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

Abstract

Disclosed herein are systems and assays for determining the amount of glial fibrillary acidic protein (GFAP) in a biological sample obtained from a subject using magneto-sensitive beads and a point-of-care device including a magnetic immunosensor.

Description

[0001] Related Application Information

[0002] This application claims priority to U.S. Application No. 63 / 356,843, filed on June 29, 2022, U.S. Application No. 63 / 402,122, filed on August 30, 2022, U.S. Application No. 63 / 433,134, filed on December 16, 2022, and U.S. Application No. 63 / 522,334, filed on June 21, 2023, the respective contents of which are incorporated herein by reference.

[0003] Sequence Listing Submission

[0004] The content of the electronic sequence listing entitled 40972_601_ST26.xml (size: 7,839 bytes; and creation date: June 28, 2023) is incorporated herein by reference in its entirety. Technical Field

[0005] 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 system and assay utilize a point-of-care device containing at least one cartridge that includes at least one magnetic immunosensor to magnetically capture, retain, and determine the amount of GFAP in the sample. The amount of GFAP in the sample can assist in diagnosing and evaluating whether a subject has suffered, may have suffered, or is suspected of having suffered a head injury, such as an acquired brain injury, such as a traumatic brain injury (TBI). Background Art

[0006] In the United States alone, more than 5 million mild traumatic brain injuries (TBIs) occur each year. Most of the evaluations and diagnoses of TBI are based on subjective data. Unfortunately, objective measurements such as head CT and Glasgow Coma Scale (GCS) are not very comprehensive or sensitive in evaluating mild TBI. In addition, head CT is negative for the vast majority of mild TBIs, is expensive, and exposes the patient to unnecessary radiation. Additionally, a negative head CT does not mean that the patient is free from concussion; rather, it only means that certain interventions (e.g., surgery) are not necessary. Clinicians and patients need objective, reliable information to accurately assess the situation to facilitate appropriate triage and rehabilitation. To date, limited data are available for using GFAP to assist in patient evaluation and management in the acute care setting.

[0007] Mild TBI or concussion is more difficult to objectively detect and is a daily challenge for emergency centers worldwide. Concussions generally do not result in gross pathology, such as bleeding, and do not appear abnormal on routine computed tomography scans of the brain, but rather result in a rapid-onset neuronal dysfunction that resolves spontaneously within days to weeks. Approximately 15% of mild TBI patients develop persistent cognitive impairment. There is an unmet need for mild TBI victims at the scene, in emergency rooms and clinics, in sports settings, and in military activities (e.g., combat).

[0008] Current algorithms for assessing the severity of brain injury include the Glasgow Coma Scale score and other measures. These measures may sometimes be sufficient to correlate acute severity, but are not sensitive enough to detect subtle pathologies that may lead to permanent deficits. The GCS and other measures also do not distinguish between injury types and may be inadequate. Thus, patients entering clinical trials grouped into a single GCS level may have very different severities and types of injury. Since outcomes vary accordingly, inappropriate classification undermines the integrity of clinical trials. Improved injury classification will enable more accurate characterization of disease severity and type in clinical trials for TBI patients.

[0009] In addition, current brain injury trials rely on outcome measures such as the Extended Glasgow Outcome Scale, which captures global phenomena but fails to assess subtle differences in outcomes. As a result, 30 consecutive trials of brain injury therapeutics have failed. Sensitive outcome measures are needed to determine how well patients are recovering from brain injury in order to test treatment methods and preventive measures. SUMMARY OF THE INVENTION

[0010] 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 comprises:

[0011] (a) contacting the sample with a cartridge that includes at least one magnetic immunosensor and: (i) at least one first specific binding partner printed on the cartridge, the at least one first specific binding partner including at least one anti-GFAP antibody that specifically binds to GFAP in the sample, wherein the at least one first specific binding partner is immobilized on at least one magnetically sensitive bead; and (ii) at least one second specific binding partner printed on the cartridge, the at least one second specific binding partner including a detectable label, thereby producing one or more complexes that include the first specific binding partner-GFAP-second specific binding partner;

[0012] (b) magnetically capturing and retaining the beads containing the complex on at least one magnetic immunosensor in the cartridge of a point-of-care device; and

[0013] (c) Use the at least one magnetic immunosensor to evaluate a signal from the complex, wherein the amount of the detectable signal from the detectable label indicates the amount of GFAP in the sample,

[0014] wherein the assay exhibits an increase in sensitivity of at least 5-fold compared to an assay that does not immobilize the first specific binding partner on the magnetosensitive beads and does not magnetically capture and retain the beads containing the complex on at least one magnetic immunosensor in a cartridge contained in a point-of-care device.

[0015] In a further aspect, the above assay further comprises the step of washing unbound sample that is not magnetically captured and retained on the at least one magnetic immunosensor.

[0016] In a further aspect of the above assay, the magnetic immunosensor comprises a sensing electrode on a substantially planar chip and a magnetic layer on the chip. More specifically, in a further aspect, the magnetic layer comprises high-field magnetic particles.

[0017] In a further aspect of the above assay, the assay further comprises measuring the amount of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) in the sample in a non-magnetic assay.

[0018] In a further aspect of the above assay, the assay exhibits an increase in sensitivity 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 compared to an assay that does not immobilize the first specific binding partner on the magnetosensitive beads and does not magnetically capture and retain the beads containing the complex on at least one magnetic immunosensor in a cartridge contained in a point-of-care device.

[0019] In a further aspect, the above assay is used to assist in diagnosing and evaluating a subject who has suffered or may have suffered a head injury. In some aspects, the subject is diagnosed with an acquired brain injury. In a further aspect, the subject is diagnosed with a traumatic brain injury. In a further aspect, treat the subject's traumatic brain injury.

[0020] In a further aspect of the above assay, the sample is collected using a microsampling device or a finger stick device. In some aspects, the sample is a venous blood sample, a capillary blood sample, a finger stick blood sample, or a combination thereof.

[0021] In another aspect, the sample is processed prior to the determination. In some aspects, the sample is processed by plasmapheresis. In some aspects, the sample is processed using a plasmapheresis device. In another aspect, the plasmapheresis device: (a) is incorporated into or operably connected to a point-of-care device; or (b) is separate from the point-of-care device.

[0022] In another aspect, the amount of GFAP is communicated by display on the device.

[0023] In another aspect, prior to displaying the amount of GFAP on the device, the determination further comprises:

[0024] a. determining the amount of GFAP in the capillary blood sample;

[0025] b. selecting a conversion factor to compare the amount of GFAP in the sample to the amount of GFAP in venous blood, wherein the conversion factor is a static correlation ratio, a dynamic ratio, or a combination thereof; and

[0026] c. normalizing the amount of GFAP in the sample using the amount of GFAP from venous blood by applying the conversion factor selected in step b) to the amount of GFAP in the sample.

[0027] In another 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.

[0028] In another aspect, if a conversion factor is employed, the amount of normalized GFAP is displayed by the device.

[0029] In another embodiment, the present disclosure relates to a system. In some aspects, the system comprises:

[0030] a determination for glial fibrillary acidic protein (GFAP), wherein the determination comprises contacting a biological sample from a subject with a cartridge, the cartridge comprising at least one magnetic immunosensor and: (i) at least one first specific binding partner printed on the cartridge, the at least one first specific binding partner comprising at least one anti-GFAP antibody that specifically binds GFAP in the sample, wherein the at least one first specific binding partner is immobilized on at least one magnetizable bead; and (ii) at least one second specific binding partner printed on the cartridge, the at least one second specific binding partner comprising a detectable label, thereby generating one or more complexes comprising the first specific binding partner-GFAP-second specific binding partner;

[0031] A point-of-care device including a cartridge, wherein the cartridge includes at least one magnetic immunosensor, wherein the device (a) determines the amount of GFAP in a sample obtained from the subject by magnetically capturing and retaining the beads containing the complex on the at least one magnetic immunosensor; and (b) evaluates a signal from the complex, wherein the amount of the detectable signal from the detectable label indicates the amount of GFAP in the sample,

[0032] wherein the assay exhibits at least a 5-fold increase in sensitivity compared to an assay in which the first specific binding partner is not immobilized on the magnetosensitive beads and the beads containing the complex are not magnetically captured and retained on at least one magnetic immunosensor in a cartridge contained in the point-of-care device.

[0033] In a further aspect, the above system further includes a step of washing unbound sample that is not magnetically captured and retained on the at least one magnetic immunosensor.

[0034] In a further aspect of the above system, the magnetic immunosensor includes a sensing electrode on a substantially planar chip and a magnetic layer on the chip. More specifically, in a further aspect, the magnetic layer comprises high-field magnetic particles.

[0035] In a further aspect of the above system, the assay further includes measuring the amount of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) in the sample in a non-magnetic assay.

[0036] In a further aspect of the above system, the assay exhibits 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 in which the first specific binding partner is not immobilized on the magnetosensitive beads and the beads containing the complex are not magnetically captured and retained on at least one magnetic immunosensor in a cartridge contained in the point-of-care device.

[0037] In a further aspect, the above system is used to assist in diagnosing and evaluating a subject who has suffered or may have suffered a head injury. In some aspects, the subject is diagnosed with an acquired brain injury. In a further aspect, the subject is diagnosed with a traumatic brain injury. In a further aspect, treating the subject's acquired brain injury. In a further aspect, treating the subject's traumatic brain injury.

[0038] In a further aspect of the above system, the sample is collected using a microsampling device or a fingerstick device. In some aspects, the sample is a venous blood sample, a capillary blood sample, a fingerstick blood sample, or a combination thereof.

[0039] In another aspect, the sample is processed prior to the determination. In some aspects, the sample is processed by plasmapheresis. In some aspects, the sample is processed using a plasmapheresis device. In another aspect, the plasmapheresis device: (a) is incorporated into or operably connected to a point-of-care device; or (b) is separate from the point-of-care device.

[0040] In another aspect, the amount of GFAP is conveyed by display on the device.

[0041] In another embodiment, the present disclosure relates to a cartridge comprising:

[0042] a magnet;

[0043] a region containing printed paramagnetic particles coated with an anti-GFAP antibody; and

[0044] a region containing a plurality of printed, detectably labeled anti-GFAP antibodies, wherein the cartridge is used in a point-of-care device.

[0045] In some aspects of the cartridge, the region containing the plurality of detectably labeled anti-GFAP antibodies is the same as the region containing the printed paramagnetic particles coated with an anti-GFAP antibody.

[0046] In other aspects of the cartridge, the region containing the plurality of detectably labeled anti-GFAP antibodies is adjacent to the region containing the printed paramagnetic particles coated with an anti-GFAP antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Showing a GFAP assay using magnetic beads as described in Example 1 and magnetically captured and retained on a magnet.

[0048] Figure 2 Showing a system for diagnosing and evaluating a subject who has suffered or may have suffered a head injury according to one aspect of the present disclosure.

