Determining subacute brain injury (TBI) in subjects who have received head computed tomography (CT) scans and are negative or not subjected to head CT scans using biomarkers

Abstract

Disclosed herein are methods of assisting in determining whether a subject has 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, wherein the subject has received a head CT scan and is negative to TBI.

Description

[0001] Related application information

[0002] This application claims priority to U.S. Application No. 63 / 294,344, filed on December 28, 2021, the contents of which are incorporated herein by reference.

[0003] Incorporation by Reference of Electronic Submissions

[0004] Incorporated herein by reference in its entirety is a computer-readable nucleotide / amino acid sequence listing, filed concurrently herewith and identified as follows: a 7,756-byte XML file created on December 28, 2022, named "40205_601_ST26.xml". Technical Field

[0005] The present disclosure relates to methods to aid in the diagnosis and evaluation of subjects who have suffered, may have suffered, or are suspected of suffering a head injury, such as a traumatic brain injury (TBI). The methods involve: detecting the level of at least one biomarker, such as glial fibrillary acid protein (GFAP), in one or more samples taken from a subject at one or more time points from about 24 hours to about 3 weeks after the subject has suffered, may have suffered, or is suspected of suffering a head injury, wherein the subject has also undergone a head computed tomography (CT) at the same time, before, or after (within a clinically relevant time frame) and is negative for TBI; and diagnosing the subject as more likely to have TBI if the level of one or more biomarkers in the sample is above a reference level. Background Art

[0006] In the United States alone, more than 5 million mild traumatic brain injuries (TBI) occur each year. Currently, there are no simple, objective, accurate measurements that can be used to aid in patient assessment. In fact, most of the evaluation and diagnosis of TBI 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. In addition, head CT fails to show mild TBI most of the time, is expensive, and exposes patients to unnecessary radiation. In addition, a negative head CT does not mean that the patient is free of concussion; rather, it only means that certain interventions (such as surgery) are unnecessary. Clinicians and patients need objective, reliable information to accurately assess this condition to facilitate appropriate classification and recovery. To date, only limited data are available for the use of UCH-L1 and GFAP to aid in patient assessment and management in an acute care setting. To date, only limited data are available for the use of UCH-L1 and GFAP to aid in patient assessment and management in an acute care setting.

[0007] Mild TBI, or concussion, is more difficult to detect objectively and is a daily challenge for emergency centers worldwide. Concussions typically do not result in gross pathology, such as bleeding, and do not appear abnormal on routine computed tomography scans of the brain, but are characterized by rapid onset of neuronal dysfunction that resolves spontaneously over days to weeks. Approximately 15% of patients with mild TBI suffer from persistent cognitive dysfunction. There is an unmet need to detect and evaluate victims of mild TBI in the field, in emergency rooms and clinics, in athletic arenas, and in military activities (e.g., combat).

[0008] Current algorithms for assessing the severity of brain injury include the Glasgow Coma Scale score and other measures. These measures may sometimes be adequate to correlate acute severity but are not sensitive enough to detect subtle pathology that may lead to permanent deficits. The GCS and other measures also do not differentiate between injury types and may be inadequate. Therefore, patients entering clinical trials grouped into a single GCS level may have very different injury severity and types. Inappropriate classification undermines the integrity of clinical trials because outcomes vary as a result. Improved injury classification will allow for more precise characterization of illness severity and type in patients with TBI in clinical trials.

[0009] Furthermore, current brain injury trials rely on outcome measures, such as the expanded Glasgow Outcome Scale, which capture global phenomena but cannot assess subtle differences in outcomes. Sensitive outcome measures are needed to determine how well patients recover from brain injury in order to test therapeutics and preventive measures. Summary of the invention

[0010] In one aspect, the present disclosure relates to an improvement in a method for aiding in the diagnosis and evaluation of a subject (such as a human subject) who has suffered, may have suffered, or is suspected of having suffered a head injury. The improved method comprises simultaneously or sequentially performing: (1) performing an assay on a sample obtained from the subject about 24 hours to about three weeks after an actual or suspected head injury to measure or detect the level of a biomarker in the sample, the biomarker comprising glial fibrillary acidic protein (GFAP); and (2) performing a head computed tomography (CT) scan on the subject within a clinically relevant time frame, wherein the improvement comprises: if the level of the biomarker is above a reference level and the head CT scan is negative for TBI, then diagnosing the subject as being more likely to have a traumatic brain injury (TBI).

[0011] In another aspect, the reference level is related to a cutoff level associated with: (a) the level in subjects who have sustained a head injury; (b) the incidence of TBI in a subject; (c) the stage of TBI in a subject, such as mild, moderate, severe, or moderate to severe; (d) the loss of consciousness in a subject; (e) an MRI that is positive for TBI, rather than negative; (f) the incidence of amnesia in a subject (i.e., the presence versus the absence of amnesia); or (g) the severity of TBI in a subject.

[0012] In another aspect, the improved method further comprises: if the level of the biomarker is above the reference level and the head CT scan is negative for TBI, monitoring the subject for TBI. In another aspect, the improved method further comprises: if the level of the biomarker is above the reference level and the head CT scan is negative for TBI, treating the subject for TBI. In another aspect, the improved method further comprises: if the level of the biomarker is above the reference level and the head CT scan is negative for TBI, treating the subject for TBI and then monitoring the subject.

[0013] In another aspect of the above-mentioned improved method, a sample may be obtained about 24.5 hours after an actual head injury or a suspected head injury. For example, a sample may be obtained within about 25 hours of an actual head injury or a suspected head injury. Alternatively, a sample may be obtained within about 26 hours of an actual head injury or a suspected head injury. Alternatively, a sample may be obtained within about 27 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within 28 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within about 29 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within 30 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within 31 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within 32 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within 33 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within 34 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within 35 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within 36 hours of an actual or suspected head injury. Alternatively, the sample may be obtained within 37 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 38 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 39 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 40 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 41 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 42 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 43 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 44 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 45 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 46 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 47 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 48 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 3 days of actual or suspected head injury. Alternatively, the sample may be obtained within 4 days of actual or suspected head injury. Alternatively, the sample may be obtained within 5 days of actual or suspected head injury. Alternatively, the sample may be obtained within 6 days of actual or suspected head injury. Alternatively, the sample may be obtained within 7 days (or 1 week) of actual or suspected head injury. Alternatively, the sample may be obtained within 2 weeks of actual or suspected head injury. Alternatively, the sample may be obtained within 3 weeks of actual or suspected head injury.

[0014] In one embodiment, using the above improved methods, the subject is assessed or evaluated as having TBI. In another embodiment, using the above improved methods, the subject is assessed or evaluated as having mild TBI. In another embodiment, using the above improved methods, the subject is assessed or evaluated as having moderate TBI. In another embodiment, using the above improved methods, the subject is assessed or evaluated as having severe TBI. In another embodiment, using the above improved methods, the subject is assessed or evaluated as having moderate to severe TBI. In another embodiment, using the above improved methods, the subject is assessed or evaluated as not having TBI.

[0015] The improved methods described above may also include treating a subject (such as a human subject) who is assessed or evaluated to have mild, moderate, severe, or moderate to severe TBI with a treatment for TBI (e.g., surgical treatment, therapeutic treatment, or a combination thereof). Any such treatment known in the art and further described herein may be used. In addition, in another embodiment, any subject treated for TBI may also be optionally monitored during and after any treatment course. Alternatively, the method may also include monitoring subjects assessed to have mild, moderate, severe, or moderate to severe TBI (such as those who may not have yet received any treatment).

[0016] In the above improved method, the reference level of GFAP is about 5pg / mL to about 115pg / mL. In the above improved method, the reference level of GFAP is about 5pg / mL to about 110pg / mL. In the above improved method, the reference level of GFAP is about 5pg / mL to about 105pg / mL. In some embodiments, the reference level of GFAP is about 5pg / mL to about 100pg / mL. In some embodiments, the reference level of GFAP is about 5pg / mL to about 90pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 80pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 70pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 60pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 50pg / mL. In other embodiments, the reference level is about 5pg / mL to about 40pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 30pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 25pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 20pg / mL.

[0017] In other aspects, the reference level of GFAP is about 10pg / mL to about 115pg / mL. In the above-mentioned improved method, the reference level of GFAP is about 10pg / mL to about 110pg / mL. In the above-mentioned improved method, the reference level of GFAP is about 10pg / mL to about 105pg / mL. In some embodiments, the reference level of GFAP is about 10pg / mL to about 100pg / mL. In some embodiments, the reference level of GFAP is about 10pg / mL to about 90pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 80pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 70pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 60pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 50pg / mL. In other embodiments, the reference level is about 10pg / mL to about 40pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 30pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 25pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 20pg / mL.

[0018] In other aspects, the reference level of GFAP is about 15pg / mL to about 115pg / mL. In the above-mentioned improved method, the reference level of GFAP is about 15pg / mL to about 110pg / mL. In the above-mentioned improved method, the reference level of GFAP is about 15pg / mL to about 105pg / mL. In some embodiments, the reference level of GFAP is about 15pg / mL to about 100pg / mL. In some embodiments, the reference level of GFAP is about 15pg / mL to about 90pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 80pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 70pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 60pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 50pg / mL. In other embodiments, the reference level is about 15pg / mL to about 40pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 30pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 25pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 20pg / mL.

[0019] In the above-mentioned improved method, the sample can be selected from the group consisting of: blood sample, urine sample, cerebrospinal fluid sample, tissue sample, body fluid sample, saliva sample, oropharyngeal specimen and nasopharyngeal specimen. The blood sample may 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 other embodiments, the sample is a serum sample. In other embodiments, the sample is a saliva sample. In other embodiments, the sample is an oropharyngeal specimen. In other embodiments, the sample is a nasopharyngeal specimen. Such a sample can be obtained in a variety of ways. For example, the subject can be subjected to a head injury caused by a blunt impact, one or more falls, explosions or shock waves or other types of blunt force trauma caused by body shaking, external mechanical forces or other forces resulting in closed or open head trauma, and obtain the sample. Alternatively, the sample can be obtained after the subject has ingested or has been exposed to a combination of fire, chemicals, toxins or chemicals and toxins. Examples of chemical substances or toxins are mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organometallics, drugs of abuse, or one or more combinations thereof. Further, samples can be obtained from subjects suffering from autoimmune diseases, metabolic disorders, brain tumors, hypoxia, viral infections (e.g., SARS-CoV-2), fungal infections, bacterial infections, meningitis, hydrocephalus, or any combination thereof.

[0020] The above-described improved method can be performed on any subject, such as a human subject, without regard to factors selected from the group consisting of: the subject's clinical condition; the subject's laboratory values; the subject's classification as suffering from mild, moderate, severe, or moderate to severe TBI; the subject exhibiting low, moderate, or high levels of GFAP; and the time of any event in which the subject has suffered or may have suffered a head injury.

[0021] In the above improved method, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In other embodiments, the assay is a clinical chemistry assay. In other embodiments, the assay is a single molecule detection assay. In other embodiments, the assay is an immunoassay, the subject is a human and the sample is whole blood. In other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is whole blood. In other embodiments, the assay is a clinical chemistry assay and the sample is whole blood. In other embodiments, the assay is a single molecule detection assay and the sample is whole blood. In other embodiments, the assay is an immunoassay, the subject is a human and the sample is serum.

[0022] In other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is serum. In other embodiments, the assay is a clinical chemistry assay and the sample is serum. In other embodiments, the assay is a single molecule detection assay and the sample is serum. In other embodiments, the assay is an immunoassay, the subject is a human and the sample is plasma. In other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is plasma. In other embodiments, the assay is a clinical chemistry assay and the sample is plasma. In other embodiments, the assay is a single molecule detection assay and the sample is plasma. In other embodiments, the assay is an immunoassay, the subject is a human and the sample is saliva. In other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is saliva. In other embodiments, the assay is a clinical chemistry assay and the sample is saliva. In other embodiments, the assay is a single molecule detection assay and the sample is saliva. In other embodiments, the assay is an immunoassay, the subject is a human and the sample is an oropharyngeal specimen or a nasopharyngeal specimen. In other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is an oropharyngeal specimen or a nasopharyngeal specimen. In other embodiments, the assay is a clinical chemistry assay and the sample is an oropharyngeal specimen or a nasopharyngeal specimen. In other embodiments, the assay is a single molecule detection assay and the sample is an oropharyngeal specimen or a nasopharyngeal specimen.

[0023] In another aspect, the present disclosure relates to a method for aiding in the diagnosis and evaluation of a subject (such as a human subject) who has suffered, may have suffered, or is suspected of having suffered a head injury. The method comprises:

[0024] a. simultaneously or sequentially: (1) performing an assay on a sample obtained from the subject about 24 hours to about 3 weeks after actual or suspected head injury to measure or detect the level of a biomarker in the sample, the biomarker comprising glial fibrillary acidic protein (GFAP); and (2) performing a head computed tomography (CT) scan on the subject within a clinically relevant time frame; and

[0025] b. If the level of the biomarker is above a reference level and the head CT scan is negative for TBI, diagnosing the subject as being more likely to have traumatic brain injury (TBI).

[0026] In another aspect, the reference level is related to a cutoff level associated with: (a) the level in subjects who have sustained a head injury; (b) the incidence of TBI in a subject; (c) the stage of TBI in a subject, such as mild, moderate, severe, or moderate to severe; (d) the loss of consciousness in a subject; (e) an MRI that is positive for TBI, rather than negative; (f) the incidence of amnesia in a subject (i.e., the presence versus the absence of amnesia); or (g) the severity of TBI in a subject.

[0027] In another aspect, the above method further comprises: if the level of the biomarker is above the reference level and the head CT scan is negative for TBI, monitoring the subject for TBI. In another aspect, the method further comprises: if the level of the biomarker is above the reference level and the head CT scan is negative for TBI, treating the subject for TBI. In another aspect, the method further comprises: if the level of the biomarker is above the reference level and the head CT scan is negative for TBI, treating the subject for TBI and then monitoring the subject.

[0028] In another aspect of the above method, the sample may be obtained about 24.5 hours after the actual or suspected head injury. For example, the sample may be obtained within about 25 hours of the actual or suspected head injury. Alternatively, the sample may be obtained within about 26 hours of the actual or suspected head injury. Alternatively, the sample may be obtained within about 27 hours of the actual or suspected head injury. Alternatively, the sample may be obtained within 28 hours of the actual or suspected head injury. Alternatively, the sample may be obtained within about 29 hours of the actual or suspected head injury. Alternatively, the sample may be obtained within 30 hours of the actual or suspected head injury. Alternatively, the sample may be obtained within 31 hours of the actual or suspected head injury. Alternatively, the sample may be obtained within 32 hours of the actual or suspected head injury. Alternatively, the sample may be obtained within 33 hours of the actual or suspected head injury. Alternatively, the sample may be obtained within 34 hours of the actual or suspected head injury. Alternatively, the sample may be obtained within 35 hours of the actual or suspected head injury. Alternatively, the sample may be obtained within 36 hours of the actual or suspected head injury. Alternatively, the sample may be obtained within 37 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 38 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 39 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 40 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 41 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 42 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 43 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 44 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 45 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 46 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 47 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 48 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 3 days of actual or suspected head injury. Alternatively, the sample may be obtained within 4 days of actual or suspected head injury. Alternatively, the sample may be obtained within 5 days of actual or suspected head injury. Alternatively, the sample may be obtained within 6 days of actual or suspected head injury. Alternatively, the sample may be obtained within 7 days (or 1 week) of actual or suspected head injury. Alternatively, the sample may be obtained within 2 weeks of actual or suspected head injury. Alternatively, the sample may be obtained within 3 weeks of actual or suspected head injury.

[0029] In the above improved method, the reference level of GFAP is about 5pg / mL to about 115pg / mL. In the above improved method, the reference level of GFAP is about 5pg / mL to about 110pg / mL. In the above improved method, the reference level of GFAP is about 5pg / mL to about 105pg / mL. In some embodiments, the reference level of GFAP is about 5pg / mL to about 100pg / mL. In some embodiments, the reference level of GFAP is about 5pg / mL to about 90pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 80pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 70pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 60pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 50pg / mL. In other embodiments, the reference level is about 5pg / mL to about 40pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 30pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 25pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 20pg / mL.

[0030] In other aspects, the reference level of GFAP is about 10pg / mL to about 115pg / mL. In the above-mentioned improved method, the reference level of GFAP is about 10pg / mL to about 110pg / mL. In the above-mentioned improved method, the reference level of GFAP is about 10pg / mL to about 105pg / mL. In some embodiments, the reference level of GFAP is about 10pg / mL to about 100pg / mL. In some embodiments, the reference level of GFAP is about 10pg / mL to about 90pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 80pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 70pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 60pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 50pg / mL. In other embodiments, the reference level is about 10pg / mL to about 40pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 30pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 25pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 20pg / mL.

[0031] In other aspects, the reference level of GFAP is about 15pg / mL to about 115pg / mL. In the above-mentioned improved method, the reference level of GFAP is about 15pg / mL to about 110pg / mL. In the above-mentioned improved method, the reference level of GFAP is about 15pg / mL to about 105pg / mL. In some embodiments, the reference level of GFAP is about 15pg / mL to about 100pg / mL. In some embodiments, the reference level of GFAP is about 15pg / mL to about 90pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 80pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 70pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 60pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 50pg / mL. In other embodiments, the reference level is about 15pg / mL to about 40pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 30pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 25pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 20pg / mL.

[0032] In one embodiment, using the above methods, the subject is assessed or evaluated as having TBI. In another embodiment, using the above methods, the subject is assessed or evaluated as having mild TBI. In another embodiment, using the above methods, the subject is assessed or evaluated as having moderate TBI. In another embodiment, using the above methods, the subject is assessed or evaluated as having severe TBI. In another embodiment, using the above methods, the subject is assessed or evaluated as having moderate to severe TBI. In another embodiment, using the above methods, the subject is assessed or evaluated as not having TBI.

[0033] The above methods may also include treating a subject (such as a human subject) who is assessed or evaluated to have mild, moderate, severe, or moderate to severe TBI with a treatment for TBI (e.g., surgical treatment, therapeutic treatment, or a combination thereof). Any such treatment known in the art and further described herein may be used. In addition, in another embodiment, any subject treated for TBI may also be optionally monitored during and after any treatment course. Alternatively, the method may also include monitoring subjects assessed to have mild, moderate, severe, or moderate to severe TBI (such as those who may not have received any treatment).

[0034] In the above method, the sample can be selected from the group consisting of: blood sample, urine sample, cerebrospinal fluid sample, tissue sample, body fluid sample, saliva sample, oropharyngeal specimen and nasopharyngeal specimen. 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 other embodiments, the sample is a serum sample. In other embodiments, the sample is a saliva sample. In other embodiments, the sample is an oropharyngeal specimen. In other embodiments, the sample is a nasopharyngeal specimen. Such a sample can be obtained in a variety of ways. For example, the subject can be subjected to a head injury caused by a body shake, an external mechanical force or other force resulting in a closed or open head trauma, one or more falls, an explosion or shock wave or other types of blunt force trauma, after which the sample is obtained. Alternatively, the sample can be obtained after the subject has ingested or has been exposed to a combination of fire, chemicals, toxins or chemicals and toxins. Examples of chemicals or toxins are mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organic metals, drugs of abuse or one or more combinations thereof. Still further, the sample can be 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.

[0035] The above method can be performed on any subject, such as a human subject, without regard to factors selected from the group consisting of: the subject's clinical condition; the subject's laboratory values; the subject's classification as suffering from mild, moderate, severe, or moderate to severe TBI; the subject exhibiting low, moderate, or high levels of GFAP; and the time of any event in which the subject has suffered or may have suffered a head injury.

[0036] In the above methods, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In other embodiments, the assay is a clinical chemistry assay. In other embodiments, the assay is a single molecule detection assay. In other embodiments, the assay is an immunoassay, the subject is a human and the sample is whole blood. In other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is whole blood. In other embodiments, the assay is a clinical chemistry assay and the sample is whole blood. In other embodiments, the assay is a single molecule detection assay and the sample is whole blood. In other embodiments, the assay is an immunoassay, the subject is a human and the sample is serum. In other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is serum. In other embodiments, the assay is a clinical chemistry assay and the sample is serum. In other embodiments, the assay is a single molecule detection assay and the sample is serum. In other embodiments, the assay is an immunoassay, the subject is a human and the sample is plasma. In other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is plasma. In other embodiments, the assay is a clinical chemistry assay and the sample is plasma. In other embodiments, the assay is a single molecule detection assay and the sample is plasma. In other embodiments, the assay is an immunoassay, the subject is a human and the sample is saliva. In other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is saliva. In other embodiments, the assay is a clinical chemistry assay and the sample is saliva. In other embodiments, the assay is a single molecule detection assay and the sample is saliva. In other embodiments, the assay is an immunoassay, the subject is a human and the sample is an oropharyngeal specimen or a nasopharyngeal specimen. In other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is an oropharyngeal specimen or a nasopharyngeal specimen. In other embodiments, the assay is a clinical chemistry assay and the sample is an oropharyngeal specimen or a nasopharyngeal specimen. In other embodiments, the assay is a single molecule detection assay, and the sample is an oropharyngeal specimen or a nasopharyngeal specimen.

[0037] In another aspect, the present disclosure relates to improvements in methods for aiding in the diagnosis and evaluation of subjects (such as human subjects) who have suffered or may have suffered a head injury. The improvements include assaying a sample obtained from the subject about 24 hours to about three weeks after an actual or suspected head injury to measure or detect the level of a biomarker in the sample, the biomarker including glial fibrillary acidic protein (GFAP); and wherein the improvements include: diagnosing the subject as more likely to have a traumatic brain injury (TBI) if the level of the biomarker is above a reference level and a head computed tomography (CT) scan of the subject performed within a clinically relevant time frame is negative for TBI or a head CT scan was not performed on the subject.

[0038] In another aspect, the reference level is related to a cutoff level associated with: (a) the level in subjects who have sustained a head injury; (b) the incidence of TBI in a subject; (c) the stage of TBI in a subject, such as mild, moderate, severe, or moderate to severe; (d) the loss of consciousness in a subject; (e) an MRI that is positive for TBI, rather than negative; (f) the incidence of amnesia in a subject (i.e., the presence versus the absence of amnesia); or (g) the severity of TBI in a subject.

[0039] In another aspect, the improved method further comprises: if the level of the biomarker is above the reference level, and optionally, if a head CT scan is performed and the head CT scan is negative for TBI, monitoring the subject for TBI. In another aspect, the improved method further comprises: if the level of the biomarker is above the reference level, and optionally, if a head CT scan is performed and the head CT scan is negative for TBI, treating the subject for TBI. In another aspect, the improved method further comprises: if the level of the biomarker is above the reference level, and optionally, if a head CT scan is performed and the head CT scan is negative for TBI, treating the subject for TBI and then monitoring the subject.

[0040] In another aspect of the above improved method, a sample may be obtained about 24.5 hours after an actual or suspected head injury. For example, a sample may be obtained within about 25 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within about 26 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within about 27 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within 28 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within about 29 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within 30 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within 31 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within 32 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within 33 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within 34 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within 35 hours of an actual or suspected head injury. Alternatively, a sample may be obtained within 36 hours of an actual or suspected head injury. Alternatively, the sample may be obtained within 37 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 38 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 39 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 40 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 41 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 42 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 43 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 44 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 45 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 46 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 47 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 48 hours of actual or suspected head injury. Alternatively, the sample may be obtained within 3 days of actual or suspected head injury. Alternatively, the sample may be obtained within 4 days of actual or suspected head injury. Alternatively, the sample may be obtained within 5 days of actual or suspected head injury. Alternatively, the sample may be obtained within 6 days of actual or suspected head injury. Alternatively, the sample may be obtained within 7 days (1 week) of actual or suspected head injury. Alternatively, the sample may be obtained within 2 weeks of actual or suspected head injury. Alternatively, the sample may be obtained within 3 weeks of actual or suspected head injury.

[0041] In the above improved method, the reference level of GFAP is about 5pg / mL to about 115pg / mL. In the above improved method, the reference level of GFAP is about 5pg / mL to about 110pg / mL. In the above improved method, the reference level of GFAP is about 5pg / mL to about 105pg / mL. In some embodiments, the reference level of GFAP is about 5pg / mL to about 100pg / mL. In some embodiments, the reference level of GFAP is about 5pg / mL to about 90pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 80pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 70pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 60pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 50pg / mL. In other embodiments, the reference level is about 5pg / mL to about 40pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 30pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 25pg / mL. In other embodiments, the reference level of GFAP is about 5pg / mL to about 20pg / mL.

