Magnetic point-of-care system and assay for determining GFAP in a biological sample

Abstract

A system and assay are disclosed herein for determining the amount of glial fibrillary acidic protein (GFAP) in a biological sample obtained from a subject using a point-of-care device that includes magnetic susceptibility beads and a magnetic immunosensor.

Description

Technical Field

[0001] This application claims the benefit of priority of U.S. Patent Application No. 63 / 356,843, filed Jun. 29, 2022; U.S. Patent Application No. 63 / 402,122, filed Aug. 30, 2022; U.S. Patent Application No. 63 / 433,134, filed Dec. 16, 2022; and U.S. Patent Application No. 63 / 522,334, filed Jun. 21, 2023, the contents of each of the above patent documents being incorporated herein by reference.

[0002] The contents of the electronic sequence listing entitled 40972_601_ST26.xml (size: 7,839 bytes; and creation date: Jun. 28, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to systems and assays for determining the amount of glial fibrillary acidic protein (GFAP) in a biological sample obtained from a subject (e.g., a blood sample such as a venous blood sample, a capillary blood sample, a finger stick blood sample, or a combination thereof). The systems and assays utilize a point-of-care device containing at least one cartridge that contains at least one magnetic immuno-sensor for magnetically capturing, retaining, and determining the amount of GFAP in the sample. The amount of GFAP in the sample can assist in the diagnosis and assessment of whether a subject has sustained, is likely to have sustained, or is suspected of having sustained an injury to the head, such as an acquired brain injury, e.g., a traumatic brain injury (TBI).

Background Art

[0004] In the United States alone, more than five million mild traumatic brain injuries (TBIs) occur every year. Much of the TBI assessment and diagnosis is based on subjective data. Unfortunately, objective measures such as head CT and Glasgow Coma Scale (GCS) are not very comprehensive or sensitive in the assessment of mild TBI. Furthermore, head CT often reveals nothing in the case of mild TBI, is expensive, and exposes patients to unnecessary radiation. In addition, a negative head CT does not necessarily mean that the patient has been clearly shown to have no concussion, but only that certain interventions such as surgery are not warranted. Physicians and patients need objective and reliable information to accurately assess this condition and facilitate appropriate triage and recovery. To date, the available data for the use of GFAP in the emergency setting to aid in patient assessment and management have been limited.

[0005] Mild TBI or concussion is much more difficult to detect objectively and is a common challenge in emergency rooms worldwide. Concussion typically does not cause gross pathological conditions such as bleeding or abnormalities on conventional computed tomography scans of the brain, but rather causes a rapidly developing type of neurological dysfunction that resolves in a spontaneous manner over a period of days to weeks. Approximately 15% of mild TBI patients suffer from persistent cognitive dysfunction. There are still unmet needs for mild TBI victims at the scene, in emergency rooms and clinics, on sports fields, and in military activities (e.g., combat).

[0006] Current algorithms for assessing the severity of brain injury include the Glasgow Coma Scale score and other criteria. These criteria may be appropriate for correlating acute severity, but are insensitive to subtle pathologies that can result in persistent deficits. The GCS and other criteria also do not allow for differentiation between types of injury and may be inappropriate. Thus, patients classified into a single GCS level entering a clinical trial may have very heterogeneous severities and types of injury. Since outcomes also vary accordingly, inappropriate classification undermines the integrity of clinical trials. Improved classification of injury would allow for a more accurate portrayal of disease severity and type for TBI patients in clinical trials. Summary of the Invention Problems to be Solved by the Invention

[0007] In addition, current brain injury trials rely on outcome criteria such as the Glasgow Outcome Scale Extended, which captures the overall phenomenon but cannot assess subtle differences in outcome. Thus, 30 consecutive trials of brain injury therapeutics have failed. Sensitive outcome criteria are needed to determine how well patients recover from brain injury in order to test therapeutics and prophylactics. Means for Solving the Problems

[0008] (Summary of the Invention) In one embodiment, the present disclosure relates to an assay for measuring the amount of glial fibrillary acidic protein (GFAP) in a biological sample obtained from a subject. In some aspects, the assay (a) Contact the sample with a cartridge containing at least one magnetic immunosensor and: (i) at least one first specific binding partner printed on the cartridge, which is at least one anti-GFAP antibody that specifically binds to GFAP in the sample and is immobilized on at least one magnetic bead; and (ii) at least one second specific binding partner containing a detectable label printed on the cartridge, thereby producing one or more complexes containing the first specific binding partner-GFAP-second specific binding partner; (b) Magnetically capture and hold the beads containing the complex on at least one magnetic immunosensor in the cartridge contained in the point-of-care device; and (c) Use at least one magnetic immunosensor to evaluate the signal from the complex, wherein the amount of the detectable signal from the detectable label indicates the amount of GFAP in the sample. The assay includes immobilizing the first specific binding partner on magnetic beads and magnetically capturing and holding the beads containing the complex on at least one magnetic immunosensor in the cartridge contained in the point-of-care device, and shows at least a five-fold increase in sensitivity compared to an assay that does not.

[0009] In a further aspect, the above assay further includes a step of washing the unbound sample that is not magnetically captured and held on at least one magnetic immunosensor.

[0010] In yet a further aspect of the above assay, the magnetic immunosensor includes a sensing electrode on a substantially planar chip and a magnetic layer on the chip. More specifically, in yet a further aspect, the magnetic layer includes high magnetic field magnetic particles.

[0011] In yet further aspects of the above assay, the assay further comprises measuring the amount of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in a sample in a non-magnetic assay.

[0012] In yet further aspects of the above assay, the assay shows an increase of at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold or at least 15-fold in sensitivity as compared to an assay where a first specific binding partner is immobilized on magnetic beads and beads containing the complex are magnetically captured and retained on at least one magnetic immunosensor in a cartridge contained in a point-of-care device.

[0013] In yet further aspects, the above assay is used to aid in the diagnosis and assessment of a subject who has suffered or is at risk of suffering head injury. In some aspects, the subject is diagnosed with an acquired brain injury. In yet further aspects, the subject is diagnosed with a traumatic brain injury. In yet further aspects, the subject is treated for a traumatic brain injury.

[0014] In yet further aspects of the above assay, the sample is collected using a microsampling device or a fingerstick device. In some aspects, the collected sample is a venous blood sample, a capillary blood sample, a fingerstick blood sample, or a combination thereof.

[0015] In yet further aspects, the sample is processed prior to performing the assay. In some aspects, the sample is processed by plasma separation. In some aspects, the sample is processed using a plasma separation device. In yet further aspects, the plasma separation device is (a) incorporated within or operably coupled to the point-of-care device; or (b) separated from the point-of-care device.

[0016] In yet a further aspect, the amount of GFAP is communicated by being displayed on a device.

[0017] In yet another aspect, prior to displaying the amount of GFAP on a device, the assay a. determines the amount of GFAP in a capillary blood sample; b. selects a conversion factor for comparing the amount of GFAP in the sample to the amount of GFAP in venous blood, the conversion factor being a static correlation ratio, a dynamic ratio, or a combination thereof; and c. normalizes the amount of GFAP in the sample by the amount of GFAP from venous blood by applying the conversion factor selected in step b) to the amount of GFAP in the sample further comprises.

[0018] In yet a further aspect, the conversion factor is from about 1.2:1.0 to about 1.0:0.5. In other aspects, the conversion factor is about 1.0:0.85.

[0019] In yet a further aspect, when the conversion factor is used, the normalized amount of GFAP is displayed by the device.

[0020] In another embodiment, the disclosure relates to a system. In some aspects, the system is an assay for glial fibrillary acidic protein (GFAP) that comprises contacting a biological sample from a subject with a cartridge comprising at least one magnetic immunosensor and: (i) at least one first specific binding partner printed on the cartridge and immobilized on at least one magnetic bead, the at least one first specific binding partner comprising at least one anti-GFAP antibody that specifically binds to GFAP in the sample; and (ii) at least one second specific binding partner printed on the cartridge and comprising a detectable label, thereby producing one or more complexes comprising the first specific binding partner - GFAP - the second specific binding partner. A point-of-care device including a cartridge, the cartridge including at least one magnetic immuno-sensor, the device determining the amount of GFAP in a sample obtained from a subject by (a) magnetically capturing and holding beads containing a complex on at least one magnetic immuno-sensor; and (b) evaluating a signal from the complex, wherein the amount of detectable signal from a detectable label indicates the amount of GFAP in the sample. Including, the assay shows at least a 5-fold increase in sensitivity compared to an assay that immobilizes a first specific binding partner on magnetic beads and magnetically captures and holds beads containing a complex on at least one magnetic immuno-sensor in a cartridge contained in the point-of-care device.

[0021] In a further aspect, the system described above further includes the step of washing unbound sample that is not magnetically captured and held on at least one magnetic immuno-sensor.

[0022] In yet a further aspect of the system described above, the magnetic immuno-sensor includes a sensing electrode on a substantially planar chip and a magnetic layer on the chip. More specifically, in yet a further aspect, the magnetic layer includes high magnetic field magnetic particles.

[0023] In yet a further aspect of the system described above, the assay further includes measuring the amount of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) in a sample in a non-magnetic assay.

[0024] In yet a further aspect of the system described above, the assay shows at least a 7-fold, at least an 8-fold, at least a 9-fold, at least a 10-fold, at least an 11-fold, at least a 12-fold, at least a 13-fold, at least a 14-fold or at least a 15-fold increase in sensitivity compared to an assay that immobilizes a first specific binding partner on magnetic beads and magnetically captures and holds beads containing a complex on at least one magnetic immuno-sensor in a cartridge contained in the point-of-care device.

[0025] In yet a further aspect, the system described above is used to assist in the diagnosis and assessment of a subject who has suffered or is at risk of suffering a head injury. In some aspects, the subject is diagnosed as having an acquired brain injury. In yet further aspects, the subject is diagnosed as having a traumatic brain injury. In yet further aspects, the subject is treated for an acquired brain injury. In yet further aspects, the subject is treated for a traumatic brain injury.

[0026] In yet a further aspect of the system described above, the sample is collected using a microsampling device or a fingerstick device. In some aspects, the collected sample is a venous blood sample, a capillary blood sample, a fingerstick blood sample, or a combination thereof.

[0027] In yet a further aspect, the sample is processed prior to performing the assay. In some aspects, the sample is processed by plasma separation. In some aspects, the sample is processed using a plasma separation device. In yet further aspects, the plasma separation device is (a) incorporated within or operably coupled to a point-of-care device; or (b) separate from the point-of-care device.

[0028] In yet further aspects, the amount of GFAP is communicated by displaying it on the device.

[0029] In another embodiment, the present disclosure relates to a magnet; a region containing printed paramagnetic particles coated with an anti-GFAP antibody; and a region containing a plurality of printed detectably labeled anti-GFAP antibodies comprising a cartridge for use in a point-of-care device.

[0030] In some embodiments of the cartridge, the region containing the plurality of detectably labeled anti-GFAP antibodies is in the same region containing printed paramagnetic particles coated with anti-GFAP antibodies.

[0031] In other embodiments of the cartridge, the region containing the plurality of detectably labeled anti-GFAP antibodies is adjacent to the region containing printed paramagnetic particles coated with anti-GFAP antibodies.

Brief Description of the Drawings

[0032]

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 3E

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

[0033] The present disclosure relates to systems and methods (e.g., assays) for determining the amount of glial fibrillary acidic protein (GFAP) in a biological sample such as a blood sample (e.g., a venous blood sample, a capillary blood sample, a finger stick blood sample, or any combination thereof) obtained from a subject (e.g., a human subject, etc.). The systems and methods described herein use at least one point-of-care device containing at least one cartridge that includes at least one magnetic immunosensor or immunosensing device.

[0034] In one aspect, the system and method involve contacting a biological sample, such as blood (e.g., venous blood sample, capillary blood sample, finger stick blood sample, or any combination thereof), with a cartridge containing at least one magnetic immunosensor and (i) at least one first specific binding partner printed on the cartridge and immobilized on at least one magnetic bead, the at least one first specific binding partner being at least one anti-GFAP antibody that specifically binds to GFAP in the sample, and (ii) at least one second specific binding partner printed on the cartridge and containing a detectable label, to produce one or more complexes comprising the first specific binding partner - GFAP - the second specific binding partner, which includes performing an assay for GFAP. The point-of-care device contains at least one cartridge used to magnetically capture and hold the beads containing the complexes using at least one magnetic immunosensor, the signal from the complexes is evaluated, and the amount of the detectable signal from the detectable label indicates the amount of GFAP in the sample. The GFAP assay implemented and used in the system described herein shows at least a five-fold increase in sensitivity compared to an assay that does not use a point-of-care device that includes immobilizing the first specific binding partner on magnetic beads and contains at least one cartridge with at least one magnetic immunosensor.

[0035] The subsection headings used in this section of the specification and the entire disclosure are for organizational purposes only and are not intended to be limiting.

[0036] 1. Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control. In the practice or testing of the present disclosure, methods and materials similar or equivalent to those described herein may be used, but the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0037] As used herein, the terms "comprise(s)", "include(s)", "having", "has", "can be", and "containing" are open-ended transitional phrases, terms, or words that do not exclude the possibility of further acts or structures. The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "include", "consist of", or "consist essentially of" the embodiments or elements presented herein, whether explicitly or not.

[0038] As used herein, in listing numerical ranges, each number with the same precision intervening between them is explicitly contemplated. For example, for the range of 6 to 9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated, and for the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0039] As used herein, "acquired brain injury" or (ABI) refers to injury to the brain caused by events that occur after birth. In other words, acquired brain injury is not genetic or congenital, but rather is the result of a neurological condition and injury. Acquired brain injury is often divided into two categories. The first category is acquired traumatic brain injury (TBI) caused by external forces such as, for example, sports injuries, falls, physical shock, blunt trauma, explosions, blast or exposure to fire. The second category is non-traumatic acquired brain injury, which in some cases is caused by internal factors and includes stroke, tumor, anoxia, infection, metabolic disorders and others. As used herein, acquired brain injury does not include or encompass injury to the brain caused by stroke (including, for example, ischemic stroke, hemorrhagic stroke or transient ischemic attack, etc.).

[0040] As used herein, "affinity matured antibody" refers to the affinity of an antibody for a target antigen (i.e., K D , k d or k a) that results in an improvement compared to the parental antibody without the ) change, and refers to an antibody with one or more changes in one or more CDRs. Exemplary affinity matured antibodies have an affinity in the nanomolar or picomolar range for the target antigen. Various procedures for making affinity matured antibodies are known in the art, including screening combinatorial antibody libraries prepared using the BioDisplay method. For example, Marks et al., BioTechnology, 10:779-783 (1992) describe affinity maturation by domain shuffling of VH and VL. Random mutagenesis of CDR residues and / or framework residues has been described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-3319 (1995) and Hawkins et al., J. Mol. Biol., 226:889-896 (1992). Site-directed mutagenesis at contact or hypervariable positions with site-directed mutagenesis positions and activity-enhancing amino acid residues has been described in U.S. Patent No. 6,914,128 B1.

[0041] As used herein, "amount" refers to a specified (e.g., large or small) content or number, e.g., this number is a level such as a position on an actual or imagined scale of the amount or content, or e.g., a concentration such as the relative amount of a given substance contained in a solution or in a specific volume of space, e.g., the amount of solute per unit volume of solution.

[0042] As used herein, the terms "antibody" and "antibodies" refer to mammalian antibodies such as monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully humanized or partially humanized antibodies), avian (e.g., duck or goose) antibodies, shark antibodies, whale antibodies, and non-primate (e.g., bovine, porcine, camel, llama, equine, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, etc.) antibodies or non-human primate (e.g., monkey, chimpanzee, etc.) antibodies, but are not limited thereto, recombinant antibodies, chimeric antibodies, single-chain Fv ("scFv"), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fv ("sdFv"), and anti-idiotype ("anti-Id") antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (for dual variable domain immunoglobulins and methods for creating them, the contents of each are incorporated herein by reference, Wu, C. et al., Nature Biotechnology, 25(11):1290~1297(2007) and PCT International Application No. 2001 / 058956), and any of the foregoing, functionally active epitope-binding fragments. An antibody includes an immunoglobulin molecule and an immunologically active fragment of an immunoglobulin molecule, i.e., a molecule containing an analyte-binding site. The immunoglobulin molecule can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or isotype. For the sake of brevity, an antibody against an analyte is herein referred to as either "anti-analyte antibody" or simply "analyte antibody" (e.g., anti-UCH-L1 antibody or UCH-L1 antibody) with high frequency.

[0043] As used herein, an "antibody fragment" refers to a portion of an intact antibody that contains an antigen-binding site or variable region. The portion does not include the heavy chain constant domain of the Fc region of the intact antibody (i.e., CH2, CH3, or CH4 depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing three CDRs of 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.

[0044] As used herein, an "aperture" refers to an opening, hole, or gap.

[0045] "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 a non-significant test. Preferred AUCs can be at least approximately 0.700, at least approximately 0.750, at least approximately 0.800, at least approximately 0.850, at least approximately 0.900, at least approximately 0.910, at least approximately 0.920, at least approximately 0.930, at least approximately 0.940, at least approximately 0.950, at least approximately 0.960, at least approximately 0.970, at least approximately 0.980, at least approximately 0.990, or at least approximately 0.995.

[0046] "Beads" and "particles" are used interchangeably herein and refer to substantially spherical solid supports. An example of a bead or particle is a microparticle. The microparticles that can be used herein can be of any type known in the art. For example, the beads or particles can be magnetosensitive (or responsive) beads or particles (e.g., see U.S. Patent Nos. 4,230,685, 4,554,088, and 4,628,037, all of which are incorporated herein by reference) or magnetic particles, which are used interchangeably herein. Another example of a bead or particle is a magnetic or magnetosensitive bead or particle.

[0047] As used herein, the term "binding protein" refers to a monomeric or multimeric protein, such as a polypeptide, antigen, compound or other molecule, or any type of substrate, that binds to a binding partner and forms a complex therewith. A binding protein binds specifically to a binding partner. Binding proteins include, in addition to antibodies, other molecules known in the art and described below herein that contain one or more antigen-binding domains that bind to these antigen-binding fragments and their other diverse forms and derivatives, as well as to antigen molecules or specific sites (epitopes) on antigen molecules. Thus, binding proteins include, but are not limited to, antibodies that are tetrameric immunoglobulins, IgG molecules, IgG1 molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, affinity-matured antibodies, and any such antibody fragments that retain the ability to bind to an antigen.

[0048] As used herein, the term "bispecific antibody" refers to a full-length antibody produced by the quadroma technique (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 KIH (knob-into-hole) method or a similar method (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14):6444-6448(1993)) that introduces mutations within the Fc region, resulting in multiple different immunoglobulin molecular species, only one of which is a functional bispecific antibody. A bispecific antibody binds to one antigen (or epitope) at one of its two binding arms (one pair of HC / LC) and to a different antigen (or epitope) at its second arm (a different pair of HC / LC). By this definition, a bispecific antibody has two significantly different antigen-binding arms (both the specific sequence and the CDR sequence) and is monovalent for each antigen to which it binds.

[0049] As used herein, the term "capillary blood sample" refers to a sample of capillary-derived blood obtained (e.g., extracted) through the skin (not a vein) of a subject using a syringe, needle, or any other suitable device or combination thereof. For example, a whole blood sample can be obtained from the skin at the fingers and / or toes, hands, feet (including heels), earlobes, arms and / or legs, chest, back, head, or any combination thereof. In other embodiments, a whole capillary blood sample is extracted from the arm or leg. In yet other embodiments, a capillary blood sample is obtained from the hand or foot. In yet other embodiments, a capillary blood sample is obtained from the chest or back. In still other embodiments, a capillary blood sample is obtained from the earlobe. In yet other embodiments, a capillary blood sample is obtained from the head.

[0050] In a further aspect, the capillary blood sample is whole blood, serum, or plasma. In other embodiments, the capillary blood sample may primarily contain capillary blood but also contain or include a small amount or percentage of interstitial fluid.

[0051] In still a further aspect, the capillary blood sample obtained from the subject is obtained without using a syringe, needle (e.g., 21-gauge needle, winged needle, etc.), or any other suitable device typically used for collecting blood (e.g., venous blood), or any combination thereof. Instead, the capillary blood sample is obtained using a blood collection device applied to oneself or another person. Examples of blood collection devices applied to oneself or another person include microsampling devices. Examples of microsampling devices that may be used herein include the devices described in U.S. Patent No. 9,113,836, which is incorporated herein by reference in its entirety, together with the TAP device available from YourBio Health, Inc. (Cambridge, MA), the Tasso+, Tasso-M20, and Tasso-ST devices available from Tasso, Inc. (Seattle, WA), the One Draw device available from Draw Bridge Health (San Diego, CA), the PBS-1000 of PreciHealth (Neuchatel, Switzerland), or the Loop blood collection device available from Loop Medical (Lausanne, Switzerland).

[0052] In yet a further aspect, the capillary blood sample is obtained or collected from the subject in a decentralized setting. For example, the capillary blood sample can be obtained or collected from an urgent care clinic, pharmacy, grocery store or other convenience store, place of residence, workplace, and / or government office.

[0053] In addition to or instead of, and in still further embodiments, capillary blood samples are obtained from a subject by a user not trained in blood collection (e.g., by a person other than a trained phlebotomist, nurse, medical assistant, and / or physician). For example, capillary blood samples may be obtained from the subject by the subject himself / herself, a relative, a friend, a colleague, a coach, a pharmacist, and / or any other individual. In still further embodiments, capillary blood samples are obtained from a subject by a robot.

[0054] In yet still further embodiments, the capillary blood sample obtained from the subject is in an amount less than about 4 mL. In some embodiments, the capillary blood sample obtained from the subject is less than about 3 mL. In some embodiments, the capillary blood sample obtained from the subject is less than about 2 mL. In some embodiments, the amount of the capillary blood sample obtained from the subject is less than about 3.9 mL, about 3.8 mL, about 3.7 mL, about 3.6 mL, about 3.5 mL, about 3.4 mL, about 3.3 mL, about 3.2 mL, about 3.1 mL, about 3.0 mL, about 2.9 mL, about 2.8 mL, about 2.7 mL, about 2.6 mL, about 2.5 mL, about 2.4 mL, about 2.3 mL, about 2.2 mL, about 2.1 mL, about 2.0 mL, about 1.9 mL, about 1.8 mL, about 1.7 mL, about 1.6 mL, about 1.5 mL, about 1.4 mL, about 1.3 mL, about 1.2 mL, about 1.1 mL, about 1.0 mL, about 0.9 mL, about 0.8 mL, about 0.7 mL, about 0.6 mL, or about 0.5 mL. In some embodiments, when the collected sample is whole blood, a greater volume of capillary blood may be obtained.

[0055] As used herein, "cartridge" refers to a hollow container and / or chip containing one or more substances and / or components (e.g., liquid, reagents (e.g., antibodies and / or antigens) and / or particles (e.g., beads or microparticles)) for insertion into a device (e.g., a point-of-care device). In some embodiments, the cartridge includes at least one chip. In other embodiments, the cartridge is a chip. In still further embodiments, the cartridge has one or more openings. In some embodiments, the cartridge is a microfluidic cartridge. In other embodiments, the cartridge contains a magnetic immunosensor or a magnetic immunosensing device.

[0056] As used herein, "coupled" or "connected" refers to two or more components held together by any suitable means. Thus, in some embodiments, the description that two or more parts or components are "coupled" shall mean that the parts are joined or operate together either directly or indirectly, e.g., via one or more intermediate parts or components.

[0057] As used herein, "CDR" refers to "complementary determining regions" within antibody variable sequences. In each of the variable regions of the heavy and light chains, there are three CDRs each. In order from the N-terminus of the heavy or light chain, these regions are designated "CDR1", "CDR2", and "CDR3" for each of the variable regions. As used herein, the term "CDR set" refers to a group of three CDRs that occur in a single variable region and that are the group of CDRs that bind to an antigen. Thus, the antigen-binding site can include six CDRs, including a CDR set from each of the heavy-chain variable region and the light-chain variable region. A polypeptide containing a single CDR (e.g., CDR1, CDR2, or CDR3) can be referred to as a "molecular recognition unit". X-ray crystallographic analysis of antigen-antibody complexes has confirmed that the amino acid residues of the CDRs form extensive contacts with the bound antigen, in which case the most extensive antigen contacts are with the heavy-chain CDR3. Thus, the molecular recognition unit can mainly contribute to the specificity of the antigen-binding site. In general, CDR residues are directly and very substantially involved in their influence on binding to the antigen.

[0058] The exact boundaries of these CDRs are defined in different ways 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 presents a clear residue numbering system applicable to any variable region of an antibody, but also presents the exact residue boundaries that define the three CDRs. These CDRs may be referred to as "Kabat CDRs". Chothia and colleagues (Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); and Chothia et al., Nature, 342:877-883 (1989)) found that certain sub-portions within the Kabat CDRs adopt conformations of nearly identical peptide backbones, despite having great diversity at the amino acid sequence level. These sub-portions were named "L1", "L2", and "L3", or "H1", "H2", and "H3", where "L" and "H" refer to the light chain region and heavy chain region, respectively. These regions may be referred to as "Chothia CDRs", and they have boundaries that overlap with the Kabat CDRs. Other boundaries that define CDRs overlapping with the Kabat CDRs are described by Padlan, FASEB J., 9:133-139 (1995); and MacCallum, J. Mol. Biol., 262(5):732-745 (1996). Still other definitions of CDR boundaries may not strictly follow the definition of the system herein, and in light of predictions or experimental findings that a particular residue or group of residues or even the entire CDR has no significant effect on binding to the antigen, they may be shorter or longer, but still overlap with the Kabat CDRs. The methods used herein may use CDRs defined according to any of these systems, but certain embodiments use CDRs defined by Kabat or CDRs defined by Chothia.

[0059] The "clinically relevant time frame" refers to the time frame (e.g., seconds, minutes, or hours) during which a careful and prudent medical practitioner (e.g., a doctor, nurse, paramedic, or others) reasonably considers the results of one or more biomarker tests in relation to an imaging procedure such as a head CT scan, in accordance with guidelines established by a monitoring entity (e.g., a standard-setting organization such as the World Health Organization (WHO), a medical review board, a regulatory approval authority such as the FDA, EMEA, or others).

[0060] "One component", "a plurality of components", or "at least one component" generally refers to capture antibodies, detection or conjugates, calibrators, controls, sensitivity panels, containers, buffers, diluents, salts, enzymes, enzyme cofactors, detection reagents, pretreatment reagents / solutions, substrates (e.g., as a solution), stop solutions, and others that may be included in a kit for the assay of a test sample such as a patient whole blood, serum, or plasma sample, in accordance with the methods described herein and other methods known in the art. Some components may be in solution or may be lyophilized for reconstitution for use in the assay.

[0061] As used herein, "correlated to something" refers to being compared to something.

[0062] As used herein, "CT scan" refers to computed tomography (CT) scan. A CT scan combines a series of X-ray images obtained from different angles and uses computer processing to create cross-sectional images or slices of bones, blood vessels, and soft tissues within the body. A CT scan can use X-ray CT, positron emission tomography (PET), single photon emission computed tomography (SPECT), computed axial tomography (CAT scan), or computer-assisted tomography. A CT scan can be a conventional CT scan or a spiral / helical CT scan. In a conventional CT scan, the scan is obtained slice by slice, and after each slice is scanned, there is a stop and a movement to the next slice, for example, from above the abdomen to the pelvis. Conventional CT scans require the patient to hold their breath to avoid artifacts due to movement. Spiral / helical CT scans are continuous scans, obtained in a spiral shape, and the scanned images are continuous, so the procedure is much faster.

[0063] If no intracranial lesion(s) are observed in an image taken from a subject who has sustained, is likely to have sustained, or is suspected of having sustained an injury to the head, the head CT scan is "negative" for TBI. To further clarify, if no lesion is found or identified, the subject's head CT scan is "negative" for TBI; however, in some aspects, a subject may still be experiencing symptoms (e.g., of TBI) even though the head CT is negative. Considering that not all injuries or lesions may be visualized by a head CT, most subjects will be negative for TBI on a head CT. As a result, the methods and assays described herein may still be used to provide an assessment or determination of a subject with a negative head CT who may still have TBI.