[0049] Figures 3A - 3E Showing Figure 2 the operating steps of the system.

[0050] Figure 4 Showing Figure 2 alternative and substitute aspects of the system.

[0051] Figure 5 Showing Figure 2 alternative and substitute aspects of the system.

[0052] Figure 6 Showing Figure 2 alternative and substitute aspects of the system.

[0053] Figure 7 Shown in Figure 2 the transfer tube used in the system.

[0054] Figure 8 Shown is a device that can be used as a plasma separation device.

[0055] Figure 9 Shown is an alternative device that can be used as a plasma separation device.

[0056] Figure 10 Shown is a device that can be used as the plasma separation device described in Example 2.

[0057] Figure 11 Shown is a device that includes a device that is operably connected, removably coupled, or in fluid communication with a sample analysis cartridge. The second end of the microchannel of the device is in fluid communication with the sample application area on the sample analysis cartridge.

[0058] Figure 12 Shown are the mean GFAP (pg / mL) concentration readings of donors and sources as described in Example 4 with a 95% confidence interval. Detailed Description

[0059] 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 obtained from a subject (such as a human subject), such as a blood sample (e.g., a venous blood sample, a capillary blood sample, a finger stick blood sample, or any combination thereof). The systems and methods described herein employ at least one point-of-care device that contains at least one cartridge that includes at least one magnetic immunosensor or immunosensing device.

[0060] In some aspects, the systems and methods involve performing an assay for GFAP that includes contacting a biological sample such as blood (e.g., a venous blood sample, a capillary blood sample, a finger stick blood sample, or any combination thereof) with a cartridge that includes at least one magnetic immunosensor and (i) at least one first specific binding partner printed on the cartridge that includes at least one anti-GFAP antibody that specifically binds GFAP in the sample, wherein the at least one first specific binding partner is immobilized on at least one magnetizable bead; and (ii) at least one second specific binding partner printed on the cartridge that includes a detectable label to produce one or more complexes that include the first specific binding partner-GFAP-second specific binding partner. A point-of-care device contains at least one cartridge that is configured to magnetically capture and retain the beads containing the complex using at least one magnetic immunosensor and to evaluate a signal from the complex, wherein the amount of the detectable signal from the detectable label indicates the amount of GFAP in the sample. The GFAP assay performed and used in the systems described herein exhibits at least a 5-fold increase in sensitivity compared to a point-of-care device that does not immobilize the first specific binding partner on magnetizable beads and uses a cartridge that includes at least one magnetic immunosensor.

[0061] The section headings used in this part and throughout this disclosure are for organizational purposes only and are not intended to be limiting.

[0062] 1. Definitions

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. 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.

[0064] As used herein, the terms "comprising," "including," "having," "has," "can," "containing," and variations thereof are open-ended conjunctive terms, terms, or words that are intended not to exclude the possibility of additional acts or structures. Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include plural referents. The present disclosure also encompasses other embodiments that "include the embodiments or elements presented herein," "consist of the embodiments or elements presented herein," and "consist essentially of the embodiments or elements presented herein," whether or not explicitly stated.

[0065] For the purposes of the description of numerical ranges herein, each intermediate number therebetween having the same degree of precision is clearly contemplated. For example, for the range 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are clearly contemplated.

[0066] "Acquired brain injury" or (ABI) as used herein refers to brain injury caused by events occurring after birth. In other words, acquired brain injury is not genetic or congenital, but rather the result of neurological conditions and injuries. Acquired brain injury is typically divided into two categories. The first category is acquired traumatic brain injury (TBI), which is caused by external forces such as sports injuries, falls, physical shaking, blunt force trauma, explosions, shock waves, or exposure to fire. The second category is non-traumatic acquired brain injury, which is caused by internal factors in some cases and includes stroke, tumors, hypoxia, infection, metabolic disorders, etc. As used herein, acquired brain injury does not include or cover brain injury caused by stroke (including ischemic stroke, hemorrhagic stroke, or transient ischemic attack, etc.).

[0067] "Affinity matured antibody" is used herein to refer to an antibody having one or more changes in one or more CDRs, said changes resulting in an increase in the affinity of the antibody for a target antigen (i.e., K D 、k d or k a)Improved. Exemplary affinity matured antibodies will have nanomolar or even picomolar affinity for the target antigen. A variety of procedures for generating affinity matured antibodies are known in the art, including screening of combinatorial antibody libraries prepared using biological display. For example, Marks et al., BioTechnology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described in Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-3319 (1995); and Hawkins et al., J. Mol. Biol., 226:889-896 (1992). Selective mutagenesis at selected mutagenic positions and by active enhancing amino acid residues at contacting or hypermutagenic positions is described in U.S. Patent No. 6,914,128 B1.

[0068] As used herein, "amount" refers to a specified amount (e.g., high or low) or number, e.g., the number is a level (such as a position on a real or imaginary scale of amount or quantity) or concentration (such as the relative amount of a given substance contained within a solution or within a particular volume of space, e.g., the amount of solute per unit volume of solution).

[0069] As used herein, "an antibody" and "antibodies" refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as but not limited to avians (e.g., duck or goose), sharks, whales, and mammals (including non - primates (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, etc.) or non - human primates (e.g., monkeys, chimpanzees, etc.)), recombinant antibodies, chimeric antibodies, single - chain Fv ("scFv"), single - chain antibodies, single - domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide - linked Fv ("sdFv"), and anti - idiotypic ("anti - Id") antibodies, dual - domain antibodies, dual - variable - domain (DVD) or triple - variable - domain (TVD) antibodies (dual - variable - domain immunoglobulins and methods for their preparation are described in Wu, C et al., Nature Biotechnology, 25(11):1290 - 1297 (2007) and PCT international application WO 2001 / 058956, the contents of each of which are incorporated herein by reference), and epitope - binding fragments of the functional activity of any of the above antibodies. Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an analyte - binding site. The immunoglobulin molecule can be of any type (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass. For simplicity, an antibody against an analyte is often referred to herein as an "anti - analyte antibody" or simply an "analyte antibody" (e.g., anti - UCH - L1 antibody or UCH - L1 antibody).

[0070] As used herein, "antibody fragment" refers to a portion of a whole antibody that contains an antigen - binding site or variable region. The portion does not include the constant heavy - chain domains of the Fc region of the whole 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 the three CDRs of the light - chain variable domain, single - chain polypeptides containing only one heavy - chain variable region, and single - chain polypeptides containing the three CDRs of the heavy - chain variable region.

[0071] As used herein, "aperture" refers to an opening, hole, or gap.

[0072] "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 meaningless test. Preferred AUCs can be at least about 0.700, at least about 0.750, at least about 0.800, at least about 0.850, at least about 0.900, at least about 0.910, at least about 0.920, at least about 0.930, at least about 0.940, at least about 0.950, at least about 0.960, at least about 0.970, at least about 0.980, at least about 0.990 or at least about 0.995.

[0073] "Bead" and "particle" are used interchangeably herein and refer to a substantially spherical solid support. 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 bead or particle can be a magnetosensitive (or responsive) bead or particle (see, e.g., U.S. Patent Nos. 4,230,685, 4,554,088, and 4,628,037, all of which are incorporated herein by reference) or a magnetic particle, as used interchangeably herein. Another example of a bead or particle is a magnetic or magnetosensitive bead or particle.

[0074] "Binding protein" is used herein to refer to a monomeric or polymeric protein that binds to a binding partner and forms a complex therewith, such as a polypeptide, an antigen, a chemical compound or other molecule, or any type of substrate. The binding protein specifically binds to the binding partner. Binding proteins include antibodies, as well as antigen-binding fragments thereof and various other forms and derivatives known in the art and described hereinafter, and other molecules that contain an antigen-binding domain that binds to one or more antigen molecules or specific sites (epitopes) on an antigen molecule. Thus, binding proteins include, but are not limited to, antibodies, tetrameric immunoglobulins, IgG molecules, IgG1 molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, affinity-matured antibodies, and fragments of any such antibodies that retain the ability to bind antigen.

[0075] "Bispecific antibody" is used herein to refer to full-length antibodies generated by the following techniques: the tetrahybridoma 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 diabody or similar methods by introducing mutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14):6444-6448 (1993)), which generate a variety of different immunoglobulin species, only one of which is a functional bispecific antibody. A bispecific antibody binds one antigen (or epitope) on one of its two binding arms (a pair of HC / LC), and a different antigen (or epitope) on its second arm (the other pair of HC / LC). By this definition, a bispecific antibody has two different antigen-binding arms (both in terms of specificity and CDR sequence) and is monovalent for each antigen it binds.

[0076] "Capillary blood sample" as used herein refers to a capillary blood sample obtained (e.g., extracted) through the skin (but 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 locations on the fingers and / or toes, hands, feet (including heels), earlobes, arms and / or legs, chest, back, head, or any combination thereof. In other aspects, a whole capillary blood sample is extracted from the arm or leg. In other aspects, a capillary blood sample is obtained from the hand or foot. In other aspects, a capillary blood sample is obtained from the chest or back. In other aspects, a capillary blood sample is obtained from the earlobe. In other aspects, a capillary blood sample is obtained from the head.

[0077] In additional aspects, the capillary blood sample is whole blood, serum, or plasma. In other embodiments, the capillary blood sample predominantly contains capillary blood but may also contain or include a small amount or a small fraction of interstitial fluid.

[0078] In another aspect, capillary blood samples obtained from a subject are obtained without using a syringe, needle (e.g., 21-gauge needle, butterfly needle, etc.), or any other suitable device or any combination thereof that is typically used to draw blood (e.g., venous blood). Instead, capillary blood samples are obtained using a self-administered or other-administered blood collection device. Examples of self-administered or other-administered blood collection devices include micro-sampling devices. Exemplary micro-sampling devices that may be used herein include the TAP device available from YourBio Health, Inc. (Cambridge, MA); and the devices described in U.S. Patent No. 9,113,836, the contents of which are incorporated herein by reference; the Tasso+, Tasso-M20, and Tasso-ST devices available from Tasso, Inc. (Seattle, WA); the Tasso-ST device available from Draw Bridge Health (San Diego, CA); the PBS-1000 from PreciHealth (Neuchatel, Switzerland); or the Loop blood collection device available from LoopMedical (Lausanne, Switzerland).

[0079] In another aspect, capillary blood samples are obtained or collected from a subject in a decentralized environment. For example, capillary blood samples may be obtained or collected from an urgent care medical clinic, pharmacy, grocery store, or other convenience store, residence, workplace, and / or government office.

[0080] In addition or alternatively, in another aspect, capillary blood samples are obtained from a subject by a user who is not trained to collect blood (e.g., by someone other than a trained phlebotomist, nurse, medical assistant, and / or physician). For example, capillary blood samples may be obtained by the subject himself / herself, a relative, a friend, a colleague, a coach, a pharmacist, and / or any other individual. In another aspect, capillary blood samples are obtained from a subject by a robot.