[0042] In other aspects, the reference level of GFAP is about 10pg / mL to about 115pg / mL. In the above-mentioned improved method, the reference level of GFAP is about 10pg / mL to about 110pg / mL. In the above-mentioned improved method, the reference level of GFAP is about 10pg / mL to about 105pg / mL. In some embodiments, the reference level of GFAP is about 10pg / mL to about 100pg / mL. In some embodiments, the reference level of GFAP is about 10pg / mL to about 90pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 80pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 70pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 60pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 50pg / mL. In other embodiments, the reference level is about 10pg / mL to about 40pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 30pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 25pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 20pg / mL.

[0043] In other aspects, the reference level of GFAP is about 15pg / mL to about 115pg / mL. In the above-mentioned improved method, the reference level of GFAP is about 15pg / mL to about 110pg / mL. In the above-mentioned improved method, the reference level of GFAP is about 15pg / mL to about 105pg / mL. In some embodiments, the reference level of GFAP is about 15pg / mL to about 100pg / mL. In some embodiments, the reference level of GFAP is about 15pg / mL to about 90pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 80pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 70pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 60pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 50pg / mL. In other embodiments, the reference level is about 15pg / mL to about 40pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 30pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 25pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 20pg / mL.

[0044] In one embodiment, using the above improved methods, the subject is assessed or evaluated as having TBI. In another embodiment, using the above improved methods, the subject is assessed or evaluated as having mild TBI. In another embodiment, using the above improved methods, the subject is assessed or evaluated as having moderate TBI. In another embodiment, using the above improved methods, the subject is assessed or evaluated as having severe TBI. In another embodiment, using the above improved methods, the subject is assessed or evaluated as having moderate to severe TBI. In another embodiment, using the above improved methods, the subject is assessed or evaluated as not having TBI.

[0045] The improved methods described above may also include treating a subject (such as a human subject) who is assessed or evaluated to have mild, moderate, severe, or moderate to severe TBI with a treatment for TBI (e.g., surgical treatment, therapeutic treatment, or a combination thereof). Any such treatment known in the art and further described herein may be used. In addition, in another embodiment, any subject treated for TBI may also be optionally monitored during and after any treatment course. Alternatively, the method may also include monitoring subjects assessed to have mild, moderate, severe, or moderate to severe TBI (such as those who may not have yet received any treatment).

[0046] In the above-mentioned improved method, the sample can be selected from the group consisting of: blood sample, urine sample, cerebrospinal fluid sample, tissue sample, body fluid sample, saliva sample, oropharyngeal specimen and nasopharyngeal specimen. 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 other embodiments, the sample is a serum sample. In other embodiments, the sample is a saliva sample. In other embodiments, the sample is an oropharyngeal specimen. In other embodiments, the sample is a nasopharyngeal specimen. Such a sample can be obtained in a variety of ways. For example, the subject can be subjected to a head injury caused by a blunt impact, one or more falls, explosions or shock waves or other types of blunt force trauma caused by body shaking, external mechanical forces or other forces resulting in closed or open head trauma, and obtain the sample. Alternatively, the sample can be obtained after the subject has ingested or has been exposed to a combination of fire, chemicals, toxins or chemicals and toxins. Examples of chemicals or toxins are mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organometallics, drugs of abuse, or one or more combinations thereof. Further, samples can be obtained from subjects suffering from autoimmune diseases, metabolic disorders, brain tumors, hypoxia, viral infections (e.g., SARS-CoV-2), fungal infections, bacterial infections, meningitis, hydrocephalus, or any combination thereof.

[0047] The above-described improved methods can be implemented on any subject, such as a human subject, without regard to factors selected from the group consisting of: the subject's clinical condition; the subject's laboratory values; the subject's classification as suffering from mild, moderate, severe, or moderate to severe TBI; the subject exhibiting low, moderate, or high levels of GFAP; and the time of any event in which the subject has suffered or may have suffered a head injury.

[0048] In the above improved method, the assay is an immunoassay. In some embodiments, the assay is a point-of-care assay. In other embodiments, the assay is a clinical chemistry assay. In other embodiments, the assay is a single molecule detection assay. In other embodiments, the assay is an immunoassay, the subject is a human and the sample is whole blood. In other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is whole blood. In other embodiments, the assay is a clinical chemistry assay, and the sample is whole blood. In other embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In other embodiments, the assay is an immunoassay, the subject is a human and the sample is serum. In other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is serum. In other embodiments, the assay is a clinical chemistry assay, and the sample is serum. In other embodiments, the assay is a single molecule detection assay, and the sample is serum. In other embodiments, the assay is an immunoassay, the subject is a human and the sample is plasma. In other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is plasma. In other embodiments, the assay is a clinical chemistry assay, and the sample is plasma. In additional embodiments, the assay is a single molecule detection assay and the sample is plasma. In other embodiments, the assay is an immunoassay, the subject is a human and the sample is saliva.

[0049] In other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is saliva. In other embodiments, the assay is a clinical chemistry assay, and the sample is saliva. In other embodiments, the assay is a single molecule detection assay, and the sample is saliva. In other embodiments, the assay is an immunoassay, the subject is a human and the sample is an oropharyngeal specimen or a nasopharyngeal specimen. In other embodiments, the assay is a point-of-care assay, the subject is a human and the sample is an oropharyngeal specimen or a nasopharyngeal specimen. In other embodiments, the assay is a clinical chemistry assay, and the sample is an oropharyngeal specimen or a nasopharyngeal specimen. In other embodiments, the assay is a single molecule detection assay, and the sample is an oropharyngeal specimen or a nasopharyngeal specimen. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 ROC analysis showing GFAP levels associated with Glasgow Coma Scale (GCS) score severity (ie, GCS mild vs. moderate / severe) for those subjects whose CT scans were negative for TBI. Samples were evaluated within two weeks of injury.

[0051] Figure 2Shown is a ROC analysis of GFAP levels associated with the Extended Glasgow Outcome Scale (GOSE) for those subjects whose CT scans were negative for TBI. Samples were evaluated within two weeks after injury.

[0052] Figure 3 Shown is a ROC analysis of GFAP levels associated with MRI results (ie, positive vs. negative) for those subjects whose CT scans were negative for TBI. Samples were evaluated within two weeks of injury. DETAILED DESCRIPTION

[0053] The present disclosure relates to improved methods for aiding in the diagnosis and evaluation of subjects (such as human subjects) who have sustained, may have sustained, or are suspected of sustaining a head injury, such as a traumatic brain injury (e.g., such as mild, moderate, severe, or moderate to severe TBI), using biomarkers such as ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, when the subject has optionally undergone at least one head computed tomography (CT) scan within a clinically relevant time frame and which was negative for TBI.

[0054] The methods described herein involve detecting one or more biomarkers in one or more samples taken from a subject (such as a human subject) at any time point from about 24 hours to about three weeks after an actual head injury or suspected head injury. Optionally, if a head CT scan is performed, the head CT scan can be performed simultaneously or sequentially in any order at the same time, before, or after detecting the level of one or more biomarkers in one or more samples taken from the subject. When the subject has optionally undergone a head CT scan and is negative for TBI, detecting the level of one or more biomarkers (such as UCH-L1, GFAP, or a combination thereof) (which is higher than a reference level) helps to accurately evaluate or diagnose the subject (such as a human subject) as being more likely to have TBI. In other words, in some aspects, the improved methods described herein allow for the identification of subjects who have already suffered from TBI but who may have otherwise been incorrectly diagnosed as not having TBI if the diagnosis was made based solely on the results of one or more head CT scans.

[0055] The section headings as used in this section and throughout the disclosure herein are for organizational purposes only and are not intended to be limiting.

[0056] 1. Definition

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. In the event of a conflict, this document (including definitions) shall prevail. Preferred methods and materials are described below, but methods and materials similar to or equivalent to those described herein may be used in practice or testing the present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and embodiments disclosed herein are merely illustrative and are not intended to be restrictive.

[0058] As used herein, the terms "comprise," "include," "having," "has," "can," "contain," and variations thereof are intended to be open-ended conjunctions, terms, or words that do not exclude the possibility of additional actions or structures. Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural references. Whether explicitly stated or not, the present disclosure also encompasses other embodiments that "comprise," "consist of," and "consist essentially of" the embodiments or elements presented herein.

[0059] For the description of numerical ranges herein, each intervening number with the same degree of precision is expressly included. For example, for the range of 6-9, in addition to 6 and 9, the numbers 7 and 8 are also included, and for the range of 6.0-7.0, 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 included.

[0060] An "affinity matured antibody" is used herein to refer to an antibody with one or more changes in one or more CDRs that result in an increase in affinity (i.e., K) for a target antigen compared to a parent antibody that does not have the changes. D , k d or k a) is increased. Exemplary affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Various procedures for generating affinity matured antibodies are known in the art, including screening of combinatorial antibody libraries prepared using biodisplay. 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 is described in Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-3319 (1995); and Hawkins et al., J. Mol. Biol., 226:889-896 (1992). Selective mutagenesis at selective mutagenesis positions and at contact or hypermutation positions by activity-enhancing amino acid residues is described in U.S. Pat. No. 6,914,128 B1.

[0061] As used herein, "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., in 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.

[0062] As used herein, "an antibody" and "antibodies" refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, birds (e.g., ducks or geese), sharks, whales, and mammals (including non-primates (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, etc.) or non-human primates (e.g., monkeys, chimpanzees, etc.)), recombinant antibodies, chimeric antibodies, single-chain Fv ("scFv"), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') ... 2Fragments, disulfide-linked Fv ("sdFv") 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 their preparation are described in Wu, C et al., Nature Biotechnology, 25(11): 1290-1297 (2007) and PCT International Application WO 2001 / 058956, the contents of each of which are incorporated herein by reference), and functionally active epitope-binding fragments of any of the above antibodies. 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), classification (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. For simplicity, antibodies directed against an analyte are often referred to herein as "anti-analyte antibodies" or simply "analyte antibodies" (eg, anti-UCH-L1 antibodies or UCH-L1 antibodies).

[0063] As used herein, "antibody fragment" refers to a portion of an intact antibody that contains the antigen binding site or variable region. The portion does not include the constant heavy chain domains (i.e., CH2, CH3 or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab') 2 Fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing three CDRs of the light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing three CDRs of the heavy chain variable region.

[0064] "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, while an AUC of 0.5 represents an insignificant test. Preferred AUCs can 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.

[0065] "Beads" and "particles" are used interchangeably herein and refer to a substantially spherical solid support. An example of a bead or particle is a microparticle. Microparticles that can be used herein can be of any type known in the art. For example, the bead or particle can be a magnetic bead or magnetic particle. The magnetic bead / particle can be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or ferrofluid. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO 2 , MnAs, MnBi, EuO and NiO / Fe. Examples of ferrimagnetic materials include NiFe 2 O 4 、CoFe 2 O 4 , Fe 3 O 4 (or FeO·Fe 2 O 3 ). The beads can have a magnetic solid core portion surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion can be a layer surrounding the non-magnetic core. The microparticles can have any size that works in the methods described herein, such as about 0.75 to about 5 nm, or about 1 to about 5 nm, or about 1 to about 3 nm.

[0066] "Binding protein" is used herein to refer to a monomeric or multimeric protein that binds to a binding partner and forms a complex therewith, such as, for example, a polypeptide, an antigen, a chemical compound or other molecule, or a substrate of any kind. The binding protein specifically binds to the binding partner. The binding protein includes antibodies, and antigen-binding fragments thereof, and other various forms and derivatives thereof known in the art and described below, and other molecules of antigen-binding domains that contain one or more binding antigen molecules or specific sites (epitopes) on antigen molecules. Therefore, binding proteins include, but are not limited to, antibodies, tetrameric immunoglobulins, IgG molecules, IgG1 molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, affinity-matured antibodies, and fragments of any such antibodies that retain the ability to bind to antigens.

[0067] "Bispecific antibody" is used herein to refer to full-length antibodies generated by the following techniques: quadroma technology (see Milstein et al., Nature, 305(5934): 537-540 (1983)); chemical conjugation of two different monoclonal antibodies (see Staerz et al., Nature, 314(6012): 628-631 (1985)); or the knob-into-hole method or similar methods of introducing mutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14): 6444-6448 (1993)), which generate multiple different immunoglobulin species, only one of which is a functional bispecific antibody. A bispecific antibody binds one antigen (or epitope) on one of its two binding arms (a pair of HC / LC) and binds a different antigen (or epitope) on its second arm (another pair of HC / LC). According to this definition, a bispecific antibody has two distinct antigen-binding arms (in terms of both specificity and CDR sequences) and is monovalent for each antigen it binds.

[0068] "CDR" is used herein to refer to the "complementarity determining region" within the variable structure sequence of an antibody. There are three CDRs in each variable region of the heavy chain and light chain. For each variable region, starting from the N-terminus of the heavy chain or light chain, these regions are represented as "CDR1", "CDR2" and "CDR3". The term "CDR group" as used herein refers to a group of three CDRs present in a single variable region that binds an antigen. Therefore, an antigen binding site can include six CDRs, which include a CDR group from each of the heavy and light chain variable regions. A polypeptide comprising a single CDR (e.g., CDR1, CDR2 or CDR3) can be referred to as a "molecular recognition unit". Crystallographic analysis of antigen-antibody complexes has shown that the amino acid residues of CDR form extensive contacts with the bound antigen, wherein the most extensive antigen contact is contact with the heavy chain CDR3. Therefore, a molecular recognition unit may be primarily responsible for the specificity of an antigen binding site. Generally speaking, CDR residues are directly and most substantially involved in influencing antigen binding.

[0069] The precise boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as "Kabat CDRs." Chothia and coworkers (Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that despite the enormous diversity at the level of amino acid sequence, certain subportions within the Kabat CDRs adopt nearly identical peptide backbone conformations. These subportions are designated "L1," "L2," and "L3," or "H1," "H2," and "H3," where "L" and "H" represent the light chain and heavy chain regions, respectively. These regions may be referred to as "Chothia CDRs." CDRs" that have boundaries that overlap with Kabat CDRs. Other boundaries that define CDRs that overlap with Kabat CDRs have been described by Padlan, FASEB J., 9:133-139 (1995) and MacCallum, J. Mol. Biol, 262(5):732-745 (1996). Still other CDR boundary definitions may not strictly follow one of the systems herein, but will still overlap with Kabat CDRs, but they may be shortened or lengthened given that particular residues or groups of residues, or even entire CDRs, do not significantly affect predictions or experimental findings of antigen binding. The methods used herein can utilize CDRs defined according to any of these systems, but certain embodiments use Kabat or Chothia defined CDRs.

[0070] "Clinically relevant time frame" means a time frame (e.g., seconds, minutes, or hours) during which a careful and prudent practitioner (e.g., a physician, nurse, paramedic, or other) would reasonably consider the results of one or more biomarker tests in connection with an imaging procedure (such as a head CT scan) or in accordance with guidelines established by an oversight entity (e.g., a standard-setting body such as the World Health Organization (WHO), a physician review committee, a regulatory approval agency such as the FDA, EMEA, or other agency, etc.).

[0071] "A component," "components," or "at least one component" generally refers to a capture antibody, a detector or conjugate, a calibrator, a control, a sensitivity panel, a container, a buffer, a diluent, a salt, an enzyme, a cofactor for an enzyme, a detection reagent, a pretreatment reagent / solution, a substrate (e.g., as a solution), a stop solution, etc. that can be included in a kit for assaying a test sample (such as a patient urine, whole blood, serum, or plasma sample) according to the methods described herein and other methods known in the art. Some components can be in solution or lyophilized for reconstitution for use in the assay.

[0072] As used herein, "associated with" means compared to.

[0073] 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 in your body. A CT scan can 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 can be a conventional CT scan or a spiral / spiral CT scan. In a conventional CT scan, the scan is performed slice by slice, and after each slice, the scan stops and moves down to the next slice, for example, from the top of the abdomen down to the pelvis. Conventional CT scans require patients to hold their breath to avoid motion artifacts. A spiral / spiral CT scan is a continuous scan that is taken in a spiral manner, and is a much faster process in which the scanned image is continuous.

[0074] A head CT scan is "negative" for TBI when no intracranial lesions are observed in images taken from a subject who has suffered, may have suffered, or is suspected of suffering a head injury. To further clarify, a subject's head CT scan is "negative" for TBI when no lesions are found or identified; however, in some aspects, a subject may still experience symptoms (e.g., symptoms of TBI) even if the head CT is negative. Given that not all injuries or lesions can be visualized by head CT, most subjects will be negative for TBI on head CT. Therefore, the methods and assays described herein can be used to provide an assessment or determination of a subject who has a negative head CT but may still have TBI.

[0075] "Determined by determination" is used herein to refer to determining a reference level by any suitable determination. In some embodiments, the determination of reference level can be achieved by the determination of the same type of determination to be applied to the subject's sample (for example, by immunoassay, clinical chemistry determination, single molecule detection determination, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, or protein immunostaining, electrophoretic analysis, protein determination, competitive binding determination, functional protein determination or chromatography or spectroscopy, such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS)). In some embodiments, the determination of reference level can be achieved by the determination of the same type and the same determination conditions to be applied to the subject's sample. As noted herein, the disclosure provides exemplary reference levels (for example, calculated by comparing the reference levels of different time points). Based on the description provided by the disclosure, it is completely within the capabilities of those of ordinary skill in the art to be disclosed herein to be suitable for other determinations to obtain the determination specific reference levels of those other determinations. For example, a set of training samples including samples obtained from human subjects known to have suffered a head injury (and more specifically, samples obtained from human subjects known to have suffered (i) a mild TBI and / or (ii) a moderate, severe, or moderate to severe TBI) and samples obtained from human subjects known not to have suffered a head injury can be used to obtain assay-specific reference levels. It should be understood that parameter levels "determined by an assay" and having recited levels of "sensitivity" and / or "specificity" are used herein to refer to reference levels that have been determined to provide the recited sensitivity and / or specificity when employed in the methods of the present disclosure. It is well within the capabilities of one of ordinary skill 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 performing repeated statistical analyses of assay data using multiple different possible reference levels.

[0076] In practice, when distinguishing between subjects with or without traumatic brain injury or subjects with mild relative to moderate, severe or moderate to severe traumatic brain injury, the technician will weigh the impact of increasing the cutoff value on sensitivity and specificity. Increasing or lowering the cutoff value will have a clear and predictable impact on sensitivity and specificity and other standard statistical measures. It is well known that increasing the cutoff value will improve specificity, but may worsen sensitivity (proportional to those patients with the disease who test positive). In contrast, lowering the cutoff value will improve specificity, but will worsen sensitivity (proportional to those who test negative without the disease). The consequences of being used to detect traumatic brain injury or determine mild relative to moderate, severe or moderate to severe traumatic brain injury will be obvious to those skilled in the art. In distinguishing whether a subject has traumatic brain injury or mild relative to moderate, severe, or moderate to severe traumatic brain injury, the higher the cutoff value, the better the specificity, because more true negatives (i.e., subjects who do not have traumatic brain injury, do not have mild traumatic brain injury, do not have moderate traumatic brain injury, do not have severe traumatic brain injury, or do not have moderate to severe traumatic brain injury) are distinguished from those who have traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate to severe traumatic brain injury. However, at the same time, increasing the cutoff value will reduce the number of cases identified as positive overall, as well as the number of true positive cases, so sensitivity will inevitably decrease. Conversely, the lower the cutoff value, the improved sensitivity, because more true positives (i.e., subjects with traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate to 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 to severe traumatic brain injury. However, at the same time, lowering the cutoff value increases the number of cases identified as positive overall, as well as the number of false positives, so specificity must decrease.

[0077] Typically, a high sensitivity value helps a technician rule out a disease or condition (such as traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate to severe traumatic brain injury), while a high specificity value helps a technician include a disease or condition. Whether a technician wants to rule out or include a disease depends on the consequences for patients of each wrong type. Therefore, without fully disclosing the basic information about how to choose the value, the exact balance used to derive the test cutoff value cannot be known or predicted. The balance between sensitivity to specificity and other factors will be different based on the situation. This is why it is sometimes preferred to provide an alternative cutoff value (e.g., a reference value) so that a physician or doctor can choose.

[0078] As used herein, the "derivative" of an antibody may refer to an antibody having one or more modifications to its amino acid sequence compared to a true or parent antibody and exhibiting a modified domain structure. Derivatives may still be able to adopt the typical domain configuration found in natural antibodies, as well as amino acid sequences that can specifically bind to a target (antigen). Typical examples of antibody derivatives are antibodies coupled to other polypeptides, rearranged antibody domains, or antibody fragments. Derivatives may also include at least one other compound, such as a protein domain, which is connected by covalent or non-covalent bonds. According to methods known in the art, the connection may be based on genetic fusion. The other domains present in the fusion protein comprising an antibody may preferably be connected by a flexible linker, advantageously a peptide linker, wherein the peptide linker includes a plurality of hydrophilic peptide-bonded amino acids, and its length is sufficient to span the distance between the C-terminus of the other protein domain and the N-terminus of the antibody, and vice versa. Antibodies may be connected to effector molecules, which have a conformation suitable for biological activity or selective binding, such as solid supports, biologically active substances (e.g., cytokines or growth hormones), chemical reagents, peptides, proteins, or drugs.

[0079] As used herein, "digital assay" refers to an assay in which an analyte is captured and the analyte molecules 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 the signal. In a digital assay, the result is 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, nanopore assays, single molecule enzyme-linked immunosorbent assays, direct capture counting assays, etc.

[0080] "Drugs of abuse" are used herein to refer to one or more additive substances (e.g., drugs) taken for non-medical reasons (e.g., recreational and / or mind-altering effects). Excessive addiction, use, or dependence on such drugs of abuse is commonly referred to as "drug abuse." Examples of drugs of abuse include alcohol, barbiturates, benzodiazepines, marijuana, cocaine, hallucinogens (such as ketamine, mescaline (peyote), PCP, psilocybin, DMT and / or LSD), methaqualone, opioids, amphetamines (including methamphetamine), anabolic steroids, inhalants (i.e., substances containing volatile substances with psychoactive properties, such as nitrites, spray paints, cleaning fluids, markers, glues, etc.), and combinations thereof.

[0081] "Dual specific antibody" is used herein to refer to a full-length antibody that can bind to two different antigens (or epitopes) in each of its two binding arms (a pair of HC / LC) (see PCT publication WO 02 / 02773). Thus, a dual specific binding protein has two identical antigen binding arms with the same specificity and the same CDR sequences, and is bivalent for each antigen it binds.

[0082] "Dual variable domains" are used herein to refer to two or more antigen binding sites on a binding protein, which may be divalent (two antigen binding sites), tetravalent (four antigen binding sites) or multivalent binding proteins. DVDs may be monospecific, i.e., capable of binding to an antigen (or a specific epitope), or multispecific, i.e., capable of binding to two or more antigens (i.e., two or more epitopes of the same antigen molecule or two or more epitopes of different target antigens). Preferred DVD binding proteins include two heavy chain DVD polypeptides and two light chain DVD polypeptides and are referred to as "DVD immunoglobulins" or "DVD-Igs". Such DVD-Ig binding proteins are therefore tetrameric and similar to IgG molecules, but provide more antigen binding sites than IgG molecules. Therefore, each half of a tetrameric DVD-Ig molecule is similar to half of an IgG molecule, and includes a heavy chain DVD polypeptide and a light chain DVD polypeptide, but unlike a pair of heavy chains and light chains that provide a single antigen binding domain of an IgG molecule, a pair of heavy chains and light chains of DVD-Ig provide two or more antigen binding sites.

[0083] Each antigen binding site of DVD-Ig binding proteins can be derived from donor (" parent ") monoclonal antibody, therefore comprises heavy chain variable domain (VH) and light chain variable domain (VL) with six CDRs in total that each antigen binding site is involved in antigen binding.Therefore, the DVD-Ig binding proteins in conjunction with two different epi-positions (that is, two different epi-positions of two different antigen molecules or two different epi-positions of the same antigen molecule) comprise the antigen binding site derived from the first parent monoclonal antibody and the antigen binding site of the second parent monoclonal antibody.