[0064] As used interchangeably herein, the "Curie point" or "Curie temperature" refers to a characteristic property of ferromagnetic materials. The Curie point of a ferromagnetic material is the temperature above which the material loses its characteristic ferromagnetic ability to possess a net (spontaneous) magnetization in the absence of an external magnetic field. At temperatures below the Curie point, the magnetic moments are partially aligned within magnetic domains in the ferromagnetic material. As the temperature increases from below the Curie point until the net magnetization becomes zero above the Curie point, thermal fluctuations gradually disrupt this alignment. Above the Curie point, the material is purely paramagnetic.

[0065] As used interchangeably herein, "decentralized", "decentralized", or "decentralization" refers to the performance of one or more medical tests and / or assays outside of traditional medical settings (e.g., hospitals, clinics, independent research sites, etc.) at one or more locations such as urgent care clinics, retail clinics, pharmacies, grocery stores or convenience stores, places of residence (e.g., houses, apartments, etc.), workplaces and / or official agencies (e.g., the U.S. Transportation Security Administration) in the context of an examination. "Hybrid decentralized" or "hybrid decentralized" refers to a situation where a subject or patient collects a sample at their place of residence and / or workplace and sends the sample to a laboratory, avoiding specialized collection sites (such as hospitals, clinics, or independent sample collection or research sites).

[0066] "Determined by an assay" is used herein to refer to the determination of a reference level by any suitable assay. The determination of the reference level can be accomplished, in some embodiments, by an assay of the same type as the assay to be applied to a sample from a subject (e.g., an immunoassay, a clinical chemistry assay, a single molecule detection assay, a 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 methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS)). The determination of the reference level can be accomplished, in some embodiments, by an assay of the same type as the assay to be applied to a sample from a subject, under the same assay conditions. As described herein, the present disclosure provides exemplary reference levels (e.g., calculated by comparing reference levels at different time points). It is well within the skill of one of ordinary skill in the art to adapt the disclosure herein for other assays and obtain assay-specific reference levels for such other assays based on the description provided by the present disclosure. For example, a set of training samples can be used that includes samples obtained from human subjects known to have suffered a head injury (more particularly, (i) a mild TBI; and / or (ii) samples obtained from human subjects known to have suffered a moderate, severe or moderate-severe TBI) and samples obtained from human subjects known not to have suffered a head injury to obtain assay-specific reference levels. "Determined by an assay", a reference level having the "sensitivity" and / or "specificity" of the recited levels is understood to be used herein to refer to a reference level determined such that when the reference level is employed in the methods of the present disclosure, it provides the method of the recited sensitivity and / or specificity. For example, it is well within the skill of one of ordinary skill in the art to determine the sensitivity and specificity associated with a given reference level in the methods of the present disclosure by repeated statistical analysis of assay data using a plurality of different possible reference levels.

[0067] Indeed, when discriminating between subjects with or without traumatic brain injury, or subjects with mild to moderate, severe or moderate-to-severe traumatic brain injury, one of ordinary skill in the art balances the effect of increasing the cutoff in terms of sensitivity and specificity. An increase or decrease in the cutoff has a well-defined and predictable impact on sensitivity and specificity as well as other standard statistical measures. It is well known that an increase in the cutoff improves specificity but may worsen sensitivity (the proportion of those with the disease for which the test result is positive). In contrast, a decrease in the cutoff improves sensitivity but worsens specificity (the proportion of those without the disease for which the test result is negative). The tradeoff for detecting traumatic brain injury or determining mild to moderate, severe or moderate-to-severe traumatic brain injury is readily apparent to one of ordinary skill in the art. In the discrimination of whether a subject has or does not have traumatic brain injury, or has mild to moderate, severe or moderate-to-severe traumatic brain injury, the higher the cutoff, the more true negatives (i.e., subjects without traumatic brain injury, without mild traumatic brain injury, without moderate traumatic brain injury, without severe traumatic brain injury or without moderate-to-severe traumatic brain injury) are distinguished from those with traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury or moderate-to-severe traumatic brain injury, so the specificity improves. However, at the same time, the increase in the cutoff must decrease the sensitivity because it decreases the number of true positives along with the total number of cases identified as positive. Conversely, the lower the cutoff, the more true positives (i.e., subjects with traumatic brain injury, with mild traumatic brain injury, with moderate traumatic brain injury, with severe traumatic brain injury or with moderate-to-severe traumatic brain injury) are distinguished from those without traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury or moderate-to-severe traumatic brain injury, so the sensitivity improves. However, at the same time, the decrease in the cutoff must decrease the specificity because it increases the number of false positives along with the total number of cases identified as positive.

[0068] Generally, a high sensitivity value helps the person skilled in the art to 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), and a high specificity value helps the person skilled in the art to include a disease or condition. Whether the person skilled in the art wishes to rule out or include a disease depends on what the outcome is for each patient for each error type. Thus, without a complete disclosure of the underlying information about how the values were selected, it is not possible to know or predict the exact balance used to derive a test cut-off. The balance of sensitivity to specificity and other factors varies on a case-by-case basis. For these reasons, it may sometimes be preferable to provide an alternative cut-off (e.g., a reference) value that the physician or practitioner can choose in such a way.

[0069] As used herein, an "antibody derivative" may refer to an antibody having one or more modifications to its amino acid sequence when compared to a native antibody or parental antibody, and may exhibit a modified domain structure. The derivative not only adopts an amino acid sequence capable of specifically binding to a target (antigen), but may also further adopt a typical domain configuration found in native antibodies. Typical examples of antibody derivatives are antibodies conjugated to other polypeptides, rearranged antibody domains or antibody fragments. The derivative may also include at least one additional compound, for example, a protein domain, and the protein domain is linked by covalent or non-covalent bonds. The linkage may be based on gene fusion according to methods known in the art. The additional domain present in the fusion protein containing the antibody may preferably be linked by a flexible linker, which is advantageously a peptide linker, in which case the peptide linker contains a plurality of hydrophilic, peptide-bonded amino acids of sufficient length to span the distance between the C-terminus of the additional protein domain and the N-terminus of the antibody or vice versa. The antibody may have a conformation suitable for biological activity or may be linked to an effector molecule that selectively binds to, for example, a solid support, a biologically active substance (such as a cytokine or growth hormone), a chemical agent, a peptide, a protein or a drug.

[0070] As used herein, "abused drug" refers to one or more additive substances (such as drugs) taken for non-medical reasons (such as for recreational and / or psychoactive effects). Excessive indulgence, use, or dependence on such abused drugs is often referred to as "substance abuse". Examples of abused drugs 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, adhesives, etc.), and combinations thereof.

[0071] As used herein, "bispecific antibody" refers to a full-length antibody (see PCT Publication No. 02 / 02773) that can bind to two different antigens (or epitopes) in each of its two binding arms (HC / LC pairs). Thus, a bispecific binding protein has two identical antigen-binding arms with the same specificity and the same CDR sequences and is bivalent for each antigen to which it binds.

[0072] As used herein, the term "dual variable domain" refers to two or more antigen-binding sites on a binding protein that can be a bivalent binding protein (two antigen-binding sites), a tetravalent binding protein (four antigen-binding sites), or a multivalent binding protein. A DVD may be monospecific, i.e., capable of binding to one antigen (or one specific epitope), or multispecific, i.e., capable of binding to two or more antigens (i.e., two or more epitopes of the same target antigen molecule or two or more epitopes of different target antigens). 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-Ig". Thus, such a DVD-Ig binding protein is a tetramer and is similar to an IgG molecule but provides more antigen-binding sites than an IgG molecule. Thus, each half of the 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 the pair of heavy and light chains of an IgG molecule, which provides a single antigen-binding domain, the pair of heavy and light chains of a DVD-Ig provides two or more antigen-binding sites.

[0073] Since each antigen-binding site of a DVD-Ig binding protein can be derived from a donor ("parent") monoclonal antibody, it can include a heavy chain variable domain (VH) and a light chain variable domain (VL) with a total of six CDRs per antigen-binding site, including the CDRs involved in binding to the antigen. Thus, a DVD-Ig binding protein that binds to two different epitopes (i.e., two different epitopes of two different antigen molecules or two different epitopes of the same antigen molecule) includes an antigen-binding site derived from a first parent monoclonal antibody and an antigen-binding site of a second parent monoclonal antibody.

[0074] The design, expression, and characterization of DVD-Ig binding molecules are described in PCT Publication No. 2007 / 024715, U.S. Patent No. 7,612,181, and Wu et al., Nature Biotech., 25:1290-1297 (2007). Preferred examples of such DVD-Ig molecules have 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 provided that it is not CH1, X2 is an Fc region, and n is 0 or 1, preferably 1] for the heavy chain; and the structural formula: 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 provided that it is not CH1, X2 does not include an Fc region, and n is 0 or 1, preferably 1] for the light chain. Such a DVD-Ig can include two such heavy chains and two such light chains, in which case each chain includes tandemly linked variable domains without a constant domain intervening between the variable regions, and the heavy and light chains associate to form a tandem functional antigen-binding site, and pairs of heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein with four functional antigen-binding sites. In another example, the DVD-Ig molecule can include a heavy chain and a light chain each including three tandemly linked variable domains (VD1, VD2, VD3) without a constant domain intervening between the variable domains, in which case pairs of heavy and light chains can associate to form three antigen-binding sites, and pairs of heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein with six antigen-binding sites.

[0075] In a preferred embodiment, the DVD-Ig binding protein not only binds to the same target molecule to which its parental monoclonal antibody binds, but also possesses one or more of one or more desired properties of its parental monoclonal antibody. Preferably, such additional properties are one or more antibody parameters of the parental monoclonal antibody. Antibody parameters that can contribute to a DVD-Ig binding protein derived from one or more of its parental 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, cross-reactivity with tissues, and binding to orthologous antigens.

[0076] The DVD-Ig binding protein binds to at least one epitope of GFAP. Non-limiting examples of DVD-Ig binding proteins include DVD-Ig binding proteins that bind to one or more epitopes of GFAP, DVD-Ig binding proteins that bind to epitopes of human GFAP and epitopes of GFAP of another species (e.g., mouse), and DVD-Ig binding proteins that bind to epitopes of human GFAP and epitopes of another target molecule.

[0077] As used herein, "dynamic range" refers to the range in which the assay readout information is proportional to the amount of target molecule or analyte in the sample being analyzed.

[0078] "Epitope" or "epitopes" or "epitope of interest" refers to a site on any molecule that can be recognized and bind to a complementary site on its specific binding partner. The molecule and 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.

[0079] As used interchangeably herein, "fingerstick blood sample" or "fingerprick blood sample" refers to a capillary blood sample obtained from a subject's finger or digit using a microneedle, lancet, microlancet, or any other suitable device or combination thereof (e.g., a fingerstick device). In some embodiments, the fingerstick blood sample is whole blood, serum, or plasma. In some aspects, the fingerstick blood sample primarily contains capillary blood, but may also contain or include a small amount or percentage of interstitial fluid.

[0080] As used herein, "Fab (fragment antigen-binding) fragment" or "Fab fragment" refers to a fragment of an antibody that binds to an antigen and contains one complete light chain and a portion of one heavy chain, which is one antigen-binding site. Fab is a monovalent fragment consisting of the VL domain, VH domain, CL domain, and CH1 domain. Fab is composed of one constant domain and one variable domain of each heavy chain and each light chain. The variable domain contains a paratope (antigen-binding site) that includes a set of complementarity-determining regions at the monomeric amino terminus. Thus, each arm of the Y binds to an epitope on the antigen. Fab fragments can be made as described in the art using, for example, papain, an enzyme that can be used to cleave an immunoglobulin monomer into two Fab fragments and an Fc fragment, or can be produced by recombinant means.

[0081] As used herein, the "F(ab’)2 fragment" refers to an antibody produced by pepsin digestion of a whole IgG antibody that removes most of the Fc region while leaving a part of the hinge region intact. The F(ab’)2 fragment has two antigen-binding F(ab) moieties that are linked together by disulfide bonds and is thus a bivalent fragment with a molecular weight of approximately 110 kDa. The bivalent antibody fragment (F(ab’)2 fragment) is smaller than the whole IgG molecule and enables good penetration into tissues, thus facilitating good antigen recognition in immunohistochemistry. The use of the F(ab’)2 fragment also avoids non-specific binding to Fc receptors or protein A / G on live cells. The F(ab’)2 fragment can bind to an antigen and precipitate it.

[0082] As used herein, "framework" (FR) or "framework sequence" may mean the remaining sequence of the variable region excluding the CDRs. Since the exact definition of the CDR sequences can be determined by different systems (e.g., see above), the meaning of the framework sequences is subject to different interpretations accordingly. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) also divide the framework regions on the light and heavy chains into four sub-regions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. As referred to by other researchers, when a particular sub-region is not designated as FR1, FR2, FR3, or FR4, the framework region represents the combination of FRs within the variable region of a single, native immunoglobulin chain. As used herein, FR represents one of the four sub-regions, and FRs represent two or more of the four sub-regions that make up the framework region.

[0083] FR sequences of human heavy and light chains that can be used as "acceptor" framework sequences (or simply, "acceptor" sequences) of heavy and light chains to humanize non-human antibodies using techniques known in the art are known in the art. In one embodiment, the acceptor sequences of human heavy and light chains are selected from framework sequences listed in publicly available databases such as V-base (hypertext transfer protocol: / / vbase.mrc-cpe.cam.ac.uk / ) or the international ImMunoGeneTics® (IMGT®) information system (hypertext transfer protocol: / / imgt.cines.fr / texts / IMGTrepertoire / LocusGenes / ).

[0084] As used herein, "functional antigen-binding site" can mean 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 to an antigen must be measurable using any one of various known methods for assaying proteins that bind to an antigen, e.g., an antibody. Further, the antigen-binding affinity of each of the antigen-binding sites of a multivalent protein, e.g., a multivalent antibody herein, need not be quantitatively the same.

[0085] "GFAP" is used herein to describe glial fibrillary acidic protein. GFAP is a protein that can be encoded and produced by the GFAP gene in humans (e.g., by recombinant means, in other species).

[0086] "GFAP status" can mean the level or amount of GFAP at a given time (such as by a single criterion of GFAP), the level or amount of GFAP relevant to monitoring (such as by repeated testing in a subject to identify an increase or decrease in GFAP amount), the level or amount of GFAP relevant to treatment for traumatic brain injury (whether it is a primary and / or secondary brain injury), or any combination thereof.

[0087] As used herein, "GCS (Glasgow Coma Scale)" or "GCS" refers to a 15 - point scale (for example, described by Graham Teasdale and Bryan Jennett, Lancet 1974; 2: 81 - 4) that provides a practical method for assessing functional impairment of the level of consciousness in a patient who has suffered a brain injury. The examination measures the best motor response, verbal response, and eye - opening response by these values: I. Best motor response (6: follows two - part command; 5: reaches for hand on stimulation of the head and neck above the clavicle; 4: bends arm sharply at the elbow but the characteristic is mainly not abnormal; 3: bends arm at the elbow, the characteristic is clearly mainly abnormal; 2: extends arm at the elbow; 1: no movement of arm / leg, no interfering factor; NT: paralyzed or other limiting factor); II. Verbal response (5: gives correct name, place, and date; 4: has no orientation but communication is consistent; 3: understands words; 2: moans / groans only; 1: no audible response, no interfering factor; NT: factor interfering with communication) and III. Eye - opening (4: opens before stimulation; 3: after verbal or shouted command; 2: after fingertip stimulation; 1: never opens eyes, no interfering factor; NT: closed by local factor). The final score is determined by adding the values of I + II+III. When the GCS score is 13 - 15, the subject is considered to have a mild TBI. When the GCS score is 9 - 12, the subject is considered to have a moderate TBI. When the GCS score is 8 or less, typically 3 - 8, the subject is considered to have a severe TBI.

[0088] As used herein, the "GOS (Glasgow Outcome Scale)" refers to an overall scale for functional outcome that grades a patient's condition into one of five categories: death, vegetative state, severe disability, moderate disability, or good recovery. The "GOSE (Extended Glasgow Outcome Scale)" or "GOSE", used interchangeably herein, presents a more detailed classification into eight types by subdividing the types of severe physical disability, moderate physical disability, and good recovery into the upper and lower types shown in Table 1.

[0089]

Table 1

[0090] As used herein, the term "hydrophilic" in reference to a "hydrophilic material" (e.g., a membrane, a film, etc.) refers to a material having a water contact angle of less than about 40 degrees.

[0091] As used herein, the term "hydrophobic" in reference to a "hydrophobic material" (e.g., a membrane, a film, etc.) refers to a material having a water contact angle of greater than about 80 degrees.

[0092] As used herein, the term "humanized antibody" is used to describe an antibody in which the heavy chain variable region sequence and the light chain variable region sequence are derived from a non-human species (e.g., mouse), but at least a part of the VH sequence and / or VL sequence has been changed to be more "human-like", i.e., more similar to human germline variable sequences. A "humanized antibody" is an antibody or a variant, derivative, analogue 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 having 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 at least one, but typically two, variable domains (Fab, Fab’, F(ab’)2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin (i.e., the donor antibody) and all or substantially all of the framework regions are the framework regions of a human immunoglobulin consensus sequence, and comprises substantially all of the variable domains. In certain embodiments, a humanized antibody also comprises an immunoglobulin constant region (Fc), typically at least a part of the Fc region of a human immunoglobulin. In some embodiments, a humanized antibody contains at least the variable domain of the heavy chain in addition to the light chain. The antibody may also comprise the CH1 region, hinge region, CH2 region, CH3 region and CH4 region of the heavy chain. In some embodiments, a humanized antibody contains only a humanized light chain. In some embodiments, a humanized antibody contains only a humanized heavy chain. In a specific embodiment, a humanized antibody contains only the humanized variable domains of the light chain and / or the humanized heavy chain.

[0093] A humanized antibody can be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including, without limitation, IgG1, IgG2, IgG3, and IgG4. A humanized antibody can contain sequences derived from more than one class or isotype, and specific constant domains can be selected using techniques well known in the art to optimize the desired effector function.

[0094] The framework regions and CDRs of a humanized antibody need not exactly correspond to the parent sequences. For example, donor antibody CDRs or consensus frameworks can be mutagenized by substitution, insertion, and / or deletion of at least one amino acid residue such that the CDR residues or framework residues at this site do not correspond to the donor antibody or consensus framework. However, in a preferred embodiment, such mutations are not extensive mutations. Typically, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the residues of the humanized antibody correspond to the residues of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region within a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related immunoglobulin sequences (see, e.g., Winnaker, "From Genes to Clones" (Verlagsgesellschaft, Weinheim, 1987)). Thus, a "consensus immunoglobulin sequence" can include a "consensus framework region" and / or a "consensus CDR". In a family of immunoglobulins, each position within the consensus sequence is occupied by the amino acid that occurs most frequently at this position within the family. When two amino acids occur with equal frequency, both can be included within the consensus sequence.

[0095] As used herein in the context of two or more polypeptide or polynucleotide sequences, "identical" or "identity" can mean that the sequences have the specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which identical residues occur in both sequences, obtaining the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the lengths of the two sequences are different or the alignment results in one or more sticky ends and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator but not the numerator of the calculation.

[0096] As used interchangeably herein, "injury to the head" or "head injury" refers to any trauma to the scalp, skull, or brain. Such injury can include only a minor bump on the skull or can be a severe brain injury. Such injury includes primary injury to the brain and / or secondary injury to the brain. Primary brain injury occurs at the time of the initial injury and is caused as a result of a displacement of the physical structure of the brain. More specifically, primary brain injury is physical damage to the parenchyma (tissue, blood vessels) that occurs during a traumatic event and results in shear and compression of the surrounding brain tissue. Secondary brain injury occurs subsequent to the primary injury and can involve a series of cellular processes. More specifically, secondary brain injury refers to changes that develop over a period (hours to days) after the primary brain injury. Secondary brain injury includes the entire cascade of changes in cells, chemicals, tissues, or blood vessels within the brain that contribute to further destruction of the brain tissue.

[0097] Head injuries can be either closed or open (penetrating). A closed head injury is an injury to the scalp, skull, or brain without penetration of the skull by an impact object. An open head injury is an injury to the scalp, skull, or brain with penetration of the skull by an impact object. Head injuries can be caused by external mechanical or other forces, cerebrovascular accidents (e.g., stroke), one or more falls (e.g., during sports or other activities), blunt impact due to an explosion or blast (collectively, "blast injury"), and other types of blunt force trauma resulting from a person's physical movement (e.g., vehicle accidents such as in a car, airplane, train, etc.; blows to the head such as with a baseball bat or from a firearm). Alternatively, head injuries can be caused by ingestion and / or exposure to fire, chemicals, toxins, or combinations of chemicals and toxins. Examples of such chemicals and / or toxins include mold, asbestos, pesticides and insecticides, organic solvents, paints, adhesives, gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organometals (such as methyl mercury, tetraethyl lead, and organotin), and / or one or more addictive drugs. Alternatively, head injuries can be caused as a result of the 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. In some cases, it may not be possible to determine whether any such event or injury has occurred or taken place. For example, there may be no medical history regarding the patient or subject, the subject may be unable to speak, the subject may be unaware of what events they were exposed to, etc. Such situations are described herein as the subject having "potentially sustained a head injury". In certain embodiments herein, closed head injuries do not include and specifically exclude cerebrovascular accidents such as stroke.

[0098] As used herein, "interstitial fluid" refers to a fluid that surrounds and / or fills the spaces between cells. Interstitial fluid can contain or include a mixture of water, ions, and small solutes that are forced out of the blood by the systolic pressure generated when the heart pumps.

[0099] As used herein, "intracranial lesion" refers to a damaged area within the brain. An intracranial lesion can be an abnormality seen in a CT scan or a brain imaging examination, such as magnetic resonance imaging (MRI). In a CT or MRI scan, a brain lesion can appear as a dark or bright spot that does not look like normal brain tissue.

[0100] As used herein, an "isolated polynucleotide" means that the isolated polynucleotide is not associated with all or part of the polynucleotides with which it is found together in nature in its origin; it is operably linked to a polynucleotide that is not linked in nature; or it is not present as part of a larger sequence in nature (for example, of genomic, cDNA or synthetic origin or a combination of these).

[0101] As used herein, "label" and "detectable label" refer to a moiety conjugated to an antibody or an analyte so as 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 detectable by visual or measurement means. A variety of labels include signal-generating substances such as chromogens, fluorescent compounds, chemiluminescent compounds, radioactive compounds, etc. Representative examples of labels include moieties that produce light, such as acridinium compounds, and moieties that produce fluorescence, such as fluorescein. Other labels are also described herein. In this regard, the moiety itself may not be detectable, but may become detectable upon reacting with yet another moiety. The term "detectably labeled" is intended to encompass such labels.

[0102] Any suitable detectable label known in the art may be used. For example, detectable labels can be radioactive labels (such as 3H, 14C, 32P, 33P, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm), enzyme labels (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, and others), chemiluminescent labels (such as acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, and others), fluorescent labels (such as fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein, 3’6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, and others), rhodamine, phycobiliprotein, R-phycoerythrin, quantum dots (e.g., cadmium selenide capped with zinc sulfide), thermometric labels, or immunopolymerase chain reaction labels. Introduction of the label, labeling procedures, and detection of the label can be found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd Edition, Springer Verlag, N.Y. (1997) and Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996) (which is a combined handbook and catalog published by Molecular Probes, Inc., Eugene, Oregon). Fluorescent labels can be used in FPIA (see, e.g., U.S. Patent Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are hereby incorporated by reference in their entirety).Acridinium compounds can be used as detectable labels in homogeneous chemiluminescence assays (see, for example, 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)).

[0103] 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, K.V. ed.; CRC Press: Boca Raton, 77-105 (2002); Adamczyk et al., Org. Lett. 5:3779-3782 (2003); and U.S. Pat. Nos. 5,468,646, 5,543,524, and 5,783,699 (each of which is incorporated herein by reference in its entirety for its teachings related thereto).

[0104] Another example of an acridinium compound is an acridinium-9-carboxylate aryl ester. An example of an acridinium-9-carboxylate aryl ester of formula II is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available from Cayman Chemical, Ann Arbor, MI). Methods for preparing acridinium 9-carboxylate aryl esters are described in McCapra et al., Photochem. Photobiol. 4:1111-1121 (1965); Razavi et al., Luminescence 15:245-249 (2000); Razavi et al., Luminescence 15:239-244 (2000); and U.S. Patent No. 5,241,070 (each of which is hereby incorporated by reference in its entirety for its teaching in this regard). Such acridinium-9-carboxylate aryl esters are efficient chemiluminescent indicators for hydrogen peroxide produced in the oxidation of an analyte by at least one oxidase, from the perspective of signal intensity and / or signal rapidity. The course of chemiluminescence emission for acridinium-9-carboxylate aryl esters is completed rapidly, i.e., in less than 1 second, while acridinium-9-carboxamide chemiluminescence emission extends beyond 2 seconds. However, acridinium-9-carboxylate aryl esters lose their chemiluminescent properties in the presence of proteins. Thus, their use requires the absence of proteins during signal generation and detection. Methods for separating or removing proteins in a sample are well known to those skilled in the art and include, but are not limited to, ultrafiltration, extraction, precipitation, dialysis, chromatography, and / or digestion (see, for example, Wells, High Throughput Bioanalytical Sample Preparation. Methods and Automation Strategies, Elsevier (2003)). The amount of protein removed or separated from the test sample can be about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.Further details regarding the acridinium-9-carboxylate aryl ester and its use are set forth in U.S. Patent Application No. 11 / 697,835, filed April 9, 2007. The acridinium-9-carboxylate aryl ester can be dissolved in any suitable solvent such as degassed anhydrous N,N-dimethylformamide (DMF) or aqueous sodium cholate.

[0105] "Linked array" or "linked peptide array" refers to one or more natural or artificial polypeptide sequences connected to a polypeptide sequence of interest (e.g., full-length sequence, sequence fragment, etc.). The term "connected" refers to the joining of the linked array to the polypeptide sequence of interest. Such polypeptide sequences are preferably joined by one or more peptide bonds. The linked array can have a length of about 4 to about 50 amino acids. Preferably, the length of the linked array is about 6 to about 30 amino acids. Natural linked arrays can be modified by amino acid substitution, addition or deletion to create artificial linked arrays. Linked arrays can be used for many purposes, including use within recombinant Fabs. Exemplary linked arrays include, but are not limited to: (i) a histidine (His) tag such as a 6×His tag having the amino acid sequence of HHHHHH (SEQ ID NO: 3), which is useful as a linked array that facilitates the isolation and purification of the polypeptide and antibody of interest; (ii) an enterokinase cleavage site such as a His tag, which is used in the isolation and purification of the protein and antibody of interest. Enterokinase cleavage sites are often used in conjunction with His tags in the isolation and purification of the protein and antibody of interest. In the art, various enterokinase cleavage sites are known. 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) other sequences can also be used to link or connect the light chain variable region and / or the heavy chain variable region of a single-chain variable region fragment. Examples of other linked arrays 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). Linked arrays can also be modified for additional functions, such as drug conjugation or conjugation to a solid support. In the context of the present disclosure, monoclonal antibodies can contain linked arrays such as, for example, His tags, enterokinase cleavage sites, or both.

[0106] As used interchangeably herein, "magnetic resonance imaging" or "MRI" refers to a medical imaging technique used in radiology to form images of anatomy and physiological processes of the body in both health and disease (e.g., interchangeably referred to herein as "MRI", "MRI procedure", or "MRI examination"). MRI is a form of medical imaging that measures the response of the atomic nuclei of body tissues to high-frequency radio waves when placed in a strong magnetic field and creates images of internal organs. MRI scanners are based on the science of nuclear magnetic resonance (NMR) and use strong magnetic fields, radio waves, and field gradients to produce images of the inside of the body.

[0107] As used herein, the term "microchannel" refers to a channel having a cross-sectional dimension (i.e., height and width) of less than about 200 μm. In some embodiments, the channel has a cross-sectional dimension of less than about 150 μm. In other embodiments, the channel has a cross-sectional dimension of less than about 100 μm.