[0081] In another aspect, the amount of capillary blood sample obtained from a subject is less than about 4 mL. In some aspects, the capillary blood sample obtained from a subject is less than about 3 mL. In some aspects, the capillary blood sample obtained from a subject is less than about 2 mL. In some aspects, the amount of capillary blood sample obtained from a 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 aspects, when the sample collected is whole blood, a larger amount of capillary blood can be obtained.

[0082] As used herein, "cartridge" refers to a hollow container and / or chip that contains one or more substances and / or components (e.g., liquids, reagents (cyclopentenyl, cyclohexenyl, cyclohept, antibodies and / or antigens) and / or particles (e.g., beads or microparticles)) and is used for insertion into a device (e.g., a point-of-care device). In some aspects, the cartridge includes at least one chip. In other aspects, the cartridge is a chip. In another aspect, the cartridge has one or more holes. In some aspects, the cartridge is a microfluidic cartridge. In other aspects, the cartridge contains a magnetic immunosensor or a magnetic immunosensing device.

[0083] As used herein, "couple" or "connect" means to secure two or more components together by any suitable means. Thus, in some embodiments, the statement 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., through one or more intermediate parts or components).

[0084] "CDR" is used herein to refer to "complementary determining regions" within the variable structural sequences of antibodies. There are three CDRs present in each variable region of the heavy and light chains. For each variable region, starting from the N-terminus of the heavy or light chain, these regions are designated as "CDR1", "CDR2", and "CDR3". The term "CDR set" as used herein refers to a set of three antigen-binding CDRs present in a single variable region. Thus, an antigen-binding site can include six CDRs, which comprise a CDR set from each of the heavy and light chain variable regions. A polypeptide containing a single CDR (e.g., CDR1, CDR2, or CDR3) may be referred to as a "molecular recognition unit". Crystallographic analysis of antigen-antibody complexes has demonstrated extensive contacts between the amino acid residues of the CDRs and the bound antigen, with the most extensive antigen contacts being with the heavy chain CDR3. Thus, the molecular recognition unit may be primarily responsible for the specificity of the antigen-binding site. In general, CDR residues are directly and most substantially involved in influencing antigen binding.

[0085] The precise boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991))) not only provides a clear residue numbering system applicable to any variable region of an antibody, but also provides the precise residue boundaries that define the three CDRs. These CDRs can 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 although there is great diversity at the level of amino acid sequences, certain subportions within the Kabat CDRs adopt nearly identical peptide backbone conformations. These subportions are designated as "L1", "L2", and "L3" or "H1", "H2", and "H3", where "L" and "H" represent the light chain region and heavy chain region, respectively. These regions can be called "Chothia CDRs", which have boundaries that overlap with the Kabat CDRs. Other boundaries that define CDRs overlapping with the Kabat CDRs have been described by Padlan, FASEB J., 9:133-139 (1995) and MacCallum, J. Mol. Biol, 262(5):732-745 (1996). Other CDR boundary definitions may not strictly follow one of the systems in this article, but will still overlap with the Kabat CDRs, and they can be shortened or lengthened given that a particular residue or group of residues or even an entire CDR does not significantly affect the prediction or experimental findings of antigen binding. The methods used in this article can utilize CDRs defined according to any of these systems, but certain embodiments use CDRs defined by Kabat or Chothia.

[0086] "Clinically relevant time frame" means a time frame (e.g., seconds, minutes, or hours) during which a careful and prudent practicing physician (e.g., doctor, nurse, paramedic, or other person) would reasonably consider the results of one or more biomarker tests relevant to an imaging procedure (such as a head CT scan) or in accordance with guidelines established by a supervisory entity (e.g., a standards-setting body such as the World Health Organization (WHO), a physician review board, a regulatory approval agency such as the FDA, the EMEA, or other agencies, etc.).

[0087] "Component", "Constituent", or "At least one component" generally refers to a capture antibody, analyte, or conjugate, calibrator, control, sensitivity set, container, buffer, diluent, salt, enzyme, cofactor of an enzyme, detection reagent, pretreatment reagent / solution, substrate (e.g., as a solution), stop solution, etc. that can be included in a kit for assaying a test sample (such as a patient whole blood, serum, or plasma sample) according to the methods described herein and other methods known in the art. Some components may be in solution or lyophilized for reconstitution for use in the assay.

[0088] "Associated with" as used herein means compared to.

[0089] "CT scan" as used herein refers to computed tomography (CT) scan. A CT scan combines a series of X-ray images taken from different angles and uses computer processing to create cross-sectional images or slices of the bones, blood vessels, and soft tissues inside your body. CT scans can use X-ray CT, positron emission tomography (PET), single photon emission computed tomography (SPECT), computed axial tomography (CAT scan), or computer-aided tomography. CT scans can be conventional CT scans or spiral / helical CT scans. In a conventional CT scan, the scanning is done slice by slice, and after each slice, the scan stops and moves down to the next slice, e.g., from the top of the abdomen down to the pelvis. Conventional CT scans require the patient to hold their breath to avoid motion artifacts. Spiral / helical CT scans are continuous scans that are taken in a spiral manner and are a faster process where the scanned images are continuous.

[0090] When no intracranial lesion is observed in an image taken from a subject who has suffered, may have suffered, or is suspected of having suffered an injury to the head, the head CT scan is "negative" for TBI. For further clarification, when no lesion is found or identified, the subject's head CT scan is "negative" for TBI; however, in some instances, even if the head CT is negative, the subject may still exhibit symptoms (e.g., symptoms of TBI). Given that not all injuries or lesions can be visualized by head CT, most subjects will have a negative head CT for TBI. Thus, the methods and assays described herein can be used to provide an assessment or determination of subjects with a negative head CT who may still have TBI.

[0091] As used interchangeably herein, "Curie point" or "Curie temperature" refers to a characteristic property of ferromagnetic materials. The Curie point of a ferromagnetic material is the temperature at which, in the absence of an external magnetic field, the material loses its characteristic ferromagnetic ability to have a net (spontaneous) magnetization. At temperatures below the Curie point, the magnetic moments are partially aligned within the magnetic domains in the ferromagnetic material. As the temperature is raised from below the Curie point, the thermal fluctuations gradually disrupt this alignment until the net magnetization becomes zero at and above the Curie point. Above the Curie point, the material is purely paramagnetic.

[0092] As used interchangeably herein, "decentralize", "decentralized", or "decentralization" in the context of testing refers to performing one or more medical tests and / or assays at one or more locations outside of a traditional medical setting (e.g., a hospital, a doctor's office, an independent laboratory site, etc.), such as an urgent care medical clinic, a retail clinic, a pharmacy, a grocery store or convenience store, a residence (e.g., a house, an apartment, etc.), a workplace, and / or a government office (e.g., the United States Transportation and Security Administration). "Hybrid decentralization" or "hybrid decentralized" refers to the situation where a subject or patient collects samples at a residence and / or workplace and transports the samples to a laboratory, bypassing a professional collection site (such as a hospital, a doctor's office, or an independent sample collection or laboratory site).

[0093] "Determined by assay" as used herein refers to determining a reference level by any suitable assay. In some embodiments, determination of the reference level can be achieved by an assay of the same type as the assay to be applied to the subject sample (e.g., by immunoassay, clinical chemistry assay, single molecule detection assay, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, or protein immunostaining, electrophoretic analysis, protein assay, competitive binding assay, functional protein assay, or chromatography or spectrometry, such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS)). In some embodiments, determination of the reference level can be achieved by an assay of the same type and under the same assay conditions as the assay to be applied to the subject sample. As noted herein, the present disclosure provides exemplary reference levels (e.g., calculated by comparing reference levels at different time points). Based on the description provided by the present disclosure, it is well within the ability of one of ordinary skill in the art to adapt the disclosure herein to other assays to obtain assay-specific reference levels for those other assays. For example, a set of training samples, which includes samples obtained from human subjects known to have suffered a head injury (and more specifically, samples obtained from human subjects known to have suffered (i) a mild TBI and / or (ii) a moderate, severe, or moderate to severe TBI) and samples obtained from human subjects known not to have suffered a head injury, can be used to obtain assay-specific reference levels. It should be understood that a parameter level that is "determined by assay" and has the listed "sensitivity" and / or "specificity" levels as used herein refers to such a reference level that has been determined to provide the listed sensitivity and / or specificity when employed in the methods of the present disclosure. For example, it is well within the ability 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 multiple different possible reference levels.

[0094] In fact, when differentiating between subjects with traumatic brain injury or without traumatic brain injury or subjects with mild versus moderate, severe, or moderate-to-severe traumatic brain injury, a person skilled in the art will weigh the impact of increasing the cut-off value on sensitivity and specificity. Increasing or decreasing the cut-off value will have a definite and predictable impact on sensitivity and specificity as well as other standard statistical measures. It is well known that increasing the cut-off value will increase specificity but may decrease sensitivity (the proportion of diseased patients who test positive). In contrast, decreasing the cut-off value will increase sensitivity but will decrease specificity (the proportion of non-diseased individuals who test negative). Detecting traumatic brain injury or determining the outcome of mild versus moderate, severe, or moderate-to-severe traumatic brain injury will be obvious to a person skilled in the art. When differentiating between whether a subject has traumatic brain injury or mild versus moderate, severe, or moderate-to-severe traumatic brain injury, the higher the cut-off value, the better the specificity, because 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. But at the same time, increasing the cut-off value will reduce the number of cases overall identified as positive, as well as the number of true positives, so sensitivity must decrease. Conversely, the lower the cut-off value, the higher the sensitivity with more true positives (i.e., differentiating 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 from subjects without traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-to-severe traumatic brain injury). But at the same time, decreasing the cut-off value will increase the number of cases overall identified as positive, as well as the number of false positives, so specificity must decrease.

[0095] Generally, high sensitivity values help a person skilled in the art to rule out diseases or conditions (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 high specificity values help a person skilled in the art to rule in diseases or conditions. Whether a person skilled in the art wishes to rule out or rule in a disease depends on the consequences for patients of each type of error. Therefore, without fully disclosing the underlying information on how to choose values, it is not possible to know or predict the precise balance used to derive the test cut-off value. The balance of sensitivity with specificity and other factors will depend on the specific circumstances. This is why sometimes alternative cut-off values (e.g., reference values) are provided so that a physician or practitioner can choose.

[0096] A "derivative" of an antibody as used herein can refer to an antibody having one or more modifications to its amino acid sequence compared to the native or parental antibody and exhibiting a modified domain structure. The derivative can still be capable of adopting the typical domain configuration found in native antibodies and of having an amino acid sequence capable of specifically binding a target (antigen). Typical examples of antibody derivatives are antibodies conjugated to other polypeptides, rearranged antibody domains, or antibody fragments. The derivative can also comprise at least one additional compound, such as a protein domain, which is linked by covalent or non-covalent bonds. According to methods known in the art, the linkage can be based on genetic fusion. Additional domains present in a fusion protein comprising an antibody can preferably be linked by a flexible linker, preferably a peptide linker, which comprises a plurality of hydrophilic peptide-bonded amino acids, the length of which is sufficient to span the distance between the C-terminus of the additional protein domain and the N-terminus of the antibody, and vice versa. An antibody can be linked to an effector molecule having a conformation suitable for a biological activity or for selective binding to, for example, a solid support, a bioactive substance (such as a cytokine or a growth hormone), a chemical reagent, a peptide, a protein, or a drug.