[0084] Descriptions of the design, expression, and characterization of DVD-Ig binding molecules are provided in PCT Publication No. WO 2007 / 024715, US Pat. No. 7,612,181, and Wu et al., Nature Biotech., 25: 1290-1297 (2007). Preferred examples of such DVD-Ig molecules include: a heavy chain comprising the structural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker (with the proviso that it is not CH1), X2 is an Fc region, and n is 0 or 1, but preferably 1; and a light chain comprising VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker (with the proviso that it is not CH1), X2 does not comprise an Fc region; and n is 0 or 1, but preferably 1. This DVD-Ig can include two such heavy chains and two such light chains, wherein each chain includes the variable domains connected in series, and there is no intervening constant region between the variable regions, wherein the heavy chain and the light chain associate to form a series of functional antigen binding sites, and a pair of heavy chains and light chains can associate to form a tetramer with four functional antigen binding sites with another pair of heavy chains and light chains. In another example, a DVD-Ig molecule can include such a heavy chain and a light chain, i.e., each of the heavy chains and light chains includes three variable domains (VD1, VD2, VD3) connected in series, and there is no constant region in between the variable domains, wherein a pair of heavy chains and light chains can associate to form three antigen binding sites, and wherein a pair of heavy chains and light chains can associate to form a tetramer with six antigen binding sites with another pair of heavy chains and light chains.

[0085] In a preferred embodiment, the DVD-Ig binding protein not only binds to the same target molecule bound to its parent monoclonal antibody, but also has one or more of the desired properties of one or more of its parent monoclonal antibodies. Preferably, this additional property is an antibody parameter of one or more of the parent monoclonal antibodies. The antibody parameters that can contribute to the DVD-Ig binding protein from one or more of its parent monoclonal antibodies include but are not limited to antigen specificity, antigen affinity, efficacy, biological function, epitope recognition, protein stability, protein solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross-reactivity and orthologous antigen binding.

[0086] 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 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 human UCH-L1, an epitope of human GFAP, and an epitope of UCH-L1 of another species (e.g., mouse), and a DVD-Ig binding protein that binds to human UCH-L1, an epitope of human GFAP, and an epitope of another target molecule.

[0087] As used herein, "dynamic range" refers to the range over which assay readouts are proportional to the amount of target molecule or analyte in the sample being analyzed.

[0088] An "epitope" or "epitopes" or "epitopes of interest" refers to a site on any molecule that is recognized and can bind to a complementary site on its specific binding partner. The molecule and the specific binding partner are part of a specific binding pair. For example, an epitope can be on a polypeptide, protein, hapten, carbohydrate antigen (such as but not limited to glycolipid, glycoprotein or lipopolysaccharide) or polysaccharide. Its specific binding partner can be, but is not limited to, an antibody.

[0089] As used herein, "fragment antigen binding fragment" or "Fab fragment" refers to an antibody fragment that binds to an antigen and contains one antigen binding site, one complete light chain and a portion of a heavy chain. Fab is a monovalent fragment consisting of VL, VH, CL and CH1 domains. Fab consists of one constant domain and one variable domain of each of the heavy and light chains. The variable domain contains a paratope (antigen binding site), which is contained in a set of complementary 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 generated as already described in the art, for example, using the enzyme papain, which can be used to cleave immunoglobulin monomers into two Fab fragments and an Fc fragment, or can be produced by recombinant methods.

[0090] As used herein, "F(ab') 2A "fragment" is an antibody produced by digesting a whole IgG antibody with pepsin to remove most of the Fc region while leaving some of the hinge region intact. F(ab') 2 The fragment has two antigen-binding F(ab) portions linked together by a disulfide bond and is therefore bivalent, with a molecular weight of approximately 110 kDa. 2 F(ab') fragments are smaller than the entire IgG molecule and are able to better penetrate tissues, thus facilitating better antigen recognition in immunohistochemistry. 2 The use of fragments also avoids nonspecific binding to Fc receptors or protein A / G on living cells. F(ab') 2 The fragments can bind and precipitate the antigen.

[0091] As used herein, "framework" (FR) or "framework sequence" may mean the remaining sequence of the variable region minus CDR. Because the precise definition of CDR sequences can be determined by different systems (e.g., see above), the meaning of framework sequences is susceptible to corresponding different interpretations. 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 chain and the heavy chain into four subregions (FR1, FR2, FR3 and FR4) on each chain, wherein CDR1 is between FR1 and FR2, CDR2 is between FR2 and FR3, and CDR3 is between FR3 and FR4. Without specifying a specific subregion as FR1, FR2, FR3 or FR4, the framework region as mentioned in other represents the combined FR in the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR represents one of the four subregions, and FR represents two or more of the four subregions constituting the framework region.

[0092] Human heavy and light chain FR sequences are known in the art and 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, human heavy and light chain acceptor sequences are selected from publicly available databases such as V-base (hypertext transfer protocol: / / vbase.mrc-cpe.cam.ac.uk / ) or the international ImMunoGene Framework sequences listed in the information system (hypertext transfer protocol: / / imgt.cines.fr / texts / IMGTrepertoire / LocusGenes / ).

[0093] As used herein, "functional antigen binding site" may refer to a site on a binding protein (e.g., an antibody) that is capable of binding to a target antigen. The antigen binding affinity of an antigen binding site may not be as strong as that of the parent binding protein, e.g., the parent antibody, from which the antigen binding site is derived, but the ability to bind antigen must be measurable using any of a variety of methods known for evaluating protein, e.g., antibody, binding to antigen. In addition, the antigen binding affinity of each antigen binding site of a multivalent protein, e.g., a multivalent antibody herein need not be quantitatively identical.

[0094] "GFAP" is used herein to describe glial fibrillary acidic protein. GFAP is a protein encoded by the GFAP gene in humans and the GFAP gene counterpart in other species, and it can be produced (eg, by recombinant means, in other species).

[0095] "GFAP status" can refer to the level or amount of GFAP at a point in time (e.g., using a single GFAP measurement), the level or amount of GFAP associated with monitoring (e.g., determining an increase or decrease in the amount of GFAP by repeated testing of a subject), the level or amount of GFAP associated with treatment of traumatic brain injury (whether primary brain injury and / or secondary brain injury), or a combination thereof.

[0096] As used herein, "Glasgow Coma Scale" or "GCS" refers to a 15-point scale (eg, described in 1974 by Graham Teasdale and Bryan Jennett, Lancet 1974; 2:81-4) that provides a practical method for assessing the impairment of the level of consciousness in patients with brain injury. The test measures the best motor response, verbal response, and eye opening response using the following values: I. Best motor response (6-complies with 2-part request; 5-places hand above clavicle for stimulation of head and neck; 4-flexes arm quickly at elbow, but features not most abnormal; 3-flexes arm at elbow, features clearly most abnormal; 2-extends arm at elbow; 1-no movement of arm / leg, no distractions; NT-paralysis or other limiting factors); II. Verbal response (5-speaks name, place and date correctly; 4-no orientation, but coherent communication; 3-understandable single words; 2-moan / sigh only; 1-no audible response, no distractions; NT-there are distractions to communication); and III. Eye opening (4-eyes open before stimulation; 3-after speaking or shouting request; 2-after fingertip stimulation; 1-eyes not open at any time, no distractions; NT-eyes closed due to local factors). The final score is determined by adding the values ​​of I+II+III. A subject is considered to have a mild TBI if the GCS score is 13-15. A subject is considered to have a moderate TBI if the GCS score is 9-12. A subject is considered to have a severe TBI if the GCS score is 8 or less, usually 3-8.

[0097] As used herein, the "Glasgow Outcome Scale" refers to an overall scale for functional outcomes that rates the patient's status into one of five categories: death, vegetative state, severe disability, moderate disability, or good recovery. The "Glasgow Outcome Scale Extended" or "GOSE," as used interchangeably herein, provides eight more detailed classifications by subdividing the categories of severe disability, moderate disability, and good recovery into low and high categories, as shown in Table 1.

[0098] Table 1

[0099]

[0100] The term "humanized antibody" is used herein to describe an antibody comprising heavy and light chain variable region sequences from a non-human species (e.g., mouse) but in which at least a portion of the VH and / or VL sequences has been made 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 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. 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 a non-human antibody CDR. A humanized antibody comprises substantially all (Fab, Fab', F(ab')) of at least one and typically two variable domains. 2 , FabC, Fv), wherein all or substantially all CDR regions correspond to those of non-human immunoglobulins (i.e., donor antibodies) and all or substantially all framework regions are those of human immunoglobulin consensus sequences. In one embodiment, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc) (usually a constant region of a human immunoglobulin). In some embodiments, the humanized antibody contains a light chain and at least a heavy chain variable domain. The antibody may also include a heavy chain CH1, hinge, CH2, CH3, and CH4 region. 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 a specific embodiment, the humanized antibody contains only a light chain and / or a humanized heavy chain humanized variable domain.

[0101] Humanized antibodies 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. Humanized antibodies may comprise sequences from more than one class or isotype, and specific constant domains may be selected using techniques well known in the art to optimize desired effector functions.

[0102] The framework region and CDR of humanized antibodies do not need to correspond exactly to the parent sequence, for example, the donor antibody CDR or the common framework can be mutated by replacing, inserting or / or deleting at least one amino acid residue so that the CDR or framework residue at the site does not correspond to the donor antibody or the common framework. However, in a preferred embodiment, such mutations will not be extensive. Usually, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parent FR and CDR sequences. As used herein, the term "common framework" refers to the framework region in the common immunoglobulin sequence. As used herein, the term "common immunoglobulin sequence" refers to the sequence formed by the most frequently occurring amino acids (or nucleotides) in the family of related immunoglobulin sequences (see, for example, Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, 1987)). Therefore, "common immunoglobulin sequence" can include "common framework region" and / or "common CDR". In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently in that position in the family. If two amino acids occur equally frequently, either one may be included in the consensus sequence.

[0103] "Identical" or "identity" as used herein in the context of two or more polypeptide or polynucleotide sequences can mean that the sequences have a specified percentage of identical residues over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions where the identical residue occurs in the two sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions within the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation but not the numerator.

[0104] As used interchangeably herein, "head injury (Injury to the head or head injury)" refers to any trauma to the scalp, skull or brain. Such injuries may include only slight impacts on the skull or may be severe brain damage. Such injuries include primary damage to the brain and / or secondary damage to the brain. Primary brain injury occurs during the initial violation and is caused by the displacement of the physical structure of the brain. More specifically, primary brain injury is the physical damage to substance (tissue, blood vessels) that occurs during the traumatic event, resulting in shearing and compression of surrounding brain tissue. Secondary brain injury occurs after primary injury and may involve a series of cellular processes. More specifically, secondary brain injury refers to the changes developed in a period of time (from hours to days) after primary brain injury. It includes the entire cascade of cell, chemical, tissue or vascular changes in the brain that contribute to further brain tissue destruction.

[0105] Head injury can be closed or open (penetrating). Closed head injury refers to the trauma of the scalp, skull or brain in which the skull is not penetrated by the impact object. Open head injury refers to the trauma of the scalp, skull or brain in which the skull is penetrated by the impact object. Head injury can be caused by the body shaking of the person, by the external mechanical or other forces (for example, such as traffic accidents in the case of cars, airplanes, trains, etc.; such as hitting the head with a baseball bat or from a firearm) resulting in closed or open head trauma, cerebrovascular accident (such as stroke), one or more falls (for example, such as sports or other activities), explosions or shock waves (collectively referred to as "blast wave injuries") and by other types of blunt force trauma. Alternatively, head injury may be caused by ingestion and / or exposure to a combination of fire, chemicals, toxins or chemicals and toxins. Examples of such chemicals and / or toxins include mold, asbestos, pesticides and insecticides, organic solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide and cyanide), organic metals (such as methylmercury, tetraethyl lead and organotin) and / or one or more drugs of abuse. Alternatively, head injury may be due to the subject suffering from 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 impossible to determine whether any such event or injury has occurred. For example, the patient or subject may not have a medical history, the subject may not be able to speak, the subject may know the events they have experienced, etc. Such situations are described herein as subjects "may have suffered head injuries." In certain embodiments herein, closed head injury does not include and particularly excludes cerebrovascular accidents, such as stroke.

[0106] As used herein, "intracranial lesions" refer to damaged areas within the brain. Intracranial lesions can be abnormalities seen in CT scans or brain imaging tests such as magnetic resonance imaging (MRI). In CT or MRI scans, brain lesions may appear as dark or bright spots that do not look like normal brain tissue.

[0107] As used herein, "isolated polynucleotide" may mean a polynucleotide (e.g., a polynucleotide of genomic, cDNA, or synthetic origin, or a combination thereof) which, depending on its origin, is not associated with all or a portion of a polynucleotide with which the "isolated polynucleotide" can be found in nature; is operably linked to a polynucleotide to which it is not linked in nature; or does not occur in nature as part of a larger sequence.

[0108] As used herein, "label" and "detectable label" refer to a moiety attached to an antibody or analyte to make the reaction between the antibody and the analyte detectable, and an antibody or analyte so labeled is referred to as "detectably labeled". A label can produce a signal that can be detected by visual or instrumental means. Various labels include substances that produce a signal, such as chromophores, fluorescent compounds, chemiluminescent compounds, radioactive compounds, etc. Representative examples of labels include: a light-generating moiety, such as an acridinium compound; and a fluorescence-generating moiety, such as fluorescein. Other labels are described herein. In this regard, the moiety itself may be undetectable, but may become detectable when reacted with another moiety. The use of the term "detectably labeled" is intended to encompass such labels.

[0109] Any suitable detectable label as known in the art can be used. For example, the detectable label can be a radioactive label (such as 3H, 14C, 32P, 33P, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho and 153Sm), an enzyme label (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, etc.), a chemiluminescent label (such as acridinium ester, thioester or sulfonamide; luminol, isoluminol, phenanthridinium ester, etc.), a fluorescent label (such as fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, etc.)), rhodamine, phycobiliprotein, R-phycoerythrin, quantum dots (e.g., cadmium selenide capped with zinc sulfide), a thermometric label, or an immunopolymerase chain reaction label. An introduction to labels, labeling procedures, and label detection is found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd Edition, Springer Verlag, NY (1997); and Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996), a combined handbook and catalog 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 hereby incorporated by reference in their entirety). 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., Biorg. Med. Chem. Lett. 14:3917--3921 (2004); and Adamczyk et al., Org. Lett. 5:3779-3782 (2003)).

[0110] In one aspect, 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., In Luminescence Biotechnology: Instruments and Applications; Dyke, KV ed.; CRC Press: Boca Raton, pp. 77-105 (2002); Adamczyk et al., Org. Lett. 5:3779-3782 (2003); and US Pat. Nos. 5,468,646, 5,543,524, and 5,783,699 (each of which is incorporated herein by reference in its entirety for its teachings in this regard).

[0111] Another example of an acridinium compound is an acridinium-9-carboxylate aryl ester. An example of an acridinium-9-carboxylate aryl ester having Formula II is 10-methyl-9-(phenoxycarbonyl)acridine fluorosulfonate (available from Cayman Chemical, Ann Arbor, MI). Methods for preparing acridinium-9-carboxylate aryl esters are described in McCapra et al., Photochem. Photobiol. 4: 1111-21 (1965); Razavi et al., Luminescence 15: 245-249 (2000); Razavi et al., Luminescence 15: 239-244 (2000); and U.S. Pat. No. 5,241,070 (each of which is incorporated herein by reference for its teachings in this regard). Such acridine-9-carboxylic acid aryl esters are chemiluminescent indicators that are efficient in signal intensity and / or signal rapidity for hydrogen peroxide produced in an analyte oxidized by at least one oxidase. The chemiluminescent emission process of acridine-9-carboxylic acid aryl esters is completed rapidly, i.e., completed within 1 second, while the chemiluminescent emission of acridine-9-carboxamides extends to 2 seconds. However, acridine-9-carboxylic acid aryl esters lose their chemiluminescent properties in the presence of protein. Therefore, its use requires the absence of protein during signal generation and detection. Methods for separating or removing proteins in samples are well known to those skilled in the art, and include but are not limited to ultrafiltration, extraction, precipitation, dialysis, chromatography and / or digestion (see, e.g., Wells, High Throughput Bioanalytical Sample Preparation. Methods and Automation Strategies, Elsevier (2003)). The amount of protein removed or separated from the test sample can be about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95%. More details about acridinium-9-carboxylic acid aryl esters and their uses are described in U.S. Patent Application No. 11 / 697,835 filed on April 9, 2007. Acridinium-9-carboxylic acid aryl esters can be dissolved in any suitable solvent, such as degassed anhydrous N,N-dimethylformamide (DMF) or aqueous sodium cholate.

[0112] "Linker sequence" or "linker peptide sequence" refers to a natural or artificial polypeptide sequence connected to one or more polypeptide sequences of interest (e.g., full length, fragments, etc.). The term "connected" refers to the joining of a linker sequence to a polypeptide sequence of interest. Such polypeptide sequences are preferably joined by one or more peptide bonds. The linker sequence may have a length of about 4 to about 50 amino acids. Preferably, the length of the linker sequence is about 6 to about 30 amino acids. The natural linker sequence may be modified by amino acid substitution, addition or deletion to produce an artificial linker sequence. The linker sequence may be used for many purposes, including for use in recombinant Fab. Exemplary linker sequences include, but are not limited to: (i) a histidine (His) tag, such as a 6X His tag, which has an amino acid sequence of HHHHHH (SEQ ID NO: 3), which may be used as a linker sequence to facilitate separation and purification of polypeptides and antibodies of interest; (ii) an enterokinase cleavage site, such as a His tag, for separation and purification of proteins and antibodies of interest. Typically, an enterokinase cleavage site is used together with a His tag to separate and purify proteins and antibodies of interest. Various enterokinase cleavage sites are known in the art. Examples of enterokinase cleavage sites include, but are not limited to, the amino acid sequence of DDDDK (SEQ ID NO: 4) and its derivatives (e.g., ADDDDK (SEQ ID NO: 5), etc.); (iii) miscellaneous sequences can be used to link / connect the light chain and / or heavy chain variable regions 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). The linking sequence can also be modified for additional functions, such as attachment of a drug or attachment to a solid support. In the context of the present disclosure, a monoclonal antibody, for example, can contain a linking sequence, such as a His tag, an enterokinase cleavage site, or both.

[0113] "Magnetic resonance imaging" or "MRI" as used interchangeably herein refers to a medical imaging technique used in radiology to form pictures of the anatomy and physiological processes of the human body in health and disease (e.g., referred to interchangeably herein as an "MRI," an "MRI procedure," or an "MRI scan"). MRI is a form of medical imaging that measures the response of the nuclei of human tissue to high-frequency radio waves when placed in a strong magnetic field and produces images of internal organs. MRI scanners, based on the science of nuclear magnetic resonance (NMR), use strong magnetic fields, radio waves, and field gradients to produce images of the inside of the human body.

[0114] "Monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible naturally occurring mutations that may be present in small amounts, the individual antibodies constituting the population are identical. Monoclonal antibodies are highly specific, being directed against a single antigen (e.g., although there may be cross-reactivity or shared reactivity). In addition, compared to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies herein specifically include "chimeric" antibodies, wherein a portion of a heavy chain and / or a light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass and a fragment of such an antibody, as long as they exhibit desired biological properties.

[0115] "Multivalent binding protein" is used herein to refer to a binding protein comprising two or more antigen binding sites (also referred to herein as "antigen binding domains"). 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 a binding protein that can bind to two or more related or unrelated targets, including binding proteins that can bind to two or more different epitopes of the same target molecule.

[0116] "Negative predictive value" or "NPV" as used interchangeably herein refers to the probability that a subject will have a negative outcome given that the subject has a negative test result.

[0117] As used herein, "reference level" refers to a cutoff value for evaluating diagnostic, prognostic or therapeutic efficacy, which has been linked or associated with various clinical parameters (e.g., the presence of a disease, the stage of a disease, the severity of a disease, the progression of a disease, non-progression or improvement, etc.) in this article. "Absolute amount" as used herein refers to the absolute value of the change or difference between at least two assay results obtained or sampled at different time points, and similar to the reference level, it has been linked or associated with various clinical parameters (e.g., the presence of a disease, the stage of a disease, the severity of a disease, the progression of a disease, non-progression or improvement, etc.). "Absolute value" as used herein refers to the size of a real number (such as, for example, the difference between two comparison levels (e.g., the level obtained at a first time point and the level obtained at a second time point)), regardless of its sign, i.e., whether it is positive or negative.

[0118] 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 can vary depending on the properties of the immunoassay (e.g., the antibodies used, reaction conditions, sample purity, etc.), and the assay can be compared and standardized. Based on the description provided by the present disclosure, it is also entirely within the capabilities of those of ordinary skill in the art to adapt the disclosure herein to other immunoassays to obtain immunoassay-specific reference levels and absolute amounts for those other immunoassays. Although the exact values ​​of reference levels and absolute amounts may vary between assays, the findings described herein should be generally applicable and can be inferred to other assays.

[0119] A "point-of-care device" refers to a device used to provide medical diagnostic tests at or near the point of care (i.e., outside a laboratory), at the time and place of patient care (such as in a hospital, physician's office, urgent or other medical care facility, patient's home, nursing home, and / or long-term care and / or hospice facility). Examples of point-of-care devices include devices produced by Abbott Laboratories (Abbott Park, IL) (e.g., i-STAT and i-STAT Alinity), ubiquitous biosensors (Rowville, Australia) (see US 2006 / 0134713), Axis-Shield PoC AS (Oslo, Norway), and clinical laboratory products (Los Angeles, USA). In some embodiments, the point-of-care device is a disposable device. The term "disposable device" or "disposable instrument" refers to a clinical diagnostic instrument that processes and performs clinical diagnostic assays on a unit-use basis (such as a disposable cartridge) for a single patient sample. A point-of-care instrument does not perform assays on more than one clinical sample simultaneously. However, a point-of-care instrument may have the ability to measure more than one parameter (e.g., more than one analyte) in a single clinical sample on a unit-use basis.

[0120]

[00136] "Positive predictive value" or "PPV" as used interchangeably herein refers to the probability that a subject will have a positive outcome given that the subject has a positive test result.

[0121] "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., one or more, such as a plurality) are typically used to establish a calibration (standard) curve to interpolate the concentration of an analyte (such as an antibody or an analyte). Alternatively, a single calibrator close to a reference level or control level (e.g., a "low," "medium," or "high" level) may be used. Multiple calibrators (i.e., more than one calibrator or different amounts of calibrators) may be used in combination to form a "sensitivity panel."

[0122] A "receiver operating characteristic" curve or "ROC" curve refers to a graph that illustrates the performance of a binary classifier system when its discrimination threshold is varied. For example, a ROC curve can be a plot of the true positive rate versus the false positive rate for different possible cutoff points for a diagnostic test. It is generated by plotting the fraction of true positives in positives (TPR = true positive rate) versus the fraction of false positives in negatives (FPR = false positive rate) at various threshold settings. TPR is also called sensitivity, and FPR is one minus specificity or true negative rate. The ROC curve shows the trade-off between sensitivity and specificity (any increase in sensitivity is accompanied by a decrease in specificity); the more closely the curve follows the left boundary of the ROC space, and then the top boundary, 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) of the test value; and the area under the curve is a measure of the test accuracy.

[0123] "Recombinant antibody" and "recombinant antibodies" refer to antibodies prepared by one or more steps, including cloning all or part of the nucleic acid sequence encoding one or more monoclonal antibodies into an appropriate expression vector by recombinant technology, and then expressing the antibody in an appropriate host cell. The term includes, but is not limited to, recombinantly produced monoclonal antibodies, chimeric antibodies, humanized antibodies (wholly or partially humanized), multi-specific or multivalent structures formed by antibody fragments, bifunctional antibodies, heterotypic conjugate Abs, And other antibodies as described in (i) herein. (Dual variable domain immunoglobulins and methods for their preparation are described in Wu, C et al., Nature Biotechnology, 25: 1290-1297 (2007)). As used herein, the term "bifunctional antibody" refers to an antibody comprising a first arm specific for one antigenic site and a second arm specific for a different antigenic site, i.e., the bifunctional antibody has dual specificity.

[0124] As used herein, "risk assessment," "risk classification," "risk identification," or "risk stratification" of a subject (e.g., a patient) refers to the evaluation of factors, including biomarkers, to predict the risk of future events, including disease onset or disease progression, so that treatment decisions regarding the subject can be made on a more informed basis.