[0108] As used herein, the term "microsampling device" refers to any device known in the art suitable for extracting capillary blood through the skin. It is understood that samples obtained through the skin using a microsampling device mainly contain capillary blood, but the samples may also contain a small amount or percentage of interstitial fluid. In some embodiments, the microsampling device can contain from about 0.1 mL to about 4 mL of capillary blood. In some other embodiments, the device contains a plurality of microneedles, lancets or microlancets, blades or microblades, microscrews, or any combination thereof. In some embodiments, the plurality of microneedles, lancets or microlancets, blades or microblades, microscrews, or any combination thereof can be rotatable. In some other embodiments, the plurality of microneedles, lancets or microlancets, blades or microblades, microscrews, or any combination thereof are non-rotatable. In some embodiments, the microsampling device creates a vacuum and / or uses a stored vacuum to draw the skin into the device and / or activate a plurality of microneedles, lancets or microlancets, blades or microblades, microscrews, or any combination thereof to cut the skin. Exemplary microsampling devices that can be used in the methods described herein include the devices described in U.S. Patent No. 9,113,836, the contents of which are incorporated herein by reference, together with the TAP device available from YourBio Health, Inc. (Cambridge, MA); the Tasso+, Tasso-M20 and Tasso-ST devices available from Tasso, Inc. (Seattle, WA); the One Draw device available from Draw Bridge Health (San Diego, CA); the PBS-1000 of PreciHealth (Neuchatel, Switzerland); or the Loop blood collection device available from Loop Medical (Lausanne, Switzerland).In other aspects, examples of micro-sampling devices include finger stick devices. In some aspects, the micro-sampling device can include a bandage, dressing, or other suitable material that can be applied to or dispensed onto the area of skin when the sample is obtained and / or when the device is removed from or detached from the skin.

[0109] As used herein, "monoclonal antibody" refers to an antibody obtained from a population of antibodies that are substantially homogeneous, i.e., the individual antibodies that make up the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are made against a single antigen. Further, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies herein include, inter alia, "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is derived from another species or belongs to another antibody class or subclass, as well as fragments of such antibodies as long as they exhibit the desired biological activity.

[0110] As used herein, the term "multivalent binding protein" is used to refer to a binding protein that includes two or more antigen-binding sites (also referred to herein as "antigen-binding domains"). The multivalent binding protein is preferably engineered to have three or more antigen-binding sites and generally is not a natural antibody. The term "multispecific binding protein" refers to a binding protein that can bind to two or more related or unrelated targets and includes binding proteins that can bind to two or more different epitopes on the same target molecule.

[0111] As used interchangeably herein, "negative predictive value" or "NPV" refers to the probability that a subject has a negative outcome when the subject has a negative test result.

[0112] As used herein, "operatively coupled" or "operatively connected" means that a number of elements or assemblies, each movable between a first position and a second position, or a first configuration and a second configuration, are coupled such that when the first element moves from one position / configuration to another, the second element moves between positions / configurations in a similar manner. Note that the first element may be "operatively coupled" to another element, and vice versa need not be true. When movement is possible between the first element and another element, and vice versa, the elements are said to be "operatively coupled to each other".

[0113] As used herein, "normalize" or "normalizing" refers to adjusting the amount of an analyte (e.g., GFAP) determined in a capillary blood sample obtained from a subject, based on the amount of the same analyte in venous blood. In some embodiments, for example, normalizing may include multiplying the amount of the analyte in the capillary blood sample by a factor (e.g., a correlation or conversion factor).

[0114] A "point-of-care device" refers to a device used to provide medical diagnostic tests at or near the point of care (i.e., typically outside the laboratory), at the time and place of patient care (such as in a hospital, clinic, emergency or other medical facility, the patient's home, nursing home and / or long-term care and / or hospice facility, etc.). Examples of point-of-care devices include devices produced by Abbott Laboratories (Abbott Park, IL) (e.g., i-STAT and i-STAT Alinity), Universal Biosensors (Rowville, Australia) (see US2006 / 0134713), Axis-Shield PoC AS (Oslo, Norway) and Clinical Lab Products (Los Angeles, USA).

[0115] As used interchangeably herein, "positive predictive value" or "PPV" refers to the probability that a subject has a positive outcome when the subject has a positive test result.

[0116] "Quality control reagents" in the context of the immunoassays and kits described herein include, but are not limited to, calibrators, controls and sensitivity panels. A "calibrator" or "standard" is typically used (e.g., one or more such as a plurality) to establish a calibration (standard) curve for interpolation of the amount of an analyte such as an antibody or analyte. Alternatively, a single calibrator near a reference level or control level (e.g., "low", "medium" or "high" level) can be used. A plurality of calibrators (i.e., two or more calibrators or calibrators of varying amounts (plural optional)) can be used together to constitute a "sensitivity panel".

[0117] As used herein, a "reactor" refers to a holder or receptacle such as a container, receptacle, tube and / or cartridge in or on which an assay is performed. In some embodiments, the reactor can have one or more openings.

[0118] The "receiver operating characteristic" curve, or "ROC" curve, refers to a graphical plot that explains the performance of a binary classifier system as its discrimination threshold is varied. For example, an ROC curve can be a plot of the true positive rate against the false positive rate for different possible cut-off points of a diagnostic test. This is created by plotting, for various threshold settings, the proportion of true positives among the positives (TPR = true positive rate), versus the proportion of false positives among the negatives (FPR = false positive rate). The TPR is also known as sensitivity, and the FPR is one minus specificity or true negative rate. The ROC curve demonstrates the trade-off between sensitivity and specificity (any increase in sensitivity is accompanied by a decrease in specificity); the closer the curve follows the left-hand boundary, then the upper boundary, of the ROC space, the more accurate the test; the closer the curve comes to the 45-degree diagonal in the ROC space, the less accurate the test; the slope of the tangent at the cut-off point yields the likelihood ratio (LR) of the test at that value; and the area under the curve is a measure of test accuracy.

[0119] "Recombinant antibody" and "recombinant antibodies" refer to antibodies prepared by one or more steps including cloning a nucleic acid sequence encoding all or part of one or more monoclonal antibodies into an appropriate expression vector by recombinant methods and then expressing the antibody in an appropriate host cell. The terms include, but are not limited to, monoclonal antibodies made by recombination, chimeric antibodies, humanized antibodies (fully humanized or partially humanized antibodies), multispecific or multivalent structures formed from antibody fragments, bispecific antibodies, heteroconjugate Abs, DVD-Ig® and other antibodies described in (i) herein (for bispecific variable domain immunoglobulins and methods for making them, see Wu, C. et al., Nature Biotechnology, 25:1290-1297 (2007)). As used herein, the term "bispecific antibody" refers to an antibody that includes a first arm having specificity for one antigenic site and a second arm having specificity for a different antigenic site, i.e., a bispecific antibody has bispecificity.

[0120] As used herein, the “reference level” refers to an assay cut-off value that is used for the evaluation of diagnosis, prognosis, or therapeutic efficacy and that is related or associated in this specification with various clinical parameters (e.g., presence of a disease, stage of a disease, severity of a disease, progression, non-progression, or improvement of a disease, etc.). As used herein, the “absolute amount” refers to the absolute value of a change or difference between at least two assay results taken or sampled at different time points, which, like the reference level, is related or associated in this specification with various clinical parameters (e.g., presence of a disease, stage of a disease, severity of a disease, progression, non-progression, or improvement of a disease, etc.). As used herein, the “absolute value” refers to the magnitude of a real number regardless of its sign, i.e., regardless of whether it is positive or negative (e.g., the difference between two compared levels such as the level taken at a first time point and the level taken at a second time point, etc.). The UCH-L1 and / or GFAP reference levels referred to in this specification are obtained from venous blood.

[0121] This disclosure provides exemplary reference levels and absolute amounts (e.g., calculated by comparing reference levels at different time points). However, it is well known that reference levels and absolute amounts can vary depending on the nature of the immunoassay (e.g., the antibody used, reaction conditions, sample purity, etc.) and that assays can be compared and standardized. Furthermore, adapting the disclosure herein for other immunoassays to obtain immunoassay-specific reference levels and absolute amounts for such other immunoassays based on the description provided by this disclosure is well within the skill of one of ordinary skill in the art. While the exact values of the reference levels and absolute amounts can vary between assays, the findings described herein should be generally applicable and should be capable of being extrapolated to other assays.

[0122] As used herein, "removably coupled" or "removably connected" means that one component is coupled to another component in an essentially temporary manner. That is, the two components are coupled in such a way that the joining or separation of the components is easy and does not damage the components. Thus, a "removably coupled" component can be easily decoupled and recoupled without damaging the component.

[0123] As used herein, "result" refers to the information obtained by performing an assay. In one aspect, the result is the amount of a biomarker (e.g., GFAP, or GFAP and UCH-L1) in a test sample (e.g., a capillary blood sample). In another aspect, the result is the identification of the presence of a biomarker (e.g., GFAP, or GFAP and UCH-L1) in the sample. The result can be visually displayed (e.g., as readout information).

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

[0125] As used herein, the "plasma separation device" refers to an apparatus or device that can be used for separating components of whole blood (e.g., red blood cells and white blood cells) from serum, plasma, or serum and plasma, using a separation system such as at least one membrane, filter, synthetic paper (e.g., micropillar scaffold), or any combination thereof. For example, the membrane and / or filter that can be used in a plasma separation device can include at least one of polycarbonate, polysulfone, polyester, polyethylene, polyurethane, and polypropylene. In some embodiments, the membrane and / or filter is pretreated (e.g., with one or more polycations, zwitterions, one or more non-covalent surface treatments (e.g., PEGMA, HEMA, BSA, O2 plasma, etc.)). In other embodiments, the membrane and / or filter is not pretreated. In still further embodiments, the filter that can be used is a gravity-assisted separation system. Examples of plasma separation devices that can be used in the methods described herein include the plasma separation device described in U.S. Patent Publication No. 2020 / 0124508, the contents of which are incorporated herein by reference. In some embodiments, the plasma separation device does not include a lateral flow device.

[0126] As used herein, the "sensitivity" of an assay refers to the proportion of subjects with a positive outcome that are correctly identified as positive (e.g., correctly identifying a subject having the disease or medical condition for which the subject is being tested). For example, this can include correctly identifying a subject as having TBI as distinct from subjects without TBI, correctly identifying a subject as having moderate, severe, or moderate-to-severe TBI as distinct from subjects with mild TBI, correctly identifying a subject as having mild TBI as distinct from subjects with moderate, severe, or moderate-to-severe TBI, correctly identifying a subject as having moderate, severe, or moderate-to-severe TBI as distinct from subjects without TBI, or correctly identifying a subject as having mild TBI as distinct from subjects without TBI, etc.

[0127] As used herein, the "specificity" of an assay refers to the ratio of subjects with a negative outcome who are correctly identified as negative (e.g., the ratio of subjects who do not have the disease or medical condition being tested for and are correctly identified as such). For example, this can include correctly identifying subjects without TBI as distinct from subjects with TBI, correctly identifying subjects without moderate, severe, or moderate-to-severe TBI as distinct from subjects with mild TBI, correctly identifying subjects without mild TBI as distinct from subjects with moderate, severe, or moderate-to-severe TBI, and the like.

[0128] A "series of calibration compositions" refers to a plurality of compositions containing known amounts of GFAP, where each of the compositions has a different amount of GFAP from the other compositions in the series.

[0129] As used interchangeably herein, "solid phase" or "solid support" refers to any material that can be used to conjugate and / or attract and immobilize (1) one or more capture agents or specific binding partners for capture or (2) one or more detection agents or specific binding partners for detection. The solid phase can be selected for its inherent ability to attract and immobilize the capture agent. Alternatively, the solid phase may have attached thereto a linker having the ability to attract and immobilize (1) a capture agent or specific binding partner for capture or (2) a detection agent or specific binding partner for detection. For example, the linker can include a charged substance that is oppositely charged relative to the capture agent (e.g., specific binding partner for capture) or detection agent (e.g., specific binding partner for detection) itself, or relative to a charged substance conjugated to (1) a capture agent or specific binding partner for capture or (2) a detection agent or specific binding partner for detection. Generally, the linker is immobilized (conjugated) on the solid phase and can be any binding partner (preferably, specific) having the ability to immobilize (1) a capture agent or specific binding partner for capture or (2) a detection agent or specific binding partner for detection via a binding reaction. The linker enables indirect binding of the capture agent to the solid phase material either before or during the performance of the assay. For example, the solid phase can be plastic, derivatized plastic, magnetic or non-magnetic metal, glass, or silicon, including, for example, test tubes, microtiter wells, sheets, beads, microparticles, chips, and other configurations known to those of skill in the art. In some embodiments, the solid support can be magnetic beads or particles.

[0130] As used herein, "specific binding" or "specifically binds to" may refer to the interaction of an antibody, protein, or peptide with a second chemical molecular species, where the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical molecular species; for example, an antibody recognizes and binds to a specific protein structure rather than proteins in general. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free A, unlabeled A) in a reaction mixture containing labeled "A" and the antibody reduces the amount of labeled A bound to the antibody.

[0131] A "specific binding partner" is a member of a specific binding pair. A specific binding pair includes two different molecules that specifically bind to each other via chemical or physical means. Thus, in addition to the antigen-antibody specific binding pair in a typical immunoassay, other specific binding pairs can include biotin and avidin (or streptavidin), carbohydrate and lectin, complementary nucleotide sequences, effector molecule and receptor molecule, cofactor and enzyme, enzyme and enzyme inhibitor, and the like. Further, a specific binding pair can include analogs of the original specific binding members, e.g., members that are analyte analogs. Immunoreactive specific binding members include antigens, antigen fragments, as well as monoclonal and polyclonal antibodies, and antibodies including isolated or recombinantly produced complexes and fragments thereof.

[0132] As used herein, "statistically significant" refers to the likelihood that the relationship between two or more variables is caused by something other than random chance. Statistical hypothesis testing is used to determine whether the results of a dataset are statistically significant. In statistical hypothesis testing, a statistically significant result is achieved whenever the p-value observed for the test statistic is less than the significance level defined for the study. The p-value is the probability of obtaining a result at least as extreme as the one observed, assuming that at least the null hypothesis is true. Examples of statistical hypothesis analysis include the Wilcoxon signed-rank test, t-test, chi-square test, or Fisher's exact test. As used herein, "remarkable" refers to a change for which it has not been determined to be statistically significant (e.g., it may not have been subjected to a statistical hypothesis test).

[0133] As used interchangeably herein, "subject" and "patient" refer to any vertebrate, including but not limited to mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice, non-human primates (e.g., monkeys such as cynomolgus or rhesus monkeys, chimpanzees, etc.) and humans). In some embodiments, the subject can be human or non-human. In some embodiments, the subject is human. The subject or patient may be undergoing other forms of treatment.

[0134] As used herein, each of "treating", "treatment of", or "treat" is used interchangeably to describe preventing, alleviating, or inhibiting the progression of one or more symptoms of a disease and / or injury or such diseases to which such terms apply. Depending on the condition of the subject, the term also refers to preventing a disease, including preventing the occurrence of a disease or preventing symptoms associated with the disease. Treatment can be carried out acutely or chronically. The term also refers to reducing the severity of a disease or symptoms associated with such a disease prior to onset of the disease. Such prevention or reduction of the severity of the disease prior to onset refers to administration of a pharmaceutical composition to a subject not suffering from the disease at the time of administration. "Preventing" also refers to preventing recurrence of a disease or one or more symptoms associated with such a disease. "Treatment" and "therapeutically" refer to the act of treating when "treating" is as defined above.

[0135] As used herein, "transfer tube" refers to a container or receptacle used for transferring a fluid (e.g., a capillary blood sample) from one location to a second location (e.g., to a reactor or from a plasma separation device).

[0136] As used interchangeably herein, "traumatic brain injury" or "TBI" refers to a complex injury with a wide range of symptoms and physical disabilities. TBI is most often an acute event similar to other injuries. TBI can be classified as "mild", "moderate", or "severe". The causes of TBI are diverse and include, for example, physical shaking by a person, motor vehicle accidents, weapon injuries, cerebrovascular attacks (e.g., stroke), falls, explosions or blasts, and other types of blunt force trauma. Other causes of TBI include oral ingestion and / or exposure to one or more fires, chemicals or toxins (such as mold, asbestos, pesticides and insecticides, organic solvents, paints, adhesives, gases (such as carbon monoxide, hydrogen sulfide and cyanide), organometals (such as methylmercury, tetraethyllead and organotin), one or more dependency drugs or combinations thereof, etc.). Alternatively, TBI can occur in subjects suffering from 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 attacks such as stroke.

[0137] As used herein, "mild TBI" refers to a head injury in which the subject may or may not experience loss of consciousness. In subjects who experience loss of consciousness, the loss of consciousness is typically brief and usually lasts only seconds or minutes. Mild TBI is also referred to as concussion, mild head trauma, mild TBI, mild brain injury, and mild head injury. MRI and CT scans are often normal, but individuals with mild TBI may have cognitive problems such as headache, difficulty thinking, memory problems, attention deficits, mood swings, and dissatisfaction.

[0138] Mild TBI is the most common type of TBI and is often missed at the time of initial injury. Typically, the subject has a GCS score between 13 - 15 (such as 13 - 15 or 14 - 15). Among people with mild TBI, 15 percent (15%) have symptoms that persist for more than three months. Common symptoms of mild TBI include fatigue, headache, visual disturbances, memory loss, decreased attention / concentration, sleep disturbances, dizziness / vertigo (loss of balance), irritability (emotional disturbances), feelings of depression, and seizures. Other symptoms associated with mild TBI include nausea, loss of smell, hypersensitivity to light and sound, mood changes, getting lost or confusion and / or slowed thinking.

[0139] As used herein, "moderate TBI" refers to a brain injury in which loss of consciousness and / or confusion and loss of orientation are between 1 - 24 hours and the subject has a Glasgow Coma Scale (GCS) score between 9 - 13 (e.g., 9 - 12 or 9 - 13). Individuals with moderate TBI may have abnormalities in brain imaging results. As used herein, "severe TBI" refers to a brain injury in which loss of consciousness exceeds 24 hours, memory loss after injury or penetrating skull injury is longer than 24 hours, and the subject has a Glasgow Coma Scale (GCS) score between 3 - 8. Deficits range from high-level cognitive dysfunction to a vegetative state. Survivors may have limitations in arm or leg function, abnormalities in speech or language, loss of thinking ability, or emotional problems. Individuals with severe injury may remain in a persistent vegetative state for a long time. In many cases of people with severe TBI, long-term rehabilitation is often required to maximize function and independence.

[0140] As used herein, "moderate to severe" TBI includes the temporal progression from moderate to severe TBI, and thus encompasses a range of brain injuries that includes (e.g., temporally) moderate TBI alone, severe TBI alone, and combined moderate to severe TBI. For example, in some clinical situations, a subject may initially be diagnosed as having moderate TBI, but over time (minutes, hours, or days) this subject may come to have severe TBI (e.g., in a situation where there is cerebral hemorrhage). Alternatively, in some clinical situations, a subject may initially be diagnosed as having severe TBI, but over time (minutes, hours, or days) this subject may come to have moderate TBI. Such subjects are considered examples of patients who can be classified as "moderate to severe." General symptoms of moderate to severe TBI include difficulties with attention, concentration, distractibility, memory, processing speed, confusion, perseveration, impulsivity, language processing and / or "executive function," lack of comprehension of spoken words (receptive aphasia), difficulty with speech production and comprehension (expressive aphasia), unclear speech, speech that is extremely fast or extremely slow, problems with reading, problems with writing, problems with the interpretation of touch, temperature, movement, limb position and fine discrimination, difficulty integrating or patterning sensory impressions into psychologically meaningful data, partial or total loss of vision, decreased extraocular muscle strength and double vision (diplopia), blurred vision, problems with distance judgment, involuntary eye movements (nystagmus), light intolerance (photophobia), hearing problems such as decreased or loss of hearing, ringing in the ears (tinnitus), hypersensitivity to sound, loss or decrease of smell (anosmia), loss or decrease of taste, seizures associated with epilepsy, which can be of several types and can be accompanied by problems with consciousness, sensory awareness or movement, bowel and bladder control, sleep disturbances, loss of energy, changes in appetite, regulation of body temperature, menstrual difficulties, dependent behavior, problems with emotional capacity or stability, lack of motivation, irritability, aggression, depression, disinhibition, or denial / absence of consciousness, including cognitive deficits. Subjects with moderate to severe TBI can have a Glasgow Coma Scale (GCS) score of 3 to 12 (including the range of 9 to 12 for moderate TBI and 3 to 8 for severe TBI).

[0141] As used herein, the term "variant" is used to describe a peptide or polypeptide whose amino acid sequence differs by amino acid insertions, deletions, or conservative substitutions, but which retains at least one biological activity. Representative examples of "biological activity" include the ability to bind to specific antibodies or the ability to promote an immune response. As used herein, the term "variant" is also used to describe a protein having an amino acid sequence that is substantially identical to a reference protein with an amino acid sequence that retains at least one biological activity. In the art, conservative substitution of amino acids, i.e., replacement of an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree of charge, and distribution of charged regions), is typically recognized in the art as involving minor changes. As understood in the art, these minor changes can be identified, in part, by considering the hydrophobicity index of the amino acid (Kyte et al., J. Mol. Biol. 157:105-132 (1982)). The hydrophobicity index of an amino acid is based on consideration of its hydrophobicity and charge. In the art, amino acids having similar hydrophobicity indices may be substituted, yet still retain the function of the protein. In one embodiment, amino acids having a hydrophobicity index of ±2 are substituted. Amino acid hydrophilicity can also be used to identify substitutions that result in a protein retaining its biological function. Examination of amino acid hydrophilicity in the context of a peptide is a useful measure that has been reported to correlate well with antigenicity and immunogenicity, and allows calculation of the maximum value of the local average hydrophilicity of this peptide (U.S. Patent No. 4,554,101, incorporated herein by reference). Substitution of amino acids having similar hydrophilicity values can result in a peptide retaining its biological activity, e.g., immunogenicity as understood in the art. 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 particular side chain of that amino acid.Amino acid substitutions that are biologically functional and compatible depend on the relative similarity of the amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties, and in particular, are understood to depend on the side chains of these amino acids, which is consistent with this observation. "Variant" can also be used to refer to a fragment of an anti-GFAP antibody that is antigenically reactive, differs from the corresponding fragment of the anti-GFAP antibody within the amino acid sequence, but is still antigenically reactive and can compete with the corresponding fragment of the anti-GFAP antibody for binding to GFAP. "Variant" can also be used to describe a polypeptide or a fragment thereof that has been processed in a different form, such as by proteolysis, phosphorylation, or other post-translational modifications, but retains its antigen reactivity.

[0142] As used herein, the term "vector" is used to describe a nucleic acid molecule capable of carrying another nucleic acid that is ligated thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated to the viral genome. Certain vectors are capable of self-replication within the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., mammalian non-episomal vectors) integrate into the genome of the host cell upon introduction into the host cell and can thereby be replicated with the host genome. Furthermore, certain vectors are capable of directing the expression of operably linked genes. As used herein, such vectors are referred to as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA methods are often in the form of plasmids. Since plasmids are most commonly used in the form of vectors, the terms "plasmid" and "vector" can be used interchangeably. However, other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), can also be used. In this regard, RNA forms of vectors (including viral RNA vectors) can also be used in the context of the present disclosure.

[0143] As used herein, "venous blood" refers to a blood sample obtained from a vein of a subject using a syringe, needle, or combination thereof, or any suitable device. In some embodiments, the venous blood sample is obtained by a trained medical clinician such as a physician, phlebotomist, nurse, laboratory technician, or combination thereof. In some embodiments, the venous blood sample is whole blood, serum, or plasma.

[0144] Unless otherwise specified, technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art. For example, the cell culture and tissue culture methods, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and the terminology used in connection with hybridization, as well as the techniques therefor, described herein are well-known terminology and techniques and are the terminology and techniques commonly used in the art. The meaning and scope of the terms shall be clear, but in the event of any potential ambiguity, the definitions presented herein shall prevail over any dictionary or external definition. Further, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

[0145] 2. Systems and assays for measuring the amount of GFAP in a biological sample obtained from a subject In one embodiment, the present disclosure relates to systems and assays (e.g., methods) for measuring the amount of glial fibrillary acidic protein (GFAP) in a biological sample. The systems and assays of the present disclosure utilize a point-of-care device containing at least one cartridge containing at least one magnetic immunosensor or immunosensing device. The magnetic immunosensor or immunosensing device is used to determine the amount of GFAP in the sample. Specifically, as described in more detail herein, the magnetic immunosensor or magnetic immunosensing device is used to capture and retain a complex containing GFAP immobilized on magnetic beads using one or more specific binding partners (e.g., one or more antibodies).

[0146] The biological samples used in the systems and assays described herein can be blood samples such as venous blood samples, capillary blood samples, finger stick blood samples, or combinations thereof. In some embodiments, the systems and assays of the present disclosure relate to measuring the amount of GFAP in a blood sample such as a venous blood sample, capillary blood sample, finger stick blood sample, or combination thereof.

[0147] In some embodiments, when the biological sample is a blood sample (e.g., a venous blood sample, capillary blood sample, finger stick blood sample, or combination thereof), the blood sample may be subjected to further or additional processing before determining the amount of GFAP. In some embodiments, the sample is processed using centrifugation. In other embodiments, the sample is processed using a plasma separation device that can include at least one filter, membrane, and / or synthetic paper. For example, in some embodiments, a plasma separation device that can be used is the device described in Section 3. The plasma separation device can separate blood into serum and / or plasma.

[0148] In other embodiments, the plasma separation device may be in fluid communication with, or operably connected, coupled, and / or removably coupled to, a microsampling device as part of a microsampling system, as described in more detail in Section 4. In other embodiments, the plasma separation device can be integrated into the microsampling device, as described in more detail in Section 4.

[0149] In still other embodiments, the plasma separation device may be in fluid communication with, or operably connected, coupled, and / or removably coupled to, an opening of a reactor, as described in more detail in Section 4. For example, in some embodiments, the reactor is a cartridge such as those used in point-of-care devices.

[0150] In still other embodiments, the plasma separation device may be in fluid communication with, or operably coupled, coupled and / or removably coupled to, the transfer tube, as described in further detail in Section 4. In these embodiments, the transfer may be in fluid communication with, or operably coupled, coupled and / or removably coupled to, the reactor, as described in further detail in Section 4. In yet further embodiments, the plasma separation device may be integrated within the transfer tube. In still other embodiments, the transfer tube includes a cap or stopper.

[0151] In some embodiments, the system and assay include obtaining a sample within about 24 hours of an actual or suspected injury to the subject and contacting the sample with an antibody to a biomarker of TBI, such as GFAP, to allow formation of a complex of the antibody and the biomarker. More specifically, the sample may be contacted with an anti-GFAP antibody. The assay also includes detecting any resulting antibody-biomarker complex.

[0152] The systems and assays described herein can assist in determining the extent of traumatic brain injury in a subject (e.g., a human subject) who has sustained, is at risk of sustaining, or is suspected of having sustained an acquired brain injury (ABI), such as a traumatic brain injury (TBI), better than the likelihood of not having sustained such an injury. As used herein, "determining whether a subject (e.g., a human subject) has an acquired brain injury (ABI)" refers to the fact that the methods described above can be used, for example, in conjunction with other information (e.g., clinical assessment data), to determine that the subject is more likely to have an ABI than not to have an ABI. The assays described herein can be performed on a sample obtained from a subject (e.g., a human subject) within about 24 hours after an actual or suspected head injury to measure or detect the level of a biomarker of ABI, such as GFAP, in the sample and to determine whether the subject (e.g., a human subject) has an ABI. In some embodiments, if the amount of the biomarker in the sample is higher than a reference level of the biomarker (e.g., GFAP), the subject is determined to have an ABI.

