hours of each other, within about 5 hours of each other, within about 6 hours of each other, within about 7 hours of each other, within about 8 hours of each other, within about 9 hours of each other, within about 10 hours of each other, within about 11 hours of each other, or within about 12 hours of each other.

[0143] In some embodiments, the methods described herein allow for identifying a subject who has or may have suffered a head injury as having a TBI based on one or more biomarker levels in a particular instance where the subject has undergone a head CT scan that is negative for TBI. The methods described herein allow for identifying subjects who have suffered a TBI but who may otherwise have been erroneously diagnosed as not having a TBI if the diagnosis was based solely on the results of one or more head CT scans.

[0144] In some embodiments, the method can include obtaining a sample about 24 hours to about 3 weeks after the 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 can also include detecting the antibody-biomarker complex thus obtained.

[0145] In some embodiments, the sample is collected about 24 hours after injury (e.g., actual injury) or suspected injury to the head, about 24.5 hours to about 3 weeks after the injury or suspected injury, about 25 hours to about 3 weeks after the injury or suspected injury, about 26 hours to about 3 weeks after the injury or suspected injury, about 27 hours to about 3 weeks after the injury or suspected injury, about 28 hours to about 3 weeks after the injury or suspected injury, about 29 hours to about 3 weeks after the injury or suspected injury, about 30 hours to about 3 weeks after the injury or suspected injury, about 31 hours to about 3 weeks after the injury or suspected injury, about 32 hours to about 3 weeks after the injury or suspected injury, about 33 hours to about 3 weeks after the injury or suspected injury, about 34 hours to about 3 weeks after the injury or suspected injury, about 35 hours to about 3 weeks after the injury or suspected injury, about 36 hours to about 3 weeks after the injury or suspected injury, about 37 hours to about 3 weeks after the injury or suspected injury, about 38 hours to about 3 weeks after the injury or suspected injury, about 39 hours to about 3 weeks after the injury or suspected injury, about 40 hours to about 3 weeks after the injury or suspected injury, about 41 hours to about 3 weeks after the injury or suspected injury, about 42 hours to about 3 weeks after the injury or suspected injury, about 43 hours to about 3 weeks after the injury or suspected injury, about 44 hours to about 3 weeks after the injury or suspected injury, about 45 hours to about 3 weeks after the injury or suspected injury, about 46 hours to about 3 weeks after the injury or suspected injury, about 47 hours to about 3 weeks after the injury or suspected injury, about 48 hours to about 3 weeks after the injury or suspected injury, about 49 hours to about 3 weeks after the injury or suspected injury, about 50 hours to about 3 5 hours to about 3 weeks, about 40 hours to about 3 weeks after the injury or suspected injury, about 48 hours to about 3 weeks after the injury or suspected injury, about 72 hours to about 3 weeks after the injury or suspected injury, about 96 hours to about 3 weeks after the injury or suspected injury, about 120 hours to about 3 weeks after the injury or suspected injury, about 6 days to about 3 weeks after the injury or suspected injury, about 7 days to about 3 weeks after the injury or suspected injury, about 8 days to about 3 weeks after the injury or suspected injury, about 9 days to about 3 weeks after the injury or suspected injury, about 10 days to about 3 weeks, about 11 days to about 3 weeks after the injury or suspected injury, within about 12 days to about 3 weeks after the injury or suspected injury, about 24.5 hours to about 2 weeks, about 25 hours to about 2 weeks after the injury or suspected injury, about 26 hours to about 2 weeks after the injury or suspected injury, about 27 hours to about 2 weeks after the injury or suspected injury, about 28 hours to about 2 weeks after the injury or suspected injury, about 29 hours to about 2 weeks after the injury or suspected injury, about 30 hours to about 2 weeks after the injury or suspected injury, about 35 hours to about 2 weeks after the injury, or within about 40 hours to about 2 weeks after the injury or suspected injury, about 48 hours to about 2 weeks after the injury or suspected injury, about 72 hours to about 2 weeks after the injury or suspected injury, about 96 hours to about 2 weeks after the injury or suspected injury, about 120 hours to about 2 weeks after the injury or suspected injury, about 6 days to about 2 weeks after the injury or suspected injury, about 7 days to about 2 weeks after the injury or suspected injury, about 8 days to about 2 weeks after the injury or suspected injury, about 9 days to about 2 weeks after the injury or suspected injury, about 10 days to about 2 weeks after the injury or suspected injury, about 24.The sample is taken from a subject, such as a human subject, within about 5 hours to about 1 week after injury or suspected injury, about 25 hours to about 1 week after injury or suspected injury, about 26 hours to about 1 week after injury or suspected injury, about 27 hours to about 1 week after injury or suspected injury, about 28 hours to about 1 week after injury or suspected injury, about 29 hours to about 1 week after injury or suspected injury, about 30 hours to about 1 week after injury or suspected injury, about 35 hours to about 1 week after injury or suspected injury, about 40 hours to about 1 week after injury or suspected injury, about 48 hours to about 1 week after injury or suspected injury, about 72 hours to about 1 week after injury or suspected injury, about 96 hours to about 1 week after injury or suspected injury, or about 120 hours to about 1 week after injury or suspected injury. For example, the sample may be taken from a subject, such as a human subject, within about 24 hours to about 1 week after injury or suspected injury to the head.5 hours, approx. 25 hours, approx. 26 hours, approx. 27 hours, approx. 28 hours, approx. 29 hours, approx. 30 hours, approx. 31 hours, approx. 32 hours, approx. 33 hours, approx. 34 hours, approx. 35 hours, approx. 36 hours, approx. 37 hours, approx. 38 hours, approx. 39 hours, approx. 40 hours, approx. 41 hours, approx. 42 hours, approx. 43 hours, approx. 44 hours, approx. 45 hours, approx. 46 hours, approx. 47 hours, approx. 48 hours, approx. 49 hours, approx. 50 hours, approx. 51 hours, approx. 52 hours, approx. 53 hours 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours, About 83 hours, about 84 hours, about 85 hours, about 86 hours, about 87 hours, about 88 hours, about 89 hours, about 90 hours, about 91 hours, about 92 hours, about 93 hours, about 94 hours, about 95 hours, about 96 hours, about 97 hours, about 98 hours, about 99 hours, about 100 hours, about 101 hours, about 102 hours, about 103 hours, about 104 hours, about 105 hours, about 106 hours, about 107 hours, about 108 hours, about 109 hours, about 110 The antibody can be harvested from a subject, such as a human subject, within about 111 hours, about 112 hours, about 113 hours, about 114 hours, about 115 hours, about 116 hours, about 117 hours, about 118 hours, about 119 hours, about 120 hours, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or about 21 days.