[0125] As used interchangeably herein, "sample," "test sample," "specimen," "sample from a subject," "biological sample," and "patient sample" can be a blood (such as whole blood (including, for example, capillary blood, venous blood, dried blood spots, etc.), serum, or plasma) sample, or tissue, saliva, urine, amniotic fluid, oropharyngeal specimen, nasopharyngeal specimen, lower respiratory tract specimen (such as, but not limited to, sputum, endotracheal aspirate, or bronchoalveolar lavage fluid), cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample can be used directly as obtained from the patient, or can be pre-treated, such as by filtration, dilution, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to modify the characteristics of the sample in some manner discussed herein or otherwise known in the art. In addition, the sample can be a nasopharyngeal or oropharyngeal sample obtained using one or more swabs, which, once obtained, is placed in a sterile tube containing viral transport medium (VTM) or universal transport medium (UTM) and retained therein or transferred to another medium for testing.

[0126] Samples can be obtained using a variety of cell types, tissues or body fluids. Such cell types, tissues and fluids can include tissue sections, such as biopsy and autopsy samples, oropharyngeal specimens, nasopharyngeal specimens, frozen sections obtained for histological purposes, blood (e.g., whole blood, dried blood spots, etc.), plasma, serum, saliva, red blood cells, platelets, interstitial fluid, cerebrospinal fluid, etc. Cell types and tissues can also include lymph, cerebrospinal fluid, or any fluid collected by suction. Tissue or cell type can be provided by removing cell samples from humans and non-human animals, but can also be completed by using previously separated cells (e.g., separated by another person, at another time and / or for another purpose). Archival tissues can also be used, such as those with treatment or outcome history. Protein or nucleotide separation and / or purification may not be required. 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 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 other embodiments, the sample is a bronchoalveolar lavage fluid. In other embodiments, the sample is a saliva sample.

[0127] 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., those subjects who are correctly identified as having the disease or medical condition being tested). For example, this may include correctly identifying subjects with TBI as different from subjects without TBI, correctly identifying subjects with moderate, severe, or moderate to severe TBI as different from subjects with mild TBI, correctly identifying subjects with mild TBI as different from subjects with moderate, severe, or moderate to severe TBI, correctly identifying subjects with moderate, severe, or moderate to severe TBI as different from subjects without TBI, or correctly identifying subjects with mild TBI as different from subjects without TBI, etc.

[0128] As used herein, the "specificity" of an assay refers to the proportion of subjects with a negative outcome that are correctly identified as negative (e.g., correctly identifying those subjects as not having the disease or medical condition being tested). For example, this may include correctly identifying subjects without TBI as being different from subjects with TBI, correctly identifying subjects without moderate, severe, or moderate-to-severe TBI as being different from subjects with mild TBI, correctly identifying subjects without mild TBI as being different from subjects with moderate, severe, or moderate-to-severe TBI, and the like.

[0129] A "calibration composition series" refers to a plurality of compositions comprising known concentrations of: UCH-L1, wherein each composition is distinguished from the other compositions in the series by the concentration of UCH-L11; and / or (2) GFAP, wherein each composition is distinguished from the other compositions in the series by the concentration of GFAP.

[0130] As used interchangeably herein, "solid phase" or "solid support" refers to any material that can be used to attach and / or attract and immobilize (1) one or more capture agents or capture specific binding partners, or (2) one or more detection agents or detection specific binding partners. The solid phase can be selected for its inherent ability to attract and immobilize capture agents. Alternatively, the solid phase can be adhered with a linker that has the ability to attract and immobilize (1) a capture agent or capture specific binding partner, or (2) a detection agent or detection specific binding partner. For example, the linker can include a charged substance that is oppositely charged relative to the capture agent (e.g., capture specific binding partner) or detection agent (e.g., detection specific binding partner) itself or relative to a charged substance conjugated to (1) the capture agent or capture specific binding partner or (2) the detection agent or detection specific binding partner. In general, the linking agent can be any binding partner (preferably specific) that is fixed to (attached to) a solid phase and has the ability to fix (1) a capture agent or capture a specific binding partner or (2) a detection agent or detect a specific binding partner through a binding reaction. The linking agent allows the capture agent to be indirectly bound to the solid phase material before or during the performance assay. For example, the solid phase can be a plastic, a derivatized plastic, a magnetic or non-magnetic metal, glass or silicon, including, for example, a test tube, a microtiter well, a sheet, a bead, a microparticle, a chip, and other structures known to those of ordinary skill in the art.

[0131] As used herein, "specific binding" or "specifically binds" may refer to an interaction of an antibody, protein or peptide with a second chemical substance, wherein the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical substance; for example, an antibody recognizes and binds to a specific protein structure, rather than binding to a protein broadly. If the antibody is specific for epitope "A", then in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (or free unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0132] "Specific binding partners" are members of specific binding pairs. Specific binding pairs contain two different molecules that specifically bind to each other by chemical or physical means. Thus, in addition to the antigen and antibody specific binding pairs of common immunoassays, other specific binding pairs may include biotin and avidin (or streptavidin); carbohydrates and lectins; complementary nucleotide sequences; effector molecules and receptor molecules; cofactors and enzymes; enzymes and enzyme inhibitors, etc. In addition, specific binding pairs may include members that are analogs of the original specific binding members, such as analyte-analogs. Immunoreactive specific binding members include isolated or recombinantly produced antigens, antigen fragments, and antibodies, including monoclonal and polyclonal antibodies, and complexes and fragments thereof.

[0133] As used herein, "statistically significant" refers to the possibility that the relationship between two or more variables is caused by factors 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, as long as the p-value of the observed test statistic is less than the significance level defined by the study, a statistically significant result is obtained. The p-value is the probability of obtaining a result that is at least as extreme as the observed result when the null hypothesis is true. Examples of statistical hypothesis analysis include Wilcoxon signed rank test, t-test, Chi-Square or Fisher's exact test. As used herein, "significant" refers to a change that has not yet been determined to be statistically significant (e.g., it may not have yet been subjected to statistical hypothesis testing).

[0134] As used herein, "subacute" refers to an injury between the acute and chronic stages (such as, for example, traumatic brain injury). In some aspects, subacute refers to an injury from about 24 hours to about three weeks after an actual head injury or suspected head injury.

[0135] "Subject" and "patient" as used interchangeably herein refer to any vertebrate, including but not limited to mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates (e.g., monkeys, such as cynomolgus or rhesus monkeys, chimpanzees, etc.) and humans). In some embodiments, the subject can be human or non-human. In some embodiments, the subject is a human. The subject or patient may be receiving other forms of treatment.

[0136] "Treat," "treating," or "treatment" are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progression of a disease and / or injury to which such term applies, or one or more symptoms of such disease. Depending on the condition of the subject, the term also refers to preventing the disease, and includes preventing the onset of the disease or preventing symptoms associated with the disease. Treatment may be performed in an acute or chronic manner. The term also refers to reducing the severity of the disease or symptoms associated with the disease prior to suffering from the disease. Such prevention of disease or reduction of disease severity prior to suffering refers to not administering the pharmaceutical composition to the subject at the time of administration when suffering from the disease. "Prevention" also refers to preventing the recurrence of a disease or one or more symptoms associated with the disease. "Treatment" and "therapeutically" refer to the act of treating, as "treating" is defined above.

[0137] "Traumatic brain injury" or "TBI" as used interchangeably herein refers to a complex injury with a wide spectrum of symptoms and disability. TBI is often an acute event similar to other injuries. TBI can be classified as "mild", "moderate", "moderate to severe" or "severe". The causes of TBI are varied and include, for example, shaking of the human body, car accidents, firearm injuries, cerebrovascular accidents (e.g., strokes), falls, explosions or shock waves, and other types of blunt force trauma. Other causes of TBI include ingestion 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 cyanide), organic metals (such as methylmercury, tetraethyl lead and organotin), one or more drugs of abuse or a combination thereof). Alternatively, TBI may occur in subjects with autoimmune diseases, metabolic disorders, brain tumors, hypoxia, viral infections (e.g., SARS-CoV-2, meningitis, etc.), fungal infections (e.g., meningitis), bacterial infections (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.

[0138] As used herein, "mild TBI" refers to a head injury in which a subject may or may not experience loss of consciousness. For subjects who experience loss of consciousness, it is usually brief, usually lasting only a few seconds or minutes. Mild TBI is also known as concussion, mild head trauma, mild TBI, mild brain injury, and mild head injury. Although MRI and CT scans are often normal, individuals with mild TBI may have cognitive problems such as headaches, difficulty thinking, memory problems, attention deficits, mood swings, and depression.

[0139] Mild TBI is the most common TBI and is often missed at the time of the initial injury. Typically, subjects have a Glasgow Coma Scale score between 13-15 (such as 13-15 or 14-15). Fifteen percent (15%) of patients with mild TBI have symptoms that persist for 3 months or longer. Common symptoms of mild TBI include fatigue, headache, visual disturbances, memory loss, poor attention / concentration, sleep disturbances, dizziness / loss of balance, irritable mood disorders, depressed mood, and seizures. Other symptoms associated with mild TBI include nausea, loss of smell, sensitivity to light and sound, mood changes, confusion or bewilderment, and / or slowed thinking.

[0140] As used herein, "moderate TBI" refers to brain injury, where loss of consciousness and / or confusion and disorientation are between 1 and 24 hours and the subject has a Glasgow Coma Scale score between 9-13 (such as 9-12 or 9-13). Individuals with moderate TBI may have abnormal brain imaging results. As used herein, "severe TBI" refers to brain injury, where loss of consciousness exceeds 24 hours and memory loss is longer than 24 hours after injury or penetrating skull injury and the subject has a Glasgow Coma Scale score between 3-8. The range of defects is from higher levels of 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 be in an unresponsive state for a long time. For many people with severe TBI, long-term rehabilitation is usually required to maximize function and independence.

[0141] As used herein, "moderate to severe" TBI refers to a spectrum of brain injuries that includes changes over time from moderate to severe TBI, and thus includes (e.g., in time) moderate TBI alone, severe TBI alone, and moderate to severe TBI in combination. For example, in some clinical situations, a subject may initially be diagnosed with moderate TBI, but over time (minutes, hours, or days), progress to severe TBI (e.g., in the case of cerebral hemorrhage). Alternatively, in some clinical situations, a subject may initially be diagnosed with severe TBI, but over time (minutes, hours, or days), progress to moderate TBI. Such a subject would be an example of a patient who may be classified as "moderate to severe." Common symptoms of moderate to severe TBI include cognitive deficits, including difficulties with attention, concentration, distractibility, memory, speed of calculation, confusion, persistent speech, impulsivity, language processing and / or "executive functioning", inability to understand spoken words (sensory aphasia), difficulty speaking and being understood (expressive aphasia), slurred speech, very fast or slow speech, problems reading, problems writing, difficulty interpreting touch, temperature, movement, limb position and fine discrimination, difficulty integrating or patterning sensory impressions into mentally meaningful data, partial or complete loss of vision, eye muscle weakness and double vision (diplopia), blurred vision, difficulty judging distances Problems with hearing loss, involuntary eye movements (nystagmus), intolerance to light (photophobia), hearing problems (such as reduced or loss of hearing, ringing in the ears (tinnitus), increased sensitivity to sound), loss or reduced sense of smell (anosmia), loss or reduced sense of taste, seizures associated with epilepsy (which can be of several types and can involve disruption of consciousness, sensory perception, or motor movement), problems with bowel and bladder control, sleep disturbances, loss of endurance, changes in appetite, problems with temperature regulation, menstrual difficulties, dependent behavior, problems with emotional capacity or stability, lack of motivation, irritability, aggression, depression, disinhibition, or rejection / lack of consciousness. Subjects with moderate to severe TBI may have a Glasgow Coma Scale score of 3-12 (which includes a range of 9-12 for moderate TBI and a range of 3-8 for severe TBI).

[0142] "Ubiquitin carboxyl-terminal hydrolase L1" or "UCH-L1" as used interchangeably herein refers to a deubiquitinating enzyme encoded by the UCH-L1 gene in humans and by the UCH-L1 gene counterparts in other species. UCH-L1 (also known as ubiquitin carboxyl-terminal esterase L1 and ubiquitin thioesterase) is a member of a gene family whose products hydrolyze small C-terminal adducts of ubiquitin to generate ubiquitin monomers.

[0143] "UCH-L1 status" can mean the level or amount of UCH-L1 at a point in time (such as, using a single measurement of UCH-L1), the level or amount of UCH-L1 associated with monitoring (such as, repeated testing of a subject to identify an increase or decrease in the amount of UCH-L1), the level or amount of UCH-L1 associated with the treatment of traumatic brain injury (whether primary brain injury and / or secondary brain injury), or a combination thereof.

[0144] "Variant" is used herein to describe a peptide or polypeptide that differs in amino acid sequence due to 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 to promote an immune response. Variant is also used herein to describe a protein having an amino acid sequence substantially identical to a reference protein, wherein the reference protein has an amino acid sequence that retains at least one biological activity. Conservative substitution of amino acids, i.e., replacing amino acids with different amino acids of similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is generally recognized in the art as generally involving minor changes. These minor changes can be identified in part by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J.Mol.Biol.157:105-132 (1982). The hydropathic index of amino acids is based on considerations of their hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids with a hydropathic index of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that will produce proteins that retain biological functions. Considering the hydrophilicity of amino acids in the context of peptides allows calculation of the maximum local average hydrophilicity of the peptide, which is a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is incorporated herein by reference in its entirety. As is known in the art, substitution of amino acids with similar hydrophilicity values ​​can produce peptides that retain biological activity (e.g., immunogenicity). Substitutions can be made with amino acids having hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the specific side chain of the amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and in particular the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties. "Variant" can also be used to refer to an antigenically reactive fragment of an anti-UCH-L1 antibody that differs in amino acid sequence from the corresponding fragment of the anti-UCH-L1 antibody, but still has antigenic reactivity and can compete with the corresponding fragment of the anti-UCH-L1 antibody for binding to UCH-L1. "Variant" may also be used to describe a polypeptide or fragment thereof that has been differentially processed (such as by proteolysis, phosphorylation or other post-translational modification) but still retains its antigenic reactivity.

[0145] "Vector" is used herein to describe a nucleic acid molecule that can transport another nucleic acid that has been connected. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which an additional DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to a viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and additional mammalian vectors containing bacterial replication origins). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, some vectors can guide the expression of the genes to which they are operably connected. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). Generally speaking, expression vectors useful in recombinant DNA technology are usually in the form of plasmids. "Plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms. However, other forms of expression vectors that function equivalently, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), can be used. In this regard, RNA versions of vectors, including RNA viral vectors, may also be used in the context of the present disclosure.

[0146] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure will have the meanings commonly understood by those of ordinary skill in the art. For example, any nomenclature used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and its technology are those well known and commonly used in the art. The meaning and scope of the term should be clear; however, if there is any implicit ambiguity, the definition provided herein takes precedence over any dictionary or external definition. In addition, unless the context requires otherwise, singular terms should include plural terms and plural terms should include singular terms.

[0147] 2. Methods to help determine traumatic brain injury (TBI) in subjects whose head computed tomography (CT) scans are negative for TBI

[0148] Among other methods, the present disclosure relates to a method of helping determine whether a subject (such as a human subject) who has suffered, may have suffered, or is suspected of suffering a head injury is more likely to suffer a traumatic brain injury (TBI), wherein the subject has undergone one or more head CT scans and is negative for TBI. Specifically, the method may include the following steps: (a) simultaneously or sequentially (in any order): (1) performing an assay on a sample obtained from the subject at least once between about 24 hours and about three weeks after the actual or suspected head injury to measure or detect the level of a biomarker in the sample, the biomarker comprising ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof; and (2) performing a head CT scan on the subject at least once within a clinically relevant time frame; and (b) diagnosing the subject as more likely to have TBI if the level of the biomarker is above a reference level and the head CT scan is negative for TBI. The sample may be a biological sample.

[0149] In another aspect, the present disclosure relates to a method for helping to identify a subject (such as a human subject) who has suffered, may have suffered, or is suspected of suffering a head injury. Specifically, the method includes measuring a sample obtained from the subject about 24 hours to about three weeks after an actual head injury or a suspected head injury to measure or detect the level of a biomarker in the sample, the biomarker including ubiquitin carboxyl terminal hydrolase L1 (UCH-L1), glial fibrillary acid protein (GFAP), or a combination thereof; and wherein the method includes: if the level of the biomarker is higher than a reference level, and a head computed tomography (CT) scan of the subject performed within a clinically relevant time frame is negative for TBI or a head CT scan is not performed on the subject, then the subject is diagnosed as being more likely to have a subacute traumatic brain injury (TBI). 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.

[0150] As mentioned herein, at least one assay and, optionally, at least one head CT scan, if performed, can be performed simultaneously or sequentially in any order. When performed sequentially, the assay and the 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.

[0151] In some aspects, the methods described herein allow for the identification of subjects who have suffered or may have suffered a head injury as having TBI based on the levels of one or more biomarkers in certain circumstances, where such subjects have undergone a head CT scan and are negative for TBI. The methods described herein allow for the identification of subjects who have suffered TBI but who may otherwise have been incorrectly diagnosed as not having TBI if the diagnosis was made based solely on the results of one or more head CT scans.

[0152] In some embodiments, the method may include obtaining a sample from the subject about 24 hours to about three weeks after the suspected injury and contacting the sample with an antibody to a TBI biomarker such as ubiquitin carboxyl terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, to allow formation of a complex of the antibody and biomarker. The method also includes detecting the resulting antibody-biomarker complex.

[0153] In some embodiments, a sample is obtained from a subject, such as a human subject, at the following times: about 24 hours after a head injury (e.g., an acute injury) or suspected head injury; about 24.5 hours to about 3 weeks after an injury or suspected injury; about 25 hours to about 3 weeks after an injury or suspected injury; about 26 hours to about 3 weeks after an injury or suspected injury; about 27 hours to about 3 weeks after an injury or suspected injury; about 28 hours to about 3 weeks after an injury or suspected injury; about 29 hours to about 3 weeks after an injury or suspected injury; about 30 hours to about 3 weeks after an injury or suspected injury; about 35 hours to about 3 weeks after an injury or suspected injury; about 40 hours to about 3 weeks after an injury or suspected injury; about 48 hours to about 3 weeks after an injury or suspected injury ; about 72 hours to about 3 weeks after injury or suspected injury; about 96 hours to about 3 weeks after injury or suspected injury; about 120 hours to about 3 weeks after injury or suspected injury; about 6 days to about 3 weeks after injury or suspected injury; about 7 days to about 3 weeks after injury or suspected injury; about 8 days to about 3 weeks after injury or suspected injury; about 9 days to about 3 weeks after injury or suspected injury; about 10 days to about 3 weeks after injury or suspected injury; about 11 days to about 3 weeks after injury or suspected injury; about 12 days to about 3 weeks after injury or suspected injury; about 24.5 hours to about 2 weeks after injury or suspected injury; about 25 hours to about 2 weeks after injury or suspected injury; about 26 hours to about 2 weeks after injury or suspected injury; about 27 hours to about 2 weeks after injury or suspected injury; injury or about 28 hours to about 2 weeks after the 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 suspected injury; 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 24.5 hours to about 1 week after an injury or suspected injury; about 25 hours to about 1 week after an injury or suspected injury; about 26 hours to about 1 week after an injury or suspected injury; about 27 hours to about 1 week after an injury or suspected injury; about 28 hours to about 1 week after an injury or suspected injury; about 29 hours to about 1 week after an injury or suspected injury; about 30 hours to about 1 week after an injury or suspected injury; about 35 hours to about 1 week after an injury or suspected injury; about 40 hours to about 1 week after an injury or suspected injury; about 48 hours to about 1 week after an injury or suspected injury; about 72 hours to about 1 week after an injury or suspected injury; about 96 hours to about 1 week after an injury or suspected injury; or about 120 hours to about 1 week after an injury or suspected injury or suspected head injury.For example, the patient may be assessed at about 24.5 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about 81 hours, about 82 hours, about 83 hours, about 84 hours hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours 0 hours, about 81 hours, about 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 The sample is obtained from a subject, such as a human subject, within about 10 days, about 109 hours, about 110 hours, 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.

[0154] Generally speaking, a reference level of a biomarker such as UCH-L1, GFAP, or a combination thereof, etc. can be used as a benchmark to evaluate the results obtained when measuring UCH-L1 in a test sample. Generally speaking, when making such a comparison, a reference level of a biomarker such as UCH-L1, GFAP, or a combination thereof, etc. is obtained by running a specific assay a sufficient number of times and under appropriate conditions so that the presence, amount, or concentration of the analyte can be linked or associated with a specific stage or endpoint of TBI or with a specific marker. Typically, a reference level of a biomarker such as UCH-L1, GFAP, or a combination thereof, etc. is obtained by assaying a reference subject (or subject population). The measured biomarkers such as UCH-L1, GFAP, or a combination thereof, etc. may include fragments thereof, degradation products thereof, and / or enzymatic cleavage products thereof.

[0155] In some aspects, the reference level of GFAP is about 5pg / mL to about 115pg / mL. In other aspects, the reference level of GFAP is about 5pg / mL to about 110pg / mL. In other aspects, the reference level of GFAP is about 5pg / mL to about 105pg / mL. In other aspects, the reference level of GFAP is about 5pg / mL to about 100pg / mL. In other aspects, the reference level of GFAP is about 5pg / mL to about 90pg / mL. In other aspects, the reference level of GFAP is about 5pg / mL to about 80pg / mL. In other aspects, the reference level of GFAP is about 5pg / mL to about 70pg / mL. In other aspects, the reference level of GFAP is about 5pg / mL to about 60pg / mL. In other aspects, the reference level of GFAP is about 5pg / mL to about 50pg / mL. In other aspects, the reference level is about 5pg / mL to about 40pg / mL. In other aspects, the reference level of GFAP is about 5 pg / mL to about 30 pg / mL. In other aspects, the reference level of GFAP is about 5 pg / mL to about 25 pg / mL. In other aspects, the reference level of GFAP is about 5 pg / mL to about 20 pg / mL.

[0156] In other aspects, the reference level of GFAP is about 10pg / mL to about 115pg / mL. In the above-mentioned improved method, the reference level of GFAP is about 10pg / mL to about 110pg / mL. In the above-mentioned improved method, the reference level of GFAP is about 10pg / mL to about 105pg / mL. In some embodiments, the reference level of GFAP is about 10pg / mL to about 100pg / mL. In some embodiments, the reference level of GFAP is about 10pg / mL to about 90pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 80pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 70pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 60pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 50pg / mL. In other embodiments, the reference level is about 10pg / mL to about 40pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 30pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 25pg / mL. In other embodiments, the reference level of GFAP is about 10pg / mL to about 20pg / mL.

[0157] In other aspects, the reference level of GFAP is about 15pg / mL to about 115pg / mL. In the above-mentioned improved method, the reference level of GFAP is about 15pg / mL to about 110pg / mL. In the above-mentioned improved method, the reference level of GFAP is about 15pg / mL to about 105pg / mL. In some embodiments, the reference level of GFAP is about 15pg / mL to about 100pg / mL. In some embodiments, the reference level of GFAP is about 15pg / mL to about 90pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 80pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 70pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 60pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 50pg / mL. In other embodiments, the reference level is about 15pg / mL to about 40pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 30pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 25pg / mL. In other embodiments, the reference level of GFAP is about 15pg / mL to about 20pg / mL.

[0158] In some embodiments, the method further comprises treating a subject (such as a human subject) suffering from a subacute traumatic brain injury with a traumatic brain injury therapy and / or monitoring the subject, as described below.

[0159] 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 immunoassay, 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 chromatography or spectroscopy, such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). Moreover, the assay can be performed in a clinical chemistry format known to those of ordinary skill in the art. Such assays are described in more detail in Sections 4-8 herein. It is known in the art that the values ​​used in the assays using specific sample types (e.g., reference levels, cutoff values, threshold values, specificity, sensitivity, concentrations of calibrators and / or controls) can be extrapolated to other assay formats using techniques known in the art (such as assay standardization). For example, one way to perform assay standardization is to apply a factor to the calibrator used in the assay to make the sample concentration reading higher or lower to obtain a slope aligned with a comparison method. Other methods of normalizing results obtained for one assay to another are well known and described in the literature (see, e.g., David Wild, Immunoassay Handbook, 4th edition, Chapter 3.5, pages 315-322, the contents of which are incorporated herein by reference).