[0153] The systems and assays described herein can aid in determining the extent of traumatic brain injury in a subject (e.g., a human subject) having an actual or suspected head injury, e.g., determining whether the subject (e.g., a human subject) has a mild traumatic brain injury, a moderate traumatic brain injury, a severe traumatic brain injury, or a moderate to severe traumatic brain injury. As used herein, "determining whether the subject (e.g., a human subject) has a mild, moderate, severe, or moderate to severe traumatic brain injury" means using the methods described above, e.g., in conjunction with other information (e.g., clinical assessment data), to determine that the subject is more likely than not to have a mild, moderate, severe, or moderate to severe traumatic brain injury. The assays described herein are performed on a sample obtained from a subject (e.g., a human subject) within about 24 hours after an actual or suspected head injury to measure or detect the level of a biomarker of traumatic brain injury, such as GFAP, in the sample, and to determine whether the subject (e.g., a human subject) has sustained a mild, moderate, severe, or moderate to severe traumatic brain injury (TBI). In some embodiments, if the amount of the biomarker in the sample is higher than a reference level of the biomarker (e.g., GFAP), the subject is determined to have a mild, moderate, severe, or moderate or severe TBI.

[0154] A. Assay In another embodiment, the present disclosure relates to an assay (e.g., a method) for measuring GFAP in a biological sample obtained from a subject. In some aspects, the biological sample is a blood sample such as a venous blood sample, a capillary blood sample, a finger stick blood sample, or a combination thereof. Generally, the assay involves performing an assay for GFAP. The assay includes contacting the biological sample with a cartridge having at least one first specific binding partner that includes at least one magnetic immunosensor and at least one anti-GFAP antibody, where the specific binding partner is immobilized on at least one magnetosensitive bead printed on the cartridge as described herein. The GFAP assays described herein can be used alone or in combination with other assays that do not use a cartridge that includes at least one magnetic immunosensor. Such assays include, for example, an assay for ubiquitin carboxy-terminal hydrolase L1 (UCH-L1).

[0155] Once one or more complexes are formed, the beads containing the immobilized one or more complexes are magnetically captured and retained using at least one magnetic immunosensor or magnetic immunodetection device in a cartridge contained within a point-of-care device. In some aspects, any unbound sample that is not magnetically captured and retained on the magnetic immunosensor can be removed by washing the immunosensor.

[0156] Once one or more complexes are captured and retained on at least one magnetic immunosensor, the signal from the one or more complexes is evaluated. Specifically, the amount of signal from the detectable label indicates the amount of GFAP in the sample.

[0157] In one aspect, the assay performed is an assay for measuring the amount of GFAP in a biological sample, such as a blood sample (e.g., a venous blood sample, a capillary blood sample, a finger stick blood sample, or a combination thereof). In this aspect, the assay involves contacting the sample with a cartridge (used in a point-of-care device) containing at least one magnetic immunosensor and: (i) at least one first specific binding partner that specifically binds to GFAP in the sample and is immobilized on at least one magnetosensitive bead printed on the cartridge; and (ii) at least one second specific binding partner printed on the cartridge and containing a detectable label, to produce one or more complexes comprising the first specific binding partner - GFAP - the second specific binding partner. After the complexes are formed, the magnetosensitive beads are captured and retained on at least one magnetic immunosensor in the cartridge in the point-of-care device. Optionally, any unbound sample can be washed from the magnetic immunosensor using routine techniques known in the art. For the complexes retained on at least one magnetic immunosensor, the signal from the complexes is evaluated. Specifically, the amount of the detectable signal from the detectable label of the complex indicates the amount of GFAP in the sample. It has been found that the assay for measuring GFAP described herein shows at least a five-fold increase in sensitivity compared to an assay for GFAP that does not immobilize the first specific binding partner on magnetosensitive beads and magnetically capture and retain the beads containing the complexes on at least one magnetic immunosensor in a cartridge contained in a point-of-care device.In some embodiments, the assay for measuring GFAP described herein shows at least a 6-fold, at least a 7-fold, at least an 8-fold, at least a 9-fold, at least a 10-fold, at least an 11-fold, at least a 12-fold, at least a 13-fold, at least a 14-fold or at least a 15-fold increase in sensitivity compared to an assay that immobilizes a first specific binding partner on magnetic beads and magnetically captures and holds the beads containing the complex on at least one magnetic immunosensor in a cartridge contained in a point-of-care device.

[0158] In some embodiments, the sample is taken from a subject (e.g., a human subject) within about 24 hours of an actual or suspected injury to the head. For example, the sample can be taken from a subject (e.g., a human subject) within about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours or within about 24 hours of an actual or suspected injury to the head.

[0159] Examples of assays that can be used to determine the amount of GFAP include immunoassays such as enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay, chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), microparticle enzyme immunoassay (MEIA), nephelometric immunoassay, and the like. In other embodiments, the assay can be, for example, a clinical chemistry assay such as photometry, spectrophotometry, absorbance, fluorescence, turbidimetry, nephelometry, potentiometry, and / or electrophoresis assay. In yet further embodiments, the assay can be a combination of an immunoassay and a clinical chemistry assay. In still other embodiments, the assay can be a single molecule detection assay.

[0160] In some embodiments, at least one assay for GFAP is performed in about 4 to about 20 minutes. In some embodiments, each of the at least one assay for GFAP is performed in about 4 minutes. In some embodiments, at least one assay for GFAP is performed in about 5 minutes. In some embodiments, at least one assay for GFAP is performed in about 6 minutes. In some embodiments, at least one assay for GFAP is performed in about 7 minutes. In some embodiments, at least one assay for GFAP is performed in about 8 minutes. In some embodiments, at least one assay for GFAP is performed in about 9 minutes. In some embodiments, at least one assay for GFAP is performed in about 10 minutes. In some embodiments, at least one assay for GFAP is performed in about 11 minutes. In some embodiments, at least one assay for GFAP is performed in about 12 minutes. In some embodiments, at least one assay for GFAP is performed in about 13 minutes. In some embodiments, at least one assay for GFAP is performed in about 14 minutes. In some embodiments, at least one assay for GFAP is performed in about 15 minutes. In some embodiments, at least one assay for GFAP is performed in about 16 minutes. In some embodiments, at least one assay for GFAP is performed in about 17 minutes. In some embodiments, at least one assay for GFAP is performed in about 18 minutes. In some embodiments, at least one assay for GFAP is performed in about 19 minutes. In some embodiments, at least one assay for GFAP is performed in about 20 minutes.

[0161] In some embodiments, the subject received a Glasgow Coma Scale score before or after the assay was performed. In some embodiments, the subject (e.g., a human subject) is suspected of having moderate, severe, or moderate-to-severe traumatic brain injury based on the Glasgow Coma Scale score. In some embodiments, the reference level of a biomarker such as GFAP correlates with subjects having moderate, severe, or moderate-to-severe traumatic brain injury. In some embodiments, the reference level of a biomarker such as GFAP correlates with a Glasgow Coma Scale score of 9 - 13 (moderate TBI). In some embodiments, the reference level of a biomarker such as GFAP correlates with a Glasgow Coma Scale score of 3 - 8 (severe TBI). In some embodiments, the reference level of a biomarker such as GFAP correlates with a Glasgow Coma Scale score of 3 - 12 (moderate, severe, or moderate-to-severe TBI). In some embodiments, the subject is suspected of having mild traumatic brain injury based on the Glasgow Coma Scale score. In some embodiments, the reference level of a biomarker such as GFAP correlates with subjects having mild traumatic brain injury. In some embodiments, the reference level of a biomarker such as GFAP correlates with a Glasgow Coma Scale score of 13 - 15 (mild TBI).

[0162] Generally, the reference level of a biomarker such as GFAP can also be used as a benchmark to evaluate the results obtained in the assay of the test sample for the biomarker such as GFAP. Generally, when making such a comparison, the reference level of a biomarker such as GFAP is obtained by running or performing a particular assay a sufficient number of times under appropriate conditions such that the presence, amount, or concentration of the analyte can be related or associated with a particular stage or endpoint of TBI or a particular sign. Typically, the reference level of a biomarker such as GFAP is obtained by assaying a reference subject (or a population of subjects). The biomarker such as GFAP to be measured can include fragments thereof, degradation products thereof, and / or enzymatically cleaved products thereof.

[0163] In certain embodiments, the reference level may correlate with a control subject (e.g., a human subject) without head injury.

[0164] In some embodiments, after the amount of GFAP in a capillary blood sample is determined using the methods described herein, a result is obtained. This result can be further processed. Specifically, this further processing includes selecting a conversion factor for comparing the amount of GFAP in the sample to the amount of GFAP in venous blood. Specifically, the conversion factor selected can be a static correlation ratio, a dynamic ratio, or a combination of a static correlation ratio and a dynamic ratio. The static correlation ratio is the ratio that exists between venous blood and capillary blood samples for GFAP such that a single ratio (i.e., a "static" correlation) exists. The static correlation ratio assumes a constant ratio, for example, 1.0× or 1.2× or 1.5× or even 0.8×, that is used to convert a reading for capillary blood to a value if GFAP were measured from venous blood collection.

[0165] The dynamic ratio is a ratio that exists between venous blood and capillary blood samples for GFAP where more than a single ratio is involved (i.e., a non-linear or variant type of relationship is involved), but instead depends on one or more factors such as sampling time, diffusion rate, etc. For example, it can be expected that the ratio of GFAP sampled by venous versus capillary blood collection for a marker such as a marker for an acquired brain injury such as traumatic brain injury (TBI) can be time-dependent (i.e., or "dynamic"), and thus, the fact that it is not in an equilibrium state means that the factor changes based on the time sampled after the event. This means that because the correlation can have a time delay factor, for example, a long-term sampling design is necessary for the development of a "correlation table" that includes, for example, for example, two factors, magnitude (of the ratio) and timing (relative to the "event"), and whatever amount in venous versus capillary blood.

[0166] In some embodiments, the conversion factor (e.g., static correlation ratio, dynamic correlation ratio, or a combination of static and dynamic correlation ratios) may be selected based on (a) the amount of GFAP in the sample; (b) a disease, disorder, condition, stage, or situation associated with GFAP; (c) whether the amount of GFAP is determined using an analog assay, a digital assay, or a combination of analog and digital assays; or (d) any combination of (a)-(c).

[0167] For example, in some embodiments, the conversion factor selected for GFAP may be the dynamic correlation ratio.

[0168] Alternatively, in yet further embodiments, when the disease, disorder, condition, stage, or situation associated with GFAP is ABI or TBI, the conversion factor selected is the dynamic correlation ratio.

[0169] In still further embodiments, the conversion factor selected is the static correlation ratio, the dynamic correlation ratio, both the static and dynamic correlation ratios, and is about 0.01, about 0.05, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, about 10.0, about 11.0, about 12.0, about 13.0, about 14.0, about 15.0, about 16.0, about 17.0, about 18.0, about 19.0, or about 20.0.

[0170] Alternatively, in some embodiments, the conversion factor for GFAP may be from about 1.2:1.0 (venous whole blood or plasma, vs. capillary whole blood or plasma) to about 1.0:0.5 (venous whole blood or plasma, vs. capillary whole blood or plasma). Alternatively, in some embodiments, the conversion factor for GFAP is about 1.0:0.85 (venous whole blood or plasma, vs. capillary whole blood or plasma). In other embodiments, the conversion factor is about 1.0:0.8 (venous whole blood or plasma, vs. capillary whole blood or plasma).

[0171] Once a conversion factor (e.g., a static correlation ratio, a dynamic correlation ratio, both a static correlation ratio and a dynamic correlation ratio) is selected, the process further involves normalizing the amount of GFAP in the capillary blood sample by the amount of GFAP from venous blood by applying the conversion factor to the amount of GFAP in the sample. For example, the amount of GFAP in the capillary blood sample can be multiplied by the conversion factor to obtain the normalized amount of GFAP in the sample.

[0172] In some embodiments, the processing of the amount of GFAP (e.g., the results) can be performed by a processing system that includes a computer processor and a non - transitory computer memory including one or more computer programs, in conjunction with the computer processor configured to select a conversion factor for comparing the amount of GFAP in the sample to the amount of GFAP in venous blood and to normalize the amount of GFAP in the sample by applying the selected conversion factor to the amount of GFAP in the sample so as to normalize the amount of GFAP in the sample by the amount of GFAP from venous blood.

[0173] Once the normalized amount of GFAP in the capillary blood sample is obtained, this normalized result can be communicated (e.g., reported) for further analysis, interpretation, processing, and / or display. The results can be communicated (e.g., reported) by a computer into a document and / or spreadsheet, onto a mobile device (e.g., a smartphone), onto a website, into an email, or any combination thereof.

[0174] In some embodiments, the results are communicated by being displayed on a device or the like. In further embodiments, the results are displayed to indicate whether the amount of GFAP in the subject is elevated, not elevated, or that the assay for GFAP should be repeated.

[0175] As described in further detail in Section 2(B), the systems and assays of the present disclosure utilize a point-of-care device. Suitable point-of-care devices for use in the systems and assays described herein include, for example, the i-STAT and i-STAT Alinity devices sold by Abbott Laboratories. Such point-of-care devices can contain a user interface that can display a determination.

[0176] In some embodiments, the results of GFAP (which can be normalized results in some embodiments) are communicated between about 4 minutes and about 40 minutes from the time the sample was collected (e.g., from the time of injury or suspected injury). In other embodiments, the results are communicated between about 4 minutes and about 30 minutes from the time the sample was collected (e.g., the time of injury or suspected injury). In yet other embodiments, the results are communicated between about 4 minutes and about 20 minutes from the time the sample was collected (e.g., from the time of injury or suspected injury). In some embodiments, the results are communicated between about 40 minutes or less, about 39 minutes or less, about 38 minutes or less, about 37 minutes or less, about 36 minutes or less, about 35 minutes or less, about 34 minutes or less, about 33 minutes or less, about 32 minutes or less, about 31 minutes or less, about 30 minutes or less, about 29 minutes or less, about 28 minutes or less, about 27 minutes or less, about 26 minutes or less, about 25 minutes or less, about 24 minutes or less, about 23 minutes or less, about 22 minutes or less, about 21 minutes or less, about 20 minutes, about 19 minutes, about 18 minutes, about 17 minutes, about 16 minutes, about 15 minutes, about 14 minutes, about 13 minutes, about 1 minutes, about 11 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes or about 4 minutes from the time the sample was collected (e.g., from the time of injury or suspected injury).

[0177] In some embodiments, the device contains software for performing one or more tasks, including the implementation of the methods and algorithms described herein. In some embodiments, the device contains software for automatically determining the next appropriate step in the methods and algorithms described herein. For example, the device can contain software for determining the amount or presence of a target analyte. The software can display this determination, such as on a graphical user interface.

[0178] In some embodiments, the device stores software that instructs a processor to perform a given task. In some embodiments, the software stores machine-readable instructions that instruct a processor to perform a given task. The machine-readable instructions can be one or more executable programs or portions (plural) of executable programs for execution by a computer. The program can be embodied in software stored on a non-transitory computer-readable storage medium such as a CD-ROM, floppy disk, hard drive, DVD, Blu-ray disk, or memory associated with the processor. Alternatively, the entire program and / or a portion thereof can instead be executed by a device other than the processor and / or can be embodied in firmware or dedicated hardware. Additionally or alternatively, the processing can be performed by one or more hardware circuits (e.g., discrete and / or integrated analog and / or digital circuits, FPGAs, ASICs, comparators, operational amplifiers (op-amps), logic circuits, etc.) structured to perform the corresponding operations without executing software or firmware.

[0179] Machine-readable instructions may be stored in one or more of a compressed format, an encrypted format, a fragmented format, a compiled format, an executable format, a packaged format, etc. The machine-readable instructions described herein may be stored as data (e.g., portions of instructions, code, representations of code, etc.) that can be used to create, manufacture, and / or produce machine-executable instructions. For example, the machine-readable instructions may be fragmented and stored on one or more storage devices and / or computer processing devices (e.g., servers). Machine-readable instructions may require one or more of installation, modification, adaptation, updating, combination, supplementation, configuration, decryption, decompression, unpacking, distribution, reassignment, compilation, etc. in order to be directly readable, interpretable, and / or executable by a computer processing device and / or other machine. For example, the machine-readable instructions may be stored in multiple portions that are individually compressed, encrypted, and stored on separate computer processing devices, and these portions, when decrypted, decompressed, and combined, form a set of executable instructions that perform a program such as those described herein.

[0180] In another example, machine-readable instructions may be stored in a situation where they can be read by a computer, but require the addition of a library (e.g., a dynamic link library (DLL)), a software development kit (SDK), an application programming interface (API), etc. in order to execute the instructions on a particular computer processing device or other device. In another example, machine-readable instructions may need to be configured (e.g., stored settings, input data, recorded network addresses, etc.) before the machine-readable instructions and / or corresponding program(s) can be executed in whole or in part. Thus, the disclosed machine-readable instructions and / or corresponding program(s) are intended to encompass such machine-readable instructions and / or program(s) regardless of the particular format or situation of the machine-readable instructions and / or program(s) when stored or otherwise during a pause or transfer.

[0181] The machine-readable instructions described herein can be represented by any past, present, or future instruction language, scripting language, programming language, etc. For example, the machine-readable instructions can be represented using any of the following languages: C, C++, Java, C#, Perl, Python, JavaScript, HyperText Markup Language (HTML), Structured Query Language (SQL), Swift, etc.

[0182] The machine-readable instructions can be stored on a non-transitory computer and / or machine-readable medium, such as a hard disk drive, flash memory, read-only memory, compact disk, digital versatile disk, cache, random access memory, and / or any other storage device or storage disk on which information is stored for any period of time (e.g., permanently, for an extended period, for a short instant, for temporary buffering and / or caching of information). As used herein, the term non-transitory computer-readable medium is clearly defined to include any type of computer-readable storage device and / or storage disk, excluding propagated signals and excluding transmission media.

[0183] In some aspects, the GFAP assay described herein is used in combination with an assay for UCH-L1 that does not use a cartridge containing a magnetic immunosensor, and the reference level for UCH-L1 is from about 320 to about 400 pg / mL. In other aspects, the reference level for UCH-L1 is about 360 pg / mL. In still further aspects, the reference level for UCH-L1 is about 400 pg / mL.

[0184] In some embodiments, the reference level for UCH-L1 is from about 320 to about 400 pg / mL, and the sample is obtained from the subject within about 24 hours. In other embodiments, the reference level for UCH-L1 is about 360 pg / mL, and the sample is obtained from the subject within about 24 hours. In yet other embodiments, the reference level for UCH-L1 is about 400 pg / mL, and the sample is obtained from the subject within about 24 hours.

[0185] In some embodiments, the reference level for GFAP is from about 25 to about 40 pg / mL. In other embodiments, the reference level for GFAP is about 30 pg / mL. In still further embodiments, the reference level for GFAP is about 35 pg / mL.

[0186] In some embodiments, the reference level for GFAP is from about 25 to about 40 pg / mL, and the sample is obtained from the subject within about 24 hours. In other embodiments, the reference level for GFAP is about 30 pg / mL, and the sample is obtained from the subject within about 24 hours. In yet other embodiments, the reference level for GFAP is about 35 pg / mL, and the sample is obtained from the subject within about 24 hours.

[0187] In some embodiments, the reference level for UCH-L1 is about 360 pg / mL and the reference level for GFAP is about 30 pg / mL. In other embodiments, the reference level for UCH-L1 is about 400 pg / mL and the reference level for GFAP is about 35 pg / mL. In still further embodiments, the reference level for UCH-L1 is about 360 pg / mL, the reference level for GFAP is about 30 pg / mL, and the sample is obtained from the subject within about 24 hours. In yet other embodiments, the reference level for UCH-L1 is about 400 pg / mL, the reference level for GFAP is about 35 pg / mL, and the sample is obtained from the subject within about 24 hours.

[0188] In some embodiments, the method includes performing at least one assay for GFAP and at least one assay for UCH-L1 in at least one sample obtained from a subject, and determining, based on the results of the assays, whether the amounts (e.g., levels) of GFAP and UCH-L1 in the subject are elevated. In some embodiments, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are elevated. In some embodiments, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are elevated if the amount of GFAP in the sample is equal to or greater than 30 pg / mL, the level of UCH-L1 is less than about 360 pg / mL, not determinable by an assay for UCH-L1, or not reported by an assay for UCH-L1. In some embodiments, the method includes determining that the amounts of GFAP and UCH-L1 in a sample obtained from the subject are elevated if the level of GFAP is equal to or greater than about 30 pg / mL and the level of UCH-L1 is equal to or greater than about 360 pg / mL. In some embodiments, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are elevated if the level of GFAP is not determinable by an assay for GFAP, not reported by an assay, and the level of UCH-L1 is equal to or greater than about 360 pg / mL.

[0189] In some embodiments, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are not elevated. In some embodiments, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are not elevated if the level of GFAP in the sample is less than about 30 pg / mL and the level of UCH-L1 in the sample is less than about 360 pg / mL.

[0190] In some embodiments, the method includes determining that the assays for GFAP and UCH-L1 should be repeated. In some embodiments, the method includes determining that the assays for GFAP and UCH-L1, or the assay for GFAP, should be repeated if the amount of GFAP is less than about 30 pg / mL and the level of UCH-L1 cannot be determined or is not reported by the assay for UCH-L1. In some embodiments, the method includes determining that the assays for GFAP and UCH-L1, or the assay for GFAP, should be repeated if the amount of GFAP cannot be determined or is not reported by the assay and the level of UCH-L1 is less than about 360 pg / mL. In some embodiments, the method includes determining that the assays for GFAP and UCH-L1, or the assay for GFAP, should be repeated if the amount of GFAP cannot be determined or is not reported by the assay for GFAP and the level of UCH-L1 cannot be determined or is not reported by the assay for UCH-L1.

[0191] In some embodiments, the method includes performing at least one assay for GFAP and at least one assay for UCH-L1 in at least one sample obtained from a subject, and determining, based on the results of the assays, whether the amounts of GFAP and UCH-L1 in the subject are elevated. In some embodiments, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are elevated. In some embodiments, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are elevated if the amount of GFAP in the sample is equal to or greater than 35 pg / mL, the amount of UCH-L1 is less than about 400 pg / mL, cannot be determined by an assay for UCH-L1, or is not reported by an assay for UCH-L1. In some embodiments, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are elevated if the amount of GFAP is equal to or greater than about 35 pg / mL and the level of UCH-L1 is equal to or greater than about 400 pg / mL. In some embodiments, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are elevated if the amount of GFAP cannot be determined by an assay for GFAP or is not reported by the assay, and the amount of UCH-L1 is equal to or greater than about 400 pg / mL.

[0192] In some embodiments, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are not elevated. In some embodiments, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are not elevated if the amount of GFAP in the sample is less than about 35 pg / mL and the amount of UCH-L1 in the sample is less than about 400 pg / mL.

[0193] In some embodiments, the method includes determining that the assay for GFAP and UCH-L1, or for GFAP, should be repeated. In some embodiments, the method includes determining that the assay for GFAP and UCH-L1, or for GFAP, should be repeated if the amount of GFAP is below about 35 pg / mL and the amount of UCH-L1 cannot be determined or is not reported by the assay for UCH-L1. In some embodiments, the method includes determining that the assay for GFAP and UCH-L1, or for GFAP, should be repeated if the amount of GFAP cannot be determined or is not reported by the assay for GFAP and the amount of UCH-L1 is below about 400 pg / mL. In some embodiments, the method includes determining that the assay for GFAP and UCH-L1, or for GFAP, should be repeated if the amount of GFAP cannot be determined or is not reported by the assay for GFAP and the amount of UCH-L1 cannot be determined or is not reported by the assay for UCH-L1.

[0194] In some embodiments, the method further includes performing a head computed tomography (CT) scan, a magnetic resonance imaging (MRI) procedure, or both a CT scan and an MRI procedure on the subject if the level of GFAP, or of GFAP and UCH-L1, in the subject is elevated. For example, in some embodiments, the method further includes performing a head CT scan on the subject if the level of GFAP, or of GFAP and UCH-L1, in the subject is elevated. As another example, in some embodiments, the method further includes performing an MRI procedure on the subject if the level of GFAP, or of GFAP and UCH-L1, in the subject is elevated. In some embodiments, the method further includes performing a head CT scan and an MRI procedure on the subject if the level of GFAP, or of GFAP and UCH-L1, in the subject is elevated.

[0195] B. Point-of-Care Device Containing a Magnetic Immuno-Sensor As previously mentioned herein, the systems and assays of the present disclosure utilize a point-of-care device. Examples of suitable point-of-care devices that can be used in the systems and assays described herein include, for example, the i-STAT and i-STAT Alinity devices sold by Abbott Laboratories.

[0196] The point-of-care device used in the systems and assays of the present disclosure contains at least one cartridge containing at least one magnetic immuno-sensor or magnetic immuno-sensing device. In some embodiments, the at least one magnetic immuno-sensor or magnetic immuno-sensing device includes a base sensor or sensing electrode. In some embodiments, the base sensor or sensing electrode may be on a substantially planar chip (e.g., an immunosensor or sensor chip), where the sensing electrode is located within a conduit for receiving a biological sample (e.g., a blood sample such as a venous blood sample, capillary blood sample, finger stick blood sample, or a combination thereof), and the biological sample is mixed with magnetically susceptible beads that can be attracted by a magnet or respond to a magnetic field.

[0197] A high magnetic field magnet, such as a permanent magnet or an electromagnet, is positioned proximate to, incorporated within, or on the chip (e.g., on top of the chip, or below or beneath the chip) to attract magnetically susceptible beads within the conduit substantially proximate to the sensing electrode. This magnetic zone functions to capture and substantially retain the beads on or near the surface of the sensing electrode, for example, during removal of any unbound sample and during washing of the electrode. As described in detail herein, the beads are coated with at least one first specific binding partner (e.g., an anti-GFAP antibody, or an anti-GFAP antibody and an anti-UCH-L1 antibody) that specifically binds to an analyte (e.g., GFAP) in the sample. In some embodiments, the at least one first specific binding partner is an anti-GFAP antibody. In another embodiment, the at least one first specific binding partner is an anti-UCH-L1 antibody.

[0198] In yet a further aspect, in addition to the magnetic immuno-sensor or magnetic immuno-sensing device, the cartridge further comprises: (a) one or more beads (e.g., paramagnetic particles) coated with at least one first specific binding partner (e.g., an anti-GFAP antibody) that specifically binds to an analyte (e.g., GFAP), printed or dispensed onto the cartridge using routine techniques known in the art and dried; and (b) one or more zones or regions containing at least one detectably labeled second specific binding partner (e.g., an anti-GFAP antibody), printed or dispensed onto the cartridge using routine techniques known in the art and dried. The one or more beads containing at least one first specific binding partner (e.g., an anti-GFAP antibody) and the at least one detectably labeled second specific binding partner (e.g., an anti-GFAP antibody) can be printed and dried in the same zone or region on the cartridge, or alternatively, in zones or regions adjacent to or in close proximity to each other.

[0199] In one aspect, during the performance of the assay, printed beads containing at least one first specific binding partner (e.g., an anti-GFAP antibody), and printed, detectably labeled at least one second specific binding partner (e.g., an anti-GFAP antibody) are reconstituted by wetting or dissolving the printed area with at least one diluent, buffer, or other liquid. Once reconstituted, the sample can be added and mixed with the beads and the detectable label. In other aspects, the printed beads and label may be reconstituted using the sample, thereby allowing reconstitution and mixing to occur simultaneously. Once the sample is mixed with the beads coated with at least one first specific binding partner and the detectably labeled at least one second specific binding partner, a complex comprising the first specific binding partner-coated beads - analyte - detectably labeled second specific binding partner is formed. Once formed, a magnet contained within or on the cartridge is used to capture and hold the beads containing the complex, and the amount of analyte (e.g., GFAP) is determined using routine techniques known in the art. In some aspects, the magnet can be located on top of the chip. In other aspects, the magnet can be located under or beneath the chip.

[0200] Moreover, in some other aspects, the point-of-care device includes a reading device or reader, a disposable cartridge containing a magnetic immuno-sensor or immunosensing device, and all of the other assay components used to analyze an analyte in a biological sample.