[0146] Generally, a reference level of a biomarker, such as UCH-L1, GFAP, or a combination thereof, can be used as a benchmark against which results obtained when assaying a test sample for UCH-L1 can be assessed. 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 be able to establish a link or association between the presence, amount, or concentration of the analyte and a particular stage or endpoint or a particular symptom of TBI. 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.

[0147] In some embodiments, the reference level for GFAP is about 5 pg / mL to about 115 pg / mL. In yet other embodiments, the reference level for GFAP is about 5 pg / mL to about 110 pg / mL. In other embodiments, the reference level for GFAP is about 5 pg / mL to about 105 pg / mL. In other embodiments, the reference level for GFAP is about 5 pg / mL to about 100 pg / mL. In other embodiments, the reference level for GFAP is about 5 pg / mL to about 90 pg / mL. In yet other embodiments, the reference level for GFAP is about 5 pg / mL to about 80 pg / mL. In yet further embodiments, the reference level for GFAP is about 5 pg / mL to about 70 pg / mL. In still other embodiments, the reference level for GFAP is about 5 pg / mL to about 60 pg / mL. In yet further aspects, the reference level for GFAP is about 5 pg / mL to about 50 pg / mL. In yet further aspects, the reference level for GFAP is about 5 pg / mL to about 40 pg / mL. In yet other embodiments, the reference level for GFAP is about 5 pg / mL to about 30 pg / mL. In yet other aspects, the reference level for GFAP is about 5 pg / mL to about 25 pg / mL. In yet other aspects, the reference level for GFAP is about 5 pg / mL to about 20 pg / mL.

[0148] In other aspects, the reference level for GFAP is about 10 pg / mL to about 115 pg / mL. In the improved method, the reference level for GFAP is about 10 pg / mL to about 110 pg / mL. In the improved method, the reference level for GFAP is about 10 pg / mL to about 105 pg / mL. In some embodiments, the reference level for GFAP is about 10 pg / mL to about 100 pg / mL. In some embodiments, the reference level for GFAP is about 10 pg / mL to about 90 pg / mL. In still other embodiments, the reference level for GFAP is about 10 pg / mL to about 80 pg / mL. In still other embodiments, the reference level for GFAP is about 10 pg / mL to about 70 pg / mL. In still other embodiments, the reference level for GFAP is about 10 pg / mL to about 60 pg / mL. In yet further embodiments, the reference level for GFAP is about 10 pg / mL to about 50 pg / mL. In yet further embodiments, the reference level for GFAP is about 10 pg / mL to about 40 pg / mL. In yet other embodiments, the reference level for GFAP is about 10 pg / mL to about 30 pg / mL. In yet other embodiments, the reference level for GFAP is about 10 pg / mL to about 25 pg / mL. In yet other embodiments, the reference level for GFAP is about 10 pg / mL to about 20 pg / mL.

[0149] In yet another aspect, the reference level for GFAP is about 15 pg / mL to about 115 pg / mL. In the improved method, the reference level for GFAP is about 15 pg / mL to about 110 pg / mL. In the improved method, the reference level for GFAP is about 15 pg / mL to about 105 pg / mL. In some embodiments, the reference level for GFAP is about 15 pg / mL to about 100 pg / mL. In some embodiments, the reference level for GFAP is about 15 pg / mL to about 90 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 80 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 70 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 60 pg / mL. In yet further embodiments, the reference level for GFAP is about 15 pg / mL to about 50 pg / mL. In yet further embodiments, the reference level for GFAP is about 15 pg / mL to about 40 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 30 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 25 pg / mL. In yet other embodiments, the reference level for GFAP is about 15 pg / mL to about 20 pg / mL.

[0150] In some embodiments, the methods further include treating the subject, such as a human subject having a subacute traumatic brain injury, with a traumatic brain injury treatment and / or monitoring the subject, as described below.

[0151] The nature of the assay used in the methods described herein is not critical, and the test can be any assay known in the art, such as, for example, immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, or protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, or chromatographic or spectroscopic methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). The assay can also be used in clinical chemistry formats as would be known to one of skill in the art. Such assays are described in further detail in Sections 4-8 herein. It is known in the art that values ​​(e.g., reference levels, cut-offs, thresholds, specificity, sensitivity, calibrator and / or control concentrations, etc.) used in 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 normalization can be performed is by applying a factor to the calibrators used in the assay to increase or decrease the sample concentration lead to obtain a slope that is consistent with the comparator method. Other methods for normalizing 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).

[0152] 3. Treatment and monitoring of subjects with head injuries The subject identified by the above method may be treated or monitored. In some embodiments, the method further comprises treating the subject, such as a human subject, 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 a subject suffering from a mild TBI, treatment may include one or more of the following: 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 of 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 and put the individual into a drug-induced coma, or pharmaceutical or biopharmaceutical drugs known or developed in the future for the treatment of TBI), one or more surgical procedures (such as, for example, removing a hematoma, repairing a skull fracture, decompressive craniectomy, etc.), protecting the airway, and one or more therapies (such as, for example, one or more of rehabilitation, cognitive behavioral therapy, anger management, counseling psychology, etc.). In some embodiments, the method further includes monitoring the subject, such as a human subject. In some embodiments, the subject may be monitored using CT scans and MRI procedures.

[0153] 4. Methods for Measuring UCH-L1 Levels In the methods described above, UCH-L1 levels can be measured by any means, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, antibody-dependent methods such as SDS-PAGE and Western blot analysis, protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, or chromatographic methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS), or spectroscopic methods. The assays can also be utilized in clinical chemistry formats that would be known to those skilled in the art.

[0154] 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.

[0155] 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.