[0160] 3. Treatment and monitoring of subjects who have suffered head injury

[0161] The subject identified in 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, treatment of traumatic brain injury may take a variety of forms depending on the severity of the head injury. For example, for a subject with mild TBI, treatment may include one or more rest periods, giving up physical exercise (such as exercise), avoiding light or wearing sunglasses in the sun, medications to relieve headaches or migraines, anti-nausea medications, etc. Treatment of patients with moderate, severe, or moderate to severe TBI may include administration of one or more suitable medications (e.g., diuretics, anticonvulsants, medications for sedation and drug-induced comas, or other pharmaceutical or biopharmaceutical drugs (known or future developments for the treatment of TBI)), one or more surgical procedures (e.g., hematoma removal, skull fracture repair, decompressive craniectomy, etc.), airway protection, and one or more therapies (e.g., one or more rehabilitation, cognitive behavioral therapy, anger management, psychological counseling, etc.). In some embodiments, the method further comprises monitoring the subject (such as a human subject). In some embodiments, the subject may be monitored using a CT scan or MRI procedure.

[0162] 4. Methods for measuring UCH-L1 levels

[0163] In the above methods, UCH-L1 levels can be measured by any means, such as antibody-dependent methods, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, protein immunostaining, electrophoretic analysis, protein analysis, competitive binding analysis, functional protein analysis, or chromatography or spectroscopy, such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). Moreover, the assay can be performed in a clinical chemistry format known to those skilled in the art, for example.

[0164] 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 includes: contacting the sample simultaneously or sequentially in any order with: (1) a capture antibody (e.g., UCH-L1 capture antibody) that binds an epitope on UCH-L1 or a UCH-L1 fragment to form a capture antibody-UCH-L1 antigen complex (e.g., UCH-L1 capture antibody-UCH-L1 antigen complex), and (2) a detection antibody (e.g., UCH-L1 detection antibody) that includes a detectable label and binds an epitope on UCH-L1 that is not bound by the capture antibody to form a UCH-L1 antigen-detection antibody complex (e.g., UCH-L1 antigen-UCH-L1 detection antibody complex), such that a capture antibody-UCH-L1 antigen-detection antibody complex (e.g., UCH-L1 capture antibody-UCH-L1 antigen-UCH-L1 detection antibody complex) is formed; and measuring the amount or concentration of UCH-L1 in the sample based on the signal generated by the detectable label in the capture antibody-UCH-L1 antigen-detection antibody complex.

[0165] In some embodiments, the first specific binding member is immobilized on a solid support. In some embodiments, the second specific binding member is immobilized on a solid support. In some embodiments, the first specific binding member is a UCH-L1 antibody as described below.

[0166] In some embodiments, the sample is diluted or undiluted. 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 about 1 to about 150 microliters or less or about 1 to about 25 microliters or less.

[0167] Some instruments other than point-of-care devices, such as the Abbott Laboratories instrument ARCHITECT and other core laboratory instruments, may be able to measure UCH-L1 levels above or greater than 25,000 pg / mL in a sample.

[0168] Other detection methods include the use of or can be adapted for use on a nanopore device or nanowell device. Examples of nanopore devices are described in International Patent Publication No. WO 2016 / 161402, which is hereby incorporated by reference in its entirety. Examples of nanowell devices are described in International Patent Publication No. WO 2016 / 161400, which is hereby incorporated by reference in its entirety.

[0169] 5. UCH-L1 Antibody

[0170] The methods described herein may use an isolated antibody that specifically binds ubiquitin carboxyl-terminal hydrolase L1 ("UCH-L1") (or a fragment thereof), referred to as a "UCH-L1 antibody." The UCH-L1 antibody may be used to assess UCH-L1 status as a measure of traumatic brain injury, to detect the presence of UCH-L1 in a sample, to quantify the amount of UCH-L1 present in a sample, or to detect the presence of UCH-L1 in a sample and quantify its amount.

[0171] a. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1)

[0172] Ubiquitin carboxyl-terminal hydrolase 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. The expression of UCH-L1 is highly specific to neurons and cells of the diffuse neuroendocrine system and their tumors. It is present in large quantities in all neurons (1%-2% of total brain protein) and is specifically expressed in neurons and testes / ovaries. 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 are responsible for its hydrolase activity.

[0173] Human UCH-L1 may have the following amino acid sequence:

[0174] MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFPLTAQHENFRKKQIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGSV LKQFLSETEKMSPEDRAKCFEKNEAIQAAHDAVAQEGQCRVDDKVNFHFILFNNVDGHLYELDGRMPFPVNHGASSEDTLLKDAAKVCREFTEREQGEVRFSAVALCKAA(SEQ ID NO:1).

[0175] Human UCH-L1 may be a fragment or variant of SEQ ID NO: 1. The length of the fragment of UCH-L1 may be between 5 and 225 amino acids, between 10 and 225 amino acids, between 50 and 225 amino acids, between 60 and 225 amino acids, between 65 and 225 amino acids, between 100 and 225 amino acids, between 150 and 225 amino acids, between 100 and 175 amino acids, or between 175 and 225 amino acids. The fragment may comprise a plurality of consecutive amino acids from SEQ ID NO: 1.

[0176] b. UCH-L1 recognition antibody

[0177] 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 F(ab') 2 , Fv or scFv fragments. Antibody derivatives can be produced by mimetic peptides. In addition, the techniques described for producing single-chain antibodies can be adapted to produce single-chain antibodies.

[0178] The anti-UCH-L1 antibody may be a chimeric anti-UCH-L1 or a humanized anti-UCH-L1 antibody. In one embodiment, both the humanized antibody and the chimeric antibody are monovalent. In one embodiment, both the humanized antibody and the chimeric antibody comprise a single Fab region connected to an Fc region.

[0179] 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 an immune response in the human body. See, for example, Funaro et al., BMC Biotechnology, 2008 (8): 85. Therefore, the antibody may be a product of a human rather than an animal library. Because it is humanized, the risk of reacting to self-antigens can be minimized. Alternatively, standard yeast display libraries and display technologies can be used to select and isolate human anti-UCH-L1 antibodies. For example, a library of natural 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.

[0180] Humanized antibodies may be antibody molecules from non-human species that bind to the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.

[0181] The antibody is different from known antibodies in that it has a biological function different from that known in the art.

[0182] (1) Epitope

[0183] The antibody can immunospecifically bind to UCH-L1 (SEQ ID NO: 1), a fragment thereof, or a variant thereof. The antibody can immunospecifically recognize and bind to at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, or at least ten amino acids within the epitope region. The antibody can immunospecifically recognize and bind to an epitope having at least three consecutive amino acids, at least four consecutive amino acids, at least five consecutive amino acids, at least six consecutive amino acids, at least seven consecutive amino acids, at least eight consecutive amino acids, at least nine consecutive amino acids, or at least ten consecutive amino acids within the epitope region.

[0184] c. Antibody Preparation / Production

[0185] Antibodies can be prepared by any of a variety of techniques (including those well known to those skilled in the art). Generally, antibodies can be produced by cell culture techniques, including by conventional techniques or by transfecting antibody genes, heavy chains and / or light chains into suitable bacteria or mammalian cell hosts to produce monoclonal antibodies, to achieve the production of antibodies, wherein the antibodies can be recombinant. The various forms of the term "transfection" are intended to encompass the various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although antibodies can be expressed in prokaryotic or eukaryotic host cells, it is preferred to express antibodies in eukaryotic cells, and most preferably in mammalian host cells, because such eukaryotic cells (particularly mammalian cells) are more likely to express, assemble, and secrete antibodies that are appropriately folded and immunologically active than prokaryotic cells.

[0186] 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)), which are used with a DHFR selective marker, such 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 period of time sufficient to allow the antibody to be expressed in the host cell or, more preferably, to secrete 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.

[0187] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It should be understood that the above procedures can be modified. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of the light chain and / or heavy chain of the antibody. Recombinant DNA technology can also be used to remove part or all of the DNA encoding one or both of the light or heavy chains that are unnecessary for binding to the antigen of interest. The antibodies also encompass molecules expressed by such truncated DNA molecules. In addition, bifunctional antibodies can be produced by cross-linking the antibody with a second antibody using standard chemical cross-linking methods, in which one heavy chain and one light chain are antibodies (i.e., binding to human UCH-L1) and the other heavy chain and the other light chain are specific for antigens other than human UCH-L1.

[0188] In a preferred system for the recombinant expression of an antibody or its antigen-binding portion thereof, a recombinant expression vector encoding an antibody heavy chain and an antibody light chain is introduced into a dhfr-CHO cell by calcium phosphate-mediated transfection. In the recombinant expression vector, the antibody heavy chain and light chain genes are each operably connected to a CMV enhancer / AdMLP promoter regulatory element to drive gene height transcription. The recombinant expression vector also carries the DHFR gene, which allows the selection / amplification of methotrexate to select the CHO cells transfected by the vector. The selected transformant host cell is cultivated to allow expression of the antibody heavy chain and light chain and complete antibodies are recovered from the culture medium. Standard molecular biological techniques are used to prepare the recombinant expression vector, transfect host cells, select transformants, cultivate host cells, and recover antibodies from the culture medium. In addition, the method for synthesizing recombinant antibodies can be by cultivating host cells in a suitable culture medium until the synthesis of recombinant antibodies. The method may further include separating the recombinant antibodies from the culture medium.

[0189] Methods for preparing monoclonal antibodies include preparing immortalized cell lines capable of producing antibodies with the desired specificity. Such cell lines can be produced by spleen cells obtained from immunized animals. Animals can be immunized with UCH-L1 or fragments and / or variants thereof. The peptide used to immunize animals can contain amino acids encoding human Fc (e.g., crystallizable fragments) or the tail region of human antibodies. Splenocytes can then be immortalized, for example, by fusion with a myeloma cell fusion partner. A variety of fusion techniques can be used. For example, spleen cells and myeloma cells can be mixed with a nonionic detergent for a few minutes and then inoculated at low density on a selective medium that supports the growth of hybrid cells rather than myeloma cells. One such technique uses hypoxanthine, aminopterin, thymidine (HAT) selection. Another technique includes electrofusion. After a sufficient time (usually about 1 to 2 weeks), colonies of hybrids are observed. Select a single colony and test its culture supernatant for binding activity to the polypeptide. Hybridomas with high reactivity and specificity can be used.

[0190] Monoclonal antibodies can be isolated from the supernatant of the hybridoma colonies grown. In addition, various techniques can be used to increase yield, such as injecting the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host (such as a mouse). The monoclonal antibodies can then be harvested from ascites or blood. Contaminants can be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation and extraction. Affinity chromatography is an example of a method that can be used to purify antibodies.

[0191] The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (the F(ab) fragments) each contain a covalent heterodimer with an intact antigen-binding site. Pepsin is able to cleave IgG molecules to provide multiple fragments containing two antigen-binding sites, including the F(ab')2 Fragment.

[0192] Fv fragments can be produced by preferential proteolytic cleavage of IgM and occasionally proteolytic cleavage of IgG or IgA immunoglobulin molecules. Fv fragments can be derived using recombinant technology. Fv fragments include non-covalent VH::VL heterodimers that contain antigen binding sites that retain many antigen recognition and binding capabilities of natural antibody molecules.

[0193] Antibodies, antibody fragments or derivatives may include a heavy chain complementary determining region ("CDR") group and a light chain complementary determining region ("CDR") group, respectively, inserted between a heavy chain framework ("FR") group and a light chain framework ("FR") group, the framework group providing support for the CDR and defining the spatial relationship of the CDR relative to each other. The CDR group may include three hypervariable regions of a heavy chain or light chain V region.

[0194] Other suitable methods for producing or isolating antibodies with the necessary specificity may be used, including but not limited to methods for selecting recombinant antibodies from peptide or protein libraries (e.g., but not limited to phage, ribosome, oligonucleotide, RNA, cDNA, yeast, etc. display libraries); for example, as known in the art, available from various commercial suppliers such as Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid / Planegg, Del.), Biovation (Aberdeen, Scotland, UK), BioInvent (Lund, Sweden). See U.S. Pat. Nos. 4,704,692, 5,723,323, 5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862. Alternative methods rely on immunization of transgenic animals capable of producing a human antibody repertoire (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 as described herein. Such techniques include, but are not limited to, ribosome display (Hanes et al. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998) Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibody production techniques (e.g., the selected lymphocyte antibody method ("SLAM") (U.S. Pat. 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 droplet 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)).

[0195] Affinity-matured antibodies can be produced by any of a number of methods known in the art. For example, see Marks et al., BioTechnology, 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described in 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 at selective mutagenesis positions and at contact or hypermutation positions with activity enhancing amino acid residues are described in US Pat. No. 6,914,128 Bl.

[0196] Antibody variants can also be prepared by delivering a polynucleotide encoding the antibody to a suitable host, such as to provide a transgenic animal or mammal that produces such antibodies in its milk, such as goats, cows, horses, sheep, etc. These methods are known in the art and are described, for example, in U.S. Pat. Nos. 5,827,690, 5,849,992, 4,873,316, 5,849,992, 5,994,616, 5,565,362 and 5,304,489.

[0197] Antibody variants can also be prepared by delivering polynucleotides to provide transgenic plants and cultivated plant cells (such as but not limited to tobacco, corn and duckweed), which produce such antibodies, specific parts or variants in plant parts or cells cultivated therefrom. For example, Cramer et al. (1999) Curr.Top.Microbiol.Immunol.240:95-118 and references cited therein describe, for example, transgenic tobacco leaves expressing a large amount of recombinant proteins using inducible promoters. Transgenic corn has been used to express mammalian proteins at commercial production levels, and its biological activity is the same as that 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 (including tobacco seeds and potato tubers), including antibody fragments, such as single-chain antibodies (scFv). See, for example, Conrad et al. (1998) Plant Mol. Biol. 38: 101-109 and references cited therein. Thus, transgenic plants can also be used to produce antibodies according to known methods.

[0198] Antibody derivatives can be produced, for example, by adding exogenous sequences to modify immunogenicity or to reduce, enhance or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life or any other suitable characteristic. Generally speaking, some or all of the non-human or human CDR sequences are maintained, while the non-human sequences of the variable and constant regions are replaced by human or other amino acids.

[0199] Small antibody fragments can be diabodies with two antigen binding sites, wherein the fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH VL). See, e.g., EP 404,097; WO93 / 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 two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and produce two antigen binding sites. See also U.S. Pat. No. 6,632,926 to Chen et al., which is incorporated herein by reference in its entirety and discloses antibody variants having one or more amino acids inserted into the hypervariable region of a parent antibody and having a binding affinity for a target antigen that is at least about twice as strong as the binding affinity of the parent antibody for the antigen.

[0200] The antibody may be a linear antibody. Procedures for preparing linear antibodies are known in the art and described in Zapata et al., (1995) Protein Eng. 8(10): 1057-1062. In short, these antibodies comprise 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.

[0201] 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.

[0202] It may be useful to detectably label antibodies. Methods for conjugating antibodies to these reagents are known in the art. For illustrative purposes only, antibodies can be labeled with detectable moieties such as radioactive atoms, chromophores, or fluorophores. Such labeled antibodies can be used for diagnostic techniques in vivo or in isolated test samples. They can be connected to cytokines, ligands, and another antibody. Suitable agents for conjugation to antibodies to achieve anti-tumor effects include cytokines, such as interleukin 2 (IL-2) and tumor necrosis factor (TNF); photosensitizers for photodynamic therapy, including aluminum (III) phthalocyanine aluminum tetrasulfonate, hematoporphyrin, and phthalocyanine; radionuclides, such as iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), technetium-99m (99mTc), rhenium-186 (186Re), and rhenium-188 (188Re); antibiotics, such as adriamycin, doxorubicin, daunorubicin, methotrexate, daunomycin, neocarbazine, 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-α toxin, cytotoxin of the Chinese cobra (Zhoushan cobra) and gelonin (a plant toxin); ribosome-inactivating proteins from plants, bacteria and fungi, such as restrictocin (ribosome-inactivating protein produced by Aspergillus restriction), saporin (ribosome-inactivating protein from soapwort) and RNase; tyrosine kinase inhibitors; ly207702 (difluoropurine nucleoside); liposomes containing anti-cystic drugs (e.g., antisense oligonucleotides, plasmids encoding toxins, methotrexate, etc.); and other antibodies or antibody fragments, such as F(ab).

[0203] Antibody production by using hybridoma technology, the selected lymphocyte antibody method (SLAM), transgenic animals and recombinant antibody libraries is described in more detail below.

[0204] (1) Anti-UCH-L1 monoclonal antibody using hybridoma technology

[0205] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridomas, recombinant and phage display techniques or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including those known in the art and taught, for example, in the following documents: Harlow et al., Antibodies: A Laboratory Manual, Second Edition, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988); Hammerling et al., In Monoclonal Antibodies and T-Cell Hybridomas, (Elsevier, NY, 1981). It should also be noted that the term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic organism, prokaryotic organism or phage clone, rather than a method for producing the antibody.

[0206] Methods for producing monoclonal antibodies and antibodies produced by the methods may include culturing hybridoma cells that secrete antibodies of the present disclosure, wherein the hybridomas are preferably produced by fusing spleen cells isolated from an animal, such as a rat or mouse, immunized with UCH-L1 with myeloma cells, and then screening the hybridomas produced by the fusion for hybridoma clones that secrete 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 an immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immunostimulatory complex). Such adjuvants may protect the polypeptide from rapid dispersion by sequestering the polypeptide in a local reservoir, or they may contain substances that stimulate the host to secrete factors that are chemotactic to macrophages and other components of the immune system. Preferably, if a polypeptide is being administered, the immunization program will involve two or more administrations of the polypeptide, spread over several weeks; however, a single administration of the polypeptide may also be used.

[0207] After immunizing an animal with a UCH-L1 antigen, antibodies and / or antibody-producing cells can be obtained from the animal. Serum containing anti-UCH-L1 antibodies 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 the anti-UCH-L1 antibodies can be purified from the serum. The serum or immunoglobulins obtained in this manner are polyclonal and therefore have a heterogeneous set of properties.

[0208] Once an immune response is detected, for example, antibodies specific for the antigen UCH-L1 are detected in the rat serum, the rat spleen is harvested and the splenocytes are isolated. The splenocytes are then fused with any suitable myeloma cells (e.g., cells from the cell line SP20 available from the 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 that secrete antibodies capable of binding to UCH-L1 by methods known in the art. Ascites, which generally contains high levels of antibodies, can be produced by immunizing rats with positive hybridoma clones.

[0209] In another embodiment, immortalized hybridomas producing antibodies can be prepared from immunized animals. After immunization, the animal is sacrificed and the spleen B cells are fused with immortalized myeloma cells, as is well known in the art. See, for example, Harlow and Lane, supra. In a preferred embodiment, the myeloma cells do not secrete immunoglobulin polypeptides (non-secreting cell line). After fusion and antibiotic selection, 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 a radioimmunoassay (RIA), preferably an ELISA. Examples of ELISA screening are provided in PCT Publication No. WO 00 / 37504.

[0210] 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 (e.g., nude mice), or cultured and expanded in vitro in cell culture. Methods for selecting, cloning, and expanding hybridomas are well known to those of ordinary skill in the art.

[0211] In a preferred embodiment, the hybridoma is a rat hybridoma. In another embodiment, the hybridoma is produced in a non-human, non-rat species such as mouse, sheep, pig, goat, cattle or horse. In yet another preferred embodiment, the hybridoma is a human hybridoma in which a human non-secretory myeloma is fused with a human cell expressing an anti-UCH-L1 antibody.

[0212] Antibody fragments that recognize a specific epitope can be generated by known techniques. For example, the fragments can be synthesized by using enzymes such as papain (to produce two identical Fab fragments) or pepsin (to produce F(ab') fragments). 2 The Fab and F(ab') fragments of the present invention are produced by proteolytic cleavage of immunoglobulin molecules. 2 Fragment. F(ab') of IgG molecule 2 The fragment retains two antigen-binding sites of the larger ("parent") IgG molecule, which includes two light chains (containing a variable light chain region and a constant light chain region), the CH1 domain of the heavy chain, and the hinge region of the parent IgG molecule that forms a disulfide bond. 2 The fragments are still able to cross-link antigen molecules, such as the parent IgG molecules.

[0213] (2) Anti-UCH-L1 monoclonal antibody using SLAM

[0214] In another aspect of the disclosure, recombinant antibodies are generated from single, isolated lymphocytes using a procedure known in the art as the Selected Lymphocyte Antibody Method (SLAM), as described in U.S. Pat. No. 5,627,052; PCT Publication No. WO 92 / 02551; and Babcook et al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996). In this method, single cells secreting antibodies of interest, such as lymphocytes derived from any immunized animal, are screened using an antigen-specific hemolytic plaque assay, in which the antigen UCH-L1, a subunit of UCH-L1, or a fragment thereof is coupled to sheep erythrocytes using a linker such as biotin, and used to identify single cells secreting antibodies specific for UCH-L1. After identifying the antibody-secreting cells of interest, heavy and light chain variable region cDNAs are rescued from the cells by reverse transcriptase-PCR (RT-PCR), and these variable regions can then be expressed in a mammalian host cell such as COS or CHO cells in the context of an appropriate immunoglobulin constant region (e.g., a human constant region). Host cells transfected with amplified immunoglobulin sequences derived from lymphocytes selected in vivo can then be further analyzed and selected in vitro, for example, by panning the transfected cells to isolate cells expressing antibodies to UCH-L1. Amplified immunoglobulin sequences can be further manipulated in vitro, such as by in vitro affinity maturation methods. See, for example, PCT Publication No. WO 97 / 29131 and PCT Publication No. WO 00 / 56772.

[0215] (3) Anti-UCH-L1 monoclonal antibodies using transgenic animals

[0216] In another embodiment of the present disclosure, antibodies are produced by immunizing a non-human animal comprising some or all of the human immunoglobulin loci with a UCH-L1 antigen. In one embodiment, the non-human animal is Transgenic mice, an engineered mouse strain that contains a large fragment of the human immunoglobulin locus and lacks mouse antibody production. See, e.g., Green et al., Nature Genetics, 7:13-21 (1994) and U.S. Pat. Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364. See also PCT Publication Nos. WO 91 / 10741, WO 94 / 02602, WO 96 / 34096, WO 96 / 33735, WO 98 / 16654, WO 98 / 24893, WO 98 / 50433, WO 99 / 45031, WO 99 / 53049, WO 00 / 09560 and WO 00 / 37504. The transgenic mice produce an adult-like fully human antibody repertoire and generate antigen-specific human monoclonal antibodies. Transgenic mice contain approximately 80% of the human antibody repertoire by introducing megabase-sized, germline-configured YAC fragments of the human heavy chain loci and x light chain loci. See Mendez et al., Nature Genetics, 15:146-156 (1997); Green and Jakobovits, J. Exp. Med., 188:483-495 (1998), the disclosures of which are incorporated herein by reference.

[0217] (4) Anti-UCH-L1 monoclonal antibodies using recombinant antibody library

[0218] The antibodies of the present disclosure can also be prepared using in vitro methods, in which 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 include methods described in, for example, U.S. Patent No. 5,223,409 (Ladner et al.); PCT Publication No. WO 92 / 18619 (Kang et al.); PCT Publication No. WO 91 / 17271 (Dower et al.); PCT Publication No. WO 92 / 20791 (Winter et al.); PCT Publication No. WO 92 / 15679 (Markland et al.); PCT Publication No. WO 93 / 01288 (Breitling et al.); PCT Publication No. WO 92 / 01047 (McCafferty et al.); PCT Publication No. WO 92 / 09690 (Garrard et al.); Fuchs et al., Bio / Technology, 9:1369-1372 (1991); Hay et al., Hum. Antibod. Hybridomas, 3:81-85 (1992); Huse et al., Science, 246:1275-1281 (1989); McCafferty et al., Nature, 348:552-554 (1990); Griffiths et al., EMBO J., 12:725-734 (1993); Hawkins et al., J. Mol. Biol., 226:889-896 (1992); Clackson et al., Nature, 352:624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992); Garrard et al., Bio / Technology, 9:1373-1377 (1991); Hoogenboom et al., Nucl. Acids Res., 19:4133-4137 (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. WO 97 / 29131, the contents of each of which are incorporated herein by reference.

[0219] The recombinant antibody library can be from a subject immunized with UCH-L1 or a portion of UCH-L1. Alternatively, the recombinant antibody library can be from a natural subject, i.e., a person who has not been immunized with UCH-L1, such as a human antibody library from a human subject who has not been immunized with human UCH-L1. The antibodies of the present disclosure are selected by screening the recombinant antibody library with a peptide comprising human UCH-L1, thereby selecting those 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 previous paragraphs. In order to select an antibody of the present disclosure having a specific binding affinity for UCH-L1, such as with a specific K off Those with a desired K for dissociation from human UCH-L1 can be selected using surface plasmon resonance methods known in the art. off In order to select antibodies of the present disclosure having specific neutralizing activity against hUCH-L1, such as having a specific IC 50 For those that do, standard methods known in the art for assessing inhibition of UCH-L1 activity can be used.