[0201] (1) High magnetic field magnet and magnetic layer In some embodiments, at least one magnetic immuno-sensor includes a sensing electrode on a substantially planar chip and has a high magnetic field magnet, such as a permanent magnet or an electromagnet, positioned proximate to (e.g., beneath) or associated with the chip. The magnetic immuno-sensor can provide a field exceeding about 0.1 Tesla and has an event horizon (e.g., a point of no return) that can efficiently collect beads from a range of about 0.05 mm to about 5 mm in the region of the sensing electrode.

[0202] High magnetic field magnets such as permanent magnets or electromagnets include any material that provides a high magnetic field (e.g., exceeding about 0.1 Tesla, exceeding 0.4 Tesla, or exceeding 1 Tesla). The magnetic field can be measured, for example, as the field remaining in a substantially flat surface area of the magnet. Examples of materials that can be used are alloys of neodymium-iron-boron (NdFeB) or Nd2Fe 14 B, although other materials may be used. For example, high magnetic field permanent magnets can include ferrite or aluminum-nickel-cobalt (AlNiCo) magnets that typically exhibit a field of 0.1 - 1 Tesla. Other high magnetic field permanent magnets composed of rare earth element alloys (e.g., neodymium alloys and samarium-cobalt (SmCo) alloys) exhibit fields exceeding 1 Tesla, e.g., exceeding 1.2 Tesla or exceeding 1.4 Tesla.

[0203] Rare earth magnets are generally brittle and also vulnerable to corrosion, and thus such materials are often plated or coated to be protected from breakage and chipping. Additionally, the Curie point of rare earth magnets substantially exceeds the temperatures encountered in the assays described herein that can be run at ambient temperature to about 50 °C. In some embodiments, the assay is at a constant temperature of 37 °C for use with blood samples (such as venous blood samples, capillary blood samples, finger stick blood samples, or combinations thereof).

[0204] In another aspect, the high magnetic field magnet includes an electromagnet in which a magnetic field is generated by the flow of an electric current. The electric current can be provided by a reader within a point-of-care device into which the magnetic immuno-sensor or magnetic immuno-sensing device is inserted and with which the magnetic immuno-sensor or magnetic immuno-sensing device is in electrical contact.

[0205] The magnetic immuno-sensor or magnetic immuno-sensing device includes sensing electrodes on a substantially planar chip and a bulk permanent high magnetic field magnet located proximate to the electrodes (e.g., under or on the opposite side of the chip). In some aspects, the bulk permanent high magnetic field magnet is located within the housing of the point-of-care device (e.g., by cutting out or grooving a plastic cartridge). In other aspects, the bulk permanent high magnetic field magnet is located within the base of the plastic cartridge housing (e.g., not in the same plane as the sensing electrodes). In other aspects, the magnet is located adjacent to or within a reading device or reader in the point-of-care device.

[0206] In some aspects, the bulk high magnetic field permanent magnet is substantially cylindrical with a diameter in the range of about 0.1 mm to about 5 mm and a length in the range of about 0.1 mm to about 5 mm and is positioned to create an event horizon within a conduit suitable for bead capture within a short period of time (e.g., 1 to 5 minutes). The conduit generally has a height in the range of about 0.2 mm to about 5 mm, a width in the range of about 0.2 mm to about 5 mm, and a cross-sectional area that can be either uniform or non-uniform. In other embodiments, the shape of the bulk magnet can be in the form of a square, rectangle, ellipse, flake, pyramid, sphere, sub-spherical form or other shape.

[0207] In another aspect, a magnetic immuno-sensor or a magnetic immuno-sensing device includes a sensing electrode on a substantially planar chip. The electrode is located within a conduit for receiving a sample mixed with one or more first specific binding partners (e.g., antibodies) immobilized on magnetic beads, one or more labeled second specific binding partners, and a magnetization layer (e.g., a microfabricated magnetic layer). The magnetization (or magnetic) layer may be included on the chip (e.g., located on the chip, directly attached, coated or patterned on any of its surfaces), or may be embedded therein (e.g., located within the chip, integrated into the chip). This configuration attracts magnetic beads substantially proximate to the electrode during removal of unbound sample and cleaning of the electrode, and substantially retains them at the electrode.

[0208] The magnetization layer can be formed from a mobile magnetic composition, such as a slurry, including particles in an immobilization or support matrix (e.g., polyimide, polyvinyl alcohol (PVA) or thermoplastic equivalents) and a material capable of sustaining a high magnetic field permanent magnetic field, such as an NdFeB alloy. The slurry is not limited by viscosity and can include any viscosity known to be suitable in the art. In some aspects, the mobile magnetic composition has a viscosity ranging from 0.3 to 300,000 CPS, such as 100 to 100,000 CPS or 1,000 to 10,000 CPS. In other aspects, the magnetic particles in the slurry have an average particle size ranging from 0.01 μm to 100 μm, such as 0.1 μm to 10 μm or 3 μm to 7 μm.

[0209] In addition to polyimide, PVA, and thermoplastic polyimide, a two-part chemically cured epoxy resin, kapton, and others may be used as a support matrix for fixing magnetic particles to a wafer. The method of curing the matrix can be based on a process initiated by light, thermally, or chemically. In certain embodiments, the immobilization matrix is composed of other photoformed matrix materials.

[0210] As described above, the slurry can be applied at various locations in or on an immunosensing device (e.g., on a wafer or chip, electrodes, housing, front or back side of a reader, etc.). For example, in some embodiments, a high magnetic field permanent magnetic material is applied to a substantially planar chip (e.g., using a mask) in a patterned manner. In certain embodiments, a high magnetic field permanent magnetic material is also applied to a microfabricated sensing electrode. In other embodiments, the slurry is applied to a layer under the sensing electrode.

[0211] Prior to the application process, the slurry may or may not be magnetized. However, after the deposition step, the magnetic layer can be magnetized to provide a directionality to the field.

[0212] (2) Sensing Electrodes The sensing electrodes can be microfabricated (e.g., current-sensing gold arrays) on a substantially planar chip (e.g., a silicon wafer) using any technique known in the art, such as those described in U.S. Pat. Nos. 5,200,051 and 7,419,821, the contents of which are incorporated herein by reference.

[0213] (3) Magnetic Sensitive Beads In some embodiments, a biological sample, e.g., a blood sample, is mixed with magnetic sensitive beads. The magnetic sensitive beads can be composed of any material known in the art that is highly sensitive to movement by a magnet (e.g., a permanent magnet or an electromagnet) utilized in or in cooperation with the devices of the present disclosure. Thus, the terms "magnetic" and "magnetic sensitive" with respect to the beads can be used interchangeably.

[0214] In some embodiments, the beads include a magnetic core that can be completely or partially coated with a coating material. The magnetic core can include ferromagnetic, paramagnetic, or superparamagnetic materials. In some embodiments, the magnetosensitive beads include a ferrite core and an outer polymer coating. However, the magnetic core can include one or more of Fe, Co, Mn, Ni, a metal containing one or more of these elements, a regular alloy of these elements, a crystal composed of these elements, a magnetic oxide structure, such as ferrite, and combinations thereof. In other embodiments, the magnetic core is magnetite (Fe3O4), maghemite (γ-Fe2O3), or of the formula Me 1-x OFe3+xO3 (wherein Me is, for example, Cu, Fe, Ni, Co, Mn, Mg or Zn or a combination of these materials, and x ranges from 0.01 to 99.) and can be composed of divalent metal-ferrite provided by

[0215] Materials suitable for the coating include synthetic and biological polymers, copolymers and polymer blends, and inorganic materials. The polymer materials can include various combinations of acrylate, siloxane, styrene, acetate, alkylene glycol, alkylene, alkylene oxide, parylene, polymers of lactic acid and glycolic acid. The biopolymer materials include starch or similar carbohydrates. The inorganic coating materials can include any combination of metals, metal alloys and ceramics. Examples of ceramic materials can include hydroxyapatite, silicon carbide, carboxylate, sulfonate, phosphate, ferrite, phosphonate, and oxides of Group IV elements of the periodic table.

[0216] In other embodiments, the magnetic beads include non-magnetic substrate beads formed from a material selected from the group consisting of, for example, polystyrene, polyacrylic acid, and dextran, on which a magnetic coating is placed.

[0217] Magnetic susceptibility beads of any correct size that can be positioned with the described high magnetic field magnet can be utilized, taking into account the dispersibility requirements of the magnetic susceptibility beads. In some embodiments, at least 50 wt%, for example, at least 75 wt% of the magnetic susceptibility beads are retained on the electrode surface. In some embodiments, the average particle size of the magnetic susceptibility beads can range from 0.01 μm to 20 μm, for example, from 0.1 μm to 10 μm, from 0.1 μm to 5 μm, or from 0.2 μm to 1.5 μm. As used herein, the term "average particle size" refers to the average longest dimension of the particles, e.g., the diameter of spherical particles determined by methods well known in the art. The particle size distribution of the magnetic susceptibility beads can be unimodal, although a multimodal distribution may also be used. Spherical magnetic susceptibility beads may be used, but other bead shapes and structures, e.g., ellipsoidal, sub-spherical, cylindrical, and other irregularly shaped particles, are within the meaning of the terms "beads" and "microparticles" as used herein.

[0218] Commercial sources of magnetic susceptibility beads include Invitrogen (trademark) by Life Technologies (trademark) (Carlsbad, Calif., U.S.A.), Ademtech (Pessac, France), Chemicell GmbH (Berlin, Germany), Bangs Laboratories, Inc. (trademark), (Fishers, Ind.), and Seradyn, Inc. (Indianapolis, Ind.). Many of the commercially available products incorporate surface functionalization that may be used to immobilize antibodies (e.g., IgG) on the bead surface. Exemplary functionalizations include carboxyl, amino, or streptavidin-modified magnetic susceptibility beads.

[0219] In some embodiments, the magnetic susceptibility beads are coated with an antibody such as an anti-GFAP antibody. Coating the beads with the antibody immobilizes the antibody on the beads.

[0220] In certain embodiments, the magnetic beads are deposited in a suitable area of the magnetic immunosensing device as a suspension in a mixture, for example, a mixture of lactitol and DEAE-dextran, such as the mixture supplied by Advanced Enzyme Technologies (Pontypool, Great Britain). Evaporation of the solvent, usually water, results in a glassy deposit in which the beads are immobilized. Lactitol / DEAE-dextran enables the beads to be localized within the device in a mechanically and biochemically stable situation, which also dissolves rapidly after contact with the sample.

[0221] In various embodiments, the beads are mobile, which enables interaction with the analyte. After binding to GFAP, magnetic force is used to concentrate the beads at the electrode for measurement and to localize the magnetic beads at the current measuring electrode for signal detection.

[0222] (4) Manufacture of a magnetic immunosensor or magnetic immunosensing device Magnetic immunosensors or immune sensing devices can be manufactured using techniques known in the art, including, for example, the techniques described in U.S. Patent Nos. 9,233,370, 9,958,440, and 10,145,843 and International Patent Publication Nos.: WO18107016, WO18107015, WO18107007, WO18107009, WO18107012, WO18107013, WO21211331, and WO21211332, the contents of which are incorporated herein by reference. For example, a silicon wafer is thermally oxidized to form an insulating oxide layer having a thickness of about 1 μm. Next, a titanium / tungsten layer is sputtered onto the oxide layer to a preferred thickness of about 100 Å to about 1000 Å, followed by a gold layer that is sputtered to a thickness of 500 Å to 1000 Å, most preferably about 800 Å. Next, photoresist is spun onto the wafer, dried, and baked. Next, the surface is exposed using a contact mask, the latent image is developed, and the wafer is exposed to a gold-etching solution. The patterned gold layer is coated with photosensitive polyimide, preferably baked, exposed using a contact mask, developed, cleaned in an oxygen plasma, and preferably imidized at 350 °C for about 5 hours. This operation leaves a number of electrode openings in the polyimide layer in a square array. In some embodiments, the square array has a diameter of, for example, about 2 μm to about 100 μm, about 5 μm to about 15 μm, or about 7 μm, and the distance between them is, for example, about 5 μm to about 100 μm, about 10 μm to about 20 μm, or about 15 μm. The area covered by these electrodes (i.e., the sensor area) is substantially circular and has a diameter of, for example, about 50 μm to about 1000 μm, about 100 μm to about 300 μm, or about 300 μm.

[0223] After the wafer is diced into individual chips, each chip is assembled into a disposable cartridge. The cartridge can be of the type described in U.S. Patent Nos. 7,419,821, 8,747,774, and 9,415,389, which are incorporated herein by reference. In one aspect, the sensor is located within a conduit for receiving a sample, and a high magnetic field magnet, such as a permanent or electromagnetic magnet, is located beneath the sensor, such as directly beneath its central region. In another aspect, the high magnetic field magnet can be located above the sensor region of the conduit. These elements can be in fixed positions within the device housing or can be adapted to an actuator that can move to and from positions relative to the immunosensor and the conduit. One or more high magnetic field magnets can be used to attract magnetic beads within the conduit (e.g., substantially proximate to the sensor) while removing the sample and washing the sensor to remove unbound or partially absorbed reagents, and to hold this in the region of the sensor. As described above, the magnetic beads are coated with an antibody (e.g., an anti-GFAP antibody) for detecting GFAP in a sample.

[0224] C. System In another embodiment, the present disclosure relates to a system for determining the amount of GFAP in a biological system obtained from a subject. In some aspects, the system utilizes the assay described in Section 2(A) that utilizes at least one magnetic bead. Additionally, the system utilizes a point-of-care device that contains at least one cartridge that includes at least one magnetic immunosensor or immune sensing device described in Section 2(B). The point-of-care device determines the amount of GFAP in a sample by magnetically capturing and holding beads containing a complex on at least one magnetic immunosensor, and evaluating the signal from the complex, whereby the amount of detectable signal from a detectable label indicates the amount of GFAP in the sample.

[0225] In a further aspect, when the system uses the GFAP assay described in Section 2A, the assay has been found to exhibit at least a 5-fold increase in sensitivity compared to an assay for GFAP that does not immobilize the first specific binding partner on magnetic beads and magnetically capture and hold the beads containing the complex on at least one magnetic immunosensor in a cartridge contained in a point-of-care device. In some aspects, the assay for measuring GFAP described in Section 2A has been found to exhibit at least a 6-fold, at least a 7-fold, at least an 8-fold, at least a 9-fold, at least a 10-fold, at least an 11-fold, at least a 12-fold, at least a 13-fold, at least a 14-fold or at least a 15-fold increase in sensitivity compared to an assay that does not immobilize the first specific binding partner on magnetic beads and magnetically capture and hold the beads containing the complex on at least one magnetic immunosensor in a cartridge contained in a point-of-care device.

[0226] 3. Plasma Separation Device As previously described, in some aspects of the systems and assays described herein, a device that is a plasma separation device can be used to process a biological sample (e.g., a blood sample). In one aspect, the device includes a hydrophobic layer that includes at least one microchannel, and an upper layer that is adjacent to, on, or above the hydrophobic layer. The hydrophobic layer can include or be constructed from at least one hydrophobic material. The hydrophobic material can be a membrane, film, fabric, fiber, filter, microfilm, screen, mesh, or any combination thereof. In one aspect, the hydrophobic layer is a membrane or a film. Hydrophobic membranes or films that can be used are hydrophobic membranes or films known in the art. Specifically, membranes or films such as those available from Adhesive Research (Glen Rock, PA), 3M (Minneapolis, Minnesota), and / or Tesa SE (Norderstadt, Germany) can be used.

[0227] The hydrophobic layer includes at least one microchannel having first and second ends that define a path for capillary fluid flow of a processed blood sample or blood product (e.g., plasma). In some embodiments, the at least one microchannel extends longitudinally along a portion of the hydrophobic layer from the first end to an opening at the second (e.g., opposite) end of the microchannel. In other embodiments, the at least one microchannel extends across the width of a portion of the hydrophobic layer from the first end to an opening at the second (e.g., opposite) end of the microchannel. In some embodiments, the microchannel contains a first opening connected to the first end of the microchannel. In these embodiments, the processed blood or blood product (e.g., plasma) can enter from the first opening into the first end of the microchannel and flow to a second opening at the second end of the microchannel.

[0228] The opening at the second end of the microchannel allows the processed blood or blood product (e.g., plasma) to flow out of the device. For example, if the fluid being processed is blood, the plasma can flow by capillary fluid flow from the first end toward the second opening at the second (e.g., opposite) end. The plasma can be collected at the second (e.g., opposite) end using a collection or other device, or if the device is operatively coupled, removably coupled, or in fluid communication with another device (such as a sample analysis cartridge (e.g., a microfluidic cartridge)), it can continue to flow directly over or through the device for further processing and / or analysis.

[0229] The microchannels can be of any length. In some embodiments, the microchannels have a length of less than about 80 mm. In other embodiments, at least one microchannel has a length of about 70 mm, at least one microchannel has a length of about 60 mm, about 55 mm, about 50 mm, about 45 mm, about 40 mm, about 35 mm, about 30 mm, about 25 mm, about 20 mm or about 15 mm. In other embodiments, at least one microchannel has a width of less than about 5 mm, less than about 4.5 mm, less than about 4 mm, about 3 mm, less than about 2.5 mm or less than about 2.0 mm.

[0230] Moreover, at least one microchannel can be located anywhere on the hydrophobic layer. For example, at least one microchannel may be placed at the center of the hydrophobic layer, slightly off-center on the hydrophobic layer, or at or near the side or edge of the hydrophobic layer.

[0231] In additional embodiments, the hydrophobic layer can have a thickness of about 50 to about 200 microns. In other embodiments, the hydrophobic layer can have a thickness of about 100 to about 200 microns. In some embodiments, the hydrophobic layer can have a thickness of about 100 to about 150 microns.

[0232] The device also includes an upper layer adjacent to, on, or over the hydrophobic layer. In some embodiments, the upper layer is adhered to the hydrophobic layer. In yet further embodiments, the surface of the upper layer facing the hydrophobic layer includes a material or is coated with a hydrophilic material. The hydrophilic material can be an adhesive substance, a membrane, a film, a fabric, a fiber, a filter, a microfilm, a screen, a mesh, or any combination thereof. In other embodiments, the entire upper layer is made or constructed from a hydrophilic material such as a membrane, a film, a fabric, a fiber, a filter, a microfilm, a screen, a mesh, or any combination thereof. For example, in one embodiment, the upper layer is a membrane or a film. In some embodiments, the membrane or film is not constructed from a hydrophilic material but is coated with a hydrophilic material. The portion of the upper layer coated with the hydrophilic material faces the hydrophobic layer. In another embodiment, the entire membrane or film is made or constructed from a hydrophilic material. The hydrophilic membranes or films that can be used are hydrophilic membranes or films known in the art. Examples of hydrophilic films that can be used are the 9984 Diagnostic Microfluidic Surfactant-Free Hydrophilic Film available from 3M (Minneapolis, MN), Kemafoil H from Coveme (S. Lazzaro di Savena, Italy), a hydrophilic-coated polyester film, Tesa 62580 from Tesa SE (Norderstadt, Germany), a hydrophilic-coated polyester film. In additional embodiments, the upper layer can have a thickness of from about 50 to about 200 microns. In other embodiments, the upper layer can have a thickness of from about 100 to about 200 microns. In some embodiments, the upper layer can have a thickness of from about 100 to about 150 microns.

[0233] When a blood sample or a blood preparation is placed on top of the upper layer of the device, the blood sample or preparation flows through the upper layer. During this process, the cellular components of the blood (e.g., red blood cells, white blood cells, platelets, and combinations thereof) are captured in the pores and / or fibers of the hydrophilic material, thereby enabling the plasma to continue to flow through the hydrophilic material, to the hydrophobic layer, and into the microchannel. Once in the microchannel, the plasma flows by capillary fluid flow from the first end to an opening at the second (e.g., opposite) end of the channel. The plasma can be collected using a collection or other device, or if the device is operably connected to, removably coupled to, or in fluid communication with another device (such as a sample analysis cartridge (e.g., a microfluidic cartridge)), it can continue to flow directly over or through the device for further processing and analysis.

[0234] In another aspect, the device optionally includes a bottom layer. The bottom layer is adjacent to, beneath, or below the hydrophobic layer. In these aspects, the device includes at least three layers - an upper layer, a hydrophobic layer, and a bottom layer. In some aspects, the bottom layer is adhered to the hydrophobic layer. In additional aspects, the upper layer and the bottom layer are each adhered to the hydrophobic layer.

[0235] In yet a further aspect, the surface of the bottom layer facing the hydrophobic layer is either material-containing or coated with a hydrophilic material. The hydrophilic material can be an adhesive substance, a membrane, a film, a fabric, a fiber, a filter, a microfilm, a screen, a mesh, or any combination thereof. In other aspects, the entire bottom layer is made or constructed from a hydrophilic material such as a membrane, a film, a fabric, a fiber, a filter, a microfilm, a screen, a mesh, or any combination thereof. For example, in one aspect, the bottom layer is a membrane or a film. In some aspects, the membrane or film is not constructed from a hydrophilic material but is coated with a hydrophilic material. The portion of the bottom layer coated with the hydrophilic material faces the hydrophobic layer. In another aspect, the entire membrane or film is made or constructed from a hydrophilic material. The hydrophilic membranes or films that can be used are hydrophilic membranes or films known in the art. Examples of hydrophilic films that can be used are the 9984 Diagnostic Microfluidic Surfactant-Free Hydrophilic Film available from 3M (Minneapolis, MN), Kemafoil H from Coveme (S. Lazzaro di Savena, Italy), a hydrophilic-coated polyester film, Tesa 62580 from Tesa SE (Norderstadt, Germany), and a hydrophilic-coated polyester film.

[0236] In additional aspects, the bottom layer can have a thickness of from about 50 to about 200 microns. In other aspects, the bottom layer can have a thickness of from about 100 to about 200 microns. In some aspects, the bottom layer can have a thickness of from about 100 to about 150 microns.

[0237] In yet a further aspect, the device can contain a protective film adjacent to, under, or below the bottom layer. In some aspects, the protective film is adhered to the bottom layer. The protective film protects the device from moisture and / or other contaminants. The protective film can be a plastic film such as an adhesive plastic film, a cardboard with an adhesive substance, a plastic sheet with an adhesive substance, or a combination thereof.

[0238] In another aspect, when the device contains an upper layer, a hydrophobic layer, and a bottom layer, the combined thickness of the three layers is between about 100 and about 600 microns. In another aspect, the combined thickness of the three layers is between about 150 and about 600 microns. In another aspect, the combined thickness of the three layers is between about 200 and about 600 microns. In yet another aspect, the combined thickness of the three layers is between about 150 and about 500 microns. In still further aspects, the combined thickness of the three layers is between about 200 and about 500 microns.

[0239] Moreover, in some aspects, when the device contains an upper layer, a hydrophobic layer, and a bottom layer, the upper layer and the bottom layer can be made of the same material. Alternatively, the upper layer and the bottom layer can be made of different materials. For example, the upper layer and the bottom layer can be made or constructed from the same hydrophilic material or different hydrophilic materials. Alternatively, the upper and bottom layers can be made or constructed from non-hydrophilic materials, but are coated with a hydrophilic material on the surface of the layer facing the hydrophobic layer. Still further, either the upper or the bottom layer can be made or constructed from a non-hydrophilic material and coated with a hydrophilic material on the surface facing the hydrophobic layer, and the other layer can be made entirely from a hydrophilic material. <I

[0240] In some aspects, the upper layer includes a sample inlet where a blood sample or a blood product is placed to initiate sample processing through the device. The sample inlet can have any shape. For example, the sample inlet can be circular, elliptical, rectangular, square, triangular, or any combination thereof. In some aspects, a hydrophobic transfer material (e.g., transfer tape, etc.) can surround the sample inlet. The hydrophobic transfer material helps prevent the blood sample or blood product from wicking or moving away from the sample inlet area.

[0241] When the upper layer includes a sample inlet, the hydrophobic layer and optionally the bottom layer include one or more openings. The openings can have any shape. For example, the openings can be circular, oval, rectangular, square, triangular, or any combination thereof. The openings can have the same shape as the sample inlet or a different shape. In some embodiments, when the device contains two layers (e.g., an upper layer having a sample inlet and a hydrophobic layer), one or more openings can be made in the hydrophobic layer. In other embodiments, when the device contains three layers, one or more openings can be made in the hydrophobic layer but not in the bottom layer. In other embodiments, when the device contains three layers, one or more openings can be made in each of the hydrophobic layer and the bottom layer. Each opening in one or more layers can be directly below the sample inlet.

[0242] For example, FIG. 8 illustrates an apparatus for plasma separation that contains a hydrophilic upper layer and a hydrophobic layer containing a microchannel having first and second ends. The upper layer is adhered to the hydrophobic layer. The hydrophilic upper layer includes a sample inlet, and the hydrophobic layer contains a first opening directly below the sample inlet. The first opening in the hydrophobic layer directly below the sample inlet is connected to the first end of the microchannel. When plasma reaches the first opening on the hydrophobic layer, the plasma flows from the opening into the first end of the microchannel and continues to flow to a second opening at the second end (e.g., the opposite end), where the plasma can be collected or can flow directly over or into a sample analysis cartridge.

[0243] Figure 9 illustrates another embodiment of an apparatus for plasma separation. In this embodiment, the apparatus includes a hydrophilic upper layer, a hydrophobic layer, and a hydrophilic bottom layer. The hydrophilic upper layer is adhered to the hydrophobic layer, and the bottom layer is adhered to the hydrophobic layer. The hydrophilic upper layer includes a hydrophobic transfer tape and a sample inlet. The hydrophobic layer contains a first opening directly below the sample inlet. The first opening in the hydrophobic layer directly below the sample inlet is connected to the first end of a microchannel. When plasma reaches the first opening on the hydrophobic layer, the plasma flows to the first end of the microchannel and continues to flow to a second opening at the second (e.g., opposite) end, where the plasma can be collected or can flow directly over or into a sample analysis cartridge.

[0244] In yet a further aspect, the sample inlet can further include a separation membrane (such as a plasma separation membrane). In some aspects, the separation membrane is a glass fiber material such as a membrane, film, fabric, fiber, filter, microfilm, screen, mesh, or any combination thereof. Additionally, the separation membrane can be of any shape. For example, the sample inlet can be circular, oval, rectangular, square, triangular, or any combination thereof. In some aspects, the separation membrane can have the same shape as the sample inlet. In other aspects, the separation membrane can have a different shape than the sample inlet.

[0245] The separation membrane can be made from any material known in the art to be useful for separating a blood sample or blood product into its components such as plasma. When a blood sample or blood product is placed on the separation membrane, the blood sample or product flows through the membrane or material. As it flows through the membrane or material, the cellular components of the blood (e.g., red blood cells, white blood cells, platelets, and combinations thereof) are trapped in the pores and / or fibers of the membrane or material, thereby enabling the plasma to continue to flow through the sample inlet to the first opening on the hydrophobic layer. When the plasma reaches the first opening on the hydrophobic layer, the first opening on the hydrophobic layer is connected to the microchannel so that the plasma flows to the first end of the microchannel and continues to flow to the second opening at the second end (e.g., the opposite end) of the microchannel. When the plasma reaches the second opening at the second end of the microchannel, the plasma can be collected using a collection or other device, or if the device is operably coupled, removably coupled, or in fluid communication with another device (such as a sample analysis cartridge (e.g., a microfluidic cartridge)), it can continue to flow directly over or through the device for further processing and analysis.

[0246] In some embodiments, when plasma reaches the first opening, the second opening, or both the first and second openings of the microchannel, a pump, such as an air pump, a bubble ring pump, a foil blister pump, a film blister pump, or any combination thereof, can be used to direct and / or disperse the plasma in the microchannel. In some embodiments, the pump can be used to direct and / or disperse the plasma from the first end of the microchannel towards the second end of the microchannel. In yet other embodiments, the pump can be used to direct and / or disperse the plasma from the second end of the microchannel towards and / or into another device (such as a sample analysis cartridge (e.g., a microfluidic cartridge), etc.). The pump can be connected at any point along the microfluidic channel, such as at the first opening, along the side, near the second end, or any combination thereof.

[0247] In some embodiments, the separation membrane can be located on, under, or within the sample inlet or placed therein. For example, in some embodiments, the separation membrane can be cut to be larger than the size of the sample inlet and simply placed on top of the sample inlet. Alternatively, the separation membrane can be cut to the same size as or slightly smaller than the size of the sample inlet and placed in the inlet. Still further, the separation membrane can be placed below the sample inlet and attached or adhered by any means known in the art, such as an adhesive material, an adhesive, etc.