[0156] In some embodiments, the sample is diluted or neat. The sample may be about 1 to about 25 microliters, about 1 to about 24 microliters, about 1 to about 23 microliters, about 1 to about 22 microliters, about 1 to about 21 microliters, about 1 to about 20 microliters, about 1 to about 18 microliters, about 1 to about 17 microliters, about 1 to about 16 microliters, about 15 microliters, or about 1 microliter, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, about 10 microliters, about 11 microliters, about 12 microliters, about 13 microliters, about 14 microliters, about 15 microliters, about 16 microliters, about 17 microliters, about 18 microliters, about 19 microliters, about 20 microliters, about 21 microliters, about 22 microliters, about 23 microliters, about 24 microliters, or about 25 microliters. In some embodiments, the sample is from about 1 to about 150 microliters or less, or from about 1 to about 25 microliters or less.

[0157] Some meters other than point-of-care devices (e.g., the Abbott Laboratories ARCHITECT meter and other core testing meters) may be capable of measuring UCH-L1 levels in samples of 25,000 pg / mL or greater.

[0158] 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.

[0159] 5.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.

[0160] 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.

[0161] Human UCH-L1 has the following amino acid sequence:

[0162] [ka] may have.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] The antibody is distinguishable from known antibodies in that it possesses a different biological function than antibodies known in the art.

[0169] (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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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)).

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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 / 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, creating two antigen-binding sites. See also U.S. Patent No. 6,632,926 to Chen et al., which is hereby incorporated by reference in its entirety, and discloses antibody variants in which one or more amino acids are inserted into the hypervariable region of a parent antibody and the binding affinity for the target antigen is at least about twice as strong as the binding affinity of the parent antibody for the antigen.

[0185] 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.

[0186] 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.

[0187] 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 (18 radionuclides such as rhenium-188 (188Re) and rhenium-188 (6Re); antibiotics such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin; diphtheria toxin, Pseudomonas aeruginosa 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 atra), 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).

[0188] 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.

[0189] (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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] (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.

[0198] (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.

[0199] (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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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).

[0204] As an alternative to screening recombinant antibody libraries with phage libraries, other methods known in the art for screening large combinatorial libraries can be applied to identify the antibodies of the present disclosure. One type of alternative expression system expresses recombinant antibody libraries 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.

[0205] 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. 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.

[0206] 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 particularly mammalian cells) are more likely than prokaryotic cells to assemble, properly fold, and secrete immunologically active antibodies.

[0207] Exemplary mammalian host cells for expressing recombinant antibodies of the disclosure include Chinese hamster ovary cells (CHO cells) (including dhfr-CHO cells, which are described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980), used with the DHFR selection marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol. 159:601-621 (1982)), NS0 myeloma cells, COS cells, and SP2 cells. When 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.

[0208] 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 and one light chain is an antibody of the present disclosure (i.e., binds human UCH-L1) and the other heavy and light chains are specific for an antigen other than human UCH-L1 may be generated by crosslinking an antibody of the present disclosure to a second antibody via standard chemical crosslinking methods.

[0209] In a preferred system for recombinantly expressing the antibody or antigen-binding portion thereof of the present disclosure, 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 and antibody light chain genes is operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells transfected with the vector using methotrexate selection / amplification. The selected transformant host cells are cultured to allow expression of the antibody heavy and light chains and for intact antibody to be recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. Still further, the present disclosure provides a method of synthesizing a recombinant antibody of the present disclosure by culturing a host cell of the present disclosure in an appropriate culture medium until a recombinant antibody of the present disclosure is synthesized. The method may further include a step of isolating the recombinant antibody from the culture medium.

[0210] (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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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).

[0218] Alternatively, the antibodies described in WO 2018 / 067468 and / or Bazarian et al., “Accuracy of a rapid GFAP / UCH-L1 test for the prediction of intracranial injuries on head CT after mild traumatic brain injury,” Acad. Emerg. Med. (August 6, 2021), the contents of which are incorporated herein by reference, may be used.

[0219] 6. Methods for Measuring GFAP Levels In the above methods, 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 or spectroscopic methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS), and the assays can also be utilized in clinical chemistry formats that would be known to one of skill in the art.

[0220] 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.

[0221] 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.

[0222] In some embodiments, the sample is diluted or neat. The sample can be about 1 to about 25 microliters, about 1 to about 24 microliters, about 1 to about 23 microliters, about 1 to about 22 microliters, about 1 to about 21 microliters, about 1 to about 20 microliters, about 1 to about 18 microliters, about 1 to about 17 microliters, about 1 to about 16 microliters, about 15 microliters or about 1 microliter, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, about 10 microliters, about 11 microliters, about 12 microliters, about 13 microliters, about 14 microliters, about 15 microliters, about 16 microliters, about 17 microliters, about 18 microliters, about 19 microliters, about 20 microliters, about 21 microliters, about 22 microliters, about 23 microliters, about 24 microliters, or about 25 microliters. In some embodiments, the sample is from about 1 to about 150 microliters or less, or from about 1 to about 25 microliters or less.

[0223] Some instruments other than point-of-care devices (eg, Abbott laboratory instruments ARCHITECT, Alinity, and other core laboratory instruments) may be able to measure GFAP levels in samples higher or greater than 25,000 pg / mL.

[0224] 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.

[0225] 7.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.

[0226] 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.

[0227] Human GFAP has the following amino acid sequence:

[0228] [ka] may have.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] 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.

[0233] A humanized antibody can be an antibody molecule derived from a non-human species that binds to a desired antigen, having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.

[0234] The antibody is distinguishable from known antibodies in that it possesses a different biological function than antibodies known in the art.

[0235] (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.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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)).

[0246] 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.

[0247] 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.

[0248] 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 containing 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.

[0249] 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.

[0250] 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.

[0251] 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.

[0252] 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.

[0253] 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. Agents suitable for conjugation to the antibodies to achieve an anti-tumor 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; radioactive materials 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; bacterial, plant, and other toxins such as diphtheria toxin, pseudomonas exotoxin A, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-alpha toxin, cytotoxin from the Taiwan cobra (naja atra), and gelonin (a plant toxin); restrictocin (Aspergillus restrictus) restrictus), saporin (a ribosome-inactivating protein from Saponaria officinalis), and ribonuclease; tyrosine kinase inhibitors; ly207702 (a difluorinated purine nucleoside); liposomes containing anticystants (e.g., plasmids encoding antisense oligonucleotides, toxins, methotrexate, etc.); and other antibodies or antibody fragments such as F(ab).

[0254] 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.

[0255] (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.