[0220] 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.

[0221] For example, various phage display methods as known in the art can also be used to generate antibodies.In phage display methods, functional antibody domains are displayed on the surface of the phage particles carrying the polynucleotide sequences encoding them.Such phages can be used to display the antigen-binding domains expressed from pedigrees or combinatorial antibody libraries (for example, people or mice).The phage expressing the antigen-binding domains in conjunction with an antigen of interest can be selected or identified with an antigen, for example, using labeled antigens or antigens combined or captured onto solid surfaces or beads.The phage used in these methods is typically a filamentous phage, which includes fd and M13 binding domains, by phage expression of the Fv antibody domains stabilized by Fab, Fv or disulfide bonds with recombinant fusions to phage gene III or gene VIII protein. Examples of phage display methods that can be used to prepare antibodies include methods disclosed in Brinkmann et al., J. Immunol. Methods, 182:41-50 (1995); Ames et al., J. Immunol. Methods, 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic et al., Gene, 187:9-18 (1997); Burton et al., Advances in Immunology, 57:191-280 (1994); PCT Publication No. WO 92 / 01047; PCT Publication No. WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0222] As described in the above references, after phage selection, the antibody coding regions can be isolated from the phage and used to generate complete antibodies, including human antibodies or any other desired antigen-binding fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described in detail below. For example, methods known in the art can also be used to employ methods for recombinant production of Fab, Fab' and F(ab') 2Fragment techniques, such as those disclosed in PCT Publication No. WO 92 / 22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); Sawai et al., Am. J. Reprod. Immunol., 34:26-34 (1995); and Better et al., Science, 240:1041-1043 (1988). Examples of techniques that can be used to produce 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).

[0223] 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 antibodies of the present disclosure. One type of alternative expression system is a system in which the recombinant antibody library is expressed as an RNA-protein fusion, as described in PCT Publication No. WO 98 / 31700 (Szostak and Roberts) and Roberts and Szostak, Proc.Natl.Acad.Sci.USA, 94:12297-12302 (1997). In this system, covalent fusions are produced between mRNA and its encoded peptide or protein by in vitro translation of a synthetic mRNA carrying puromycin (a peptide-based receptor antibiotic) at its 3' end. Therefore, specific mRNA can be enriched from a complex mixture of mRNA (e.g., a recombinant library) based on the properties of the encoded peptide or protein (e.g., antibody) or a part thereof, such as an antibody or a part thereof and a bispecific antigen. The nucleic acid sequence encoding the antibody or a portion thereof recovered from the screening of such libraries can be expressed by recombinant means as described above (e.g., in a mammalian host cell), and in addition, can be further affinity matured by additional rounds of mRNA-peptide fusion screening (wherein mutations have been introduced into the initially selected sequence) or by other in vitro affinity maturation methods for recombinant antibodies, as described above. A preferred example of this method is the PROfusion display technology.

[0224] In another approach, antibodies can also be generated using yeast display methods known in the art. In yeast display methods, antibody domains are tethered to yeast cell walls using genetic methods and displayed on yeast surfaces. Such yeast can be used to display antigen binding domains expressed from a library or combinatorial antibody library (e.g., human or mouse). Examples of yeast display methods that can be used to prepare antibodies include methods disclosed in U.S. Patent No. 6,699,658 (Wittrup et al.), which is incorporated herein by reference.

[0225] d. Production of recombinant UCH-L1 antibody

[0226] Antibodies can be produced by any of a variety of techniques known in the art. For example, expressed from a host cell, wherein one or more expression vectors encoding 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 variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although it is possible to express antibodies of the present disclosure in prokaryotic or eukaryotic host cells, it is preferred to express antibodies in eukaryotic cells and most preferably mammalian host cells, because such eukaryotic cells (and particularly mammalian cells) are more likely to assemble and secrete antibodies that are properly folded and immunologically active than prokaryotic cells.

[0227] Exemplary mammalian host cells for expressing the recombinant antibodies of the present disclosure include: Chinese hamster ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216-4220 (1980)), which are used with a DHFR selective marker, for example 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 period of time sufficient to allow the antibody to be expressed in the host cell or, more preferably, secreted 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.

[0228] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It should be understood that the above procedures can be modified. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of the light chain and / or heavy chain of the antibody of the present disclosure. Recombinant DNA technology can also be used to remove part or all of the DNA encoding one or both of the light chain or heavy chain that is unnecessary for binding to the antigen of interest. The antibodies of the present disclosure also encompass molecules expressed by such truncated DNA molecules. In addition, bifunctional antibodies can be produced by cross-linking the antibodies of the present disclosure with a second antibody using standard chemical cross-linking methods, wherein one heavy chain and one light chain are antibodies of the present disclosure (i.e., binding to human UCH-L1) and the other heavy chain and the other light chain are specific for antigens other than human UCH-L1.

[0229] In a preferred system for recombinantly expressing the antibody of the present disclosure, or its antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into a dhfr-CHO cell by calcium phosphate-mediated transfection. In the recombinant expression vector, each of the antibody heavy chain and light chain genes is operably connected to a CMV enhancer / AdMLP promoter regulatory element to drive gene high transcription. The recombinant expression vector also carries a DHFR gene, which allows the selection / amplification of methotrexate to select CHO cells transfected by the vector. The selected transformant host cell is cultured to allow expression of the antibody heavy chain and light chain and complete antibodies are recovered from the culture medium. The recombinant expression vector is prepared using standard molecular biology techniques, the host cell is transfected, the transformant is selected, the host cell is cultured, and the antibody is recovered from the culture medium. In addition, the present disclosure provides a method for synthesizing the recombinant antibody disclosed herein, the method being carried out by culturing the host cell of the present disclosure in a suitable culture medium until the recombinant antibody disclosed herein is synthesized. The method may further include separating the recombinant antibody from the culture medium.

[0230] (1) Humanized Antibodies

[0231] Humanized antibodies can be antibodies or variants, derivatives, analogs or parts thereof, which immunospecifically bind to an antigen of interest and comprise a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. Humanized antibodies can be derived from non-human species antibodies that bind to a desired antigen, having one or more complementary determining regions (CDRs) from non-human species and a framework region from a human immunoglobulin molecule.

[0232] As used herein, the term "substantially" in the context of a CDR refers to a CDR whose amino acid sequence 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 and typically two variable domains (Fab, Fab', F(ab') 2 , FabC, Fv), wherein all or substantially all CDR regions correspond to those of non-human immunoglobulins (i.e., donor antibodies) and all or substantially all framework regions are those of human immunoglobulin consensus sequences. According to one aspect, humanized antibodies also comprise at least a portion of an immunoglobulin constant region (Fc) (usually a constant region of a human immunoglobulin). In some embodiments, humanized antibodies contain both a light chain and at least a heavy chain variable domain. The antibody may also include a heavy chain CH1, hinge, CH2, CH3, and CH4 region. In some embodiments, humanized antibodies contain only humanized light chains. In some embodiments, humanized antibodies contain only humanized heavy chains. In specific embodiments, humanized antibodies contain only humanized variable domains of light and / or heavy chains.

[0233] Humanized antibodies 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. Humanized antibodies may comprise sequences from more than one class or isotype, and specific constant domains may be selected using techniques well known in the art to optimize desired effector functions.

[0234] The framework region and CDR region of humanized antibodies do not need to correspond exactly to the parent sequence, for example, the donor antibody CDR or the common framework can be mutated by replacing, inserting or / or deleting at least one amino acid residue so that the CDR or framework residue at the site does not correspond to the donor antibody or the common framework. However, in one embodiment, such mutations will not be extensive. Usually, at least 90%, at least 95%, at least 98% or at least 99% of the humanized antibody residues will correspond to those of the parent FR and CDR sequences. As used herein, the term "common framework" refers to the framework region in the common immunoglobulin sequence. As used herein, the term "common immunoglobulin sequence" refers to the sequence formed by the most frequently occurring amino acid (or nucleotide) in the family of related immunoglobulins (see, for example, Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, 1987)). In the immunoglobulin family, each position in the common sequence is occupied by the amino acid that most frequently occurs in that position in the family. If two amino acids occur equally frequently, then either one may be included in the common sequence.

[0235] Humanized antibodies can be designed to minimize the unwanted immune response to rodent anti-human antibodies, which limits the duration and effectiveness of the therapeutic application of those parts in human recipients. Humanized antibodies can have one or more amino acid residues introduced therein from non-human sources. These non-human residues are often referred to as "import" residues, which are usually taken from variable domains. Humanization can be carried out by replacing the corresponding sequence of human antibodies with hypervariable region sequences. Therefore, this type of "humanized" antibody is a chimeric antibody, in which significantly less than complete human variable domains have been replaced by corresponding sequences from non-human species. For example, referring to U.S. Patent number 4,816,567, the content of the patent is incorporated herein by reference. Humanized antibodies can be human antibodies, in which some hypervariable region residues and possible FR residues are replaced by residues of similar sites in rodent antibodies. Humanization or engineering of the antibodies of the disclosure may be performed 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.

[0236] Humanized antibodies can retain high affinity for UCH-L1 and other favorable biological properties. Humanized antibodies can be prepared by analytical methods of parental sequences and various conceptual humanized products using three-dimensional models of parental sequences and humanized sequences. Three-dimensional immunoglobulin models are generally available. Computer programs that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. Inspection of these displays allows analysis of the possible role of residues in the function of candidate immunoglobulin sequences, i.e., analysis of residues that affect the ability of candidate immunoglobulins to bind to their antigens. In this way, FR residues can be selected and combined from acceptor and input sequences to achieve desired antibody characteristics, such as increased affinity for UCH-L1. In general, hypervariable region residues may be directly and most substantially involved in affecting antigen binding.

[0237] As an alternative to humanization, human antibodies (also referred to herein as "fully human antibodies") can be generated. For example, it is possible to isolate human antibodies from libraries via PROfusion and / or yeast-related technologies. Transgenic animals (e.g., mice) can also be generated that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production after immunization. For example, in chimeric and germline mutant mice, the antibody heavy chain joining region (J H ) gene homozygous deletion leads to complete suppression of endogenous antibody production. In this germline mutant mouse, transfer of human germline immunoglobulin gene array will lead to the production of human antibodies after antigen attack. 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 which are incorporated herein by reference.

[0238] e. Anti-UCH-L1 antibody

[0239] Anti-UCH-L1 antibodies can be generated using the above techniques as well as using conventional techniques known in the art. In some embodiments, the anti-UCH-L1 antibody can be an unconjugated UCH-L1 antibody, such as UCH-L1 available from UnitedState Biological (Catalog No.: 031320); Cell Signaling Technology (Catalog No.: 3524); Sigma-Aldrich (Catalog No.: HPA005993); Santa Cruz Biotechnology, Inc. (Catalog No.: sc-58593 or sc-58594); R&D Systems (Catalog No.: MAB6007); Novus Biologicals (Catalog No.: NB600-1160); Biorbyt (Catalog No.: orb33715); Enzo Life Sciences, Inc. (catalog number: ADI-905-520-1); Bio-Rad (catalog number: VMA00004); BioVision (catalog number: 6130-50); Abcam (catalog number: ab75275 or ab104938); Invitrogen Antibodies (catalog number: 480012); ThermoFisher Scientific (catalog number: MA1-46079, MA5-17235, MA1-90008 or MA1-83428); EMD Millipore (catalog number: MABN48); or Sino Biological Inc. (catalog number: 50690-R011). The anti-L1 antibody may be conjugated to a fluorophore, such as the conjugated UCH-L1 antibody available from BioVision (Catalog No. 6960-25) or Aviva Systems Biology (Catalog No. OAAF01904-FITC).

[0240] Alternatively, the antibodies described in WO 2018 / 067468 and / or Bazarian et al., “Accuracy of arapid GFAP / UCH-L1 test for the prediction of intracranial injuries on head CT after mild traumatic brain injury”, Acad. Emerg. Med., (August 6, 2021) may also be used, the contents of which are incorporated herein by reference.

[0241] 6. Methods for measuring GFAP levels

[0242] In the above methods, the GFAP substance level can be measured by any means, such as antibody-dependent methods, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, protein immunostaining, electrophoretic analysis, protein analysis, competitive binding analysis, functional protein analysis, or chromatography or spectroscopy, such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). Moreover, the determination can be performed in a clinical chemistry format known to those skilled in the art, for example.

[0243] In some embodiments, measuring the level of GFAP includes 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 GFAP includes: contacting the sample with the following items simultaneously or sequentially in any order: (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 includes a detectable label and binds to an epitope on GFAP that is not bound by the capture antibody to form a GFAP antigen-detection antibody complex (e.g., a GFAP antigen-GFAP detection antibody complex), so that a capture antibody-GFAP antigen-detection antibody complex (e.g., a GFAP capture antibody-GFAP antigen-GFAP detection antibody complex) is formed; and measuring the amount or concentration of GFAP in the sample based on the signal generated by the detectable label in the capture antibody-GFAP antigen-detection antibody complex.

[0244] 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.

[0245] In some embodiments, the sample is diluted or undiluted. 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 about 1 to about 150 microliters or less or about 1 to about 25 microliters or less.

[0246] Some instruments other than point-of-care devices, such as Abbott Laboratories instruments ARCHITECT, Alinity, and other core lab instruments, may be able to measure GFAP levels above or greater than 25,000 pg / mL in a sample.

[0247] Other detection methods include the use of or can be adapted for use on a nanopore device or nanowell device. Examples of nanopore devices are described in International Patent Publication No. WO 2016 / 161402, which is hereby incorporated by reference in its entirety. Examples of nanowell devices are described in International Patent Publication No. WO 2016 / 161400, which is hereby incorporated by reference in its entirety.

[0248] 7.GFAP Antibody

[0249] The methods described herein may use isolated antibodies that specifically bind to glial fibrillary acid protein ("GFAP") (or a fragment 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 quantify the amount of GFAP present in a sample, or to detect the presence of GFAP in a sample and quantify its amount.

[0250] a. Glial fibrillary acidic protein (GFAP)

[0251] Glial fibrillary acid protein (GFAP) is a 50kDa intracytoplasmic filamentous protein that constitutes a part of the cytoskeleton in astrocytes and has been shown to be the most specific marker of astrocyte origin cells. GFAP protein is encoded by the GFAP gene of people. GFAP is the main intermediate filament of mature astrocytes. In the central rod-shaped domain of the molecule, GFAP has considerable structural homology with other intermediate filaments. GFAP participates in the activity and shape of astrocytes by providing structural stability for astrocyte processes. Glial fibrillary acid protein and its decomposition products (GFAP-BDP) are brain-specific proteins released into the blood as part of the pathophysiological response after traumatic brain injury (TBI). After trauma, genetic disorders or chemicals cause damage to human CNS, astrocytes proliferate and show extensive hypertrophy of cell bodies and processes, and GFAP is significantly upregulated. In contrast, as astrocytes increase malignantly, there is a progressive loss of GFAP production. GFAP can also be detected in Schwann cells, enteric glial cells, salivary gland neoplasms, metastatic renal carcinoma, epiglottic cartilage, pituitary cells, immature oligodendrocytes, papillary meningiomas, and mammary myoepithelial cells.

[0252] Human GFAP may have the following amino acid sequence:

[0253] MERRRITAARRSYVSSGEMMVGGLAPGRRLGPGTRLSLARMPPPLPTRVDFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEP TKLADVYQAELRELRLRLDQLTANSARLEVERDNLAQDLATVRQKLQDETNLRLEAENNLAAYRQEADEATLARLDLERKIESLEEEIRFLRKIHEEEVRELQEQLARQ QVHVELDVAKPDLTAALKEIRTQYEAMASSNMHEAEEWYRSKFADLTDAAARNAELLRQAKHEANDYRRQLQSLTCDLESLRGTNESLERQMREQEERHVREAASYQEA LARLEEEGQSLKDEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKESKQEHKDVM(SEQ ID NO:2).

[0254] Human GFAP may be a fragment or variant of SEQ ID NO: 2. The length of 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. The fragment may comprise a plurality of consecutive amino acids from SEQ ID NO: 2. The human GFAP fragment or variant of SEQ ID NO: 2 may be a GFAP degradation product (BDP). GFAP BDP may be 38 kDa, 42 kDa (weak 41 kDa), 47 kDa (weak 45 kDa); 25 kDa (weak 23 kDa); 19 kDa or 20 kDa.

[0255] b. GFAP recognition antibody

[0256] 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 F(ab') 2 , Fv or scFv fragments. Antibody derivatives can be produced by mimetic peptides. In addition, the techniques described for producing single-chain antibodies can be adapted to produce single-chain antibodies.

[0257] The anti-GFAP antibody may be a chimeric anti-GFAP or a humanized anti-GFAP antibody. In one embodiment, both the humanized antibody and the chimeric antibody are monovalent. In one embodiment, both the humanized antibody and the chimeric antibody comprise a single Fab region linked to an Fc region.

[0258] 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 an immune response in the human body. See, for example, Funaro et al., BMC Biotechnology, 2008 (8): 85. Therefore, the antibody may be a product of a human rather than an animal library. Because it is humanized, the risk of reacting to autoantigens can be minimized. Alternatively, standard yeast display libraries and display techniques can be used to select and isolate human anti-GFAP antibodies. For example, a natural human single-chain variable fragment (scFv) library can be used to select human anti-GFAP antibodies. Transgenic animals can be used to express human antibodies.

[0259] Humanized antibodies may be antibody molecules from non-human species that bind to the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.

[0260] The antibody is different from known antibodies in that it has a biological function different from that known in the art.

[0261] (1) Epitope

[0262] The antibody can immunospecifically bind to GFAP (SEQ ID NO: 2), a fragment thereof, or a variant thereof. The antibody can immunospecifically recognize and bind to at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, or at least ten amino acids within the epitope region. The antibody can immunospecifically recognize and bind to an epitope having at least three consecutive amino acids, at least four consecutive amino acids, at least five consecutive amino acids, at least six consecutive amino acids, at least seven consecutive amino acids, at least eight consecutive amino acids, at least nine consecutive amino acids, or at least ten consecutive amino acids in the epitope region.

[0263] c. Antibody Preparation / Production

[0264] Antibodies can be prepared by any of a variety of techniques (including those well known to those skilled in the art). Generally speaking, antibodies can be generated by cell culture techniques, including by conventional techniques or by transfecting antibody genes, heavy chains and / or light chains into suitable bacteria or mammalian cell hosts to generate monoclonal antibodies to achieve the production of antibodies, wherein the antibodies can be recombinant. The various forms of the term "transfection" are intended to encompass the various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although antibodies can be expressed in prokaryotic or eukaryotic host cells, it is preferred to express antibodies in eukaryotic cells, and most preferably in mammalian host cells, because such eukaryotic cells (particularly mammalian cells) are more likely to express, assemble and secrete antibodies that are appropriately folded and immunologically active than prokaryotic cells.

[0265] 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)), which are used with a DHFR selective marker, for example 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 period of time sufficient to allow the antibody to be expressed in the host cell or, more preferably, to secrete 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.

[0266] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It should be understood that the above procedures can be modified. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of the light chain and / or heavy chain of the antibody. Recombinant DNA technology can also be used to remove part or all of the DNA encoding one or both of the light chain or heavy chain that is unnecessary for binding to the antigen of interest. The antibody also encompasses molecules expressed by such truncated DNA molecules. In addition, bifunctional antibodies can be produced by cross-linking the antibody with a second antibody using a standard chemical cross-linking method, in which one heavy chain and one light chain are antibodies (i.e., binding to human GFAP) and the other heavy chain and the other light chain have specificity for antigens other than human GFAP.

[0267] In a preferred system for the recombinant expression of an antibody or its antigen-binding portion thereof, a recombinant expression vector encoding an antibody heavy chain and an antibody light chain is introduced into a dhfr-CHO cell by calcium phosphate-mediated transfection. In the recombinant expression vector, the antibody heavy chain and light chain genes are each operably connected to a CMV enhancer / AdMLP promoter regulatory element to drive gene height transcription. The recombinant expression vector also carries the DHFR gene, which allows the selection / amplification of methotrexate to select the CHO cells transfected by the vector. The selected transformant host cell is cultivated to allow expression of the antibody heavy chain and light chain and complete antibodies are recovered from the culture medium. Standard molecular biological techniques are used to prepare the recombinant expression vector, transfect host cells, select transformants, cultivate host cells, and recover antibodies from the culture medium. In addition, the method for synthesizing recombinant antibodies can be by cultivating host cells in a suitable culture medium until the synthesis of recombinant antibodies. The method may further include separating the recombinant antibodies from the culture medium.

[0268] The method for preparing monoclonal antibodies includes preparing immortalized cell lines capable of producing antibodies with the desired specificity. Such cell lines can be produced by spleen cells obtained from immunized animals. Animals can be immunized with GFAP or its fragments and / or variants. The peptide used to immunize animals can contain amino acids encoding human Fc (e.g., crystallizable fragments) or the tail region of human antibodies. Splenocytes can then be immortalized by, for example, fusion with a myeloma cell fusion partner. A variety of fusion techniques can be used. For example, spleen cells and myeloma cells can be mixed with non-ionic detergents for a few minutes and then inoculated at low density on a selective medium that supports hybrid cells rather than myeloma cell growth. One such technique uses hypoxanthine, aminopterin, thymidine (HAT) selection. Another technique includes electrofusion. After a sufficient time (usually about 1 to 2 weeks), colonies of hybrids are observed. Select a single colony and test its culture supernatant for binding activity to the polypeptide. Hybridomas with high reactivity and specificity can be used.

[0269] Monoclonal antibodies can be isolated from the supernatant of the hybridoma colonies grown. In addition, various techniques can be used to increase yield, such as injecting the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host (such as a mouse). The monoclonal antibodies can then be harvested from ascites or blood. Contaminants can be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation and extraction. Affinity chromatography is an example of a method that can be used to purify antibodies.

[0270] The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (the F(ab) fragments) each contain a covalent heterodimer with an intact antigen-binding site. Pepsin is able to cleave IgG molecules to provide multiple fragments containing two antigen-binding sites, including the F(ab') 2 Fragment.

[0271] Fv fragments can be produced by preferential proteolytic cleavage of IgM and occasionally proteolytic cleavage of IgG or IgA immunoglobulin molecules. Fv fragments can be derived using recombinant technology. Fv fragments include non-covalent VH:VL heterodimers that contain an antigen binding site that retains many antigen recognition and binding capabilities of natural antibody molecules.

[0272] Antibodies, antibody fragments or derivatives may include a heavy chain complementary determining region ("CDR") group and a light chain complementary determining region ("CDR") group, respectively, inserted between a heavy chain framework ("FR") group and a light chain framework ("FR") group, the framework group providing support for the CDR and defining the spatial relationship of the CDR relative to each other. The CDR group may include three hypervariable regions of a heavy chain or light chain V region.

[0273] Other suitable methods for producing or isolating antibodies with the necessary specificity may be used, including but not limited to methods for selecting recombinant antibodies from peptide or protein libraries (e.g., but not limited to phage, ribosome, oligonucleotide, RNA, cDNA, yeast, etc. display libraries); for example, as known in the art, available from various commercial suppliers such as Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid / Planegg, Del.), Biovation (Aberdeen, Scotland, UK), BioInvent (Lund, Sweden). See U.S. Pat. Nos. 4,704,692, 5,723,323, 5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862. Alternative methods rely on immunization of transgenic animals capable of producing a human antibody repertoire (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 as described herein. Such techniques include, but are not limited to, ribosome display (Hanes et al. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998) Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibody production techniques (e.g., the selected lymphocyte antibody method ("SLAM") (U.S. Pat. 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 droplet 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)).

[0274] Affinity-matured antibodies can be produced by any of a number of methods known in the art. For example, see Marks et al., BioTechnology, 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described in 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 at selective mutagenesis positions and at contact or hypermutation positions with activity enhancing amino acid residues are described in US Pat. No. 6,914,128 Bl.

[0275] Antibody variants can also be prepared by delivering a polynucleotide encoding the antibody to a suitable host, such as to provide a transgenic animal or mammal that produces such antibodies in its milk, such as goats, cows, horses, sheep, etc. These methods are known in the art and are described, for example, in U.S. Pat. Nos. 5,827,690, 5,849,992, 4,873,316, 5,849,992, 5,994,616, 5,565,362 and 5,304,489.