[0248] When the hydrophobic layer and / or the bottom layer contains one or more openings, the hydrophobic layer and / or the bottom layer can further contain one or more separation membranes located on, under, or within the openings in the layer. The separation membrane can be constructed from the same material as that used for the sample inlet or from a different material. In yet a further aspect, the separation membrane can be located on, under, or within one or more openings. For example, in some aspects, the separation can be cut to be larger than the size of the opening and simply placed on top of the opening. Alternatively, the separation membrane can be cut to the same size as the opening or slightly smaller than the opening and placed at the inlet. Still further, the separation membrane can be placed under the opening and attached or adhered by any means known in the art, such as an adhesive substance, an adhesive, etc.

[0249] In yet a further aspect, one or more hydrophilic meshes or hydrophilic films can be adjacent to the separation membrane at the sample inlet and / or at one or more openings. For example, the hydrophilic mesh or hydrophilic film can be located on or above the separation membrane to facilitate the spreading of a blood sample or a blood product into the device. Alternatively, the hydrophilic mesh or hydrophilic film can be located under or below the separation membrane to help facilitate the continuous movement of the blood sample or blood product when being processed up to the hydrophobic layer and the first opening connected to the first end of the microchannel.

[0250] In some aspects, the device further includes an upper substrate material including the sample inlet. The upper substrate material is adjacent to, on, or above the upper layer. In some aspects, the upper layer is adhered to the upper substrate. The upper substrate material can be a membrane, a film, a fabric, a fiber, a filter, a microfilm, a screen, a mesh, or any combination thereof. In some aspects, the upper substrate material is made of a hydrophilic material. In other aspects, the upper substrate material is made of a hydrophobic material.

[0251] The upper substrate material includes a sample inlet where a blood sample or a blood preparation is placed to initiate sample processing. The sample inlet can have any shape. For example, the sample inlet can be circular, oval, rectangular, square, triangular, or any combination thereof. In some embodiments, a hydrophobic transfer material (such as transfer tape, etc.) can surround the sample inlet. The hydrophobic transfer material helps prevent the blood sample or blood preparation from wicking or moving away from the sample inlet area.

[0252] In yet further embodiments, the sample inlet can include separation. In some embodiments, the separation membrane includes a glass fiber material such as a membrane, film, fabric, fiber, filter, microfilm, screen, mesh, or any combination thereof. The separation membrane can be made from any material known in the art to be useful for separating a blood sample or a blood preparation into its components such as plasma. The separation membrane in the upper substrate functions in the same way as the separation membrane used at the sample inlet of the upper layer.

[0253] In some embodiments, the separation membrane can be located or placed above, below, or within the sample inlet. For example, in some embodiments, the separation membrane can be cut to be larger than the size of the sample inlet and simply placed on top of the sample inlet. Alternatively, the separation membrane can be cut to the same size as the sample inlet or slightly smaller and placed in the inlet. Still further, the separation membrane can be placed below the sample inlet and attached or adhered by any means known in the art, such as an adhesive substance, an adhesive, etc.

[0254] When there is an upper substrate and a sample inlet, one or more openings are made in each of the upper layer and the hydrophobic layer, or in each of the upper layer, the hydrophobic layer, and optionally the bottom layer. The openings can have any shape. For example, the openings can be circular, oval, rectangular, square, triangular, or any combination thereof. In some embodiments, the openings are the same shape as the sample inlet. In other embodiments, the openings are a different shape than the sample inlet. In some embodiments, when the device contains an upper substrate, one or more openings can be made in the upper layer and the hydrophobic layer, but not in the bottom layer. In other embodiments, one or more openings can be made in each of the upper layer, the hydrophobic layer, and the bottom layer. Each opening in one or more layers can be directly below the sample inlet. One or more openings in each of the upper layer, the hydrophobic layer, and / or the bottom layer can also contain one or more separation membranes located above, below, or within the opening in the layer. The separation membrane can be constructed from the same material as that used for the sample inlet, or from a different material. Additionally, one or more hydrophilic meshes or films can be adjacent to the separation membrane at the sample inlet and / or at one or more openings. For example, the hydrophilic mesh or hydrophilic film can be located above or below the separation membrane as previously described herein.

[0255] In yet another embodiment, the upper layer, the hydrophobic layer, the bottom layer, the hydrophilic mesh or hydrophilic film, the separation membrane, or any combination thereof is either ubiquitous for any analyte or specific for an analyte or a class of analytes.

[0256] In yet another aspect, the device further includes at least one aggregating agent for aggregating red blood cells to form red blood cell aggregates to improve separation and produce cleaner plasma. In some aspects, the aggregating agent is coated on or incorporated into one or more of the upper layer, hydrophobic layer, bottom layer, upper substrate, separation membrane, hydrophilic mesh or hydrophilic film, or any combination thereof. Examples of aggregating agents that can be used include lectins (e.g., soybean lectin), Merquat-100, concanavalin A, DEAE-dextran, poly-L-lysine, polyvinylpyrrolidone, poly(2-(dimethylamino)ethyl methacrylate), or any combination thereof.

[0257] In still another aspect, the upper layer, hydrophobic layer, bottom layer, upper substrate, separation membrane, hydrophilic mesh or hydrophilic film, or any combination thereof is coated with a coating such as a surfactant, hydrophilic coating, or any combination thereof to improve or increase the speed or rate of separation of plasma and / or serum from whole blood when passing through any of the layer, substrate, membrane, mesh, file, or any combination thereof.

[0258] The plasma produced using the device disclosed herein need not be pure plasma, but rather may be plasma depleted of one or more components of blood (e.g., red blood cells, white blood cells, platelets, and combinations thereof). In some aspects, the plasma contains about 5% or less by volume of red blood cells, white blood cells, and / or platelets. In other aspects, the plasma contains about 5% or less by volume of red blood cells. In other aspects, the plasma contains about 4% or less by volume of red blood cells. In still other aspects, the plasma contains about 3% or less by volume of red blood cells. In still other aspects, the plasma contains about 2% or less by volume of red blood cells. In yet further aspects, the plasma contains about 1% or less by volume of red blood cells.

[0259] Another aspect relates to a device. In one aspect, the device includes an apparatus previously described herein and at least one sample analysis cartridge (e.g., a microfluidic cartridge) having a sample application area to which a test sample is applied. In one aspect, the device is configured with the sample analysis cartridge such that a microchannel is operably connected to, removably coupled to, or in fluid communication with the sample application area. Specifically, as shown in FIG. 11, the second end of the microchannel may be operably connected to, removably coupled to, or in fluid communication with the sample analysis cartridge at the sample application area. In another aspect, the device is included as part of or incorporated into a clip such as a mobile clip, an extended mobile clip, etc., and configured, coupled, or integrated with the sample analysis cartridge such that a microchannel is operably connected to, removably coupled to, or in fluid communication with the sample application area.

[0260] In another aspect, the plasma separation device includes a pre-vacuumed container or tube, each having a closed inlet and outlet end. A pressure difference exists between the inlet and outlet ends of the container or tube. The inlet end of the container or tube includes a cap or septum that can be penetrated by a needle or blood collection needle assembly used to obtain a whole blood sample from a subject. The outlet end includes a serum holding chamber (e.g., a filtrate container) that receives serum and / or plasma (e.g., filtrate) produced by the device.

[0261] The inlet end of the container or tube containing a cap or septum adapted to be penetrated by a needle or standard blood collection needle assembly defines the first end of a blood holding chamber that can freely receive a whole blood sample from the needle or blood collection needle assembly for filtration.

[0262] In some embodiments, the filter assembly is adjacent to the second end of the blood holding chamber. The filter assembly captures the cellular components of the blood (e.g., red blood cells) and allows the passage of serum and / or plasma components through the assembly to the serum holding chamber at the outlet end. In some embodiments, the filter assembly covers the entire cross-sectional area of the container or tube. In some embodiments, the filter assembly allows the passage of particles or molecules smaller than about 0.7 microns, about 0.6 microns, about 0.5 microns, about 0.4 microns, or about 0.3 microns, and functions similarly to size exclusion chromatography (SEC, also known as gel filtration), where smaller sized particles and molecules (e.g., serum and / or plasma) pass through the filter assembly faster than larger sized molecules (e.g., red blood cells). The filter assembly can be made of any material or combination of materials that can be used to separate the components of the blood based on size and allow the passage of serum and / or plasma components through the assembly towards the serum holding chamber. For example, in some embodiments, the filter assembly includes one or more microfiber membranes and / or glass fiber filter materials (e.g., low density fiber filter materials, etc.). For example, Micro-Strand glass microfibers from Johns Manville (Fruita, CO) may be used. In some embodiments, highly hydrophilic, highly porous materials such as those available from Porex Filtration Group (South Chesterfield, VA), such as POR 410 or POR4711, may be used in the filter assembly. In yet further embodiments, the filter assembly is terminated and held in place near the center of the container or tube by a screen member or other perforated material.

[0263] Optionally, in some embodiments, the flow regulator may be placed adjacent to a cap or septum and can be used to regulate the rate of flow of blood to the filter assembly. In other embodiments, the flow regulator can be included as part of the blood collection needle assembly.

[0264] Since the container or tube has been pre-vacuumed, a pressure difference exists in the area around the filter assembly. The pressure at the top of the container or tube in the blood holding chamber is higher than the pressure at the bottom of the container in the serum holding chamber. Due to this pressure difference, the whole blood sample in the blood holding chamber moves towards and through the filter assembly, where the larger red blood cells are captured and entrapped in the filter assembly, and the serum and / or plasma (e.g., filtrate) move through the filter assembly at a much faster rate and into the serum holding chamber, where they are collected.

[0265] The serum holding chamber has a hollow space sized to hold the serum and / or plasma resulting from the filter assembly. The serum holding chamber is removably attached to the container or tube (e.g., detachable) and can be removed from the container or tube to enable further processing and / or analysis of the serum or plasma. For example, the serum holding chamber can be removed from the container or tube by turning or sliding the serum holding chamber clockwise or counterclockwise to break the vacuum. When the serum holding chamber is removed from the container or tube, the serum or plasma sample can be used for further processing and / or analysis in another device and / or apparatus. For example, the serum or plasma sample in the serum holding chamber can be pipetted into another container or device (e.g., a cartridge) for further processing and / or analysis.

[0266] In other embodiments, plasma separation can occur in a container or tube that has not been pre-vacuumed. In such embodiments, plasma separation is carried out using any suitable means, for example, any means including using size exclusion chromatography, to separate the molecules or components in the blood by size differences as they pass through a material (such as a filter assembly) contained within the container or tube. For example, any material that helps preferentially slow the movement of red blood cells compared to plasma may be used so that plasma can be recovered (e.g., glass or porous beads, membranes, one or more filters, glass or other fibrous materials, or any combination thereof). Such methods involve using means other than a pressure difference. For example, gravity-fed separation may be used without using any pressure difference.

[0267] Examples of pre-vacuumed containers or tubes having the above-described components that can be used as plasma separation devices in the methods described in this disclosure include the pre-vacuumed containers or tubes described in U.S. Patent No. 9,427,707, which is incorporated herein by reference in its entirety.

[0268] In yet other embodiments, the above-described plasma separation device can be used in connection with capillary blood samples collected (1) in a decentralized setting; (2) without using a syringe, standard needle, or combination thereof; (3) by a user not trained in collecting blood samples from a subject; (4) by a robot; (5) by a blood collection device that administers to oneself or another; or (6) by any combination thereof.

[0269] In yet a further aspect, the plasma separation device described above can be used in connection with a blood sample obtained from a subject collected by use of a syringe, a standard needle, or a combination thereof. Such samples can be collected in a decentralized or centralized setting (e.g., in a traditional medical setting (hospital, examination room, independent research site, etc.)) by a user trained in blood collection or a combination thereof. In some aspects, a blood sample obtained from a subject collected by use of a syringe, a standard needle, or a combination thereof is a venous blood sample. In other aspects, a blood sample obtained from a subject collected by use of a syringe, a standard needle, or a combination thereof is a capillary blood sample. For example, in some aspects, a blood sample (e.g., a venous blood sample and / or a capillary blood sample) can be obtained from a subject by use of a syringe, a standard needle, or a combination thereof (and optionally, in a centralized setting and / or by a user trained in blood sample collection from the subject) and processed using the plasma separation device described above prior to and / or in connection with performing an assay for GFAP.

[0270] 4. Micro-sampling system for determining the amount of GFAP using a capillary blood sample Referring to FIG. 2, a micro-sampling system 10 for determining the amount of GFAP is provided. The amount of GFAP can be used to assist in the diagnosis and / or assessment of a subject who has sustained or is at risk of sustaining an injury to the head. In one aspect, the micro-sampling system 10 includes a micro-sampling device 14, a reactor 22, and an instrument, such as instrument 26 (e.g., a point-of-care device). Note that although the reactor 22 shown in FIG. 2 is rectangular in shape, the shape is not of critical significance. For example, in some aspects, the reactor 22 can be in the shape of a tube. In a further aspect, the reactor 22 can be a microfluidic cartridge.

[0271] In some additional embodiments, the system can further include a plasma separation device 18 (FIG. 2). When the plasma separation device 18 is present, the microsampling device 14 collects a capillary blood sample from the subject, and the plasma separation device 18 generates a processed capillary blood sample (e.g., serum or plasma) from the capillary blood sample.

[0272] In yet further additional embodiments, the system can further include a transfer tube 78 (FIG. 7). The transfer tube 78 can include a cap or stopper 79. The transfer tube 78 also has an opening 80. The opening 80 enables the reaction tube to receive the capillary blood sample or the processed capillary blood sample.

[0273] In some embodiments, the reactor 22 or the transfer tube 78 receives the processed capillary blood sample from the plasma separation device 18.

[0274] The instrument 26 analyzes the reactor 22 to provide a determination of the amount of GFAP in the subject. In some embodiments, the determination of the amount is communicated as a result. This result can be communicated for further analysis, interpretation, processing, and / or display. The amount can be communicated by a computer in a document and / or spreadsheet, on a mobile device (e.g., a smartphone), on a website, in an email, or any combination thereof.

[0275] In some embodiments, the communicated amount of GFAP can be displayed on a device, such as on the instrument 26. For example, the amount of GFAP can be displayed as elevated, not elevated, or that the test (e.g., assay) should be repeated. The various operating steps by the system 10 described with respect to FIGS. 3A - 3E and FIGS. 4A - 4C are described in further detail herein.

[0276] Referring to FIG. 3A, the microsampling device 14 is coupled to the subject 30 and a capillary blood sample is collected from the subject 30. In the described embodiment, the microsampling device 14 includes a housing 34 and a receptacle 38 coupled to the housing 34. In the described embodiment, the capillary blood sample is collected in the receptacle 38. In other aspects, the receptacle 38 is removably coupled to the housing 34. For example, once the capillary blood sample has been collected, the receptacle 38 can be separated from the housing 34. Still in other aspects, the receptacle 38 is the reactor 22. In these aspects, when the receptacle is the reactor, the receptacle can be removed from the microsampling device 14 and directly inserted into the instrument 26.

[0277] The microsampling device 14 further includes a microneedle, lancet, microlancet, blade, microblade, microscrew, or any combination thereof coupled to the housing. In some aspects, the microsampling device 14 includes a plurality of microneedles. In some aspects, the microsampling device 14 further includes an actuator movable relative to the housing 34. The actuator can drive the microneedle or similar component into the skin of the subject to initiate collection of a capillary blood sample from the subject 30.

[0278] Referring to FIG. 3B, the system 10 can further include a cap 42 coupled to the receptacle 38. In some embodiments, the cap 42 is attached to the receptacle 38 after the receptacle has been removed from the housing 34. In aspects, the cap 42 seals the capillary blood sample within the container 38. For example, a threaded configuration can couple the cap 42 to the receptacle 38. In some embodiments, the cap 42 is part of the interface between the receptacle 38 and the plasma separation device 18.

[0279] Referring to FIG. 3C, the plasma separation device 18 includes an inlet 46 for receiving a capillary blood sample from the microsampling device 14 and an outlet 50 through which the processed capillary blood sample exits the plasma separation device 18. In the illustrated embodiment, the inlet 46 receives the capillary blood sample from the receptacle 38. In some aspects, the plasma separation device 18 is integrated within the receptacle 38 (e.g., the plasma separation device 18 is not intended to be removed by the user from the receptacle 38).

[0280] In some other aspects, the plasma separation device 18 is formed separately from the reactor 22 or the transfer tube 78 (i.e., the plasma separation device 18 is movable and separable from the reactor 22 or the transfer tube 78). In other aspects, the plasma separation device 18 is integrated with the reactor 22 or the transfer tube 78. For example, in some aspects, the plasma separation device 18 is integrated within the housing 54 of the reactor 22 (i.e., the plasma separation device 18 is not intended to be removed by the user from the reactor 22 or the transfer tube 78).

[0281] The plasma separation device 18 includes a filter, a membrane, synthetic paper, or any combination thereof. In one aspect, the plasma separation device 18 is removably coupled to the reactor 22 or the transfer tube 78 by a removable coupling 58. In some aspects, the removable coupling 58 includes a threaded configuration. In other embodiments, the removable coupling 58 includes a retaining member located on the reactor 22 or the transfer tube 78 for retaining the plasma separation device 18 in position once the plasma separation device 18 is attached onto the reactor 22 or the transfer tube 78. The outlet 50 of the plasma separation device 18 is placed in fluid communication with an opening 62 in the reactor 22 or an opening 80 in the transfer tube 78. In other words, the processed capillary blood sample flows from the outlet 50 of the plasma separation device 18 into the opening 62 of the reactor 22 or the opening 80 of the transfer tube 78.

[0282] In yet a further aspect, the receptacle 38 is removably coupled to the plasma separation device 18 by a removable coupling 66 (FIG. 3C). In some aspects, after the plasma separation device 18 is coupled to the reactor 22 or the transfer tube 78, the receptacle 38 is coupled to the plasma separation device 18. In yet a further aspect, the cap 42 is removed before coupling the receptacle 38 to the plasma separation device 18. In some aspects, the cap 42 remains in place when the receptacle 38 is coupled to the plasma separation device 18. In some aspects, when the receptacle 38 is coupled to the plasma separation device 18, the cap 42 is penetrated to allow capillary blood sample to flow into the plasma separation device 18.

[0283] In yet a further aspect, the plasma separation device 18 is removably coupled to the reactor 22 or the transfer tube 78, and the receptacle 38. Referring to FIG. 3D, the plasma separation device 18 is positioned between the receptacle 38 and the reactor 22. In other aspects, the plasma separation device 18 is positioned between the receptacle 38 and the transfer tube 78. In some aspects, the receptacle 38 is squeezed by a user or an operator to push the capillary blood sample through the plasma separation device 18 into the reactor 22 or the transfer tube 78. In other aspects, the receptacle 38 includes a plunger for pushing the capillary blood sample through the plasma separation device 18 into the reactor 22 or the transfer tube 78. In other embodiments, the capillary blood sample is gravity-fed through the plasma separation device 18 into the reactor 22 or the transfer tube 78.

[0284] Referring to FIG. 3D, the reactor 22 in some aspects is a microfluidic cartridge. The processed capillary blood sample flows from the plasma separation device 18 to the reactor 22 where an assay is performed. In some aspects, the assay is for GFAP.

[0285] In yet a further aspect, referring to FIG. 4, the plasma separation device 18 is placed in fluid communication with the aperture 62 at any point along the reactor 22 (e.g., a microfluidic cartridge). In some aspects, the plasma separation device 18 is placed in fluid communication with the aperture 62 at one end, on the side, or in the center of the reactor 22 (e.g., a microfluidic cartridge). In some aspects, as shown in FIG. 5, the plasma separation device 18 is placed in fluid communication with the aperture 62 (e.g., a microfluidic cartridge) at an angle such as, for example, about 5 degrees, about 10 degrees, about 15 degrees, about 20 degrees, about 25 degrees, about 30 degrees, about 35 degrees, about 40 degrees, about 45 degrees, about 50 degrees, about 55 degrees, about 60 degrees, about 65 degrees, about 70 degrees, about 75 degrees, about 80 degrees, about 85 degrees, or about 90 degrees (e.g., perpendicular to form an L or J shape) at the end or side of the reactor 22. In other aspects, as shown in FIG. 6, the plasma separation device 18 is placed in fluid communication with at least one aperture 62 at one end or side of the reactor 22 (e.g., a microfluidic cartridge).

[0286] In one aspect, referring to FIG. 3E, the reactor 22 is inserted into the instrument 26 along with the processed capillary blood sample loaded into the reactor 22. In the illustrated embodiment, the reactor 22 is inserted into the bottom portion 70 of the handle 74. The instrument 26 includes a display 75 configured to communicate the results of the GFAP determined in the sample. For example, the display 75 on the instrument 26 can display the results indicating that the amount of GFAP in the subject is elevated, not elevated, or that the test should be repeated. In some aspects, the results are provided by visual, auditory, or tactile feedback.

[0287] In some embodiments, the results are communicated in about 4 minutes from the time the sample is collected (e.g., from the time of injury or suspected injury). In some embodiments, the results are communicated in about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes from the time the sample is collected (e.g., from the time of injury or suspected injury). In some embodiments, the results are communicated within a range of about 4 minutes to about 40 minutes from the time the sample is collected (e.g., from the time of injury or suspected injury). In some embodiments, the results are communicated within a range of about 4 minutes to about 30 minutes from the time the sample is collected (e.g., from the time of injury or suspected injury). In some embodiments, the results are communicated within a range of about 4 minutes to about 20 minutes from the time the sample is collected (e.g., from the time of injury or suspected injury).

[0288] Advantageously, portions of system 10 are usable in a decentralized setting. In other words, system 10 is portable. Advantageously, system 10 or portions thereof are reusable for multiple samples and / or subjects. In some embodiments, the microsampling device 14, the plasma separation device 18, and the reactor 22 or transfer tube 78 are disposable components (i.e., usable for a single test in a single subject); however, the instrument 26 is reusable for the analysis of multiple reactors 22.

[0289] 5. Treatment and Monitoring of Subjects Subjects identified in the methods described above can be treated or monitored. In some embodiments, the method further comprises treating a subject, such as a human subject, with an acquired brain injury or traumatic brain injury treatment, such as any treatment known in the art. For example, the treatment of an acquired brain injury or traumatic brain injury can take various forms depending on the severity of the injury to the head. For example, in a subject suffering from mild TBI, the treatment can include rest, refraining from physical activity such as sports, avoiding light or wearing sunglasses when outside in bright places, one or more of medications to reduce headache or migraine, antiemetics, etc. Treatments for patients suffering from moderate, severe, or moderate-severe TBI can include one or more appropriate drug therapies (e.g., diuretics, antispasmodic drug therapies, drug therapies for sedation and putting the individual in a state of drug-induced coma, or other pharmaceuticals or biopharmaceutical drug therapies (known or to be developed in the future for the treatment of TBI), etc.), one or more surgical procedures (e.g., removal of hematoma, repair of skull fracture, decompressive craniectomy, etc.), airway protection, and administration of one or more therapies (e.g., one or more rehabilitations, cognitive behavioral therapy, anger management, counseling psychology, etc.). In some embodiments, the method further comprises monitoring a subject, such as a human subject. In some embodiments, the subject can be monitored by CT scan or MRI procedure.

[0290] 6. Method for measuring the level of GFAP In the methods described above, the GFAP 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, or protein immunostaining, electrophoretic analysis, protein assay, competitive binding assay, functional protein assay, or chromatography or spectroscopic methods, such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). Also, the assay can be used in a clinical chemistry format as known to those skilled in the art.

[0291] 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, the step of measuring the level of GFAP comprises contacting the sample, simultaneously or sequentially and in any order, with (1) a capture antibody (e.g., a GFAP capture antibody) that binds to an epitope on or in 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 comprises a detectable label and that 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), thereby forming a capture antibody-GFAP antigen-detection antibody complex (e.g., a GFAP capture antibody-GFAP antigen-GFAP detection antibody complex), and measuring the amount or concentration of GFAP in the sample based on the signal generated by the detectable label in the capture antibody-GFAP antigen-detection antibody complex.

[0292] 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.

[0293] In some embodiments, the sample is diluted or undiluted. The sample is from about 1 to about 25 microliters, about 1 to about 24 microliters, about 1 to about 23 microliters, about 1 to about 22 microliters, about 1 to about 21 microliters, about 1 to about 20 microliters, about 1 to about 18 microliters, about 1 to about 17 microliters, about 1 to about 16 microliters, about 15 microliters or about 1 microliter, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, about 10 microliters, about 11 microliters, about 12 microliters, about 13 microliters, about 14 microliters, about 15 microliters, about 16 microliters, about 17 microliters, about 18 microliters, about 19 microliters, about 20 microliters, about 21 microliters, about 22 microliters, about 23 microliters, about 24 microliters or about 25 microliters. In some embodiments, the sample is from about 1 to about 150 microliters or less or from about 1 to about 25 microliters or less.

[0294] Some devices other than point-of-care devices (e.g., Abbott Laboratories device ARCHITECT® and other core laboratory devices, etc.) may be capable of measuring the level of GFAP in samples higher or greater than 25,000 pg / mL.

[0295] Other detection methods may include or be adapted for use in the use of nanopore devices or nanowell devices. Examples of nanopore devices are described in International Patent Publication No. WO2016 / 161402, which is hereby incorporated by reference in its entirety. Examples of nanowell devices are described in International Patent Publication No. WO2016 / 161400, which is hereby incorporated by reference in its entirety.

[0296] 7. GFAP Antibody The methods described herein can use an isolated antibody that specifically binds to glial fibrillary acidic protein ("GFAP") (or a fragment thereof), referred to as a "GFAP antibody". The GFAP antibody can be used to assess the GFAP status as a measure of traumatic brain injury, detect the presence of GFAP in a sample, quantify the amount of GFAP present in a sample, or detect the presence of GFAP in a sample and quantify the amount of GFAP in the sample.

[0297] a. Glial fibrillary acidic protein (GFAP) Glial fibrillary acidic protein (GFAP) is a 50 kDa cytoplasmic fibrous protein that constitutes a part of the cytoskeleton within astrocytes and has been demonstrated to be the most specific marker for cells derived from astrocytes. The GFAP protein is encoded by the GFAP gene in humans. GFAP is the major intermediate filament of mature astrocytes. In the rod domain at the molecular center, GFAP shares significant structural homology with other intermediate filaments. GFAP is involved in the motility and shape of astrocytes by providing structural stability to astrocytic processes. Glial fibrillary acidic protein and its degradation product (GFAP-BDP) are brain-specific proteins released into the blood as part of the pathophysiological response after traumatic brain injury (TBI). Following injury to the human CNS caused by trauma, genetic disorders, or chemicals, astrocytes proliferate, show significant hypertrophy of the cell body and cell processes, and GFAP is significantly upregulated. In contrast, as astrocytic malignancies increase, a progressive loss of GFAP production is seen. GFAP can also be detected in Schwann cells, gastrointestinal glial cells, salivary gland neoplasms, metastatic renal carcinomas, laryngeal cartilage, pituitary cells, immature oligodendrocytes, papillary meningiomas, and myoepithelial cells of the breast.

[0298] Human GFAP can have the following amino acid sequence:

[0299]

Chemical formula

[0300] Human GFAP can be a fragment or variant of SEQ ID NO: 2. Fragments of GFAP can be between 5 and 400 amino acids, between 10 and 400 amino acids, between 50 and 400 amino acids, between 60 and 400 amino acids, between 65 and 400 amino acids, between 100 and 400 amino acids, between 150 and 400 amino acids, between 100 and 300 amino acids, or between 200 and 300 amino acids in length. The fragment can contain contiguous amino acids derived from SEQ ID NO: 2. Fragments or variants of human GFAP of SEQ ID NO: 2 can be GFAP degradation products (BDP). GFAP BDP can be 38 kDa, 42 kDa (weak band: 41 kDa), 47 kDa (weak band: 45 kDa); 25 kDa (weak band: 23 kDa); 19 kDa or 20 kDa. In some embodiments, the human GFAP fragment or variant can be a GFAP BDP that includes between 5 and 25 amino acids, between 5 and 50 amino acids, between 5 and 100 amino acids, or between 5 and 200 amino acids.