[0256] Methods for producing monoclonal antibodies, as well as antibodies produced by the methods, may include culturing hybridoma cells secreting the antibodies of the present disclosure, preferably generated by fusing splenocytes 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 that secrete antibodies capable of binding to the polypeptides of the present disclosure. Briefly, rats may 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 adjuvants, RIBI (muramyl dipeptides), or ISCOM (immunostimulating complexes). Such adjuvants may protect the polypeptide from rapid dispersal by encapsulating it in localized deposits, or such adjuvants may contain substances that stimulate the host to secrete factors that 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.

[0257] 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.

[0258] 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.

[0259] In another embodiment, antibody-producing immortalized hybridomas may be prepared from the immunized animal. After immunization, the animal is 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.

[0260] 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.

[0261] 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.

[0262] 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 generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to generate two identical Fab fragments) or pepsin (to generate F(ab')2 fragments). The F(ab')2 fragment of an IgG molecule retains the two antigen-binding sites of the larger ("parent") IgG molecule, including both light chains (containing the light chain variable 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.

[0263] (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 known in the art as the selective lymphocyte antibody method (SLAM), as described in U.S. Patent No. 5,627,052; WO 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.

[0264] (3) Anti-GFAP monoclonal antibody using transgenic animals In another aspect of the disclosure, the antibodies are produced by immunizing a non-human animal that contains a portion or all of the human immunoglobulin locus with the GFAP antigen. In one embodiment, the non-human animal is a XENOMOUSE® transgenic mouse, an engineered mouse strain that contains a large fragment of the human immunoglobulin locus 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.

[0265] (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, and 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.

[0266] 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 a peptide comprising human 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. A particular K off To select an antibody of the present disclosure having a particular binding affinity for GFAP, such as an antibody that dissociates from human GFAP with a rate constant of the desired K off Antibodies with specific IC 50 To select antibodies of the disclosure having specific neutralizing activity against GFAP, such as antibodies having the following standard methods known in the art for assessing inhibition of GFAP activity, one may use.

[0267] In one aspect, the disclosure relates to an isolated antibody, or an 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.

[0268] 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.

[0269] 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).

[0270] As an alternative to screening recombinant antibody libraries by phage display, other methods known in the art for screening large combinatorial libraries can 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.

[0271] 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. 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.

[0272] 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 is preferred, with expression in mammalian host cells being most preferred, since such eukaryotic cells (and, in particular, mammalian cells) are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.

[0273] Exemplary mammalian host cells for expressing recombinant antibodies of the disclosure include Chinese hamster ovary cells (CHO cells) (including dhfr-CHO cells, which are described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980), used with the DHFR selection marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol. 159:601-621 (1982)), NS0 myeloma cells, COS cells, and SP2 cells. When 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.

[0274] The host cells can also be used to produce 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 this disclosure. Recombinant DNA technology can also be used to remove some or all of the DNA encoding one 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 this disclosure. Furthermore, bifunctional antibodies, in which one heavy and one light chain is an antibody of this disclosure (i.e., binds human GFAP) and the other heavy and light chains are specific for an antigen other than human GFAP, can be made by crosslinking the antibody of this disclosure to a second antibody by standard chemical crosslinking methods.

[0275] In a preferred system for recombinant expression of 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 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 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. Additionally, the present disclosure 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 include isolating the recombinant antibody from the culture medium.

[0276] (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.

[0277] 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.

[0278] The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. The humanized antibody may contain 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.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] 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.

[0283] 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).

[0284] 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.

[0285] 8. 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)).

[0286] 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-L1 and / or GFAP antibodies in an immunoassay. The presence or amount of an analyte (e.g., UCH-L1 and / or GFAP) can be determined using an antibody to detect specific binding to the analyte (e.g., UCH-L1 and / or GFAP). For example, an antibody or an antibody fragment thereof can specifically bind to the analyte (e.g., UCH-L1 and / or GFAP). If desired, one or more of the antibodies can be used in combination with one or more commercially available monoclonal / polyclonal antibodies. Such antibodies are commercially available from companies such as R&D Systems, Inc. (Minneapolis, MN) and Enzo Life Sciences International, Inc. (Plymouth Meeting, PA).

[0287] The presence or amount of an analyte (e.g., UCH-L1 and / or GFAP) present in a body sample can be readily determined using immunoassays such as sandwich immunoassays (e.g., monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonal sandwich immunoassays, including radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme immunometric assay (ELISA) (e.g., Quantikine ELISA Assay, R&D Systems, Minneapolis, MN)). An example of a point-of-care device that can be used is i-STAT (Abbott, Laboratories, Abbott Park, IL). Other methods that can be used are chemiluminescent microparticle immunoassays, particularly those using, for example, the ARCHITECT or the Alinity automated analyzer (Abbott Laboratories, Abbott Other methods include, for example, mass spectrometry and immunohistochemistry (e.g., involving sections from tissue biopsies) using anti-analyte (e.g., anti-UCH-L1 and / or anti-GFAP) antibodies (monoclonal, polyclonal, chimeric, humanized, human, etc.) against the analyte (e.g., UCH-L1 and / or GFAP) or antibody fragments thereof. Other detection methods are described, for example, in U.S. Patent Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 6,020,631; 6,120,291; 6,220,292; 6,311,142; 6,113,855; 6,019,944; 5,985,579; 6,311,142; 6,220,292; 6,311,14 ... The detection methods described in the following publications include those described in: 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524 and 5,480,792. Specific immunological binding of an antibody to an analyte (e.g., UCH-L1 and / or GFAP) can be detected via a direct label, such as a fluorescent or luminescent tag, metal or radionuclide, or via an indirect label, such as alkaline phosphatase or horseradish peroxidase, conjugated to the antibody.

[0288] The use of immobilized antibodies or fragments thereof can be incorporated into immunoassays. Antibodies can be immobilized to a variety of supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (such as a microtiter plate), or a small piece of solid substrate material. An assay strip can be prepared by coating an antibody or antibodies in an array on a solid support. The strip can then be dipped into the test sample and rapidly processed through washing and detection steps to generate a measurable signal, such as a colored spot.