[0097] "Abused drug" is used herein to refer to one or more additive substances (such as drugs) taken for non-medical reasons (such as for recreational and / or hallucinogenic effects). Excessive indulgence, use, or dependence on such abused drugs is generally referred to as "drug abuse". Examples of abused drugs include alcohol, barbiturates, benzodiazepines, cannabis, cocaine, hallucinogens (such as ketamine, mescaline (peyote), PCP, psilocybin, DMT, and / or LSD), methaqualone, opioids, amphetamine (including methamphetamine), anabolic steroids, inhalants (i.e., substances containing volatile substances having psychoactive properties, such as nitrites, spray paints, cleaning fluids, markers, glues, etc.), and combinations thereof.

[0098] "Bispecific antibody" is used herein to refer to a full-length antibody that can bind two different antigens (or epitopes) in each of its two binding arms (a pair of HC / LC) (see PCT publication WO 02 / 02773). 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.

[0099] "Dual variable domain" as used herein refers to two or more antigen-binding sites on a binding protein, which binding protein can be bivalent (two antigen-binding sites), tetravalent (four antigen-binding sites), or a multivalent binding protein. The DVD can be monospecific, i.e., capable of binding one antigen (or one specific epitope), or multispecific, i.e., capable of binding two or more antigens (i.e., two or more epitopes of the same target antigen molecule or two or more epitopes of different target antigens). Preferred DVD-binding proteins comprise two heavy chain DVD polypeptides and two light chain DVD polypeptides and are referred to as "DVD immunoglobulins" or "DVD-Ig". Such DVD-Ig binding proteins are thus tetrameric and resemble IgG molecules, but provide more antigen-binding sites than IgG molecules. Thus, each half of the tetrameric DVD-Ig molecule resembles half of an IgG molecule and comprises a heavy chain DVD polypeptide and a light chain DVD polypeptide, but unlike the pair of heavy and light chains of an IgG molecule that provide a single antigen-binding domain, the pair of heavy and light chains of a DVD-Ig provide two or more antigen-binding sites.

[0100] Each antigen-binding site of a DVD-Ig binding protein can be derived from a donor ("parent") monoclonal antibody and thus comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) having a total of six CDRs each of which participates in antigen binding. Thus, a DVD-Ig binding protein that binds two different epitopes (i.e., two different epitopes of two different antigen molecules or two different epitopes of the same antigen molecule) comprises an antigen-binding site derived from a first parent monoclonal antibody and an antigen-binding site derived from a second parent monoclonal antibody.

[0101] Descriptions of the design, expression, and characterization of DVD-Ig binding molecules are provided in PCT Publication No. WO 2007 / 024715, U.S. Patent No. 7,612,181, and Wu et al., Nature Biotech., 25:1290-1297 (2007). Preferred examples of such DVD-Ig molecules include heavy chains containing the structural formula VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker (provided that it is not CH1), X2 is an Fc region, and n is 0 or 1, but preferably 1; and light chains containing the formula VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker (provided that it is not CH1, and X2 does not contain an Fc region); and n is 0 or 1, but preferably 1. Such a DVD-Ig can comprise two such heavy chains and two such light chains, where each chain contains tandemly linked variable domains with no intervening constant regions between the variable regions, where the heavy and light chains associate to form tandem functional antigen-binding sites, and where a pair of heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein with four functional antigen-binding sites. In another example, a DVD-Ig molecule can comprise such heavy and light chains, each of the heavy and light chains containing three tandemly linked variable domains (VD1, VD2, VD3) with no intervening constant regions between the variable domains, where a pair of heavy and light chains can associate to form three antigen-binding sites, and where a pair of heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein with six antigen-binding sites.

[0102] In a preferred embodiment, the DVD-Ig binding protein not only binds the same target molecule as its parental monoclonal antibody binds, but also has one or more of the desired properties of one or more of its parental monoclonal antibodies. Preferably, such additional property is an antibody parameter of one or more of the parental monoclonal antibodies. Antibody parameters that may contribute to a DVD-Ig binding protein from one or more parental monoclonal antibodies include, but are not limited to, antigen specificity, antigen affinity, potency, biological function, epitope recognition, protein stability, protein solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross-reactivity, and orthologous antigen binding.

[0103] The DVD-Ig binding protein binds to at least one epitope of GFAP. Non-limiting examples of the DVD-Ig binding protein include a DVD-Ig binding protein that binds to one or more epitopes of GFAP, a DVD-Ig binding protein that binds to an epitope of human GFAP and an epitope of GFAP of another species (e.g., mouse), and a DVD-Ig binding protein that binds to an epitope of human GFAP and an epitope of another target molecule.

[0104] As used herein, "dynamic range" refers to the range in which the assay readings are proportional to the amount of the target molecule or analyte in the sample being assayed.

[0105] "An epitope" or "epitopes" or "epitopes of interest" refers to a site on any molecule that is recognized and can bind to a complementary site on its specific binding partner. The molecule and the specific binding partner are part of a specific binding pair. For example, an epitope can be on a polypeptide, protein, hapten, carbohydrate antigen (such as but not limited to glycolipid, glycoprotein or lipopolysaccharide) or polysaccharide. Its specific binding partner can be but is not limited to an antibody.

[0106] As used interchangeably herein, "fingerstick blood sample" or "fingerprick sample" refers to a capillary blood sample obtained from a subject's finger or toe using a microneedle, lancet, micro-lancet or any other suitable device or combination thereof (e.g., a fingerstick blood collection device). In some embodiments, the fingerstick blood sample is whole blood, serum or plasma. In some aspects, the fingerstick blood sample mainly contains capillary blood, but may also contain or include a small amount or a small fraction of interstitial fluid.

[0107] As used herein, "fragment antigen binding fragment" or "Fab fragment" refers to an antibody fragment that binds to an antigen and contains one antigen binding site, a complete light chain and a portion of a heavy chain. Fab is a monovalent fragment composed of the VL, VH, CL and CH1 domains. Fab is composed of one constant domain and one variable domain of each of the heavy and light chains. The variable domain contains a paratope (antigen binding site) at the amino terminus of the monomer, which contains a set of complementarity determining regions. Thus, each arm of Y binds to an epitope on the antigen. Fab fragments can be generated as described in the art, for example, using the enzyme papain, which can be used to cleave an immunoglobulin monomer into two Fab fragments and an Fc fragment, or can be produced by recombinant methods.

[0108] As used herein, "F(ab')2 fragment" refers to an antibody generated by pepsin digestion of an entire IgG antibody to remove most of the Fc region while leaving some hinge regions intact. The F(ab')2 fragment has two antigen-binding F(ab) portions linked together by disulfide bonds and is thus divalent, with a molecular weight of approximately 110 kDa. The divalent antibody fragment (F(ab')2 fragment) is smaller than the entire IgG molecule and can better penetrate tissues, thereby facilitating better antigen recognition in immunohistochemistry. The use of F(ab')2 fragments also avoids non-specific binding to Fc receptors or protein A / G on live cells. F(ab')2 fragments can bind and precipitate antigens.

[0109] As used herein, "framework" (FR) or "framework sequence" can mean the remaining sequence of the variable region minus the CDRs. Since the precise definition of CDR sequences can be determined by different systems (e.g., see above), the meaning of framework sequences is subject to corresponding different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) also divide the framework regions on the light 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. Without designating a specific sub-region as FR1, FR2, FR3, or FR4, the framework regions as otherwise referred to represent the combined FRs within the variable region of a single naturally occurring 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. Human heavy and light chain FR sequences are known in the art and can be used as heavy and light chain "recipient" framework sequences (or simply "recipient" sequences) to humanize non-human antibodies using techniques known in the art. In one embodiment, the human heavy and light chain recipient sequences 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 Information System (hypertext transfer protocol: / / imgt.cines.fr / texts / IMGTrepertoire / LocusGenes / ).

[0110] 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 the antigen-binding site may not be as strong as that of the parental binding protein from which the antigen-binding site is derived, e.g., the parental antibody, but the ability to bind the antigen must be measurable using any of a variety of methods known for evaluating the binding of a protein, e.g., an antibody, to an antigen. In addition, the antigen-binding affinity of each antigen-binding site of a multivalent protein, e.g., a multivalent antibody, herein need not be the same quantitatively.

[0111] "GFAP" is used herein to describe glial fibrillary acidic protein. GFAP is a protein encoded by the GFAP gene in humans and it can be produced (e.g., recombinantly, in other species).

[0112] "GFAP status" can represent the level or amount of GFAP at a given point in time (e.g., using a single GFAP measurement), the level or amount of GFAP associated with monitoring (e.g., an increase or decrease in GFAP amount determined by repeated testing of a subject), the level or amount of GFAP associated with the treatment of traumatic brain injury (whether primary brain injury and / or secondary brain injury), or a combination thereof. As used herein, "Glasgow Coma Scale" or "GCS" refers to a 15-point scale (e.g., as described in Graham Teasdale and Bryan Jennett, Lancet 1974; 2:81-4) that provides a practical method for assessing the level of impairment of consciousness in a patient with a brain injury. The test measures the best motor response, verbal response, and eye opening response using the following values: I. Best motor response (6 - obeys two-part command; 5 - places hand above clavicle in response to stimulus to head / neck; 4 - bends arm at elbow rapidly but with no major abnormal features; 3 - bends arm at elbow with major abnormal features evident; 2 - extends arm at elbow; 1 - no movement of arm / leg, no confounding factors; NT - paralysis or other restrictive factor); II. Verbal response (5 - correctly states name, location, and date; 4 - not oriented but communicates coherently; 3 - single understandable words; 2 - only groans / sighs; 1 - no audible response, no confounding factors; NT - factors confounding communication); and III. Eye opening (4 - eyes open before stimulus; 3 - after speech or shouted command; 2 - after fingertip stimulus; 1 - never opens eyes, no confounding factors; NT - eyes closed due to local factors). The final score is determined by adding the values of I + II + III. If the GCS score is 13 - 15, the subject is considered to have a mild TBI. If the GCS score is 9 - 12, the subject is considered to have a moderate TBI. If the GCS score is 8 or lower, typically 3 - 8, the subject is considered to have a severe TBI.