[0301] b. GFAP recognition antibody The antibody is an antibody that binds to GFAP, this fragment, an epitope of GFAP, or this variant. The antibody can be a fragment or this variant or derivative of an anti-GFAP antibody. The antibody can be a polyclonal or monoclonal antibody. The antibody can be an antibody fragment such as a chimeric antibody, single-chain antibody, affinity matured antibody, human antibody, humanized antibody, fully human antibody, or Fab fragment, or a mixture thereof. Fragments or derivatives of the antibody can include F(ab’)2 fragments, Fv fragments, or scFv fragments. Derivatives of the antibody can be made by peptidomimetics. Further, techniques described for making single-chain antibodies can be adapted to make single-chain antibodies.

[0302] The anti-GFAP antibody can be a chimeric anti-GFAP or 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 include a single Fab region linked to the Fc region.

[0303] Human antibodies can be derived from phage display technology or transgenic mice expressing human immunoglobulin genes. Human antibodies can be produced and isolated as a result of the immune response in humans in vivo. See, for example, Funaro et al., BMC Biotechnology, 2008(8):85. Thus, the antibody can be a human product and may not be an animal repertoire. Since the antibody is of human origin, the risk of reactivity to self-antigens can be minimized. Alternatively, standard yeast display libraries and yeast display technology can be used to select and isolate human anti-GFAP antibodies. For example, a library of naive human single-chain variable fragments (scFv) can be used to select human anti-GFAP antibodies. Transgenic animals can be used to express human antibodies.

[0304] A humanized antibody can be an antibody molecule derived from a non-human species antibody that binds to a desired antigen and has one or more complementarity-determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule.

[0305] The antibody is distinguishable from known antibodies in that it possesses a biological function different from that of antibodies known in the art.

[0306] (1) Epitope The antibody can immunospecifically bind to GFAP (SEQ ID NO: 2), this fragment or this variant. The antibody can immunospecifically recognize and bind to at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids or at least 10 amino acids within the epitope region. The antibody can immunospecifically recognize and bind to an epitope having at least 3 consecutive amino acids, at least 4 consecutive amino acids, at least 5 consecutive amino acids, at least 6 consecutive amino acids, at least 7 consecutive amino acids, at least 8 consecutive amino acids, at least 9 consecutive amino acids or at least 10 consecutive amino acids within the epitope region.

[0307] c. Preparation / Production of Antibodies The antibody can be prepared by any of a variety of techniques including techniques well known to those skilled in the art. Generally, the antibody can be produced by cell culture techniques including the production of monoclonal antibodies by conventional techniques or by transfection of antibody genes, heavy and / or light chains into suitable bacterial or mammalian cell hosts to enable the production of recombinant antibodies. The various forms of the term "transfection" are intended to encompass the many and diverse techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. It is possible to express the antibody in prokaryotic or eukaryotic host cells, but such eukaryotic cells (and especially mammalian cells) are more likely to secrete antibodies that assemble, fold properly and are immunologically active than prokaryotic cells. Therefore, expression of the antibody in eukaryotic cells is preferred, and expression in mammalian host cells is most preferred.

[0308] Exemplary mammalian host cells for expressing recombinant antibodies include, for example, Chinese hamster ovary (CHO cells) used with a DHFR selection marker as described in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982) (including dhfr-CHO cells described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a time sufficient to allow expression of the antibody in the host cell or, more preferably, secretion of the antibody into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0309] Host cells can also be used to generate functional antibody fragments, such as Fab fragments or scFv molecules. It is understood that variations to the above procedure can be implemented. For example, it may be desirable to transfect a host cell with DNA encoding a functional fragment of the light and / or heavy chain of an antibody. Recombinant DNA techniques can also be used to remove some or all of the DNA encoding one or both of the light and heavy chains that are not necessary for binding to the antigen of interest. Also, molecules expressed from such truncated DNA molecules are also included within the antibody. In addition, a bifunctional antibody in which one heavy chain and one light chain are an antibody (i.e., bind to human GFAP) and the other heavy chain and light chain are specific for an antigen other than human GFAP can be made by cross-linking the antibody to a second antibody via standard chemical cross-linking methods.

[0310] In a preferred system for the recombinant expression of an antibody or an antigen-binding portion thereof, recombinant expression vectors encoding both the antibody heavy chain and the antibody light chain are introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy chain and light chain genes are each operably linked to a CMV enhancer / AdMLP promoter regulatory element for driving high-level transcription of the genes. The recombinant expression vector also carries a DHFR gene that enables selection of CHO cells transfected with the vector using methotrexate selection / amplification. The selected transformant host cells are cultured to enable expression of the antibody heavy chain and light chain, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select the transformants, culture the host cells, and recover the antibody from the culture medium. Even further, a method of synthesizing a recombinant antibody can be made by culturing the host cells in a suitable culture medium until the recombinant antibody is synthesized. The method can further comprise isolating the recombinant antibody from the culture medium.

[0311] Methods for preparing monoclonal antibodies include the preparation of immortal cell lines capable of producing antibodies with the desired specificity. Such cell lines can be generated from spleen cells obtained from immunized animals. The animals can be immunized with GFAP or this fragment and / or variants thereof. The peptides used to immunize the animals can include amino acids encoding human Fc, for example, the fragment crystallizable region or the tail region of a human antibody. Subsequently, the spleen cells can be immortalized, for example, by fusion with a myeloma cell fusion partner. Various fusion methods can be utilized. For example, spleen cells and myeloma cells can be combined with a non-ionic surfactant for several minutes and then seeded at low density on a selective medium that supports the growth of hybrid cells but not the growth of myeloma cells. One such technique uses hypoxanthine, aminopterin, thymidine (HAT) selection. Another technique involves electrofusion. After a sufficient period of time, usually about 1-2 weeks later, colonies of hybrids are observed. A single colony is selected and its culture supernatant is tested for binding activity against the polypeptide. Hybridomas with high reactivity and specificity can be used.

[0312] Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. In addition, various techniques such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host such as a mouse can be used to enhance the yield. Next, the monoclonal antibodies can be collected 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 in the process for purifying antibodies.

[0313] Papain, a proteolytic enzyme, preferentially cleaves IgG molecules into several fragments, such that each of two of these (F(ab) fragments) results in a fragment containing a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules to result in several fragments, including an F(ab’)2 fragment that includes both antigen-binding sites.

[0314] Fv fragments can be generated by preferential proteolytic cleavage of IgM, and, less commonly, by preferential proteolytic cleavage of IgG immunoglobulin molecules or IgA immunoglobulin molecules. Fv fragments can be derived using recombinant methods. An Fv fragment contains a non-covalent VH:VL heterodimer that includes an antigen-binding site that retains much of the antigen recognition and binding ability of the native antibody molecule.

[0315] An antibody, antibody fragment or derivative can each contain a set of heavy-chain and light-chain complementarity-determining regions (“CDRs”) inserted between a set of heavy-chain and light-chain frameworks (“FRs”) that provide support for the CDRs and define the spatial relationship of the CDRs to each other. The set of CDRs can contain three hypervariable regions consisting of the heavy-chain V region or the light-chain V region.

[0316] Other suitable methods for making or isolating antibodies having the requisite specificity, including but not limited to methods using libraries of peptides or proteins (e.g., display libraries such as bacteriophage libraries, ribosome libraries, oligonucleotide libraries, RNA libraries, cDNA libraries, yeast libraries, etc., but not limited thereto) commercially available from a variety of sources such as Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid / Planegg, Del.), Biovation (Aberdeen, Scotland, UK), BioInvent (Lund, Sweden), etc., and selecting recombinant antibodies therefrom using methods known in the art. 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 (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) capable of generating a repertoire of human antibodies, which are known in the art and / or 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); antibody production techniques using single cells (e.g., selected lymphocyte antibody method ("SLAM") (U.S. Patent No. 5,627,052, Wen et al. (1987) J. Immunol., 17:887-892; Babcock et al. (1996), Proc. Natl. Acad. Sci. USA, 93:7843-7848); gel microdroplets and flow cytometry (Powell et al. (1990), Biotechnol., 8:333-337; One Cell Systems (Cambridge, Mass); Gray et al. (1995), J. Imm. Meth., 182:155-163; Kenny et al. (1995), Bio / Technol., 13:787-790); selection of B cells (Steenbakkers et al. (1994), Molec. Biol. Reports, 19:125-134 (1994)).

[0317] An affinity matured antibody can be produced by any one of a number of procedures known in the art. For example, see; Marks et al., BioTechnology, 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier 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 selected mutagenesis positions and contact or high frequency mutagenesis positions having activity enhancing amino acid residues is described in U.S. Patent No. 6,914,128B1.

[0318] Antibody variants may be prepared using methods that deliver polynucleotides encoding the antibody to a suitable host to provide, for example, transgenic animals or mammals that produce such antibodies in their milk, such as goats, cows, horses, sheep, and others. Such methods are known in the art and are described, for example, in U.S. Patent Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.

[0319] Variants of antibodies may also be prepared by delivering polynucleotides such that such antibodies, designated portions, or variants are produced in plant parts or cultured cells derived therefrom, resulting in transgenic plants and cultured plant cells (such as, but not limited to, tobacco, corn, and duckweed). For example, Cramer et al. (1999), Curr. Top. Microbiol. Immunol., 240:95-118 and references cited therein describe, for example, the production of transgenic tobacco leaves expressing large amounts of recombinant protein using inducible promoters. Transgenic corn has been used to express mammalian proteins with biological activities equivalent to those produced in other recombinant systems or purified from natural sources at levels suitable for commercial production. See, for example, Hood et al., Adv. Exp. Med. Biol. (1999), 464:127-147 and references cited therein. Antibody variants have also been produced in large quantities from transgenic plant seeds containing antibody fragments such as single-chain antibodies (scFv), including tobacco seeds and potato tubers. See, for example, Conrad et al. (1998), Plant Mol. Biol., 38:101-109 and references cited therein. Thus, antibodies may also be made using transgenic plants according to known methods.

[0320] Antibody derivatives can be produced, for example, by adding foreign sequences to modify immunogenicity or by reducing, enhancing or modifying binding, affinity, on-rate, off-rate, avidity, specificity, half-life or any other suitable characteristic. Generally, 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 with human or other amino acids.

[0321] Small antibody fragments can be diabodies having two antigen-binding sites, where the fragment can be a diabody that includes a heavy chain variable domain (VH) connected to a light chain variable domain (VL) (VH VL) in the same polypeptide chain. See, for example, EP404,097; WO93 / 1161 and Hollinger et al. (1993), Proc. Natl. Acad. Sci. USA, 90:6444-6448. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain to create two antigen-binding sites. Also see U.S. Patent No. 6,632,926 to Chen et al., which is hereby incorporated by reference in its entirety and which discloses antibody variants in which one or more amino acids are inserted into the hypervariable region of the parent antibody and the binding affinity for the target antigen is at least about two-fold stronger than the binding affinity of the parent antibody for this antigen.

[0322] Antibodies can be linear antibodies. Procedures for making linear antibodies are known in the art and are described in Zapata et al. (1995), Protein Eng., 8(10):1057-1062. Briefly, these antibodies include pairs of tandem Fd segments (VH-CH1-VH-CH1) that form pairs of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0323] 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, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") may be used for purification.

[0324] Detectably labeling an antibody can be useful. Methods for conjugating antibodies to these agents are known in the art. By way of example only, an antibody can be labeled with a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, etc. Such labeled antibodies can be used for diagnostic methods in vivo or in an isolated test sample. Such labeled antibodies can be linked to cytokines, ligands, another antibody. Agents suitable for coupling to an antibody to achieve an anti-tumor effect include cytokines such as interleukin 2 (IL-2) and tumor necrosis factor (TNF); photosensitizers for use in photodynamic therapy including aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrin and phthalocyanine; radionuclides such as iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), technetium-99m (99mTc), rhenium-186 (186Re) and rhenium-188 (188Re); antibiotics such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin and carboplatin; bacterial, plant and other toxins such as diphtheria toxin, Pseudomonas exotoxin A, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-alpha toxin, cytotoxins from Chinese cobra (Naja atra) and gelonin (plant toxin); ribosome-inactivating proteins from plants, bacteria and fungi such as restrictocin (a ribosome-inactivating protein produced by Aspergillus restrictus), saporin (a ribosome-inactivating protein from Saponaria officinalis) and RNase; tyrosine kinase inhibitors; ly207702 (purine nucleoside difluoride); liposomes containing anti-cyst agents (e.g., toxins, antisense oligonucleotides encoding methotrexate, plasmids, etc.); and other antibodies or antibody fragments including, for example, F(ab).

[0325] The production of antibodies via hybridoma technology, the selected lymphocyte antibody method (SLAM), transgenic animals, and the use of recombinant antibody libraries is described in more detail below.

[0326] (1) High GFAP monoclonal antibodies using hybridoma technology Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma techniques known in the art, including, for example, those taught in Harlow et al., Antibodies: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988); Hammerling et al., In Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, N.Y., 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, prokaryotic, or phage clone, and does not refer to the method by which it is produced.

[0327] The monoclonal antibodies produced by the method and the method for producing antibodies may include the step of culturing hybridoma cells that secrete the antibodies of the present disclosure. In this case, the hybridoma is obtained by fusing spleen cells isolated from an animal immunized with GFAP, such as a rat or a mouse, with myeloma cells, and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete antibodies capable of binding to the polypeptide of the present disclosure. Briefly, a rat can be immunized with the GFAP antigen. In a preferred embodiment, the GFAP antigen is administered together with an adjuvant so as to stimulate an immune response. Such adjuvants include Freund's complete adjuvant or Freund's incomplete adjuvant, RIBI (muramyl dipeptide), or ISCOM (immunostimulating complex). Such adjuvants can protect the polypeptide from rapid dispersion by encapsulating it within local deposits, or such adjuvants can contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. When the polypeptide is administered, the immunization schedule preferably includes more than one administration of the polypeptide spread over several weeks, although a single administration of the polypeptide can also be used.

[0328] After immunization of the animal with the 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 the animal or by sacrificing the animal. The serum can be used as obtained from the animal, the immunoglobulin fraction can be obtained from the serum, or the anti-GFAP antibodies can be purified from the serum. The serum or immunoglobulin thus obtained is polyclonal and thus has a range of heterogeneous properties.

[0329] Once an immune response is detected, for example, if an antibody specific for the antigen GFAP is detected in rat serum, the rat spleen is removed and spleen cells are isolated. The spleen cells are then fused, by well-known techniques, to any suitable myeloma cells, such as cells derived from SP20, a cell line commercially available from the American Type Culture Collection (ATCC, Manassas, Va., US). Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed, by methods known in the art, for cells that secrete antibodies capable of binding to GFAP. By immunizing rats with positive hybridoma clones, ascites fluid containing generally high levels of antibody can be produced.

[0330] In another embodiment, antibody-producing immortalized hybridomas can be prepared from immunized animals. After immunization, the animals are sacrificed and spleen B cells are fused to 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 selection with antibiotics, the hybridomas are screened using GFAP or a portion thereof or cells expressing GFAP. In a preferred embodiment, the initial screening is performed using an enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), preferably ELISA. An example of ELISA screening is presented in PCT Publication No. 00 / 37504.

[0331] Hybridomas producing anti-GFAP antibodies are selected, cloned, and further screened for desired characteristics, including robust hybridoma growth, high levels of antibody production, and the desired antibody characteristics. Hybridomas can be cultured and expanded in syngeneic animals in vivo, animals lacking an immune system, such as nude mice, or in cell cultures in vitro. Methods for selecting, cloning, and expanding hybridomas are well known to those of skill in the art.

[0332] In a preferred embodiment, the hybridoma is a rat hybridoma. In another embodiment, the hybridoma is produced in a species other than human and other than rat, such as mouse, sheep, pig, goat, cow 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-GFAP antibody.

[0333] Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, the Fab and F(ab’)2 fragments of the present disclosure can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (yielding two identical Fab fragments) or pepsin (yielding an F(ab’)2 fragment). The F(ab’)2 fragment of an IgG molecule is the two antigen-binding sites of the large (“parent”) IgG molecule, and retains the antigen-binding sites that include both light chains (containing the variable and constant regions of the light chain), the CH1 domain of the heavy chain, and the disulfide-forming hinge region of the parent IgG molecule. Thus, the F(ab’)2 fragment, like the parent IgG molecule, is capable of cross-linking antigen molecules.

[0334] (2) Anti-GFAP monoclonal antibody using SLAM In another aspect of the present disclosure, recombinant antibodies are made from a single isolated lymphocyte using a procedure known in the art as the selected lymphocyte antibody method (SLAM), as described in U.S. Patent No. 5,627,052; PCT Publication No. 92 / 02551 and Babcook et al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996). In this method, an antigen-specific hemolytic plaque assay is used to screen for a single cell secreting the antibody of interest, e.g., a lymphocyte derived from any one of immunized animals, in which a linker such as biotin is used to couple the antigen, GFAP, a subunit of GFAP, or a fragment thereof, to sheep red blood cells and is used to identify single cells secreting antibodies specific for GFAP. After identifying the antibody-secreting cell of interest, the cDNAs of the heavy and light chain variable regions are rescued from the cell by reverse transcriptase PCR (RT-PCR), and then these variable regions can be expressed in the context of appropriate immunoglobulin constant regions (e.g., human constant regions) in mammalian host cells such as COS cells or CHO cells. The host cells transfected with the amplified immunoglobulin sequences derived from the in vivo selected lymphocytes can then be further analyzed and selected in vitro, e.g., by panning the transfected cells to isolate cells expressing an antibody against GFAP. The amplified immunoglobulin sequences can be further manipulated in vitro, e.g., by in vitro affinity maturation methods. See, e.g., PCT Publication No. 97 / 29131 and PCT Publication No. 00 / 56772.

[0335] (3) Anti-GFAP monoclonal antibodies using transgenic animals In another embodiment of the present disclosure, the antibody is produced by immunizing a non-human animal comprising part or all of the human immunoglobulin locus with a GFAP antigen. In certain embodiments, the non-human animal is a XENOMOUSE® transgenic mouse, which is an engineered mouse strain that contains a large fragment of the human immunoglobulin locus and is deficient in the production of mouse antibodies. See, for example, Green et al., Nature Genetics, 7:13-21 (1994) and U.S. Patent Nos. 5,916,771; 5,939,598; 5,985,615; 5,998,209; 6,075,181; 6,091,001; 6,114,598 and 6,130,364. See also PCT Publication Nos. 91 / 10741; 94 / 02602; 96 / 34096; 96 / 33735; 98 / 16654; 98 / 24893; 98 / 50433; 99 / 45031; 99 / 53049; 00 / 09560 and 00 / 37504. The XENOMOUSE® transgenic mouse produces an adult-like human repertoire of fully human antibodies and generates antigen-specific human monoclonal antibodies. The XENOMOUSE® transgenic mouse contains approximately 80% of the human antibody repertoire by introduction of megabase-sized, germline-configured YAC fragments of the human heavy chain locus and the λ light chain locus. See Mendez et al., Nature Genetics, 15:146-156 (1997); Green and Jakobovits, J. Exp. Med., 188:483-495 (1998). The disclosures of the foregoing documents are incorporated herein by reference.

[0336] (4) Anti-GFAP monoclonal antibody using a recombinant antibody library In vitro methods can also be used to generate the antibodies of the present disclosure, in which case an antibody library is screened to identify antibodies having the desired GFAP binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include, for example, U.S. Patent No. 5,223,409 (Ladner et al.); PCT Publication No. 92 / 18619 (Kang et al.); PCT Publication No. 91 / 17271 (Dower et al.); PCT Publication No. 92 / 20791 (Winter et al.); PCT Publication No. 92 / 15679 (Markland et al.); PCT Publication No. 93 / 01288 (Breitling et al.); PCT Publication No. 92 / 01047 (McCafferty et al.); PCT Publication No. 92 / 09690 (Garrard et al.); Fuchs et al., Bio / Technology, 9:1369-1372 (1991); Hay et al., Hum. Antibod. Hybrodomas, 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); the methods described in U.S. Patent Application Publication No. 2003 / 0186374 and PCT Publication No. 97 / 29131. The content of each of the foregoing references is incorporated herein by reference.

[0337] The recombinant antibody library can be derived from a subject immunized with GFAP or a portion of GFAP. Alternatively, the recombinant antibody library can be derived from a naive subject, i.e., a subject not immunized with GFAP, such as a human antibody library derived from a human subject not immunized with human GFAP. The antibodies of the present disclosure are selected by screening a recombinant antibody library having a peptide containing GFAP, thereby selecting an antibody that recognizes GFAP. Methods for performing such screening and selection are well known in the art, such as the methods described in the references in the preceding paragraph. For selecting an antibody of the present disclosure having a specific binding affinity for GFAP and dissociating with a specific K off dissociation rate constant, such as an antibody that dissociates with a specific K off dissociation rate constant, the surface plasmon resonance method known in the art can be used to select an antibody having the desired K 50 dissociation rate constant. For selecting an antibody of the present disclosure having a specific neutralizing activity against GFAP, such as an antibody having a specific IC

[0338] standard methods known in the art for evaluating the inhibition of GFAP activity can be used.

[0339] For example, antibodies may be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequence encoding them. Such phages can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or mouse). Phages expressing an antigen-binding domain that binds to the antigen of interest can be selected or identified by the antigen, for example, using a labeled antigen or an antigen bound or captured on a solid surface or bead. The phages used in these methods are typically filamentous phages that contain fd and M13 binding domains expressed from the phage, and Fab, Fv, or disulfide-stabilized Fv antibody domains are recombinantly fused to either phage gene III or gene VIII protein. Examples of phage display methods that can be used to generate antibodies are described in Brinkmann et al., J. Immunol. Methods, 182:41-50 (1995); Ames et al., J. Immunol. Methods, 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic et al., Gene, 187:9-18 (1997); Burton et al., Advances in Immunology, 57:191-280 (1994); PCT Publication No. WO92 / 01047; PCT Publication No. WO90 / 02809; WO91 / 10737; WO92 / 01047; WO92 / 18619; WO93 / 11236; WO95 / 15982; WO95 / 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.

[0340] As described in the above references, after phage selection, the antibody-coding region derived from the phage is isolated and used for the production of the whole antibody, including human antibodies or any other desired antigen-binding fragment, and can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described in detail later. For example, techniques for recombinantly producing Fab, Fab’ and F(ab’)2 fragments are known in the art and may be used, such as the methods disclosed in PCT Publication No. WO92 / 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 for the production of single-chain Fv and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88 (1991); Shu et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999 (1993); and Skerra et al., Science, 240:1038-1041 (1988).

[0341] Other methods known in the art for screening large combinatorial libraries, which are alternative to screening recombinant antibody libraries by phage display, can also be applied to the identification of the antibodies of the present disclosure. One type of alternative expression system is an expression system in which a recombinant antibody library is expressed as an RNA-protein fusion, as described in PCT Publication No. 98 / 31700 (Szostak and Roberts) and Roberts and Szostak, Proc. Natl. Acad. Sci. USA, 94: 12297-12302 (1997). In this system, by in vitro translation of synthetic mRNA carrying puromycin, a peptidyl acceptor antibiotic, at the 3' end, a covalent fusion is created between the mRNA and the peptide or protein encoded by the mRNA. Thus, specific mRNA can be enriched from a complex mixture of mRNAs (e.g., a combinatorial library) based on the properties of the encoded peptide or protein, such as the binding of the antibody or portion thereof to a bispecific antigen. The nucleic acid sequence encoding the antibody or portion thereof recovered from screening such a library can be expressed by recombinant means as described above (e.g., in a mammalian host cell), and further subjected to further affinity maturation by additional rounds of screening of mRNA-peptide fusions in which mutations have been introduced into the originally selected sequence, or by other methods for affinity maturation of recombinant antibodies in vitro as described above. A preferred example of this methodology is PROfusion display technology.

[0342] In another approach, antibodies may be generated using yeast display methods known in the art. In yeast display methods, genetic methods are used to tether antibody domains to the yeast cell wall and display them on the surface of the yeast. Such yeast can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or mouse). Examples of yeast display methods that can be used to generate antibodies include the methods disclosed in U.S. Patent No. 6,699,658 (Wittrup et al.), which is incorporated herein by reference.

[0343] d. Production of Recombinant GFAP Antibodies Antibodies can be made by any of a number of techniques known in the art. For example, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques for expression from the host cells. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although the antibodies of the present disclosure can be expressed in prokaryotic or eukaryotic host cells, such eukaryotic cells (and in particular, mammalian cells) are more likely to secrete antibodies that assemble, fold properly, and are immunologically active than prokaryotic cells. Thus, expression of the antibodies in eukaryotic cells is preferred, and expression in mammalian host cells is most preferred.

[0344] Exemplary mammalian host cells for expressing the recombinant antibodies of the present disclosure include, for example, Chinese hamster ovary (CHO cells) (including dhfr-CHO cells described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)) used with the DHFR selectable marker 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 sufficient to allow expression of the antibody in the host cell or, more preferably, secretion of the antibody into the culture medium in which the host cell was grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0345] Host cells can also be used to generate functional antibody fragments, such as Fab fragments or scFv molecules. It is understood that variations to the above procedures can be implemented. For example, it may be desirable to transfect a host cell with DNA encoding a functional fragment of the light and / or heavy chain of an antibody of the present disclosure. Recombinant DNA techniques can also be used to remove some or all of the DNA encoding one or both of the light and heavy chains that are not necessary for binding to the antigen of interest. Also, molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the present disclosure. In addition, a bifunctional antibody in which one heavy chain and one light chain are an antibody of the present disclosure (i.e., bind to human GFAP) and the other heavy chain and light chain are specific for an antigen other than human GFAP can be made by cross-linking an antibody of the present disclosure to a second antibody via standard chemical cross-linking methods.

[0346] In a preferred system for recombinantly expressing the antibody or antigen-binding portion thereof of the present disclosure, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, each of the antibody heavy chain gene and the antibody light chain gene is operably linked to a CMV enhancer / AdMLP promoter regulatory element so as to drive high-level gene transcription. The recombinant expression vector also carries a DHFR gene that enables the selection of CHO cells transfected with a vector using selection / amplification with methotrexate. The host cells of the selected transformants are cultured such that expression of the heavy and light chains of the antibody is enabled and intact antibody is recovered from the culture medium. Standard molecular biology methods are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. Even further, the present invention provides a method for synthesizing a recombinant antibody of the present disclosure by culturing the host cells of the present disclosure in an appropriate culture medium until the recombinant antibody of the present disclosure is synthesized. The method may further include the step of isolating the recombinant antibody from the culture medium.

[0347] (1) Humanized antibody A humanized antibody can be an antibody or a variant, derivative, analogue or portion thereof that immunospecifically binds to an antigen of interest and comprises a framework (FR) region substantially having the amino acid sequence of a human antibody and a complementarity determining region (CDR) substantially having the amino acid sequence of a non-human antibody. A humanized antibody can be a non-human species antibody that binds to a desired antigen and is derived from a non-human species antibody having one or more complementarity determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule.

[0348] As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises at least one, but typically two, variable domains (Fab, Fab’, F(ab’)2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin (i.e., the donor antibody), and all or substantially all of the framework regions are the framework regions of a human immunoglobulin consensus sequence, and comprises substantially all of the variable domains. According to one aspect, a humanized antibody also comprises at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains both a light chain and at least the variable domain of a heavy chain. The antibody can also include the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, a humanized antibody contains only a humanized light chain. In some embodiments, a humanized antibody contains only a humanized heavy chain. In certain embodiments, a humanized antibody contains only the humanized variable domains of the light chain and / or heavy chain.

[0349] A humanized antibody can be selected from any class of immunoglobulins including IgM, IgG, IgD, IgA and IgE, and any isotype including, without limitation, IgG1, IgG2, IgG3 and IgG4. A humanized antibody can include sequences from more than one class or isotype, and specific constant domains can be selected to optimize desired effector functions using techniques well known in the art.