[0276] Antibody variants can also be prepared by delivering polynucleotides to provide transgenic plants and cultivated plant cells (such as but not limited to tobacco, corn and duckweed), which produce such antibodies, specific parts or variants in plant parts or cells cultivated therefrom. For example, Cramer et al. (1999) Curr.Top.Microbiol.Immunol.240:95-118 and references cited therein describe, for example, transgenic tobacco leaves expressing a large amount of recombinant proteins using inducible promoters. Transgenic corn has been used to express mammalian proteins at commercial production levels, and its biological activity is the same as that 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 (including tobacco seeds and potato tubers), including antibody fragments, such as single-chain antibodies (scFv). See, for example, Conrad et al. (1998) Plant Mol. Biol. 38: 101-109 and references cited therein. Thus, transgenic plants can also be used to produce antibodies according to known methods.

[0277] Antibody derivatives can be produced, for example, by adding exogenous sequences to modify immunogenicity or to reduce, enhance or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life or any other suitable characteristic. Generally speaking, some or all of the non-human or human CDR sequences are maintained, while the non-human sequences of the variable and constant regions are replaced by human or other amino acids.

[0278] Small antibody fragments can be diabodies with two antigen binding sites, wherein the fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH VL). See, e.g., EP 404,097; WO93 / 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 two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and produce two antigen binding sites. See also U.S. Pat. No. 6,632,926 to Chen et al., which is incorporated herein by reference in its entirety and discloses antibody variants having one or more amino acids inserted into the hypervariable region of a parent antibody and having a binding affinity for a target antigen that is at least about twice as strong as the binding affinity of the parent antibody for the antigen.

[0279] The antibody can be a linear antibody. Procedures for preparing linear antibodies are known in the art and described in Zapata et al. (1995) Protein Eng. 8 (10): 1057-1062. In short, 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.

[0280] 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.

[0281] It may be useful to detectably label antibodies. Methods for conjugating antibodies to these reagents are known in the art. For illustrative purposes only, antibodies can be labeled with detectable moieties such as radioactive atoms, chromophores, or fluorophores. Such labeled antibodies can be used for diagnostic techniques in vivo or in isolated test samples. They can be connected to cytokines, ligands, and another antibody. Suitable agents for conjugation to antibodies to achieve anti-tumor effects include cytokines, such as interleukin 2 (IL-2) and tumor necrosis factor (TNF); photosensitizers for photodynamic therapy, including aluminum (III) phthalocyanine aluminum tetrasulfonate, hematoporphyrin, and phthalocyanine; radionuclides, such as iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), technetium-99m (99mTc), rhenium-186 (186Re), and rhenium-188 (188Re); antibiotics, such as adriamycin, doxorubicin, daunorubicin, methotrexate, daunomycin, neocarbazine, 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-α toxin, cytotoxin of the Chinese cobra (Zhoushan cobra) and gelonin (a plant toxin); ribosome-inactivating proteins from plants, bacteria and fungi, such as restrictocin (ribosome-inactivating protein produced by Aspergillus restriction), saporin (ribosome-inactivating protein from soapwort) and RNase; tyrosine kinase inhibitors; ly207702 (difluoropurine nucleoside); liposomes containing anti-cystic drugs (e.g., antisense oligonucleotides, plasmids encoding toxins, methotrexate, etc.); and other antibodies or antibody fragments, such as F(ab).

[0282] Antibody production by using hybridoma technology, the selected lymphocyte antibody method (SLAM), transgenic animals and recombinant antibody libraries is described in more detail below.

[0283] (1) Anti-GFAP monoclonal antibody using hybridoma technology

[0284] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridomas, recombinant and phage display techniques or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including those known in the art and taught, for example, in the following documents: Harlow et al., Antibodies: A Laboratory Manual, Second Edition, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988); Hammerling et al., In Monoclonal Antibodies and T-Cell Hybridomas, (Elsevier, NY, 1981). It should also be noted that the term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic organism, prokaryotic organism or phage clone, rather than a method for producing the antibody.

[0285] Methods for generating monoclonal antibodies and antibodies generated by the methods may include culturing hybridoma cells that secrete antibodies of the present disclosure, wherein the hybridomas are preferably generated by fusing spleen cells isolated from animals immunized with GFAP, such as rats or mice, with myeloma cells, and then screening hybridoma clones that secrete antibodies capable of binding to the polypeptides of the present disclosure from the hybridomas generated by the fusion. In short, rats can be immunized with GFAP antigens. In a preferred embodiment, the GFAP antigen is administered with an adjuvant to stimulate an immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide) or ISCOM (immunostimulatory complex). Such adjuvants can protect the polypeptide from rapid dispersion by isolating the polypeptide in a local reservoir, or they can contain substances that stimulate the host to secrete factors that are chemotactic to macrophages and other components of the immune system. Preferably, if the polypeptide is being administered, the immunization program will involve two or more administrations of the polypeptide, spread over several weeks; however, a single administration of the polypeptide may also be used.

[0286] After immunizing an animal with a GFAP antigen, antibodies and / or antibody-producing cells can be obtained from the animal. Serum containing anti-GFAP antibodies 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-GFAP antibodies can be purified from the serum. The serum or immunoglobulins obtained in this manner are polyclonal and therefore have a heterogeneous set of properties.

[0287] Once an immune response is detected, for example, antibodies specific for the antigen GFAP are detected in rat serum, the rat spleen is harvested and splenocytes are isolated. The splenocytes are then fused with any suitable myeloma cells (e.g., cells from the cell line SP20 available from the American Type Culture Collection (ATCC, Manassas, Va., US)) by well-known techniques. Hybridomas are selected and cloned by limiting dilution. Cells secreting antibodies capable of binding to GFAP are then determined in the hybridoma clones by methods known in the art. Ascites, which generally contains high levels of antibodies, can be produced by immunizing rats with positive hybridoma clones.

[0288] In another embodiment, the immortalized hybridoma producing antibodies can be prepared from an immunized animal. After immunization, the animal is sacrificed and the spleen B cells are fused with immortalized myeloma cells, as is well known in the art. See, for example, Harlow and Lane, supra. In a preferred embodiment, the myeloma cells do not secrete immunoglobulin polypeptides (non-secreting cell lines). After fusion and antibiotic selection, GFAP, or a portion thereof, or cells expressing GFAP are used to screen hybridomas. In a preferred embodiment, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), preferably ELISA, is used for initial screening. Examples of ELISA screening are provided in PCT Publication No. WO 00 / 37504.

[0289] 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 (e.g., nude mice), or cultured and expanded in vitro in cell culture. Methods for selecting, cloning, and expanding hybridomas are well known to those of ordinary skill in the art.

[0290] In a preferred embodiment, the hybridoma is a rat hybridoma. In another embodiment, the hybridoma is produced in a non-human, non-rat species such as a mouse, sheep, pig, goat, cattle or horse. In another preferred embodiment, the hybridoma is a human hybridoma in which a human non-secretory myeloma is fused with a human cell expressing an anti-GFAP antibody.

[0291] Antibody fragments that recognize a specific epitope can be generated by known techniques. For example, the fragments can be synthesized by using enzymes such as papain (to produce two identical Fab fragments) or pepsin (to produce F(ab') fragments). 2 The Fab and F(ab') fragments of the present invention are produced by proteolytic cleavage of immunoglobulin molecules. 2 Fragment. F(ab') of IgG molecule 2 The fragment retains two antigen-binding sites of the larger ("parent") IgG molecule, which includes two light chains (containing a variable light chain region and a constant light chain region), the CH1 domain of the heavy chain, and the hinge region of the parent IgG molecule that forms a disulfide bond. 2 The fragments are still able to cross-link antigen molecules, such as the parent IgG molecules.

[0292] (2) Anti-GFAP monoclonal antibody using SLAM

[0293] In another aspect, recombinant antibodies are generated from single, isolated lymphocytes using a procedure known in the art as the Selected Lymphocyte Antibody Method (SLAM), as described in U.S. Pat. No. 5,627,052; PCT Publication No. WO 92 / 02551; and Babcook et al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996). In this method, single cells secreting antibodies of interest, such as lymphocytes derived from any one immunized animal, 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 specific for GFAP. After identifying the antibody-secreting cells of interest, heavy and light chain variable region cDNAs are rescued from the cells by reverse transcriptase-PCR (RT-PCR), and these variable regions can then be expressed in a mammalian host cell such as COS or CHO cells in the context of appropriate immunoglobulin constant regions (e.g., human constant regions). Host cells transfected with amplified immunoglobulin sequences derived from lymphocytes selected in vivo can then be further analyzed and selected in vitro, for example, by panning the transfected cells to isolate cells expressing antibodies to GFAP. Amplified immunoglobulin sequences can be further manipulated in vitro, such as by in vitro affinity maturation methods. See, for example, PCT Publication No. WO 97 / 29131 and PCT Publication No. WO 00 / 56772.

[0294] (3) Anti-GFAP monoclonal antibodies using transgenic animals

[0295] In another embodiment of the present disclosure, antibodies are produced by immunizing a non-human animal comprising some or all of the human immunoglobulin loci with a GFAP antigen. In one embodiment, the non-human animal is Transgenic mice, an engineered mouse strain that contains a large fragment of the human immunoglobulin locus and lacks mouse antibody production. See, e.g., Green et al., Nature Genetics, 7:13-21 (1994) and U.S. Pat. Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364. See also PCT Publication Nos. WO 91 / 10741, WO 94 / 02602, WO 96 / 34096, WO 96 / 33735, WO 98 / 16654, WO 98 / 24893, WO 98 / 50433, WO 99 / 45031, WO 99 / 53049, WO 00 / 09560 and WO 00 / 37504. The transgenic mice produce an adult-like fully human antibody repertoire and generate antigen-specific human monoclonal antibodies. Transgenic mice contain approximately 80% of the human antibody repertoire by introducing megabase-sized, germline-configured YAC fragments of the human heavy chain loci and x light chain loci. See Mendez et al., Nature Genetics, 15:146-156 (1997); Green and Jakobovits, J. Exp. Med., 188:483-495 (1998), the disclosures of which are incorporated herein by reference.

[0296] (4) Anti-GFAP monoclonal antibodies using recombinant antibody library

[0297] The antibodies of the present disclosure can also be prepared using in vitro methods, in which 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 include methods described in, for example, U.S. Patent No. 5,223,409 (Ladner et al.); PCT Publication No. WO 92 / 18619 (Kang et al.); PCT Publication No. WO 91 / 17271 (Dower et al.); PCT Publication No. WO 92 / 20791 (Winter et al.); PCT Publication No. WO 92 / 15679 (Markland et al.); PCT Publication No. WO 93 / 01288 (Breitling et al.); PCT Publication No. WO 92 / 01047 (McCafferty et al.); PCT Publication No. WO 92 / 09690 (Garrard et al.); Fuchs et al., Bio / Technology, 9:1369-1372 (1991); Hay et al., Hum. Antibod. Hybridomas, 3:81-85 (1992); Huse et al., Science, 246:1275-1281 (1989); McCafferty et al., Nature, 348:552-554 (1990); Griffiths et al., EMBO J., 12:725-734 (1993); Hawkins et al., J. Mol. Biol., 226:889-896 (1992); Clackson et al., Nature, 352:624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992); Garrard et al., Bio / Technology, 9:1373-1377 (1991); Hoogenboom et al., Nucl. Acids Res., 19:4133-4137 (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. WO 97 / 29131, the contents of each of which are incorporated herein by reference.

[0298] The recombinant antibody library can be from a subject immunized with GFAP or a portion of GFAP. Alternatively, the recombinant antibody library can be from a natural subject, i.e., a human who has not been immunized with GFAP, such as a human antibody library from a human subject who has not been 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 those antibodies that recognize GFAP. Methods for performing such screening and selection are well known in the art, such as those described in the references in the previous paragraphs. In order to select antibodies of the present disclosure that have a specific binding affinity for GFAP, such as with a specific K off Those with a desired K rate constant for dissociation from human GFAP can be selected using surface plasmon resonance methods known in the art. off In order to select antibodies of the present disclosure having specific neutralizing activity against GFAP, such as having a specific IC 50 For those that do, standard methods known in the art for assessing inhibition of GFAP activity can be used.

[0299] 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.

[0300] For example, various phage display methods as known in the art can also be used to generate antibodies.In phage display methods, functional antibody domains are displayed on the surface of the phage particles carrying the polynucleotide sequences encoding them.Such phages can be used to display the antigen-binding domains expressed from pedigrees or combinatorial antibody libraries (for example, people or mice).The phage expressing the antigen-binding domains in conjunction with an antigen of interest can be selected or identified with an antigen, for example, using labeled antigens or antigens combined or captured onto solid surfaces or beads.The phage used in these methods is typically a filamentous phage, which includes fd and M13 binding domains, by phage expression of the Fv antibody domains stabilized by Fab, Fv or disulfide bonds with recombinant fusions to phage gene III or gene VIII protein. Examples of phage display methods that can be used to prepare antibodies include methods disclosed in Brinkmann et al., J. Immunol. Methods, 182:41-50 (1995); Ames et al., J. Immunol. Methods, 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic et al., Gene, 187:9-18 (1997); Burton et al., Advances in Immunology, 57:191-280 (1994); PCT Publication No. WO 92 / 01047; PCT Publication No. WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0301] As described in the above references, after phage selection, the antibody coding regions can be isolated from the phage and used to generate complete antibodies, including human antibodies or any other desired antigen-binding fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described in detail below. For example, methods known in the art can also be used to employ methods for recombinant production of Fab, Fab' and F(ab') 2Fragment techniques, such as those disclosed in PCT Publication No. WO 92 / 22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); Sawai et al., Am. J. Reprod. Immunol., 34:26-34 (1995); and Better et al., Science, 240:1041-1043 (1988). Examples of techniques that can be used to produce 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).

[0302] 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 antibodies of the present disclosure. One type of alternative expression system is a system in which the recombinant antibody library is expressed as an RNA-protein fusion, as described in PCT Publication No. WO 98 / 31700 (Szostak and Roberts) and Roberts and Szostak, Proc.Natl.Acad.Sci.USA, 94:12297-12302 (1997). In this system, covalent fusions are produced between mRNA and its encoded peptide or protein by in vitro translation of a synthetic mRNA carrying puromycin (a peptide-based receptor antibiotic) at its 3' end. Therefore, specific mRNA can be enriched from a complex mixture of mRNA (e.g., a recombinant library) based on the properties of the encoded peptide or protein (e.g., antibody) or a part thereof, such as an antibody or a part thereof and a bispecific antigen. The nucleic acid sequence encoding the antibody or a portion thereof recovered from the screening of such libraries can be expressed by recombinant means as described above (e.g., in a mammalian host cell), and in addition, can be further affinity matured by additional rounds of mRNA-peptide fusion screening (wherein mutations have been introduced into the initially selected sequence) or by other in vitro affinity maturation methods for recombinant antibodies, as described above. A preferred example of this method is the PROfusion display technology.

[0303] In another approach, antibodies can also be generated using yeast display methods known in the art. In yeast display methods, antibody domains are tethered to yeast cell walls using genetic methods and displayed on yeast surfaces. Such yeast can be used to display antigen binding domains expressed from a library or combinatorial antibody library (e.g., human or mouse). Examples of yeast display methods that can be used to prepare antibodies include methods disclosed in U.S. Patent No. 6,699,658 (Wittrup et al.), which is incorporated herein by reference.

[0304] d. Production of recombinant GFAP antibodies

[0305] Antibodies can be produced by any of a variety of techniques known in the art. For example, expressed from a host cell, wherein one or more expression vectors encoding 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 variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although it is possible to express antibodies of the present disclosure in prokaryotic or eukaryotic host cells, it is preferred to express antibodies in eukaryotic cells and most preferably mammalian host cells, because such eukaryotic cells (and particularly mammalian cells) are more likely to assemble and secrete antibodies that are properly folded and immunologically active than prokaryotic cells.

[0306] Exemplary mammalian host cells for expressing the recombinant antibodies of the present disclosure include: Chinese hamster ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216-4220 (1980)), which are used with a DHFR selective marker, for example 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 period of time sufficient to allow the antibody to be expressed in the host cell or, more preferably, secreted 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.

[0307] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It should be understood that the above procedures can be modified. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of the light chain and / or heavy chain of the antibody of the present invention. Recombinant DNA technology can also be used to remove part or all of the DNA encoding one or both of the light chain or heavy chain that is unnecessary for binding to the antigen of interest. The antibodies of the present invention also encompass molecules expressed by such truncated DNA molecules. In addition, bifunctional antibodies can be produced by cross-linking the antibodies of the present invention with a second antibody using standard chemical cross-linking methods, wherein one heavy chain and one light chain are antibodies of the present invention (i.e., binding to human GFAP) and the other heavy chain and the other light chain are specific for antigens other than human GFAP.

[0308] In a preferred system for recombinantly expressing the antibody of the present disclosure, or its antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into a dhfr-CHO cell by calcium phosphate-mediated transfection. In the recombinant expression vector, each of the antibody heavy chain and light chain genes is operably connected to a CMV enhancer / AdMLP promoter regulatory element to drive gene high transcription. The recombinant expression vector also carries a DHFR gene, which allows the selection / amplification of methotrexate to select CHO cells transfected by the vector. The selected transformant host cell is cultured to allow expression of the antibody heavy chain and light chain and complete antibodies are recovered from the culture medium. The recombinant expression vector is prepared using standard molecular biology techniques, the host cell is transfected, the transformant is selected, the host cell is cultured, and the antibody is recovered from the culture medium. In addition, the present disclosure provides a method for synthesizing the recombinant antibody disclosed herein, the method being carried out by culturing the host cell of the present disclosure in a suitable culture medium until the recombinant antibody disclosed herein is synthesized. The method may further include separating the recombinant antibody from the culture medium.

[0309] (1) Humanized Antibodies

[0310] Humanized antibodies can be antibodies or variants, derivatives, analogs or parts thereof, which immunospecifically bind to an antigen of interest and comprise a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. Humanized antibodies can be derived from non-human species antibodies that bind to a desired antigen, having one or more complementary determining regions (CDRs) from non-human species and a framework region from a human immunoglobulin molecule.

[0311] As used herein, the term "substantially" in the context of a CDR refers to a CDR whose amino acid sequence 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 and typically two variable domains (Fab, Fab', F(ab') 2 , FabC, Fv), wherein all or substantially all CDR regions correspond to those of non-human immunoglobulins (i.e., donor antibodies) and all or substantially all framework regions are those of human immunoglobulin consensus sequences. According to one aspect, humanized antibodies also comprise at least a portion of an immunoglobulin constant region (Fc) (usually a constant region of a human immunoglobulin). In some embodiments, humanized antibodies contain both a light chain and at least a heavy chain variable domain. The antibody may also include a heavy chain CH1, hinge, CH2, CH3, and CH4 region. In some embodiments, humanized antibodies contain only humanized light chains. In some embodiments, humanized antibodies contain only humanized heavy chains. In specific embodiments, humanized antibodies contain only humanized variable domains of light and / or heavy chains.

[0312] Humanized antibodies 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. Humanized antibodies may comprise sequences from more than one class or isotype, and specific constant domains may be selected using techniques well known in the art to optimize desired effector functions.

[0313] The framework region and CDR region of humanized antibodies do not need to correspond exactly to the parent sequence, for example, the donor antibody CDR or the common framework can be mutated by replacing, inserting or / or deleting at least one amino acid residue so that the CDR or framework residue at the site does not correspond to the donor antibody or the common framework. However, in one embodiment, such mutations will not be extensive. Usually, at least 90%, at least 95%, at least 98% or at least 99% of the humanized antibody residues will correspond to those of the parent FR and CDR sequences. As used herein, the term "common framework" refers to the framework region in the common immunoglobulin sequence. As used herein, the term "common immunoglobulin sequence" refers to the sequence formed by the most frequently occurring amino acid (or nucleotide) in the family of related immunoglobulins (see, for example, Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, 1987)). In the immunoglobulin family, each position in the common sequence is occupied by the amino acid that most frequently occurs in that position in the family. If two amino acids occur equally frequently, then either one may be included in the common sequence.

[0314] Humanized antibodies can be designed to minimize the unwanted immune response to rodent anti-human antibodies, which limits the duration and effectiveness of the therapeutic application of those parts in human recipients. Humanized antibodies can have one or more amino acid residues introduced therein from non-human sources. These non-human residues are often referred to as "import" residues, which are usually taken from variable domains. Humanization can be carried out by replacing the corresponding sequence of human antibodies with hypervariable region sequences. Therefore, this type of "humanized" antibody is a chimeric antibody, in which significantly less than complete human variable domains have been replaced by corresponding sequences from non-human species. For example, referring to U.S. Patent number 4,816,567, the content of the patent is incorporated herein by reference. Humanized antibodies can be human antibodies, in which some hypervariable region residues and possible FR residues are replaced by residues of similar sites in rodent antibodies. Humanization or engineering of the antibodies of the disclosure may be performed 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.

[0315] Humanized antibodies can retain high affinity for GFAP and other favorable biological properties. Humanized antibodies can be prepared using three-dimensional models of parental sequences and humanized sequences, by analytical methods of parental sequences and various conceptual humanized products. Three-dimensional immunoglobulin models are usually available. Computer programs that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. These displayed inspections allow the possible effects of residues on the function of candidate immunoglobulin sequences to be analyzed, i.e., residues that affect the ability of candidate immunoglobulins to bind their antigens to be analyzed. In this way, FR residues can be selected and combined from recipient and input sequences to achieve desired antibody characteristics, such as increased affinity for GFAP. Generally speaking, hypervariable region residues may be directly and most substantially involved in affecting antigen binding.

[0316] As an alternative to humanization, human antibodies (also referred to herein as "fully human antibodies") can be generated. For example, it is possible to isolate human antibodies from libraries via PROfusion and / or yeast-related technologies. Transgenic animals (e.g., mice) can also be generated that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production after immunization. For example, in chimeric and germline mutant mice, the antibody heavy chain joining region (JH ) gene homozygous deletion leads to complete suppression of endogenous antibody production. In this germline mutant mouse, transfer of human germline immunoglobulin gene array will lead to the production of human antibodies after antigen attack. 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 which are incorporated herein by reference.

[0317] e. Anti-GFAP antibody

[0318] Anti-GFAP antibodies can be generated using the techniques described above and using routine techniques known in the art. In some embodiments, the anti-GFAP antibody can be an unconjugated GFAP antibody, such as the GFAP antibodies available from Dako (Catalog No.: M0761); ThermoFisher Scientific (catalog number: MA5-12023, A-21282, 13-0300, MA1-19170, MA1-19395, MA5-15086, MA5-16367, MA1-35377, MA1-06701 or MA1-20035); AbCam (catalog number: ab10062, ab4648, ab68428, ab33922, ab207165, ab190288, ab115898 or ab21837); EMD Millipore (catalog number: FCMAB257P, MAB360, MAB3402, 04-1031, 04-1062, MAB5628); Santa Cruz (Catalog Nos. sc-166481, sc-166458, sc-58766, sc-56395, sc-51908, sc-135921, sc-71143, sc-65343, or sc-33673); Sigma-Aldrich (Catalog Nos. G3893 or G6171); Sino Biological Inc. (Catalog No. 100140-R012-50). The anti-GFAP antibody can be conjugated to a fluorophore, such as the conjugated GFAP antibodies available from ThermoFisher Scientific (Catalog Nos. A-21295 or A-21294), EMD Millipore (Catalog Nos. MAB3402X, MAB3402B, MAB3402B, or MAB3402C3), or AbCam (Catalog Nos. ab49874 or ab194325).

[0319] Alternatively, the antibodies described in WO 2018 / 067474 and / or Bazarian et al., “Accuracy ofarapid GFAP / UCH-L1 test for the prediction of intracranial injuries on headCT after mild traumatic brain injury”, Acad. Emerg. Med., (August 6, 2021), the contents of which are incorporated herein by reference, may also be used.

[0320] 8. Method Variations

[0321] The disclosed method of determining the presence or amount of an analyte of interest (UCH-L1 and / or GFAP) present in a sample can be as described herein. The method can also be adapted according to other methods for analyzing analytes. Examples of well-known variations include, but are not limited to, immunoassays, such as sandwich immunoassays (e.g., monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonal sandwich immunoassays, including enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay) (ELISA), competitive inhibition immunoassays (e.g., forward and reverse), enzyme amplified immunoassay technique (EMIT), competitive binding assays, bioluminescence resonance energy transfer (BRET), one-step antibody detection assays, homogeneous assays, heterogeneous assays, real-time capture assays (capture on the fly assays), etc.