[0113] As used herein, the "Glasgow Outcome Scale" refers to a global scale for functional outcome that rates a patient's status into one of the following five categories: death, vegetative state, severe disability, moderate disability, or good recovery. The "Extended Glasgow Outcome Scale" or "GOSE", which may be used interchangeably herein, provides a more detailed eight-category classification by subdividing the categories of severe disability, moderate disability, and good recovery into lower and higher categories, as shown in Table 1.

[0114] Table 1

[0115]

[0116] As used herein, the term "hydrophilic", such as with respect to a "hydrophilic material" (e.g., a separator, a membrane, etc.), refers to those materials having a water contact angle of less than about 40 degrees.

[0117] As used herein, the term "hydrophobic", such as with respect to a "hydrophobic material" (e.g., a separator, a membrane, etc.), refers to those materials having a water contact angle greater than about 80 degrees.

[0118] The term "humanized antibody" is used herein to describe an antibody that contains heavy and light chain variable region sequences from non-human species (e.g., mouse) but in which at least a portion of the VH and / or VL sequences has been made more "human-like", i.e., more similar to human germline variable sequences. A "humanized antibody" is an antibody or its variant, derivative, analogue, or fragment that immunospecifically binds to an antigen of interest and contains a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR means 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 contains at least one and usually both variable domains substantially in their entirety (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., the donor antibody) and all or substantially all of the framework regions are those of human immunoglobulin consensus sequences. In one embodiment, a humanized antibody also contains at least a portion of an immunoglobulin constant region (Fc) (usually the constant region of a human immunoglobulin). In some embodiments, a humanized antibody contains a light chain as well as at least the variable domain of the heavy chain. An antibody may 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 the humanized heavy chain.

[0119] The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, as well as any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4. The humanized antibody can contain sequences 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 functions.

[0120] The framework regions and CDRs of the humanized antibody need not exactly correspond to the parental sequences. For example, the donor antibody CDR or consensus framework can be mutagenized by substituting, inserting, and / or deleting at least one amino acid residue so that the CDR or framework residue at that site does not correspond to the donor antibody or consensus framework. However, in a preferred embodiment, such mutations will not be extensive. Generally, 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 will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework regions in consensus immunoglobulin sequences. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed by the amino acids (or nucleotides) that occur most frequently in a related family of 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 an immunoglobulin family, each position in the consensus sequence is occupied by the amino acid that occurs most frequently in that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

[0121] As used herein, "identical" or "identity" in the context of two or more polypeptide or polynucleotide sequences can mean that the sequences have a specified percentage of identical residues over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residues occur in both sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences have different lengths or the alignment produces one or more staggered ends and the specified region to be compared includes only a single sequence, the residues of the single sequence are included in the denominator but not the numerator of the calculation.

[0122] "Injury to the head" or "head injury", as used interchangeably herein, refers to any trauma to the scalp, skull, or brain. Such injuries can include only minor impacts on the skull or can be severe brain injuries. Such injuries include primary brain injury and / or secondary brain injury. Primary brain injury occurs during the initial insult and is caused by the displacement of the physical structures of the brain. More specifically, primary brain injury is the physical injury to the parenchyma (tissue, blood vessels) that occurs during a traumatic event, resulting in shear and compression of the surrounding brain tissue. Secondary brain injury occurs after the primary injury and can involve a series of cellular processes. More specifically, secondary brain injury refers to changes that develop over a period of time (from hours to days) after the primary brain injury. It includes a whole cascade of cellular, chemical, tissue, or vascular changes in the brain that lead to further destruction of the brain tissue.

[0123] A head injury can be closed or open (penetrating). A closed head injury is a trauma to the scalp, skull, or brain in which the skull is not penetrated by the impacting object. An open head injury is a trauma to the scalp, skull, or brain in which the skull is penetrated by the impacting object. Injury to the head can be caused by the shaking of a person's body, blunt impact produced by external mechanical or other forces that result in closed or open head trauma (e.g., traffic accidents such as in the case of cars, airplanes, trains, etc.; blows to the head such as with a baseball bat or from a firearm), cerebrovascular accident (e.g., stroke), one or more falls (e.g., as in sports or other activities), explosion or shock wave (collectively referred to as "blast injury"), and other types of blunt force trauma. Alternatively, injury to the head may be caused by ingestion and / or exposure to fire, chemicals, toxins, or a combination of chemicals and toxins. Examples of such chemicals and / or toxins include mold, asbestos, pesticides and insecticides, organic solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organometals (such as methylmercury, tetraethyllead, and organotin), and / or one or more abused drugs. Alternatively, a head injury may be due to a subject having an autoimmune disease, metabolic disorder, brain tumor, hypoxia, viral infection (e.g., SARS-CoV-2), fungal infection, bacterial infection, meningitis, hydrocephalus, or any combination thereof. In some cases, it is not possible to determine whether any such event or injury has occurred. For example, the patient or subject may have no medical history, the subject may be unable to speak, the subject may know the events they have experienced, etc. Such cases are described herein as the subject "may have suffered a head injury". In certain embodiments herein, closed head injury does not include and specifically excludes cerebrovascular accidents such as stroke.

[0124] As used herein, "interstitial fluid" refers to the fluid that surrounds and / or fills the spaces between cells. Interstitial fluid may contain or comprise a mixture of water, ions, and small solutes that are exuded from the blood by the hydrostatic pressure generated when the heart pumps blood.

[0125] As used herein, "intracranial lesion" refers to an area of injury within the brain. An intracranial lesion may be an abnormality seen on a CT scan or a brain imaging test such as magnetic resonance imaging (MRI). On a CT or MRI scan, a brain lesion may appear as a dark or bright spot that does not look like normal brain tissue.

[0126] As used herein, "isolated polynucleotide" may mean a polynucleotide (e.g., a polynucleotide of genomic, cDNA, or synthetic origin or a combination thereof) that, depending on its source, is not associated with all or a portion of the polynucleotides with which the "isolated polynucleotide" is found in nature; is operably linked to a polynucleotide to which it is not linked in nature; or does not exist in nature as part of a larger sequence.

[0127] As used herein, "label" and "detectable label" refer to a moiety attached to an antibody or an analyte to make the reaction between the antibody and the analyte detectable, and an antibody or analyte so labeled is referred to as "detectably labeled". A label can generate a signal that can be detected by visual or instrumental means. A variety of labels include substances that generate signals such as chromophores, fluorescent compounds, chemiluminescent compounds, radioactive compounds, etc. Representative examples of labels include moieties that produce light such as acridine compounds, and moieties that produce fluorescence such as fluorescein. Other labels are described herein. In this regard, the moiety itself may be undetectable but may become detectable upon reaction with another moiety. The use of the term "detectably labeled" is intended to encompass such labels.

[0128] Any suitable detectable label known in the art can be used. For example, the detectable label can be a radioactive label (such as 3H, 14C, 32P, 33P, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm), an enzyme label (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, etc.), a chemiluminescent label (such as acridinium ester, thioester, or sulfonamide; luminol, isoluminol, phenanthridinium ester, etc.), a fluorescent label (such as fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein, 3′6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, etc.)), rhodamine, phycobiliprotein, R-phycoerythrin, quantum dots (such as cadmium selenide capped with zinc sulfide), a thermometric label, or an immunopolymerase chain reaction label. An introduction to labels, labeling procedures, and label detection 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, for example, 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 chemiluminescent assays (see, for example, Adamczyk et al., Bioorg. Med Ghem. 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)).

[0129] In one aspect, the acridine compound is acridine-9-carboxamide. Methods for preparing acridine 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, pp. 77-105 (2002); Adamczyk et al., Org. Lett. 5:3779-3782 (2003); and U.S. Patent Nos. 5,468,646, 5,543,524, and 5,783,699 (the teachings of each of which with respect to this aspect are hereby incorporated by reference in their entirety).

[0130] Another example of an acridine compound is an aryl acridine-9-carboxylate. An example of an aryl acridine-9-carboxylate having Formula II is 10-methyl-9-(phenoxycarbonyl) acridine fluorosulfonate (available from Cayman Chemical, Ann Arbor, MI). Methods for preparing aryl acridine-9-carboxylates are described in McCapra et al., Photochem. Photobiol. 4:1111-21 (1965); Razavi et al., Luminescence 15:245-249 (2000); Razavi et al., Luminescence 15:239-244 (2000); and U.S. Patent No. 5,241,070 (the teachings of each of which with respect to this aspect are incorporated herein by reference in their entirety). Such aryl acridine-9-carboxylates are chemiluminescent indicators that are efficient in terms of both signal intensity and / or signal rapidity in the production of hydrogen peroxide generated by the oxidation of an analyte by at least one oxidase. The chemiluminescent emission process of aryl acridine-9-carboxylates is rapidly completed, i.e., within 1 second, while the chemiluminescent emission of acridine-9-carboxamide continues for up to 2 seconds. However, aryl acridine-9-carboxylates 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 from 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 a 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 aryl acridine-9-carboxylates and their use are set forth in U.S. Patent Application No. 11 / 697,835, filed April 9, 2007. Aryl acridine-9-carboxylates can be dissolved in any suitable solvent, such as degassed anhydrous N,N-dimethylformamide (DMF) or sodium cholate hydrate.

[0131] "Linker sequence" or "linker peptide sequence" refers to a natural or artificial polypeptide sequence that is linked to one or more polypeptide sequences of interest (e.g., full-length, fragment, etc.). The term "linked" refers to the conjugation of the linker sequence to the polypeptide sequence of interest. Such polypeptide sequences are preferably conjugated via one or more peptide bonds. The linker sequence can have a length of about 4 to about 50 amino acids. Preferably, the linker sequence has a length of about 6 to about 30 amino acids. Natural linker sequences can be modified by amino acid substitution, addition, or deletion to produce artificial linker sequences. Linker sequences can be used for many purposes, including in recombinant Fabs. Exemplary linker sequences include, but are not limited to: (i) histidine (His) tags, such as 6X His tags, which have the amino acid sequence HHHHHH (SEQ ID NO:3), and can be used as linker sequences to facilitate the isolation and purification of polypeptides and antibodies of interest; (ii) enterokinase cleavage sites, such as His tags, for the isolation and purification of proteins and antibodies of interest. Often, enterokinase cleavage sites are used in conjunction with His tags for the isolation and purification of proteins and antibodies of interest. Various enterokinase cleavage sites are known in the art. Examples of enterokinase cleavage sites include, but are not limited to, the amino acid sequence DDDDK (SEQ ID NO:4) and its derivatives (e.g., ADDDDK (SEQ ID NO:5), etc.); (iii) miscellaneous sequences can be used to link or connect the light chain and / or heavy chain variable regions of single-chain variable region fragments. Examples of other linker sequences can be found in Bird et al., Science 242:423-426 (1988); Huston et al., PNAS USA 85:5879-5883 (1988); and McCafferty et al., Nature 348:552-554 (1990). Linker sequences can also be modified for additional functions, such as attachment of drugs or attachment to solid supports. In the context of the present disclosure, monoclonal antibodies can, for example, contain linker sequences, such as His tags, enterokinase cleavage sites, or both.