[0350] The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences. For example, the donor antibody CDR or consensus framework can be mutagenized by substitution, insertion, and / or deletion of at least one amino acid residue such that the CDR residues or framework residues at this site do not correspond to the donor antibody or consensus framework. However, in one embodiment, such mutations are not extensive mutations. Typically, at least 90%, at least 95%, at least 98%, or at least 99% of the residues of the humanized antibody correspond to the residues of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region within a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related immunoglobulin sequences (see, e.g., Winnaker, "From Genes to Clones" (Verlagsgesellschaft, Weinheim, Germany, 1987)). In a family of immunoglobulins, each position within the consensus sequence is occupied by the amino acid that occurs most frequently at this position within the family. If two amino acids occur with equal frequency, both can be included within the consensus sequence.

[0351] Humanized antibodies can be designed to minimize unwanted immunological responses against anti-human rodent antibodies that limit the duration and effectiveness of the therapeutic application of these portions in human recipients. A humanized antibody can have one or more amino acid residues introduced from a non-human source into the humanized antibody. These non-human residues are typically often referred to as "import" residues, which are taken from the variable domains. Humanization can be carried out by substituting the hypervariable region sequences with the corresponding sequences of human antibodies. Thus, such a "humanized" antibody is a chimeric antibody in which the human variable domains, which are substantially intact, are replaced by the corresponding sequences derived from non-human species. See, for example, U.S. Patent No. 4,816,567. The content of the said patent document is incorporated herein by reference. A humanized antibody can be a human antibody in which some of the hypervariable region residues and perhaps some of the FR residues are replaced by residues derived from homologous sites within the rodent antibody. The humanization or engineering of the antibodies of the present disclosure can be carried out using any known method, such as those described in U.S. Patent Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539 and 4,816,567, but not limited thereto.

[0352] Humanized antibodies can retain high affinity for GFAP and other suitable biological properties. Humanized antibodies can be prepared by analyzing the parental and humanized sequences using three-dimensional models for the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available. Computer programs are available that illustrate and present the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays enables analysis of the likely role of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from recipient and import sequences such that desired antibody characteristics, such as an increase in affinity for GFAP, are achieved. Generally, hypervariable region residues can be directly and most substantially involved in affecting antigen binding.

[0353] As an alternative to humanization, human antibodies (also referred to herein as "fully human antibodies") can be made. For example, human antibodies can be isolated from libraries via PROfusion and / or yeast-related technologies. It is also possible to generate transgenic animals (e.g., mice that, when immunized, can generate a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production). For example, in chimeric mice and germline mutant mice, the antibody heavy chain joining region (J H)Deletion of gene homozygosity results in complete inhibition of endogenous antibody production. Transfer of such germline mutant mice of the human germline immunoglobulin gene array results in the production of human antibodies upon antigen administration. Humanized antibodies 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 content of each of the above patent documents is incorporated herein by reference.

[0354] e. Anti-GFAP antibody Anti-GFAP antibodies can be made using the techniques described above as well as routine techniques known in the art. In some embodiments, the anti-GFAP antibody is a non-conjugated GFAP antibody such as a GFAP antibody commercially available from Dako (Catalog No.: M0761), ThermoFisher Scientific (Catalog No.: MA5-12023, A-21282, 13-0300, MA1-19170, MA1-19395, MA5-15086, MA5-16367, MA1-35377, MA1-06701 or MA1-20035), AbCam (Catalog No.: ab10062, ab4648, ab68428, ab33922, ab207165, ab190288, ab115898 or ab21837), EMD Millipore (Catalog No.: FCMAB257P, MAB360, MAB3402, 04-1031, 04-1062, MAB5628), Santa Cruz (Catalog No.: sc-166481, sc-166458, sc-58766, sc-56395, sc-51908, sc-135921, sc-71143, sc-65343 or sc-33673), Sigma-Aldrich (Catalog No.: G3893 or G6171) or Sino Biological Inc. (Catalog No.: 100140-R012-50). The anti-GFAP antibody can be conjugated to a fluorophore, such as a conjugated GFAP antibody commercially available from ThermoFisher Scientific (Catalog No.: A-21295 or A-21294), EMD Millipore (Catalog No.: MAB3402X, MAB3402B, MAB3402B or MAB3402C3) or AbCam (Catalog No.: ab49874 or ab194325).

[0355] Alternatively, antibodies described in WO2018 / 067474, WO2018 / 081649, U.S. Patent No. 11,078,298, U.S. Patent Application Publication No. 2019 / 0502127, and / or Bazarian et al., "Accuracy of a rapid GFAP / UCH-L1 test for the prediction of intracranial injuries on head CT after mild traumatic brain injury", Acad. Emerg. Med. (August 6, 2021), the contents of which are incorporated herein by reference, may be used.

[0356] 8. Methods for measuring levels of analytes other than GFAP (e.g., UCH-L1) In the methods described above, the magnetic point-of-care assay for GFAP may be combined with other (i.e., non-magnetic point-of-care assays) for other analytes, such as UCH-L1. UCH-L1 levels may 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 assays, competitive binding assays, functional protein assays, or chromatographic or spectrometric methods, such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). Also, the assay may be used in a clinical chemistry format as known to those of skill in the art. For example, such methods for measuring UCH-L1 are described in, for example, U.S. Patent Nos. 10,877,038, 10,877,048, 10,849,548, 11,016,105, and 11,022,617, the contents of which are incorporated herein by reference.

[0357] In some embodiments, measuring the level of UCH-L1 involves 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, the step of measuring the level of UCH-L1 involves contacting the sample, either simultaneously or sequentially and in any order, with (1) a capture antibody (e.g., a UCH-L1 capture antibody) that binds to an epitope on or on a UCH-L1 fragment to form a capture antibody-UCH-L1 antigen complex (e.g., a UCH-L1 capture antibody-UCH-L1 antigen complex), and (2) a detection antibody (e.g., a UCH-L1 detection antibody) that includes a detectable label and that binds to an epitope on UCH-L1 that is not bound by the capture antibody to form a UCH-L1 antigen-detection antibody complex (e.g., a UCH-L1 antigen-UCH-L1 detection antibody complex), thereby forming a capture antibody-UCH-L1 antigen-detection antibody complex (e.g., a UCH-L1 capture antibody-UCH-L1 antigen-UCH-L1 detection antibody complex), and measuring the amount or concentration of UCH-L1 in the sample based on the signal generated by the detectable label in the capture antibody-UCH-L1 antigen-detection antibody complex.

[0358] 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.

[0359] In some embodiments, the sample is diluted or undiluted. The sample can be from about 1 to about 25 microliters, about 1 to about 24 microliters, about 1 to about 23 microliters, about 1 to about 22 microliters, about 1 to about 21 microliters, about 1 to about 20 microliters, about 1 to about 18 microliters, about 1 to about 17 microliters, about 1 to about 16 microliters, about 15 microliters or about 1 microliter, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, about 10 microliters, about 11 microliters, about 12 microliters, about 13 microliters, about 14 microliters, about 15 microliters, about 16 microliters, about 17 microliters, about 18 microliters, about 19 microliters, about 20 microliters, about 21 microliters, about 22 microliters, about 23 microliters, about 24 microliters or about 25 microliters. In some embodiments, the sample is from about 1 to about 150 microliters or less or from about 1 to about 25 microliters or less.

[0360] 9. UCH-L1 antibody The methods described herein can use an isolated antibody that specifically binds to ubiquitin carboxyl-terminal hydrolase L1 (“UCH-L1”) (or a fragment thereof), referred to as a “UCH-L1 antibody”. The UCH-L1 antibody can be used to assess the UCH-L1 status as a measure of traumatic brain injury, or 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 to quantify the amount of UCH-L1 in the sample. a. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) 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 for neurons and cells of the diffuse neuroendocrine system and their tumors. UCH-L1 is abundantly present in all neurons (accounting for 1-2% of total brain proteins), and is expressed particularly in neurons and the testis / ovary. The catalytic triad of UCH-L1 contains cysteine at position 90, aspartic acid at position 176, and histidine at position 161, which contribute to its hydrolase activity.

[0361] Human UCH-L1 can have the following amino acid sequence:

[0362]

Chemical formula

[0363] Human UCH-L1 can be a fragment or variant of SEQ ID NO: 1. Fragments of UCH-L1 can be between 5 and 225 amino acids, between 10 and 225 amino acids, between 50 and 225 amino acids, between 60 and 225 amino acids, between 65 and 225 amino acids, between 100 and 225 amino acids, between 150 and 225 amino acids, between 100 and 225 amino acids, or between 175 and 225 amino acids in length. The fragment can contain consecutive numbered amino acids derived from SEQ ID NO: 1.

[0364] b. UCH-L1 recognition antibody The antibody is an antibody that binds to UCH-L1, a fragment thereof, an epitope of UCH-L1, or variants thereof. The antibody can be a fragment of an anti-UCH-L1 antibody or a variant or derivative thereof. The antibody can be a polyclonal or monoclonal antibody. The antibody can 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 can include an F(ab’)2, Fv or scFv fragment. The antibody derivative can be produced by a peptidomimetic. Further, the techniques described for the production of single-chain antibodies can be adapted for the production of single-chain antibodies.

[0365] The anti-UCH-L1 antibody can be a chimeric anti-UCH-L1 or 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 include a single Fab region linked to the Fc region.

[0366] Human antibodies can be derived by phage display technology or transgenic mice that express human immunoglobulin genes. Human antibodies can be made and isolated as a result of an immune response in humans in vivo. See, for example, Funaro et al., BMC Biotechnology, 2008(8):85. Thus, the antibody can be a human product and may not be an animal repertoire. Since the antibody is of human origin, the risk of reactivity against self-antigens can be minimized. Alternatively, standard yeast display libraries and yeast display technology can be used to select and isolate human anti-UCH-L1 antibodies. For example, a library of naive human single-chain variable fragments (scFv) can be used to select human anti-UCH-L1 antibodies. Transgenic animals can be used to express human antibodies.

[0367] A humanized antibody can be an antibody molecule derived from a non-human species antibody that binds to a desired antigen and has one or more complementarity-determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule.

[0368] An antibody is distinguishable from known antibodies in that it possesses a biological function different from that of antibodies known in the art.

[0369] (1) Epitope The antibody can immunospecifically bind to UCH-L1 (SEQ ID NO: 1), this fragment or this variant. The antibody can immunospecifically recognize and bind to at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids or at least 10 amino acids within the epitope region. The antibody can immunospecifically recognize and bind to an epitope having at least 3 consecutive amino acids, at least 4 consecutive amino acids, at least 5 consecutive amino acids, at least 6 consecutive amino acids, at least 7 consecutive amino acids, at least 8 consecutive amino acids, at least 9 consecutive amino acids or at least 10 consecutive amino acids within the epitope region.

[0370] c. Antibody preparation and production and anti-UCH-L1 antibody Anti-UCH-L1 antibodies can be generated or prepared using routine techniques and methods known in the art, such as those described in U.S. Patent Nos. 10,877,038, 10,877,048, 10,849,548, 11,016,105, and 11,022,617, the contents of each of which are incorporated herein by reference. In some embodiments, the anti-UCH-L1 antibody is a non-conjugated UCH-L1 antibody such as an anti-UCH-L1 antibody commercially available from United State 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 No.: ADI-905-520-1), Bio-Rad (Catalog No.: VMA00004), BioVision (Catalog No.: 6130-50), Abcam (Catalog No.: ab75275 or ab104938), Invitrogen Antibodies (Catalog No.: 480012), ThermoFisher Scientific (Catalog No.: MA1-46079, MA5-17235, MA1-90008, or MA1-83428), EMD Millipore (Catalog No.: MABN48), or Sino Biological Inc. (Catalog No.: 50690-R011). The anti-UCH-L1 antibody can be conjugated to a fluorophore, such as a conjugated UCH-L1 antibody commercially available from BioVision (Catalog No.: 6960-25) or Aviva Systems Biology (Catalog No.: OAAF01904-FITC).

[0371] 10. Other factors As described above, the methods of diagnosing, prognosing and / or evaluating can further include using other factors for diagnosis, prognosing and evaluation. In some embodiments, traumatic brain injury can be diagnosed using the Glasgow Coma Scale. Other tests, scales or indices can also be used alone or in combination with the Glasgow Coma Scale (GCS). An example is the Ranchos Los Amigos Scale (RLAS). The RLAS measures levels of consciousness, cognition, behavior and interaction with the environment. The RLAS includes Level I: No Response; Level II: Generalized Response; Level III: Localized Response; Level IV: Confused (Agitated); Level V: Confused (Inappropriate); Level VI: Confused (Appropriate); Level VII: Automatic (Appropriate) and Level VIII: Purposeful (Appropriate). Another example is the Rivermead Post-Concussion Symptoms Questionnaire, a self-report scale for measuring the severity of post-concussion symptoms following TBI. Patients are asked to rate how severe each of 16 symptoms (e.g., headache, dizziness, nausea, vomiting) has been in the past 24 hours. In each case, the symptoms are compared to how severe they were before the injury occurred (pre-onset). These symptoms are reported on a scale of 0-4 severity: No Experience, No Problem at All, Mild Problem, Moderate Problem, and Severe Problem.

[0372] 11. Sample In some embodiments, a sample is obtained after a subject, such as a human subject, has suffered head injury caused by physical oscillation, blunt impact by external mechanical or other forces resulting in closed or open head trauma, one or more falls, explosions or blasts, or other types of blunt force trauma. In some embodiments, the sample is obtained after a subject, such as a human subject, has ingested or been exposed to fire, chemicals, toxins, or a combination of fire, chemicals and toxins. Examples of such chemicals and / or toxins include mold, asbestos, pesticides and insecticides, organic solvents, paints, adhesives, gases (such as carbon monoxide, hydrogen sulfide and cyanide), organometals (such as methyl mercury, tetraethyl lead and organotin) and / or one or more addictive drugs. In some embodiments, the sample is obtained from a subject, such as a human 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.

[0373] In yet another embodiment, the methods described herein may be used to determine whether a subject has or is at risk of developing a TBI (such as a mild TBI, moderate TBI, severe TBI, or moderate-to-severe TBI) by determining the levels of UCH-L1 and / or GFAP in a subject using an anti-UCH-L1 and / or anti-GFAP antibody or antibody fragment described hereinafter, using a sample. Thus, in certain embodiments, the present disclosure also provides a method for determining whether a subject having or at risk of having a traumatic brain injury described herein and known in the art is a candidate for a treatment or therapy. Generally, the subject has at least: (i) experienced an injury to the head; (ii) ingested and / or 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 has been actually diagnosed as having or being at risk of having a TBI (e.g., 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 demonstrates an adverse (i.e., clinically undesirable) concentration or amount of UCH-L1 and / or GFAP or UCH-L1 and / or GFAP fragments as described herein.

[0374] a. Control It may be desirable to include a control sample. The control sample can be analyzed simultaneously with the sample from the subject described above. The results obtained from the subject sample can be compared with the results obtained from the control sample. A standard curve may be provided and used to compare assay results for the samples. Such a standard curve presents the level of the marker as a function of assay units, i.e., fluorescence signal intensity if a fluorescent label is used. Using samples collected from multiple donors, the standard curve can be provided for the "at-risk" level of GFAP in tissues collected from donors that may have one or more of the characteristics shown above, along with the reference level of GFAP in normal healthy tissue.

[0375] Accordingly, in view of the above, a method is provided for determining the presence, amount or concentration of GFAP in a test sample. The method includes, for example, using at least one capture antibody that binds to an epitope in GFAP and at least one detection antibody that binds to an epitope in GFAP that is different from the epitope for the capture antibody and optionally includes a detectable label, and comparing the signal generated by the detectable label as a direct or indirect indicator of the presence, amount or concentration of GFAP in the test sample with the signal generated as a direct or indirect indicator of the presence, amount or concentration of GFAP in a calibrator, by an immunoassay that includes assaying the test sample for GFAP. The calibrator is optionally, and preferably, part of a series of calibrators, each of which has a different concentration of GFAP from the other calibrators in the series.

[0376] The present disclosure has a plurality of aspects illustrated by the following non-limiting examples.

Example

[0377] [Example 1] Figure 1 shows a GFAP assay using paramagnetic microparticles (e.g., magnetic beads) that are magnetically captured and retained on a magnet (e.g., a magnetic immunosensor). A monoclonal antibody pair was used, for example, antibody A as the capture monoclonal antibody and antibody B as the detection monoclonal antibody. Antibody A and antibody B are exemplary anti-GFAP antibodies developed within Abbott Laboratories (Abbott Park, IL). The biotin linker is conjugated to antibody A using routine techniques known in the art. Antibody A containing the biotin linker is likewise coated (e.g., immobilized) onto the paramagnetic particles using routine techniques known in the art. The paramagnetic particles used were Invitrogen Dynabeads™ MyOne™ Streptavidin T1 available from ThermoFisher (Waltham, MA), which are uniform superparamagnetic beads containing a monolayer of recombinant streptavidin covalently coupled to the surface of the beads. The paramagnetic particles coated with antibody A were printed and dried onto the sensor chip in the cartridge for use in a point-of-care device. Antibody B was labeled with a detectable label (e.g., alkaline phosphatase) using routine techniques known in the art. At least one detectably labeled antibody B was printed and dried onto the same sensor chip containing the printed and dried paramagnetic particles coated with antibody A.

[0378] Fingerstick blood samples (whole blood) were obtained from normal subjects. The samples were inserted into cartridges for use with a point-of-care device. Printed paramagnetic particles coated with antibody A and a biotin linker, and detectably labeled antibody B were reconstituted and mixed with the whole blood samples. A complex containing paramagnetic particles coated with antibody A - GFAP - detectably labeled antibody B was formed. A magnet contained in the sensor chip (i.e., under the chip that collectively includes the magnetic immunosensor) captured and retained the complex, and the amount of GFAP was determined using an i-STAT Alinity instrument (or an i-STAT1 instrument). Techniques described in U.S. Patent Nos. 9,233,370, 9,958,440, 10,145,843, and International Patent Publication Nos.: WO18107016, WO18107015, WO18107007, WO18107009, WO18107012, WO18107013, WO21211331, and WO21211332, the contents of which are incorporated herein by reference, were also used. The total assay time was about 10 minutes. The quantification limit of the magnetic GFAP assay was ≤10 pg / mL. In contrast, the quantification limit of a commercially available i-STAT® TBI plasma assay (Abbott Laboratories) for use on an i-STAT® Alinity instrument is about 23 pg / mL.

[0379] [Example 2] Figure 10 shows an apparatus that can be used as a plasma separation device in the methods and systems described herein. The apparatus includes a hydrophilic top layer, a hydrophobic layer having a microchannel with first and second ends, a hydrophilic bottom layer, and a protective film. The hydrophilic top layer is adhered to the hydrophobic layer, the protective film is adhered to the bottom hydrophilic layer, and the bottom hydrophilic layer is adhered to the hydrophobic layer. The top layer includes a sample inlet. The sample inlet is surrounded by an annular hydrophobic transfer tape. On top of the hydrophobic transfer tape is a plasma separation membrane (PSM) sandwiched between hydrophilic meshes on both its top and bottom surfaces. The hydrophilic mesh located on or above the PSM aids in spreading the sample. The hydrophilic mesh located below or beneath the PSM aids in facilitating the processing of the blood sample or blood product onto the hydrophobic layer. A containment ring for aiding in containing the sample encompasses the PSM, the hydrophilic meshes, and the transfer tape. The hydrophobic layer contains an opening (e.g., a first opening) directly below the sample inlet. The opening in the hydrophobic layer is connected to the first end of the microchannel. Plasma flows from the opening (e.g., the first opening) at the first end towards a second opening at the second (e.g., opposite) end. The apparatus also contains an air pump for circulating air within the device.

[0380] [Example 3] Blood samples were obtained from 6 donors over 4 different test days. Each blood sample was manipulated to obtain several different samples. Specifically, the samples were 1. Tested without any manipulation. 2. Enhanced with native GFAP antigen. 3. Spiked with recombinant GFAP antigen.

[0381] Samples were tested at nominal hematocrit (37 - 45% packed cell volume (PCV)) and modified hematocrit (low: 30, 33% PCV; high: 44, 50, 52, 54, and 55% PCV).

[0382] Samples were collected in 6 mL K2-EDTA tubes, 1. Centrifugation; or 2. Plasma was separated using a pre-vacuumed tube having an inlet and an outlet end. The tube had a blood holding chamber at the inlet end and a serum holding chamber at the outlet end. The tube also had a filter assembly between the blood holding chamber at the inlet end and the serum holding chamber at the outlet end. An example of such a device that can be used is described in U.S. Patent No. 9,427,707.

[0383] Once the plasma was obtained, it was placed in the sample well of a cartridge and tested in a point-of-care device such as the i-STAT® device of Abbott Laboratories (Abbott Park, IL).

[0384] The results confirmed that both native and recombinant GFAP can be detected by a point-of-care device using plasma produced using a pre-vacuumed tube.

[0385] [Example 4] This study was designed to compare the levels of GFAP in capillary and ven...

Claims

1. An assay for measuring the amount of glial fibrillary acidic protein (GFAP) in a biological sample obtained from a subject, comprising: performing an assay for GFAP, said assay comprising: (a) contacting the sample with a cartridge comprising at least one magnetic immunosensor and (i) at least one first specific binding partner immobilized on at least one magnetosensitive bead, said at least one first specific binding partner being printed on the cartridge and specifically binding to GFAP in the sample; and (ii) at least one second specific binding partner comprising a detectable label printed on the cartridge, thereby producing one or more complexes comprising said first specific binding partner-GFAP-second specific binding partner; (b) using said at least one magnetic immunosensor to magnetically capture and hold said beads containing said complex; and (c) evaluating a signal from said complex, wherein the amount of detectable signal from said detectable label indicates the amount of GFAP in said sample. The assay, wherein the cartridge is used in a point-of-care device, and further, the assay shows at least a 5-fold increase in sensitivity compared to an assay that immobilizes the first specific binding partner on magnetosensitive beads and magnetically captures and holds the beads containing the complex on at least one magnetic immunosensor in a cartridge used in a point-of-care device. An assay.

2. The assay according to claim 1, further comprising the step of washing an unbound sample that is not magnetically captured and held on said at least one magnetic immunosensor.

3. The assay according to claim 1 or claim 2, wherein said magnetic immunosensor comprises a sensing electrode on a substantially planar chip and a magnetic layer on said chip.

4. The assay according to claim 3, wherein said magnetic layer comprises high magnetic field magnetic particles.

5. The assay according to any one of claims 1 to 4, further comprising measuring the amount of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in said sample using a non-magnetic assay.

6. The assay according to any one of claims 1 to 5, wherein the first specific binding partner is immobilized on a magnetic bead, and the beads containing the complex are magnetically captured and retained on at least one magnetic immunosensor in a cartridge contained in a point-of-care device, showing an increase of at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times or at least 15 times in sensitivity as compared to an assay that does not do so.

7. The assay according to any one of claims 1 to 6, which is used to assist in the diagnosis and assessment of a subject who has suffered or is likely to have suffered an injury to the head.

8. The assay according to claim 7, wherein the subject is diagnosed with having a traumatic brain injury.

9. The assay according to claim 8, wherein the subject is treated for the traumatic brain injury.

10. The assay according to any one of claims 1 to 9, wherein the sample is collected using a microsampling device or a fingerstick device.

11. The assay according to any one of claims 1 to 10, wherein the sample is venous blood, capillary blood, fingerstick blood, or a combination thereof.

12. The assay according to any one of claims 1 to 11, wherein the sample is processed before the assay is performed.

13. The assay according to claim 12, wherein the sample is processed by plasma separation.

14. The assay according to claim 13, wherein the sample is processed using a plasma separation device.

15. The assay according to claim 14, wherein the plasma separation device is (a) incorporated into or operably connected to the point-of-care device; or (b) separated from the point-of-care device.

16. The assay according to any one of claims 5 to 15, wherein the amount of GFAP is communicated by being displayed on the device.

17. A system, wherein An assay for glial fibrillary acidic protein (GFAP), comprising contacting a biological sample from a subject with a cartridge comprising at least one magnetic immunosensor and (i) at least one first specific binding partner printed on the cartridge and specifically binding to GFAP in the sample, the at least one first specific binding partner being immobilized on at least one magnetic bead; and (ii) at least one second specific binding partner printed on the cartridge and comprising a detectable label, thereby producing one or more complexes comprising the first specific binding partner - GFAP - the second specific binding partner. A point-of-care device comprising the cartridge, wherein the device determines the amount of GFAP in a sample obtained from the subject by magnetically capturing and holding the beads containing the complex on the at least one magnetic immunosensor; and (b) evaluating a signal from the complex, wherein the amount of the detectable signal from the detectable label indicates the amount of GFAP in the sample. Including, the assay immobilizes the first specific binding partner on magnetic beads, and compared with an assay that does not magnetically capture and hold the beads containing the complex on at least one magnetic immunosensor in a cartridge contained in the point-of-care device, shows at least a five-fold increase in sensitivity. System.

18. The system according to claim 17, wherein the magnetic immunosensor comprises a sensing electrode on a substantially planar chip and a magnetic layer on the chip.

19. The system according to claim 18, wherein the magnetic layer comprises high magnetic field magnetic particles.

20. The system according to any one of claims 17 to 19, further comprising an assay for measuring the amount of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the sample using a non-magnetic assay.

21. The system according to any one of claims 17 to 20, wherein the assay shows an increase of at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold or at least 15-fold in sensitivity as compared to an assay that immobilizes the first specific binding partner on magnetic beads and magnetically captures and holds the beads containing the complex on at least one magnetic immunosensor in a cartridge contained in a point-of-care device.

22. The system according to any one of claims 17 to 21, wherein the assay is used to assist in the diagnosis and assessment of a subject who has suffered or is likely to have suffered an injury to the head.

23. The system according to claim 22, wherein the subject is diagnosed with having a traumatic brain injury.

24. The system according to claim 23, wherein the subject is treated for the traumatic brain injury.

25. The system according to any one of claims 17 to 24, wherein the sample is collected using a microsampling device or a fingerstick device.

26. The system according to any one of claims 17 to 25, wherein the sample is venous blood, capillary blood, fingerstick blood, or a combination thereof.

27. The system according to any one of claims 17 to 26, wherein the sample is processed prior to the performance of the assay.

28. The assay according to claim 27, wherein the sample is processed by plasma separation.

29. The system according to claim 28, wherein the sample is processed using a plasma separation device.

30. The system according to claim 29, wherein the plasma separation device is (a) incorporated within or operatively coupled to the point-of-care device; or (b) separated from the point-of-care device.

31. The system according to any one of claims 20 to 30, wherein the amount of GFAP is communicated by displaying it on the device.

32. a. A magnet; b. An area containing printed paramagnetic particles coated with an anti-GFAP antibody; and c. An area containing a plurality of printed detectably labeled anti-GFAP antibodies A cartridge for use in a point-of-care device, the cartridge comprising: **Claim 33** The cartridge according to claim 32, wherein the region containing the plurality of detectably labeled anti-GFAP antibodies is in the same region containing the printed paramagnetic particles coated with the anti-GFAP antibody. **Claim 34** The cartridge according to claim 32, wherein the region containing the plurality of detectably labeled anti-GFAP antibodies is adjacent to the region containing the printed paramagnetic particles coated with the anti-GFAP antibody. **Claim 35** Before displaying the amount of the GFAP on the device, a. determining the amount of the GFAP in the capillary blood sample; b. selecting a conversion factor for comparing the amount of the GFAP in the sample with the amount of the GFAP in venous blood, the conversion factor being a static correlation ratio, a dynamic ratio, or a combination thereof; and c. normalizing the amount of the GFAP in the sample by the amount of the GFAP from venous blood by applying the conversion factor selected in step b) to the amount of the GFAP in the sample The assay according to claim 16, further comprising. **Claim 36** The assay according to claim 35, wherein the normalized amount of the GFAP is displayed by the device. **Claim 37** The assay according to any one of claims 35 or 36, wherein the conversion factor is from about 1.2:1.0 to about 1.0:0.

5. **Claim 38** The assay according to claim 35 or 36, wherein the conversion factor is about 1.0:0.85.

Images