[0289] Homogeneous formats may be used. For example, a mixture may be prepared after a test sample is obtained from a subject. The mixture contains the test sample to be evaluated for an analyte (e.g., UCH-L1 and / or GFAP), a first specific binding partner, and a second specific binding partner. The order in which the test sample, the first specific binding partner, and the second specific binding partner are added to form the mixture is not critical. The test sample is contacted with the first specific binding partner and the second specific binding partner simultaneously. In some embodiments, the first specific binding partner and any UCH-L1 and / or GFAP contained in the test sample can form a first specific binding partner-analyte (e.g., UCH-L1 and / or GFAP) antigen complex, and the second specific binding partner can form a first specific binding partner-analyte of interest (e.g., UCH-L1 and / or GFAP)-second specific binding partner complex. In some embodiments, the second specific binding partner and any UCH-L1 and / or GFAP contained in the test sample can form a second specific binding partner-analyte (e.g., UCH-L1) antigen complex, and the first specific binding partner can form a first specific binding partner-analyte of interest (e.g., UCH-L1 and / or GFAP)-second specific binding partner complex. The first specific binding partner can be an anti-analyte antibody (e.g., an anti-UCH-L1 antibody that binds to an epitope having an amino acid sequence comprising at least three consecutive amino acids of SEQ ID NO:1 or an anti-GFAP antibody that binds to an epitope having an amino acid sequence comprising at least three consecutive amino acids of SEQ ID NO:2). The second specific binding partner can be an anti-analyte antibody (e.g., an anti-UCH-L1 antibody that binds to an epitope having an amino acid sequence comprising at least three consecutive amino acids of SEQ ID NO:1 or an anti-GFAP antibody that binds to an epitope having an amino acid sequence comprising at least three consecutive amino acids of SEQ ID NO:2). Additionally, the second specific binding partner is labeled with or contains a detectable label, as described above.

[0290] Heterogeneous formats may be used. For example, a first mixture may be prepared after a test sample is obtained from a subject. The mixture contains the test sample to be evaluated for an analyte (e.g., UCH-L1 and / or GFAP) and a first specific binding partner, where the first specific binding partner and any UCH-L1 and / or GFAP contained in the test sample form a first specific binding partner-analyte (e.g., UCH-L1 and / or GFAP) antigen complex. The first specific binding partner may be an anti-analyte antibody (e.g., an anti-UCH-L1 antibody that binds to an epitope having an amino acid sequence that includes at least 3 consecutive amino acids of SEQ ID NO: 1 or an anti-GFAP antibody that binds to an epitope having an amino acid sequence that includes at least 3 consecutive amino acids of SEQ ID NO: 2). The order in which the test sample and the first specific binding partner are added to form the mixture is not critical.

[0291] The first specific binding partner may be immobilized on a solid phase. The solid phase used in the immunoassay (for the first specific binding partner and, optionally, the second specific binding partner) may be any solid phase known in the art, including, but not limited to, magnetic particles, beads, test tubes, microtiter plates, cuvettes, membranes, scaffolding molecules, thin films, filter paper, disks and chips. In embodiments where the solid phase is a bead, the bead may be a magnetic bead or a magnetic particle. The magnetic bead / particle may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic or magnetic fluid. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO2, MnAs, MnBi, EuO and NiO / Fe. Examples of ferrimagnetic materials include NiFe2O4, CoFe2O4, Fe3O4 (or FeO.Fe2O3). The beads may be magnetic and have a solid core portion surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion may be a layer around a non-magnetic core. The solid support on which the first specific binding member is immobilized may be stored in dry form or in liquid. The magnetic beads may be placed under a magnetic field before or after contacting the sample with the magnetic beads on which the first specific binding member is immobilized.

[0292] After the mixture containing the first specific binding partner-analyte (e.g., UCH-L1 or GFAP) antigen complex is formed, any unbound analyte (e.g., UCH-L1 and / or GFAP) is removed from the complex using any technique known in the art. For example, unbound analyte (e.g., UCH-L1 and / or GFAP) can be removed by washing. However, it is desirable that the first specific binding partner be present in excess of any analyte (e.g., UCH-L1 and / or GFAP) present in the test sample, so that the first specific binding partner binds to all analytes (e.g., UCH-L1 and / or GFAP) present in the test sample.

[0293] After any unbound analyte (e.g., UCH-L1 and / or GFAP) is removed, a second specific binding partner is added to the mixture to form a first specific binding partner-analyte of interest (e.g., UCH-L1 and / or GFAP)-second specific binding partner complex. The second specific binding partner can be an anti-analyte antibody (e.g., an anti-UCH-L1 antibody that binds to an epitope having an amino acid sequence comprising at least 3 consecutive amino acids of SEQ ID NO:1 or an anti-GFAP antibody that binds to an epitope having an amino acid sequence comprising at least 3 consecutive amino acids of SEQ ID NO:2). Additionally, the second specific binding partner is labeled with or contains a detectable label, as described above.

[0294] The use of immobilized antibodies or fragments thereof can be incorporated into immunoassays. Antibodies can be immobilized to a variety of supports, such as magnetic particles or chromatographic matrix particles (such as magnetic beads), latex particles or surface-modified latex particles, polymers or polymer films, plastics or plastic films, planar substrates, the surface of an assay plate (such as a microtiter plate), or small pieces of solid substrate material. An assay strip can be prepared by coating an antibody or multiple antibodies in an array on a solid support. The strip can then be dipped into the test sample and rapidly processed through washing and detection steps to generate a measurable signal, such as a colored spot.

[0295] In some embodiments, it may be possible to select other antibodies that may also help maintain the dynamic range and low-end sensitivity of the immunoassay. For example, it may be useful to select at least one first antibody (such as a capture antibody or first specific binding partner) that binds to an epitope near the N-terminus of the 38 kDa BDP and at least one second antibody (such as a detection antibody or second specific binding partner) that binds to an epitope near the center of the 38 kDa BDP, e.g., near the center of the 38 kDa BDP, and does not overlap with the first antibody. It is believed that other variants are possible and that one skilled in the art can easily test other variants, such as by checking the binding of antibodies to different epitopes by examining binding to short peptides, and then screening the antibody pairs using low calibrator concentrations. Furthermore, the selection of antibodies with different affinities to GFAP can also help maintain or increase the dynamic range of the assay. GFAP antibodies have been described in the literature and are commercially available.

[0296] (1) Sandwich immunoassay Sandwich immunoassays measure the amount of antigen between two layers of antibodies (i.e., at least one capture antibody) and detection antibodies (i.e., at least one detection antibody). The capture antibody and detection antibody bind to different epitopes on the analyte of interest, such as an antigen, e.g., UCH-L1 and / or GFAP. It is desirable that the binding of the capture antibody to the epitope does not interfere with the binding of the detection antibody to the epitope. Either monoclonal or polyclonal antibodies can be used as the capture antibody and detection antibody in sandwich immunoassays.