[0322] a. Immunoassay

[0323] The analyte of interest and / or its peptide or fragment (e.g., UCH-L1 and / or GFAP, and / or its peptide or fragment, i.e., UCH-L1 and / or GFAP fragment) can be analyzed in an immunoassay using UCH-L1 and / or GFAP antibodies. The presence or amount of the analyte (e.g., UCH-L1 and / or GFAP) can be determined using antibodies and detecting specific binding to the analyte (e.g., UCH-L1 and / or GFAP). For example, the antibody or its antibody fragment can specifically bind to the analyte (e.g., UCH-L1 and / or GFAP). If desired, one or more antibodies can be used in combination with one or more commercially available monoclonal / polyclonal antibodies. Such antibodies can be obtained from companies such as R&D Systems, Inc. (Minneapolis, MN) and Enzo LifeSciences International, Inc. (Plymouth Meeting, PA).

[0324] The presence or amount of an analyte (e.g., UCH-L1 and / or GFAP) present in a body sample can be readily determined using an immunoassay, such as a sandwich immunoassay (e.g., monoclonal-monoclonal sandwich immunoassay, monoclonal-polyclonal sandwich immunoassay, including radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay ELISA) (e.g., Quantikine ELISAassays, R&D Systems, Minneapolis, MN)). An example of a point-of-care device that can be used is the i-STAT (Abbott, Laboratories, Abbott Park, IL). Other methods that can be used include, for example, chemiluminescent microparticle immunoassays, particularly methods using ARCHITECT or Alinity automated analyzers (Abbott Laboratories, Abbott Park, IL). Other methods include, for example, mass spectrometry, and immunohistochemistry (e.g., using sections from tissue biopsies) using anti-analyte (e.g., anti-UCH-L1 and / or anti-GFAP) antibodies (monoclonal, polyclonal, chimeric, humanized, human, etc.) or antibody fragments thereof to the analyte (e.g., UCH-L1 and / or GFAP). Other detection methods include those described in U.S. Pat. Nos. 6,143,576, 6,113,855, 6,019,944, 5,985,579, 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, each of which is hereby incorporated by reference in its entirety. Specific immunological binding of the antibody to the analyte (eg, UCH-L1 and / or GFAP) can be detected via direct labels such as fluorescent or luminescent tags, metals and radionuclides attached to the antibody, or via indirect labels such as alkaline phosphatase or horseradish peroxidase.

[0325] The use of fixed antibodies or antibody fragments thereof can be incorporated into immunoassays. Antibodies can be fixed on a variety of supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (such as a microtiter well), a sheet of solid substrate material, etc. An assay strip can be prepared by coating the antibody or multiple antibodies in an array on a solid support. This strip can then be immersed in a test sample and rapidly processed by washing and detection steps to generate a measurable signal, such as a stain.

[0326] A homogeneous form can be used. For example, a mixture is prepared after obtaining a test sample from a subject. The mixture contains a test sample, a first specific binding partner, and a second specific binding partner of an analyte to be evaluated (e.g., UCH-L1 and / or GFAP). The order in which the test sample, the first specific binding partner, and the second specific binding partner are added to form a 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 heterosexual 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 (3) 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 (3) 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 (3) 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 (3) amino acids of SEQ ID NO: 2). Furthermore, the second specific binding partner is labeled with or contains a detectable label as described above.

[0327] A heterogeneous format may be used. For example, a first mixture is prepared after obtaining a test sample from a subject. The mixture contains a test sample of an analyte to be evaluated (e.g., UCH-L1 and / or GFAP) and a first specific binding partner, wherein 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 of at least three (3) consecutive amino acids comprising SEQ ID NO: 1; or an anti-GFAP antibody that binds to an epitope having an amino acid sequence of at least three (3) consecutive amino acids comprising 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.

[0328] The first specific binding partner can be fixed on a solid phase. The solid phase used in the immunoassay (for the first specific binding partner and optionally the second specific binding partner) can be any solid phase known in the art, such as but not limited to magnetic particles, beads, test tubes, microtiter plates, colorimetric tubes, membranes, scaffold molecules, films, filter paper, discs and chips. In those embodiments where the solid phase is a bead, the bead can be a magnetic bead or a magnetic particle. The magnetic bead / particle can be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic or ferrofluid. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO 2 , MnAs, MnBi, EuO and NiO / Fe. Examples of ferrimagnetic materials include NiFe 2 O 4 、CoFe 2 O 4 , Fe 3 O 4 (or FeO·Fe 2 O 3 ). The beads may have a magnetic solid core portion and be surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion may be a layer surrounding the non-magnetic core. The solid support having the first specific binding member fixed thereon may be stored in a dry form or in a liquid. The magnetic beads may be subjected to a magnetic field before or after contact with a sample having the magnetic beads having the first specific binding member fixed thereon.

[0329] After forming a mixture containing a first specific binding partner-analyte (e.g., UCH-L1 or GFAP) antigen complex, 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 is present in an amount that exceeds any analyte (e.g., UCH-L1 and / or GFAP) present in the test sample so that all analytes (e.g., UCH-L1 and / or GFAP) present in the test sample are bound by the first specific binding partner.

[0330] After removing any unbound analyte (e.g., UCH-L1 and / or GFAP), 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 three consecutive (3) 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 (3) amino acids of SEQ ID NO: 2). In addition, the second specific binding partner is labeled with or contains a detectable label as described above.

[0331] The use of fixed antibodies or antibody fragments thereof can be incorporated into immunoassays. Antibodies can be fixed on a variety of supports, such as magnetic 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, surfaces of assay plates (such as microtiter wells), sheets of solid substrate material, etc. Assay strips can be prepared by coating the antibody or multiple antibodies in an array on a solid support. This strip can then be immersed in a test sample and rapidly processed by washing and detection steps to generate a measurable signal, such as a stain.

[0332] In some respects, it is possible to select other antibodies, which can similarly 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 (e.g., capture antibody or first specific binding partner) that binds to an epitope near the N-terminus of 38kDa BDP and at least one second antibody (e.g., detection antibody or second specific binding partner) that binds to an epitope near the middle of 38kDa BDP (e.g., near the middle of 38kDa BDP) and does not overlap with the first antibody. Other variants are possible and can be easily tested by ordinary technicians, such as confirming the combination of antibodies to different epitopes by checking the combination with short peptides, and then using low calibrator concentrations to screen antibody pairs. In addition, selecting antibodies with different affinities for 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.

[0333] (1) Sandwich immunoassay

[0334] 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 the detection antibody bind to different epitopes on the antigen (e.g., an analyte of interest, such as UCH-L1 and / or GFAP). Desirably, the binding of the capture antibody to the epitope does not interfere with the binding of the detection antibody to the epitope. Both monoclonal and polyclonal antibodies can be used as capture antibodies and detection antibodies in sandwich immunoassays.

[0335] Generally, at least two antibodies are employed to separate and quantify the analyte (e.g., UCH-L1 and / or GFAP) in a test sample. More specifically, at least two antibodies bind to certain epitopes of the analyte (e.g., UCH-L1 and / or GFAP), thereby forming an immune complex, which is referred to as 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 one or more "capture" antibodies) and one or more antibodies may be used to bind a detectable (i.e., quantifiable) label to the sandwich (these antibodies are often referred to as one or more "detection" antibodies). In a sandwich assay, the binding of an antibody to its epitope is ideally not attenuated by the binding of any other antibody to its corresponding epitope in the assay. The antibodies are selected so that one or more first antibodies contacted with a test sample suspected of containing an analyte (e.g., UCH-L1 and / or GFAP) do not bind to all or a portion of the epitope recognized by the second or subsequent antibodies, thereby interfering with the ability of one or more second detection antibodies to bind to the analyte (e.g., UCH-L1 and / or GFAP).

[0336] The antibody can 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 antibodies of the present disclosure, the immunoassay can include a second antibody that immunospecifically binds to an epitope that is not recognized or bound by the first antibody.

[0337] A test sample suspected of containing an analyte (e.g., UCH-L1 and / or GFAP) can be contacted with at least one first capture antibody (or multiple antibodies) and at least one second detection antibody simultaneously or sequentially. 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 specific 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 multiple capture antibody-UCH-L1 and / or GFAP antigen complex is formed. In a sandwich assay, antibodies, preferably at least one capture antibody, are used in a molar excess of the maximum amount of the expected analyte (e.g., UCH-L1 and / or GFAP) in the test sample. For example, about 5 μg / mL to about 1 mg / mL of antibody can be used per ml of microparticle coating buffer.

[0338] i. Anti-UCH-L1 capture antibody

[0339] Optionally, before 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 in the form of wells, tubes, or beads (such as microparticles) made of polymeric materials. One (or more) antibodies may be bound to a solid support by adsorption, by covalent bonding using a chemical coupling agent, or by other means known in the art, provided that such binding does not interfere with the ability of the antibody to bind to the analyte (e.g., UCH-L1 and / or GFAP). In addition, if desired, the solid support may be derivatized to allow reaction 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.

[0340] Thereafter, the test sample suspected of containing the analyte (e.g., UCH-L1 and / or GFAP) is incubated to allow the formation of a first capture antibody (or multiple antibodies)-analyte (e.g., UCH-L1 and / or GFAP) complex. The incubation can be performed at a pH of about 4.5 to about 10.0, at a temperature of about 2° C. to about 45° C., and for a period of at least about one (1) minute to about eighteen (18) hours, about 2-6 minutes, about 7-12 minutes, about 5-15 minutes, or about 3-4 minutes.

[0341] ii. Detection of antibodies

[0342] After the 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 the formation of the 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. If 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. As with the first antibody, when at least a second (and subsequent) antibody is contacted with the first antibody-analyte (e.g., UCH-L1 and / or GFAP) complex, a period of incubation under conditions similar to those described above is required to form 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. A detectable label may be bound to at least one of the second antibodies before, simultaneously with, or after formation of the first / multiple antibody-analyte (eg, UCH-L1 and / or GFAP)-second / multiple antibody complex. Any detectable label known in the art may be used.

[0343] Chemiluminescent assays can be performed according to the methods described in Adamczyk et al., Anal. Chim. Acta 579 (1): 61-67 (2006). Although any suitable assay format can be used, a microplate chemiluminometer (Mithras LB-940, Berthold Technologies USA, LLC, Oak Ridge, TN) enables rapid determination of multiple small volume samples. When using a 96-well black polystyrene microplate (Costar # 3792), the chemiluminometer can be equipped with multiple reagent injectors. Each sample can be added to a separate well, and then other reagents as determined by the assay type used are added simultaneously / sequentially. Desirably, for example, by acidification, pseudobases are avoided in neutral or alkaline solutions using acridinium aryl esters. The chemiluminescent response is then recorded hole by hole. In this regard, the time for recording the chemiluminescent response depends in part on the delay between the addition of reagents and the specific acridinium used.

[0344] The order in which the test sample and one or more specific binding partners are added to form a mixture for chemiluminescent assay is not critical. If 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, if a second specific binding partner is used and the second specific binding partner is detectably labeled with an acridinium compound, a detectably labeled first specific binding partner-analyte (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).

[0345] The hydrogen peroxide may be generated in situ in the mixture, or provided or supplied to the mixture before, simultaneously with or after the addition of the acridinium compound described above.The hydrogen peroxide may be generated in situ in a variety of ways, such as will be apparent to those skilled in the art.

[0346] 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 hydrogen peroxide solution may simply be added.

[0347] After adding at least one alkaline solution to the sample simultaneously or successively, a detectable signal indicating the presence of an analyte (e.g., UCH-L1 and / or GFAP), i.e., a chemiluminescent signal, is generated. The alkaline solution contains at least one base and has a pH greater than or equal to 10, preferably greater than or equal to 12. Examples of alkaline 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 alkaline solution added to the sample depends on the concentration of the alkaline solution. Based on the concentration of the alkaline solution used, a person skilled in the art can easily determine the amount of alkaline solution added to the sample. Other labels other than chemiluminescent labels can be used. For example, enzyme labels (including but not limited to alkaline phosphatase) can be used.

[0348] The generated chemiluminescent signal or other signal can be detected using conventional 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 (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 with a standard curve of the analyte (e.g., UCH-L1 and / or GFAP) or by comparing with a reference standard. A standard curve can be generated using a serial dilution or solution of a known concentration of the analyte (e.g., UCH-L1 and / or GFAP) by mass spectrometry, gravimetric methods, and other techniques known in the art.

[0349] (2) Forward competitive inhibition assay

[0350] In a forward competition format, aliquots of labeled analytes of interest (e.g., analytes with fluorescent labels (tags attached to cleavable linkers) (e.g., UCH-L1 and / or GFAP), etc.) of known concentrations are used to compete with the analyte of interest (e.g., UCH-L1 and / or GFAP) in the test sample for binding to antibodies to the analyte of interest (e.g., UCH-L1 and / or GFAP antibodies).

[0351] In a forward competition assay, a fixed specific binding partner (such as an antibody) can be contacted with a test sample and a labeled analyte of interest, a fragment of an analyte of interest, or a variant of an analyte of interest in sequence or simultaneously. The peptide of the analyte of interest, the fragment of the analyte of interest, or the variant of the analyte of interest can be labeled with any detectable label, including a detectable label composed of a label attached with a cleavable linker. In this assay, the antibody can be fixed on a solid support. Alternatively, the antibody can be coupled to an antibody, such as an anti-species antibody, that has been fixed on a solid support (such as a microparticle or a planar substrate).

[0352] The labeled analyte of interest, test sample and antibody are incubated under conditions similar to those described above for the sandwich assay format. Antibody-analyte of interest complexes of two different species can then be produced. Specifically, one of the antibody-analyte of interest complexes produced contains a detectable label (e.g., fluorescent label, etc.), while the other antibody-analyte of interest does not contain a detectable label. The antibody-analyte of interest complex can but need not be separated from the remainder of the test sample before quantitative detectable labeling. Regardless of whether the antibody-analyte of interest complex is separated from the remainder of the test sample, the amount of detectable markers in the antibody-analyte of interest complex is then quantitatively determined. The concentration of the analyte of interest (such as membrane-related analyte of interest, soluble analyte of interest, fragments of soluble analyte of interest, variants of analyte of interest (membrane-related or soluble analyte of interest) or any combination thereof) in the test sample can then be determined, for example, as described above.

[0353] (3) Reverse competitive inhibition assay

[0354] In a reverse competition assay, the immobilized analyte of interest (eg, UCH-L1 and / or GFAP) can be contacted with a test sample and at least one labeled antibody, either sequentially or simultaneously.

[0355] The analyte of interest may be bound to a solid support, such as those discussed above with respect to the sandwich assay format.

[0356] The fixed analyte of interest, test sample and at least one labeled antibody are incubated under conditions similar to those described above for the sandwich assay format. The analyte of interest-antibody complex of two different species is then produced. Specifically, one of the analyte of interest-antibody complex produced is fixed and contains a detectable label (e.g., fluorescent label, etc.), while the other analyte of interest-antibody is not fixed and contains a detectable label. By techniques known in the art, such as washing, the unfixed analyte of interest-antibody complex and the remainder of the test sample are removed from the presence of the fixed analyte of interest-antibody complex. Once the unfixed analyte of interest-antibody complex is removed, the amount of detectable label in the fixed analyte of interest-antibody complex is then quantitatively determined after the cleavage label. The concentration of the analyte of interest in the test sample can then be determined by comparing the number of detectable labels as described above.

[0357] (4) One-step immunoassay or “instant capture” assay

[0358] In an instant capture immunoassay, a solid substrate is pre-coated with a fixative. The capture agent, analyte (e.g., UCH-L1 and / or GFAP) and detector are added to the solid substrate together, followed by a washing step and then detection. The capture agent can bind to the analyte (e.g., UCH-L1 and / or GFAP) and comprises a ligand for the fixative. The capture agent and the detector can be antibodies or any other part capable of capture or detection as described herein or known in the art. The ligand can comprise a peptide tag and the fixative can comprise an anti-peptide tag antibody. Alternatively, the ligand and the fixative can be any reagent pair (e.g., a specific binding pair, and other reagent pairs as known in the art) that can be combined together for instant capture assays. More than one analyte can be measured. In some embodiments, a solid substrate can be coated with an antigen, and the analyte to be analyzed is an antibody.

[0359] In certain other embodiments, in a one-step immunoassay or "instant capture", a solid support (such as microparticles) pre-coated with a fixative (such as biotin, streptavidin, etc.) and at least a first specific binding member and a second specific binding member (used as a capture agent and a detection agent, respectively) are used. The first specific binding member comprises a ligand of the fixative (e.g., if the fixative on the solid support is streptavidin, the ligand on the first specific binding member can be biotin) and also binds to an analyte of interest (e.g., UCH-L1 and / or GFAP). The second specific binding member comprises a detectable label and binds to an 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 (sequentially or simultaneously) can be added to the test sample. The ligand on the first specific binding member binds to the fixative 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 is combined with the solid support / first specific binding member / analyte complex, and detectable label is detected. Optional washing steps can be used before detection. In certain embodiments, in a one-step assay, more than one analyte can be measured. In certain other embodiments, more than two specific binding members can be used. In certain other embodiments, multiple detectable labels can be added. In certain other embodiments, multiple analytes of interest can be detected, or their amount, level or concentration can be measured, determined or evaluated.

[0360] The use of real-time capture assays can be performed in a variety of formats as described herein and known in the art. For example, the format can be a sandwich assay as described above, but can alternatively be a competition assay, can employ a single specific binding member, or use other variations such as are known.

[0361] 9. Other factors

[0362] The method for diagnosis, prognosis and / or assessment as described above may further include the use of other factors for diagnosis, prognosis and assessment. In some embodiments, the Glasgow Coma Scale or the Glasgow Outcome Scale (GOSE) expanded may be used to diagnose traumatic brain injury. Other tests, scales or indexes may also be used alone or in combination with the Glasgow Coma Scale. An example is the Ranchos Los Amigos Scale. The Ranchos Los Amigos Scale measures the level of consciousness, cognition, behavior and interaction with the environment. The Ranchos Los Amigos Scale includes: Grade I: No response; Grade II Generalized Response; Grade III: Local reaction; Grade IV: Confusion-agitation; Grade V: Confused-inappropriate; Grade VI: Confused-appropriate; Grade VII: Automatic-appropriate; and Grade VIII: Purposeful-appropriate. Another example is the Rivermead Post-Concussion Symptoms Questionnaire, which is a self-report scale that measures the severity of post-concussion symptoms after TBI. Patients are asked to rate the severity of each of the 16 symptoms (e.g., headache, dizziness, nausea, vomiting) they have had in the past 24 hours. In each case, the symptoms are compared to their severity before the injury occurred (pre-onset). These symptoms are reported on a 0 to 4 severity scale: not experienced, no longer a problem, mild problem, moderate problem, and severe problem.

[0363] 10. Samples

[0364] In some embodiments, the sample is obtained after the subject (such as a human subject) suffers from a head injury caused by a blunt impact, one or more falls, explosions or shock waves or other types of blunt force trauma caused by body shaking, external mechanical forces or other forces that cause closed or open head trauma. In some embodiments, the sample is obtained after the subject (such as a human subject) is ingested or exposed to a combination of fire, chemicals, toxins or fire, chemicals and toxins. Examples of such chemicals and / or toxins include mold, asbestos, pesticides and insecticides, organic solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide and cyanide), organometallics (such as methylmercury, tetraethyl lead and organotin) and / or one or more drugs of abuse. In some embodiments, samples are obtained from subjects (such as human subjects) suffering from autoimmune diseases, metabolic disorders, brain tumors, hypoxia, viral infections (e.g., SARS-CoV-2), fungal infections, bacterial infections, meningitis, hydrocephalus or any combination thereof.

[0365] In another embodiment, the samples used by the methods described herein can also be used to determine whether a subject has TBI (such as mild TBI, moderate TBI, severe TBI, or moderate to severe TBI) or is at risk of developing it by determining the level of UCH-L1 and / or GFAP in the subject using anti-UCH-L1 and / or anti-GFAP antibodies or antibody fragments thereof as described below. Thus, in certain embodiments, the present disclosure also provides a method for determining whether a subject having a traumatic brain injury as described herein and known in the art or at risk of a traumatic brain injury as described herein and known in the art is a candidate for therapy or treatment. Typically, the subject is at least one of the following: (i) has experienced a head injury; (ii) has ingested and / or has been exposed to one or more chemicals and / or toxins; (iii) suffers 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; or (iv) any combination of (i)-(iii); or, in fact, has been diagnosed with or is at risk of TBI (such as, for example, 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) and / or exhibits an unfavorable (i.e., clinically undesirable) concentration or amount of UCH-L1 and / or GFAP or a UCH-L1 and / or GFAP fragment as described herein.

[0366] a. Test or biological sample

[0367] As used herein, "sample", "test sample", "biological sample" refers to a fluid sample containing or suspected of containing UCH-L1 and / or GFAP. The sample can be derived from any suitable source. In some cases, the sample may include a liquid, a flowing particulate solid, or a fluid suspension of solid particles. In some cases, the sample...

Claims

1. An improvement in a method for aiding in the diagnosis and evaluation of a subject who has sustained or may have sustained a head injury, the method comprising simultaneously or sequentially: (1) performing an assay on a sample obtained from the subject about 24 hours to about three weeks after an actual or suspected head injury to measure or detect the level of a biomarker in the sample, the biomarker comprising glial fibrillary acidic protein (GFAP); and (2) performing a head computed tomography (CT) scan on the subject within a clinically relevant time frame, wherein the improvement comprises: If the level of the biomarker is above a reference level and the head CT scan is negative for TBI, the subject is diagnosed as being more likely to have a traumatic brain injury (TBI).

2. An improvement in a method for aiding in the diagnosis and evaluation of a human subject who has sustained or may have sustained a head injury, the method comprising performing an assay on a sample obtained from the subject from about 24 hours to about three weeks after an actual or suspected head injury to measure or detect the level of a biomarker in the sample, the biomarker comprising glial fibrillary acidic protein (GFAP); and wherein the improvement comprises: If the level of the biomarker is above a reference level and a head computed tomography (CT) scan performed on the subject within a clinically relevant time frame is negative for TBI or a head CT scan was not performed on the subject, the subject is diagnosed as being more likely to have traumatic brain injury (TBI).

3. The improvement of claim 1 or claim 2, further comprising: If the level of the biomarker is above a reference level, and optionally, if a head CT scan was performed and the head CT scan is negative for TBI, the subject is treated for TBI.

4. The improvement of any one of claims 1-3, wherein the reference level is associated with a cutoff level that is correlated with: (a) the level of subjects who have sustained a head injury; (b) the incidence of TBI in a subject; (c) the stage of TBI in a subject, such as mild, moderate, severe, or moderate to severe; (d) the loss of consciousness in a subject; (e) an MRI that is positive for TBI, rather than negative; (f) the incidence of amnesia in a subject (i.e., the presence of amnesia versus the absence of amnesia); or (g) the severity of TBI in a subject.

5. The improvement of any one of claims 1-4, wherein the sample is obtained at: (a) about 24.5 hours to about three weeks after the actual or suspected head injury; (b) about 25 hours to about three weeks; (c) about 1 week; (d) about two weeks; or (e) about three weeks.

6. The improvement of any one of claims 1-5, wherein measuring the level of GFAP is performed by immunoassay or clinical chemistry assay.

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

8. The improvement of any one of claims 1-7, 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 of any one of claims 1-8, wherein the sample is obtained after the subject has or may have suffered an actual head injury caused by a body shake, blunt impact from external mechanical or other forces resulting in closed or open head trauma, one or more falls, an explosion or blast wave, or other type of blunt force trauma.

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

11. The improvement of claim 10, wherein the chemical or toxin is mold, asbestos, pesticides, insecticides, organic solvents, paints, glues, gases, organometallics, drugs of abuse, or one or more combinations thereof.

12. The improvement of any one of claims 1-9, 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 one of claims 1-12, wherein the method can be performed on any subject without regard to factors selected from the group consisting of: the subject's clinical condition; the subject's laboratory values; the subject's classification as having mild, moderate, severe, or moderate to severe traumatic brain injury; the subject's display of low, moderate, or high levels of GFAP; and the time of any event in which the subject has or may have suffered a head injury.

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

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

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

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

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 one of claims 17-19, wherein the reference level is from about 5 pg / mL to about 50 pg / mL.

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