[0132] As used interchangeably herein, "magnetic resonance imaging" or "MRI" refers to a medical imaging technique used in radiology to form pictures of the anatomical structures and physiological processes in the human body in health and disease (e.g., interchangeably referred to herein as "MRI", "MRI procedure", or "MRI scan"). MRI is a form of medical imaging that measures the response of the atomic nuclei in human tissues to high-frequency radio waves when in a strong magnetic field and produces images of internal organs. MRI scanners, based on the science of nuclear magnetic resonance (NMR), use strong magnetic fields, radio waves, and field gradients to generate images of the inside of the human body.

[0133] 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 aspects, the cross-sectional dimension of the channel is less than about 150 μm. In other aspects, the cross-sectional dimension of the channel is less than about 100 μm.

[0134] As used herein, a "microsampling device" refers to any device known in the art suitable for extracting capillary blood through the skin. It should be understood that although the sample obtained through the skin using a microsampling device mainly contains capillary blood, the sample may also contain a small amount or a small fraction of interstitial fluid. In some aspects, the microsampling device can contain from about 0.1 mL to about 4 mL of capillary blood. In some other aspects, the device contains multiple microneedles, lancets or micro-lancets, blades or micro-blades, micro-screws or any combination thereof. In some aspects, the multiple microneedles, lancets or micro-lancets, blades or micro-blades, micro-screws or any combination thereof can be rotatable. In other aspects, the multiple microneedles, lancets or micro-lancets, blades or micro-blades, micro-screws or any combination thereof are non-rotatable. In some aspects, the microsampling device creates a vacuum and / or uses a stored vacuum to draw the skin into the device and / or actuate the multiple microneedles, lancets or micro-lancets, blades or micro-blades, micro-screws or any combination thereof to cut the skin. Exemplary microsampling devices that can be used in the methods described herein include the TAP device available from YourBio Health, Inc. (Cambridge, MA); and the devices described in U.S. Patent No. 9,113,836, the content of which is incorporated herein by reference; the Tasso+, Tasso-M20 and Tasso-ST devices available from Tasso, Inc. (Seattle, WA); the Tasso-ST device available from Draw Bridge Health (San Diego, CA); the PBS-1000 from PreciHealth (Neuchatel, Switzerland); or the Loop blood collection device available from Loop Medical (Lausanne, Switzerland). In other aspects, examples of microsampling devices include finger prick devices. In some aspects, the microsampling device can include a bandage, a dressing or other suitable material that can be applied or dispensed to the skin area once the sample has been obtained and / or the device has been removed and / or disengaged from the skin.

[0135] As used herein, a "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. A monoclonal antibody is highly specific for a single antigen. In addition, unlike polyclonal antibody preparations, which generally include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0136] "Multivalent binding protein" is used herein to refer to a binding protein that contains two or more antigen-binding sites (also referred to herein as "antigen-binding domains"). Multivalent binding proteins are preferably engineered to have three or more antigen-binding sites and are generally not naturally occurring antibodies. The term "multispecific binding protein" refers to a binding protein that can bind two or more related or unrelated targets, including a binding protein that is capable of binding two or more different epitopes of the same target molecule.

[0137] As used herein, "negative predictive value" or "NPV" is used interchangeably and refers to the probability that a subject has a negative outcome, assuming they have a negative test result.

[0138] As used herein, "operatively coupled" or "operatively connected" means coupling a plurality of elements or components that are capable of moving between a first position and a second position or between a first configuration and a second configuration such that when the first element moves from one position / configuration to another position / configuration, the second element also moves between positions / configurations. It should be noted that a first element can be "operatively coupled" to another element, but not vice versa. Where elements are capable of moving between a first element and another element and vice versa, the elements are said to be "operatively coupled to each other".

[0139] 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 aspects, for example, normalizing can involve multiplying the amount of the analyte in the capillary blood sample by a factor (e.g., a correlation or conversion factor).

[0140] A "point-of-care device" refers to a device for providing medical diagnostic tests at or near the point of care (i.e., typically outside of a laboratory), at the time and place of patient care (such as in a hospital, physician's office, urgent or other medical care facility, patient's home, nursing home, and / or long-term care and / or hospice facility). Examples of point-of-care devices include those manufactured 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).

[0141] As used interchangeably herein, "positive predictive value" or "PPV" refers to the probability that a subject has a positive outcome given that they have a positive test result.

[0142] "Quality control reagents" in the context of the immunoassays and kits described herein include, but are not limited to, calibrators, controls, and sensitivity sets. "Calibrators" or "standards" (e.g., one or more, such as multiple) are typically used to establish a calibration (standard) curve for interpolating the amount of an analyte (such as an antibody or analyte). Alternatively, a single calibrator close to a reference level or control level (e.g., "low", "medium", or "high" level) can be used. Multiple calibrators can be used in combination (i.e., more than one calibrator or different amounts of calibrators) to form a "sensitivity set".

[0143] As used herein, a "reaction vessel" refers to a holder or receptacle in which an assay is performed, such as a container, reservoir, tube, and / or cartridge. In some aspects, the reaction vessel can have one or more wells.

[0144] The "Receiver Operating Characteristic" curve or "ROC" curve is a graph that illustrates the performance of a binary classifier system as its discrimination threshold varies. For example, an ROC curve can be a plot of the true positive rate versus the false positive rate for different possible cut-off points of a diagnostic test. It is generated by plotting the fraction of true positives among the positives (TPR = true positive rate) against the fraction of false positives among the negatives (FPR = false positive rate) at various threshold settings. The TPR is also known as sensitivity, and the FPR is one minus specificity or the 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 boundary of the ROC space and then the top boundary, the more accurate the test; the closer the curve is to the 45-degree diagonal of the ROC space, the less accurate the test; the slope of the tangent at the cut-off point gives the likelihood ratio (LR) of the test value; and the area under the curve is a measure of the text accuracy.

[0145] "Recombinant antibody" and "multiple recombinant antibodies" refer to antibodies prepared by one or more steps, including cloning nucleic acid sequences encoding all or part of one or more monoclonal antibodies into a suitable expression vector by recombinant techniques and subsequently expressing the antibodies in a suitable host cell. The terms include, but are not limited to, recombinantly produced monoclonal antibodies, chimeric antibodies, humanized antibodies (fully or partially humanized), multi-specific or multivalent structures formed from antibody fragments, bispecific antibodies, heteroconjugate Abs, and other antibodies described herein (i). (Dual variable domain immunoglobulins and methods for their preparation are described in Wu, C et al., Nature Biotechnology, 25:1290 - 1297 (2007)). As used herein, the term "bispecific antibody" refers to an antibody that includes a first arm having specificity for one antigen site and a second arm having specificity for a different antigen site, i.e., a bispecific antibody has dual specificity.

[0146] As used herein, "reference level" refers to a determination threshold value used to evaluate diagnostic, prognostic, or therapeutic efficacy, which has been associated or correlated herein with various clinical parameters (e.g., presence of disease, disease stage, disease severity, disease progression, non - progression, or improvement, etc.). As used herein, "absolute amount" refers to the absolute value of the change or difference between at least two determination results obtained or sampled at different time points, and similar to the reference level, it has been associated or correlated herein with various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression, non - progression, or disease improvement, etc.). As used herein, "absolute value" refers to the magnitude of a real number without considering its sign (e.g., the difference between two comparison levels (e.g., the level collected at a first time point and the level collected at a second time point)), i.e., regardless of whether it is positive or negative. The UCH - L1 and / or GFAP reference levels mentioned herein are from venous blood.

[0147] The present disclosure provides exemplary reference levels and absolute amounts (e.g., calculated by comparing reference levels at different time points). However, it is well known that reference levels and absolute amounts can vary depending on the nature of the immunoassay (e.g., antibodies used, reaction conditions, sample purity, etc.), and the assays can be compared and standardized. Based on the description provided in the present disclosure, it is entirely within the ability of a person of ordinary skill in the art to adapt the disclosure herein to other immunoassays to obtain immunoassay - specific reference levels and absolute amounts for those other immunoassays. Although the exact values of the reference levels and absolute amounts may vary between assays, the findings described herein should be generally applicable and inferable to other assays.

[0148] As used herein, "removably coupled" or "removably connected" means that one component is coupled to another component in a substantially 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, components that are "removably coupled" can be easily decoupled and re - coupled without damaging the components.

[0149] As used herein, "result" refers to an item of information obtained by performing a determination. 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 a readout).

[0150] As used herein, "risk assessment", "risk classification", "risk identification", or "risk stratification" of a subject (e.g., a patient) refers to the evaluation of factors, including biomarkers, to predict the risk of future events, including disease onset or disease progression, so that treatment decisions regarding the subject can be made on a more informed basis.

[0151] As used herein, a "plasmapheresis device" refers to a device or apparatus that can be used to separate whole blood components (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 diaphragm, filter, synthetic paper (e.g., microcolumn support), or any combination thereof. For example, the diaphragm and / or filter that can be used in a plasmapheresis device can include at least one of polycarbonate, polysulfone, polyester, polyethylene, polyurethane, and polypropylene. In some aspects, the diaphragm 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 aspects, the diaphragm and / or filter is not pretreated. In additional aspects, the filter that can be used is a gravity-assisted separation system. Examples of plasmapheresis devices that can be used in the methods described herein include the devices described in U.S. Patent Publication No. 2020 / 0124508, the content of which is incorporated herein by reference. In some aspects, the plasmapheresis device does not include a lateral flow device.

[0152] As used herein, the "sensitivity" of a measurement refers to the proportion of subjects with a positive outcome who are correctly identified as positive (e.g., those subjects who are correctly identified as having the disease or medical condition being tested). For example, this may include correctly identifying subjects with TBI as distinct from subjects without TBI, correctly identifying subjects with moderate, severe, or moderate-to-severe TBI as distinct from subjects with mild TBI, correctly identifying subjects with mild TBI as distinct from subjects with moderate, severe, or moderate-to-severe TBI, correctly identifying subjects with moderate, severe, or moderate-to-severe TBI as distinct from subjects without TBI, or correctly identifying subjects with mild TBI as distinct from subjects without TBI, etc.

[0153] As used herein, the “specificity” of a measurement refers to the proportion of subjects with a negative outcome who are correctly identified as negative (e.g., those subjects correctly identified as not having the disease or medical condition being tested). For example, this may 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 so on.

[0154] A “series of calibration compositions” refers to a plurality of compositions containing known amounts of GFAP, where each composition differs from the other compositions in the series by the amount of GFAP.