[0297] Generally, at least two antibodies are utilized to separate and quantify an analyte (e.g., UCH-L1 and / or GFAP) in a test sample. More specifically, at least two antibodies bind to a certain epitope of the analyte (e.g., UCH-L1 and / or GFAP) to form an immune complex called a "sandwich". One or more antibodies may be used to capture the analyte (e.g., UCH-L1 and / or GFAP) in the test sample (these antibodies are often referred to as "capture" antibodies or "capture" antibodies), and one or more antibodies are used to bind a detectable (i.e., quantifiable) label to the sandwich (these antibodies are often referred to as "detection" antibodies or "detection" antibodies). In a sandwich assay, the binding of an antibody to its epitope is not attenuated by the binding of any other antibody in the assay to its respective epitope. The antibodies are selected such that one or more first antibodies, when contacted with a test sample suspected of containing an analyte (e.g., UCH-L1 and / or GFAP), do not bind to all or part of the epitope recognized by a second antibody or a subsequent antibody, thereby not interfering with the ability of one or more second detection antibodies to bind to the analyte (e.g., UCH-L1 and / or GFAP).

[0298] The antibody may be used as a first antibody in the immunoassay. The antibody immunospecifically binds to an epitope on the analyte (e.g., UCH-L1 and / or GFAP). In addition to the antibody of the present disclosure, the immunoassay may include a second antibody that immunospecifically binds to an epitope not recognized or bound by the first antibody.

[0299] A test sample suspected of containing an analyte (e.g., UCH-L1 and / or GFAP) can be contacted simultaneously or sequentially with at least one first capture antibody (or antibodies) and at least one second detection antibody. In a sandwich assay format, a test sample suspected of containing an analyte (e.g., UCH-L1 and / or GFAP) is first contacted with at least one first capture antibody that specifically binds to a particular epitope under conditions that allow the formation of a first antibody-analyte (e.g., UCH-L1 and / or GFAP) antigen complex. If more than one capture antibody is used, a first plurality of capture antibodies-UCH-L1 and / or GFAP antigen complexes are formed. In a sandwich assay, an antibody, preferably at least one capture antibody, is used in molar excess to the maximum amount of analyte (e.g., UCH-L1 and / or GFAP) expected in the test sample. For example, about 5 μg / ml to about 1 mg / ml of antibody per ml of microparticle coating buffer can be used.

[0300] i. Anti-UCH-L1 capture antibody Optionally, prior to contacting the test sample with the at least one first capture antibody, the at least one first capture antibody may be bound to a solid support that facilitates separation of the first antibody-analyte (e.g., UCH-L1 and / or GFAP) complex from the test sample. Any solid support known in the art may be used, including, but not limited to, solid supports made of polymeric materials in the form of wells, tubes, or beads (such as microparticles). The antibodies (or antibodies) may be bound to the solid support by adsorption, by covalent binding using chemical coupling agents, or by other means known in the art, provided that such binding does not interfere with the ability of the antibody to bind to the analyte (e.g., UCH-L1 and / or GFAP). Furthermore, if necessary, the solid support may be derivatized to allow reactivity with various functional groups on the antibody. Such derivatization requires the use of certain coupling agents, such as, but not limited to, maleic anhydride, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0301] The test sample suspected of containing the analyte (e.g., UCH-L1 and / or GFAP) is then incubated to allow for the formation of a first capture antibody (or antibodies)-analyte (e.g., UCH-L1 and / or GFAP) complex. The incubation may be carried out at a pH of about 4.5 to about 10.0, at a temperature of about 2° C. to about 45° C., for a time of at least about 1 minute to about 18 hours, about 2-6 minutes, about 7-12 minutes, about 5-15 minutes, or about 3-4 minutes.

[0302] ii. Detection antibody After formation of the first / multiple capture antibody-analyte (e.g., UCH-L1 and / or GFAP) complex, the complex is then contacted with at least one second detection antibody (under conditions that allow for the formation of a first / multiple antibody-analyte (e.g., UCH-L1 and / or GFAP) antigen-second antibody complex). In some embodiments, the test sample is contacted with the detection antibody simultaneously with the capture antibody. When the first antibody-analyte (e.g., UCH-L1 and / or GFAP) complex is contacted with more than one detection antibody, a first / multiple capture antibody-analyte (e.g., UCH-L1 and / or GFAP)-multiple antibody detection complex is formed. When at least the second (and subsequent) antibody is contacted with the first antibody-analyte (e.g., UCH-L1 and / or GFAP) complex, an incubation time under the same conditions as described above as for the first antibody is required for the formation of the first / multiple antibody-analyte (e.g., UCH-L1 and / or GFAP)-second / multiple antibody complex. Preferably, at least one second antibody contains a detectable label. The detectable label can be attached to at least one second antibody before, simultaneously with, or after the formation of the first / multiple antibody-analyte (e.g., UCH-L1 and / or GFAP)-second / multiple antibody complex. Any detectable label known in the art can be used.

[0303] Chemiluminescence assays can be performed according to the method described in Adamczyk et al., Anal. Chim. Acta, 579(1):61-67 (2006). While any suitable assay format can be used, a microplate chemiluminometer (Mithras LB-940, Berthold Technologies USA, LLC, Oak Ridge, TN) allows for rapid assay of multiple samples in small volumes. The chemiluminometer can be equipped with multiple reagent injectors using 96-well black polystyrene microplates (Costar, model no. 3792). Each sample can be added to an individual well, followed by simultaneous / sequential addition of other reagents as determined by the type of assay used. It is desirable to avoid the formation of pseudobases in neutral or basic solutions that utilize acridinium aryl esters, such as by acidification. The chemiluminescence response is then recorded for each well. In this regard, the time to record a chemiluminescent response will depend, in part, on the delay between the addition of the reagent utilized and the addition of the particular acridinium.

[0304] The order in which the test sample and the specific binding partner are added to form a mixture for chemiluminescence assay is not critical. When the first specific binding partner is detectably labeled with an acridinium compound, a detectably labeled first specific binding partner-antigen (e.g., UCH-L1 and / or GFAP) complex is formed. Alternatively, when a second specific binding partner is used and the second specific binding partner is detectably labeled with an acridinium compound, a detectably labeled first specific binding partner-analyte (e.g., UCH-L1 and / or GFAP)-second specific binding partner complex is formed. Any unbound specific binding partner, whether labeled or unlabeled, can be removed from the mixture using any technique known in the art, such as washing.