[0155] As used interchangeably herein, “solid phase” or “solid support” refers to any material that can be used to attach and / or attract and immobilize (1) one or more capture agents or capture-specific binding partners, or (2) one or more detection agents or detection-specific binding partners. The solid phase can be selected based on its inherent ability to attract and immobilize the capture agent. Alternatively, the solid phase can have a linker adhered thereto, the linker having the ability to attract and immobilize (1) the capture agent or capture-specific binding partner, or (2) the detection agent or detection-specific binding partner. For example, the linker can include a charged substance that is oppositely charged relative to the capture agent (e.g., capture-specific binding partner) or detection agent (e.g., detection-specific binding partner) itself or relative to a charged substance conjugated to (1) the capture agent or capture-specific binding partner, or (2) the detection agent or detection-specific binding partner. Generally, the linker can be any binding partner (preferably heterologous) that is immobilized (attached to) the solid phase and has the ability to immobilize (1) the capture agent or capture-specific binding partner, or (2) the detection agent or detection-specific binding partner through a binding reaction. The linker enables the capture agent to indirectly bind to the solid phase material 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, slides, beads, microparticles, chips, and other configurations known to those of ordinary skill in the art. In some aspects, the solid support can be a magnetically sensitive bead or particle.

[0156] "Specific binding" or "binds specifically" as used herein can refer to the interaction of an antibody, protein, or peptide with a second chemical species, where the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than binding proteins generally. If an antibody is specific for epitope "A", then in a reaction containing labeled "A" and the antibody, the presence of molecules containing epitope A (or free unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0157] A "specific binding partner" is a member of a specific binding pair. A specific binding pair consists of two different molecules that bind specifically to each other either chemically or physically. Thus, in addition to the antigen-antibody specific binding pairs of common immunoassays, 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, etc. In addition, a specific binding pair can include members that are analogs of the original specific binding partners, such as analyte-analogs. Immunoreactive specific binding partners include isolated or recombinantly produced antigens, antigen fragments, and antibodies, including monoclonal and polyclonal antibodies and their complexes and fragments.

[0158] "Statistically significant" as used herein refers to the likelihood that the relationship between two or more variables is caused by factors other than random chance. Statistical hypothesis testing is used to determine whether the results of a data set are statistically significant. In statistical hypothesis testing, a statistically significant result is obtained whenever the p-value of the observed test statistic is less than the significance level defined by the study. The p-value is the probability of obtaining a result at least as extreme as the observed result, assuming 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, "significant" refers to a change that has not been determined to be statistically significant (e.g., it may not have been subjected to statistical hypothesis testing).

[0159] "Subject" and "patient" as used interchangeably herein refer to any vertebrate, including but not limited to mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice, non-human primates (e.g., monkeys such as cynomolgus or rhesus monkeys, chimpanzees, etc.), and humans). In some embodiments, the subject can be human or non-human. In some embodiments, the subject is human. The subject or patient can undergo other forms of treatment.

[0160] "Treat / treating / treatment" are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progression of a disease and / or injury to which such term applies, or one or more symptoms of such disease. Depending on the condition of the subject, the term also refers to preventing a disease and includes preventing the onset of a disease or preventing symptoms associated with a disease. Treatment can be carried out in an acute or chronic manner. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to being afflicted with the disease. Such prevention of a disease or reduction of disease severity prior to affliction refers to administering a pharmaceutical composition to a subject at a time when the subject is not afflicted with the disease. "Prevention" also refers to preventing the recurrence of a disease or one or more symptoms associated with such disease. "Treat" and "therapeutically" refer to the act of treating, as "treat" is defined above.

[0161] As used herein, a "transfer tube" refers to a container or reservoir for transferring a fluid (e.g., a capillary blood sample) from one location to a second location (e.g., to a reaction vessel or from a plasma separation device).

[0162] "Traumatic brain injury" or "TBI", which are used interchangeably herein, refers to a complex injury with a broad spectrum of symptoms and disabilities. TBI is many times 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, shaking of a person's body, motor vehicle accidents, firearm injuries, cerebrovascular accidents (e.g., stroke), falls, explosions or shock waves, and other types of blunt force trauma. Other causes of TBI include ingestion and / or exposure to one or more fires, chemicals, or toxins (such as mold, asbestos, pesticides and insecticides, organic solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organometals (such as methylmercury, tetraethyllead, and organotin), one or more abused drugs, or combinations thereof). Alternatively, TBI may occur in subjects with autoimmune diseases, metabolic disorders, brain tumors, hypoxia, viral infections (e.g., SARS-CoV-2, meningitis, etc.), fungal infections (e.g., meningitis), bacterial infections (e.g., meningitis), or any combination thereof. Young and old people are the age groups at highest risk for TBI. In certain embodiments herein, traumatic brain injury or TBI does not include and specifically excludes cerebrovascular accidents, such as stroke.

[0163] "Mild TBI" as used herein refers to a head injury in which the subject may or may not experience loss of consciousness. For subjects who experience loss of consciousness, it is usually brief, typically lasting only a few seconds or minutes. Mild TBI is also known as concussion, minor head trauma, minor TBI, minor brain injury, and minor head injury. Although MRI and CT scans are often normal, individuals with mild TBI may have cognitive problems such as headache, difficulty thinking, memory problems, attention deficits, mood swings, and depression.

[0164] Mild TBI is the most common type of TBI and is often missed at the time of the initial injury. Typically, the subject has a Glasgow Coma Scale score between 13 - 15 (such as 13 - 15 or 14 - 15). Symptoms persist for 3 months or longer in fifteen percent (15%) of mild TBI patients. Common symptoms of mild TBI include fatigue, headache, vision problems, memory loss, poor attention / concentration, sleep disturbances, dizziness / loss of balance, stress-related mood disorders, depressive affect, and seizures. Other symptoms associated with mild TBI include nausea, loss of smell, sensitivity to light and sound, mood changes, confusion or disorientation, and / or slowed thinking.

[0165] "Moderate TBI" as used herein refers to a brain injury in which loss of consciousness and / or confusion and disorientation are present for between 1 and 24 hours and the subject has a Glasgow Coma Scale score between 9 - 13 (such as 9 - 12 or 9 - 13). Individuals with moderate TBI may have abnormal brain imaging findings. "Severe TBI" as used herein refers to a brain injury in which loss of consciousness exceeds 24 hours and memory loss persists for more than 24 hours following an injury or penetrating skull injury and the subject has a Glasgow Coma Scale score between 3 - 8. The range of deficits can be from higher levels of cognitive function impairment to a comatose state. Survivors may have limited arm or leg function, speech or language abnormalities, loss of thinking ability, or emotional problems. Individuals with severe injuries may be in a non-responsive state for an extended period. For many people with severe TBI, long-term rehabilitation is typically required to maximize function and independence.

[0166] As used herein, "moderate to severe" TBI refers to a range of brain injuries that includes changes from moderate to severe TBI over time and thus includes (e.g., over time) isolated moderate TBI, isolated severe TBI, and combined moderate to severe TBI. For example, in some clinical situations, a subject may initially be diagnosed with moderate TBI but over time (minutes, hours, or days) progress to having severe TBI (e.g., in the case of intracerebral hemorrhage). Alternatively, in some clinical situations, a subject may initially be diagnosed with severe TBI but over time (minutes, hours, or days) progress to having moderate TBI. Such subjects would be examples of patients classifiable as "moderate to severe". Common symptoms of moderate to severe TBI include cognitive deficits, including difficulties with attention, concentration, distractibility, memory, processing speed, confusion, perseveration, impulsivity, language processing, and / or "executive function", difficulty understanding spoken words (sensory aphasia), difficulty speaking and being understood (expressive aphasia), slurred speech, speaking very fast or very slow, reading problems, writing problems, difficulty interpreting touch, temperature, movement, limb position, and fine discrimination, integrating or patternizing sensory impressions into psychologically meaningful data, partial or total vision loss, weak eye muscles and double vision (diplopia), blurred vision, problems judging distance, involuntary eye movements (nystagmus), intolerance to light (photophobia), hearing problems (such as reduced or lost hearing, ringing in the ears (tinnitus), increased sensitivity to sound), loss or reduction of smell (anosmia), loss or reduction of taste, seizures associated with epilepsy, which may be several types and may involve consciousness, sensory perception, or motor movement, problems with control of the bowel and bladder, insomnia, loss of endurance, changes in appetite, problems with temperature regulation, menstrual difficulties, dependent behavior, problems with emotional capacity or stability, lack of motivation, irritability, aggression, depression, disinhibition, or denial / lack of awareness. Subjects with moderate to severe TBI may have a Glasgow Coma Scale score of 3 - 12 (which includes the range of 9 - 12 for moderate TBI and the range of 3 - 8 for severe TBI).

[0167] "Variant" is used herein to describe a peptide or polypeptide that differs in amino acid sequence due to an insertion, deletion, or conservative substitution of an amino acid, but retains at least one biological activity. Representative examples of "biological activity" include the ability to bind to a specific antibody or to promote an immune response. Variant is also used herein to describe a protein having an amino acid sequence that is substantially the same as a reference protein, the reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitution of an amino acid, i.e., replacement of an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is well recognized in the art as generally involving minimal changes. As understood in the art, these minimal changes can be identified in part by considering the hydrophilicity-hydrophobicity index of the amino acids. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydrophilicity index of an amino acid is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids having similar hydrophilicity indices can be substituted and still retain protein function. In one aspect, amino acids with a hydrophilicity index of ±2 are substituted. The hydrophilicity of an amino acid can also be used to reveal substitutions that will result in a protein that retains biological function. Considering the hydrophilicity of amino acids in the context of a peptide allows calculation of the maximal local average hydrophilicity of the peptide, which is a useful measure that has been reported to be well correlated with antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is hereby incorporated by reference in its entirety. As understood in the art, substitution of amino acids having similar hydrophilicity values can result in a peptide that retains biological activity (e.g., immunogenicity). Substitutions can be made with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the specific side chain of that amino acid. Consistent with the observations, amino acid substitutions that are compatible with biological function are understood to depend on the amino acids, and in particular the relative similarity of the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties. "Variant" can also be used to refer to an antigen-reactive fragment of an anti-GFAP antibody that differs in amino acid sequence from the corresponding fragment of the anti-GFAP antibody, but still has antigen reactivity and can compete with the corresponding fragment of the anti-GFAP antibody used to bind to GFAP. "Variant" can also be used to describe a polypeptide or a fragment thereof that has been differentially processed (such as by proteolysis, phosphorylation, or other post-translational modifications), but still retains its antigen reactivity.

[0168] "Vector" is used herein to describe a nucleic acid molecule that can transport another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Some vectors can replicate autonomously in the host cells into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell after being introduced into the host cell and are thereby replicated along with the host genome. In addition, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). Generally speaking, expression vectors useful in recombinant DNA technology generally take the form of plasmids. "Plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vectors. However, other forms of expression vectors that perform equivalent functions can be used, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). In this regard, RNA forms of vectors (including RNA viral vectors) can also be used in the context of the present disclosure.

[0169] "Venous blood" as used herein 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 health care 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.