[0305] Hydrogen peroxide can be generated in situ in the mixture or can be administered or provided to the mixture prior to, simultaneously with, or after the addition of the acridinium compound described above. Hydrogen peroxide can be generated in situ in a number of ways, such as those apparent to those skilled in the art.

[0306] Alternatively, a source of hydrogen peroxide may simply be added to the mixture. For example, the source of hydrogen peroxide may be one or more buffers or other solutions known to contain hydrogen peroxide. In this regard, a solution of hydrogen peroxide may simply be added.

[0307] Upon addition of at least one basic solution to the sample, either simultaneously or subsequently, a detectable signal, i.e., a chemiluminescent signal, is generated that indicates the presence of the analyte (e.g., UCH-L1 and / or GFAP). The basic solution contains at least one base and has a pH of 10 or more, preferably 12 or more. Examples of basic solutions include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide, calcium carbonate, and calcium bicarbonate. The amount of basic solution added to the sample depends on the concentration of the basic solution. Based on the concentration of the basic solution used, one skilled in the art can easily determine the amount of basic solution added to the sample. Labels other than chemiluminescent labels can also be used. For example, enzyme labels (including but not limited to alkaline phosphatase) can be used.

[0308] The generated chemiluminescent or other signal can be detected using routine techniques known to those skilled in the art. Based on the intensity of the generated signal, the amount of the analyte of interest (e.g., UCH-L1 and / or GFAP) in the sample can be quantified. Specifically, the amount of the analyte of interest (e.g., UCH-L1 and / or GFAP) in the sample is proportional to the intensity of the generated signal. The amount of the analyte (e.g., UCH-L1 and / or GFAP) present can be quantified by comparing the amount of light generated to a calibration curve for the analyte (e.g., UCH-L1 and / or GFAP) or by comparison to a reference standard. A calibration curve can be generated by mass spectrometry, gravimetry, and other techniques known in the art using serial dilutions or solutions of known concentrations of the analyte (e.g., UCH-L1 and / or GFAP).

[0309] (2) Forward competitive inhibition assay In the forward competition format, an aliquot of a known concentration of labeled analyte of interest (e.g., an analyte (e.g., UCH-L1 and / or GFAP) having a fluorescent label, a tag conjugated with a cleavable linker, etc.) is used to compete with the analyte of interest (e.g., UCH-L1 and / or GFAP) in a test sample for binding to an analyte of interest antibody (e.g., a UCH-L1 and / or GFAP antibody).

[0310] In a forward competitive assay, an immobilized specific binding partner (such as an antibody) can be contacted sequentially or simultaneously with the test sample and the labeled analyte of interest, fragment of the analyte of interest, or variant of the analyte of interest. The analyte of interest peptide, fragment of the analyte of interest, or variant of the analyte of interest can be labeled with any detectable label, including detectable labels composed of a tag conjugated with a cleavable linker. In this assay, the antibody can be immobilized to a solid support. Alternatively, the antibody can be linked to an antibody, such as an antispecies antibody, immobilized on a solid support, such as a microparticle or a planar substrate.

[0311] The labeled analyte of interest, the test sample and the antibody are incubated under conditions similar to those described above in relation to the sandwich assay format. Two different molecular species of antibody-analyte of interest complexes can then be generated. Specifically, one of the generated antibody-analyte of interest complexes contains a detectable label (such as a fluorescent label), while the other antibody-analyte of interest complex does not contain a detectable label. The antibody-analyte of interest complex can be, but does not have to be, separated from the rest of the test sample before quantifying the detectable label. The amount of detectable label in the antibody-analyte of interest complex is then quantified, regardless of whether the antibody-analyte of interest complex is separated from the rest of the test sample. The concentration of the analyte of interest (such as a membrane-associated analyte of interest, a soluble analyte of interest, a fragment of a soluble analyte of interest, a mutant of the analyte of interest (either a membrane-associated analyte of interest or a soluble analyte of interest) or any combination thereof) in the test sample can then be determined, for example, as described above.

[0312] (3) Reverse competitive inhibition assay In a reverse competition assay, an immobilized analyte of interest (eg, UCH-L1 and / or GFAP) can be contacted sequentially or simultaneously with the test sample and at least one labeled antibody.

[0313] The analyte of interest is bound to a solid support, such as those solid supports discussed above in connection with the sandwich assay format.

[0314] The immobilized analyte of interest, the test sample and at least one labeled antibody are incubated under conditions similar to those described above in connection with the sandwich assay format. Two different molecular species of analyte of interest-antibody complexes can then be generated. Specifically, one of the generated analyte of interest-antibody complexes is immobilized and contains a detectable label (such as a fluorescent label), while the other analyte of interest-antibody complex is not immobilized and does not contain a detectable label. The non-immobilized analyte of interest-antibody complex and the remaining test sample are removed from the presence of the immobilized analyte of interest-antibody complex through techniques known in the art, such as washing. Then, once the non-immobilized analyte of interest-antibody complex is removed, the amount of detectable label in the immobilized analyte of interest-antibody complex after cleavage of the tag is quantified. The concentration of the analyte of interest in the test sample can then be determined by comparing the quantity of detectable label as described above.

[0315] (4) One-step immunoassay or "capture-on-the-fly" assay In a capture-on-the-fly assay, the solid substrate is pre-coated with an immobilizing agent. The capture agent, analyte (e.g., UCH-L1 and / or GFAP) and detection agent are added together to the solid substrate, followed by a washing step before detection. The capture agent is capable of binding to the analyte (e.g., UCH-L1 and / or GFAP) and includes a ligand for the immobilizing agent. The capture agent and detection agent can be an antibody or any other moiety capable of capture or detection, as described herein or known in the art. The ligand can include a peptide tag, and the immobilizing agent can include an anti-peptide tag antibody. Alternatively, the ligand and immobilizing agent can be any pair of agents capable of binding together (e.g., specific binding pairs and other pairs known in the art, etc.) as utilized for the capture-on-the-fly assay. More than one analyte can be measured. In some embodiments, the solid substrate can be coated with an antigen, and the analyte being analyzed is an antibody.

[0316] In certain other embodiments of one-step immunoassays or "capture on the fly", a solid support (such as a microparticle) pre-coated with an immobilizing agent (such as biotin, streptavidin, etc.) and at least a first specific binding member and a second specific binding member (functioning as a capture reagent and a detection reagent, respectively) are used. The first specific binding member comprises a ligand for the immobilizing agent (e.g., if the immobilizing agent on the solid support is streptavidin, the ligand on the first specific binding member can be biotin) and also binds to the analyte of interest (e.g., UCH-L1 and / or GFAP). The second specific binding member comprises a detectable label and binds to the analyte of interest (e.g., UCH-L1 and / or GFAP). The solid support and the first specific binding member and the second specific binding member can be added (sequentially or simultaneously) to the test sample. The ligand on the first specific binding member binds to the immobilizing agent on the solid support to form a solid support / first specific binding member complex. Any analyte of interest present in the sample binds to the solid support / first specific binding member complex to form a solid support / first specific binding member / analyte complex. The second specific binding member binds to the solid support / first specific binding member / analyte complex and the detectable label is detected. An optional wash step may be utilized prior to detection. In certain embodiments in a one-step assay, more than one analyte may be measured. In certain other embodiments, more than two specific binding members may be utilized. In certain other embodiments, multiple detectable labels may be added. In certain other embodiments, multiple analytes of interest may be detected or their amounts, levels or concentrations may be measured, determined or assessed.

[0317] The use of capture on the fly assays can be in a variety of formats, as described herein and known in the art, for example the format can be a sandwich assay, such as those described above, but can alternatively be a competitive assay, can utilize monospecific binding members, or can use other variations, such as those known in the art.

[0318] 9. Other Factors The above-mentioned diagnostic, prognostic and / or ...

Claims

1. An improved method for aiding in the diagnosis and assessment of a subject who has suffered or may have suffered a head injury, the method comprising: (1) an assay on a sample obtained from the subject from about 24 hours to about 3 weeks after the actual or suspected head injury to measure or detect a level of a biomarker in the sample, the biomarker including glial fibrillary acidic protein (GFAP); and (2) simultaneously or sequentially performing a head computed tomography (CT) scan on the subject within a clinically relevant time frame, and diagnosing the subject as more likely than not to have traumatic brain injury (TBI) if the level of the biomarker is higher than a reference level and the head CT scan is negative for TBI.

2. An improvement in a method for aiding in the diagnosis and assessment of a human subject who has suffered or may have suffered a head injury, the method comprising performing an assay on a sample obtained from the subject from about 24 hours to about 3 weeks after the actual or suspected head injury to measure or detect a level of a biomarker in the sample, the biomarker comprising glial fibrillary acidic protein (GFAP), and diagnosing the subject as more likely than not to have traumatic brain injury (TBI) if the level of the biomarker is higher than a reference level and a head computed tomography (CT) scan of the subject within a clinically relevant time frame is negative for TBI or if a head CT scan is not performed on the subject, the improvement comprises diagnosing the subject as more likely than not to have traumatic brain injury (TBI).

3. The improvement of claim 1 or claim 2, further comprising treating the subject for TBI if the level of the biomarker is higher than the reference level and, optionally, if performed, a head CT scan is negative for TBI.

4. The improvement of any of claims 1 to 3, wherein the reference level correlates with a cut-off level related to: (a) a level in a subject suffering from a head injury; (b) occurrence of TBI in a subject; (c) a stage of TBI in a subject, such as mild, moderate, severe, or moderate-severe; (d) loss of consciousness in a subject; (e) a positive rather than negative MRI for TBI; (f) occurrence of amnesia in a subject (i.e. amnesia is present vs. not present); or (g) a severity of TBI in a subject.

5. The improvement of any of claims 1 to 4, wherein the sample is taken from about (a) 24.5 hours to about 3 weeks; (b) 25 hours to about 3 weeks; (c) 1 week; (d) 2 weeks; or (e) 3 weeks after actual or suspected trauma to the head.

6. The improvement according to any one of claims 1 to 5, wherein the measurement of the level of GFAP is carried out by immunoassay or clinical chemistry assay.

7. The improvement according to any one of claims 1 to 6, wherein the assay is carried out using a point-of-care assay or single molecule detection.

8. The improvement according to any one of claims 1 to 7, wherein the sample is selected from the group consisting of a blood sample, a urine sample, a cerebrospinal fluid sample, a tissue sample, a body fluid sample, a saliva sample, an oropharyngeal specimen, and a nasopharyngeal specimen.

9. The improvement according to any of claims 1 to 8, wherein the sample is obtained after the subject has suffered or may have suffered physical shaking, blunt impact from an external mechanical or other force resulting in closed or open head injury, one or more falls, actual injury to the head caused by an explosion or blast wave, or other type of blunt trauma.

10. The improvement according to any one of claims 1 to 9, wherein the sample is obtained after a subject has ingested or been exposed to fire, a chemical, a toxin or a combination of fire, a chemical and a toxin.

11. 11. The improvement of claim 10, wherein the chemical or toxin is mold, asbestos, a pesticide, an insecticide, an organic solvent, a paint, an adhesive, a gas, an organometallic, a drug of abuse, or a combination of one or more thereof.

12. The improvement of any of claims 1 to 9, 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.

13. The improvement of any of claims 1 to 12, wherein the method can be performed on any subject, regardless of factors selected from the group consisting of the subject's clinical condition, the subject's clinical laboratory values, the classification of the subject as suffering from mild, moderate, severe, or moderate-severe traumatic brain injury, the subject's presentation of low, moderate, or high levels of GFAP, and the timing of any events in which the subject sustained or may have sustained an injury to the head.

14. The improvement of any one of claims 1 to 13, further comprising monitoring the subject.

15. The improvement according to any one of claims 1 to 14, wherein the blood sample is whole blood, serum or plasma.

16. The improvement according to any one of claims 1 to 15, wherein the subject is a human subject.

17. The improvement of any of claims 1 to 16, wherein the reference level is from about 5 pg / mL to about 115 pg / mL.

18. 18. The improvement of claim 17, wherein the reference level is from about 5 pg / mL to about 110 pg / mL.

19. The improvement of claim 17 or claim 18, wherein the reference level is from about 5 pg / mL to about 90 pg / mL.

20. The improvement of any of claims 17 to 19, wherein the reference level is from about 5 pg / mL to about 50 pg / mL.

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