[0170] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure will have the meanings commonly understood by one of ordinary skill in the art. For example, any nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The meanings and scopes of the terms should be clear; however, if any implicit ambiguities exist, the definitions provided herein will prevail over any dictionary or extrinsic definition. In addition, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular.

[0171] 2. Systems and assays for measuring the amount of GFAP in a biological sample obtained from a subject

[0172] 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 that includes at least one cartridge, the cartridge including 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 will be described in more detail herein, the magnetic immunosensor or magnetic immunosensing device is used to capture and retain a complex containing GFAP that has been immobilized on a magnetically sensitive bead using one or more specific binding partners (e.g., one or more antibodies).

[0173] The biological sample used in the systems and assays described herein can be a blood sample, such as a venous blood sample, a capillary blood sample, a finger stick blood sample, or a combination thereof. In some aspects, 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, a capillary blood sample, a finger stick blood sample, or a combination thereof.

[0174] In some aspects, when the biological sample is a blood sample (e.g., a venous blood sample, a capillary blood sample, a finger stick blood sample, or a combination thereof), the blood sample can be further or additionally processed before determining the amount of GFAP. In some aspects, the sample is processed using centrifugation. In other aspects, the sample is processed using a plasma separation device that includes at least one filter, membrane, and / or synthetic paper. For example, in some aspects, the plasma separation device that can be used is the device described in Section 3. The plasma separation device can separate the blood into serum and / or plasma.

[0175] In other aspects, the plasma separation device can be in fluid communication with, or operatively connected, coupled, and / or removably coupled to, a micro-sampling device, as part of a micro-sampling system, as further described in detail in Section 4. In other aspects, the plasma separation device can be integrated into the micro-sampling device, as further described in detail in Section 4.

[0176] In other aspects, the plasma separation device can be in fluid communication with, or operatively connected, coupled, and / or removably coupled to, a well of a reaction vessel, as further described in detail in Section 4. For example, in some aspects, the reaction vessel is a cartridge such as used in a point-of-care device.

[0177] In other aspects, the plasma separation device can be in fluid communication with or operatively connected, coupled, and / or removably coupled to the transfer tube, as described in further detail in Section 4. In these aspects, the transfer tube can be in fluid communication with or operatively connected, coupled, and / or removably coupled to the reaction vessel, as described in further detail in Section 4. In additional aspects, the plasma separation device can be integrated into the transfer tube. In other aspects, the transfer tube includes a lid or a plug.

[0178] In some aspects, the system and assay include obtaining a sample within about 24 hours of an actual or suspected injury to a 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 can be contacted with an anti-GFAP antibody. The assay also includes detecting the resulting antibody-biomarker complex.

[0179] 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 suffered, may have suffered, or is suspected of having suffered a head injury and who is likely to have suffered an acquired brain injury (ABI), such as traumatic brain injury (TBI). 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 ABI. The assays described herein can be performed on a sample obtained from a subject (e.g., a human subject) within about 24 hours of an actual or suspected head injury to measure or detect the level of an ABI biomarker (such as GFAP) in the sample and to determine whether the subject (e.g., a human subject) has ABI. In some aspects, when the amount of the biomarker in the sample is higher than a reference level of the biomarker (e.g., GFAP), it is determined that the subject has ABI.

[0180] The systems and assays described herein can help determine the extent of traumatic brain injury in a subject (e.g., a human subject) with an actual or suspected head injury, e.g., determine 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, "determine whether the subject (e.g., a human subject) has a mild traumatic brain injury, a moderate traumatic brain injury, or a moderate-to-severe traumatic brain injury" refers to the fact that the above methods can be used, for example, in conjunction with other information (e.g., clinical assessment data) to determine that the subject is more likely not to have a mild traumatic brain injury, a moderate traumatic brain injury, a severe traumatic brain injury, or a moderate-to-severe traumatic brain injury. 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 traumatic brain injury (such as GFAP) in the sample and determine whether the subject (e.g., a human subject) has suffered a mild, moderate, severe, or moderate-to-severe traumatic brain injury (TBI). In some aspects, when the amount of the biomarker in the sample is higher than the reference level of the biomarker (e.g., GFAP), the subject is determined to have a mild, moderate, severe, or moderate or severe TBI.

[0181] A. Assay

[0182] 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 prick blood sample, or a combination thereof. Generally, the assay involves performing an assay for GFAP. The assay involves contacting the biological sample with a cartridge having at least one magnetic immunosensor and at least one first specific binding partner comprising at least one anti-GFAP antibody, wherein the specific binding partner is immobilized on at least one magnetosensitive bead, and the bead is printed on the cartridge, as will be described herein. The GFAP assay described herein can be used alone or in combination with other assays that do not employ a cartridge comprising at least one magnetic immunosensor. Such assays include, for example, an assay for ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1).

[0183] Once one or more complexes are formed, at least one magnetic immunosensor or magnetic immunosensing device in the cartridge contained in a point-of-care device is used to magnetically capture and retain the beads containing the immobilized one or more complexes. In some aspects, any unbound sample that is not magnetically captured and retained on the magnetic immunosensor can be removed by washing the immunosensor.

[0184] 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.

[0185] 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 prick blood sample, or a combination thereof). In this aspect, the assay comprises contacting the sample with a cartridge (used in a point-of-care device), the cartridge comprising at least one magnetic immunosensor and: (i) at least one first specific binding partner comprising an anti-GFAP antibody that specifically binds to GFAP in the sample, wherein the at least one first specific binding partner is immobilized on at least one magneto-sensitive bead, and the magneto-sensitive bead is printed on the cartridge; and (ii) at least one second specific binding partner printed on the cartridge, which comprises a detectable label, to produce one or more complexes comprising the first specific binding partner-GFAP-second specific binding partner. After complex formation, the magneto-sensitive 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 off the magnetic immunosensor using conventional 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 detectable signal from the detectable label of the complexes indicates the amount of GFAP in the sample. It has been found that the assay for measuring GFAP as described herein exhibits at least a 5-fold increase in sensitivity compared to an assay in which the first specific binding partner is not immobilized on the magneto-sensitive bead and the beads containing the complexes are not magnetically captured and retained on at least one magnetic immunosensor in the cartridge contained in the point-of-care device. In some aspects, it has been found that the assay for measuring GFAP as described herein exhibits 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 in which the first specific binding partner is not immobilized on the magneto-sensitive bead and the beads containing the complexes are not magnetically captured and retained on at least one magnetic immunosensor in the cartridge contained in the point-of-care device.

[0186] In some aspects, the sample is obtained 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 obtained from the 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, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after an actual or suspected injury to the head.

[0187] 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 aspects, the assay can be a clinical chemistry assay such as photometry, spectrophotometry, absorbance measurement, fluorescence measurement, nephelometry, turbidimetry, potentiometry, and / or electrophoresis assay. In additional aspects, the assay can be a combination of an immunoassay and a clinical chemistry assay. In other aspects, the assay can be a single molecule detection assay.

[0188] In some aspects, at least one determination of GFAP is performed in about 4 to about 20 minutes. In some aspects, at least one determination of GFAP is each performed in about 4 minutes. In some aspects, at least one determination of GFAP is performed in about 5 minutes. In some aspects, at least one determination of GFAP is performed in about 6 minutes. In some aspects, at least one determination of GFAP is performed in about 7 minutes. In some aspects, at least one determination of GFAP is performed in about 8 minutes. In some aspects, at least one determination of GFAP is performed in about 9 minutes. In some aspects, at least one determination of GFAP is performed in about 10 minutes. In some aspects, at least one determination of GFAP is performed in about 11 minutes. In some aspects, at least one determination of GFAP is performed in about 12 minutes. In some aspects, at least one determination of GFAP is performed in about 13 minutes. In some aspects, at least one determination of GFAP is performed in about 14 minutes. In some aspects, at least one determination of GFAP is performed in about 15 minutes. In some aspects, at least one determination of GFAP is performed in about 16 minutes. In some aspects, at least one determination of GFAP is performed in about 17 minutes. In some aspects, at least one determination of GFAP is performed in about 18 minutes. In some aspects, at least one determination of GFAP is performed in about 19 minutes. In some aspects, at least one determination of GFAP is performed in about 20 minutes.

[0189] In some aspects, the subject has received a Glasgow Coma Scale score before or after the determination. In some aspects, based on the Glasgow Coma Scale score, the subject (e.g., a human subject) is suspected of having moderate, severe, or moderate-to-severe traumatic brain injury. In some aspects, a reference level of a biomarker (such as GFAP) is associated with a subject having moderate, severe, or moderate-to-severe traumatic brain injury. In some aspects, a reference level of a biomarker (such as GFAP) is associated with a Glasgow Coma Scale score of 9 - 13 (moderate TBI). In some aspects, a reference level of a biomarker (such as GFAP) is associated with a Glasgow Coma Scale score of 3 - 8 (severe TBI). In some aspects, a reference level of a biomarker (such as GFAP) is associated with a Glasgow Coma Scale score of 3 - 12 (moderate, severe, or moderate-to-severe TBI). In some aspects, based on the Glasgow Coma Scale score, the subject is suspected of having mild traumatic brain injury. In some aspects, a reference level of a biomarker (such as GFAP) is associated with a subject having mild traumatic brain injury. In some aspects, a reference level of a biomarker (such as GFAP) is associated with a Glasgow Coma Scale score of 13 - 15 (mild TBI).

[0190] Generally speaking, the reference level of a biomarker (such as GFAP) can also be used as a benchmark to evaluate the results obtained when determining a biomarker (such as GFAP) in a test sample. Generally speaking, when making such a comparison, the reference level of a biomarker (such as GFAP) is obtained by running or performing a specific assay a sufficient number of times and under appropriate conditions so that the presence, amount, or concentration of the analyte can be associated or correlated with a specific stage or endpoint of TBI or with a specific marker. Typically, the reference level of a biomarker (such as GFAP) is obtained by assaying a reference subject (or group of subjects). The measured biomarker (such as GFAP) can include fragments thereof, degradation products thereof, and / or enzymatically cleaved products thereof.

[0191] In some aspects, the reference level can be associated with a control subject (e.g., a human subject) who has not suffered a head injury.

[0192] In some aspects, after determining the amount of GFAP in a capillary blood sample using the methods described herein, a result is obtained. This result can be further processed. Specifically, this further processing involves selecting a conversion factor in order to compare the amount of GFAP in the sample with the amount of GFAP in venous blood. Specifically, the selected conversion factor 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 between the GFAP in venous blood and the capillary blood sample such that there is a simple ratio (i.e., a "static" correlation). The static correlation ratio assumes a constant ratio, which can be, for example, 1.0x, or 1.2x, or 1.5x, or even 0.8x, for converting a reading related to capillary blood to a reading of GFAP measured from the drawn venous blood.

[0193] The dynamic ratio is the ratio between the GFAP in venous blood and the capillary blood sample, which does not involve just a simple ratio (i.e., involves a non-linear or variable relationship), but depends on one or more factors such as sampling time, diffusion rate, etc. For example, for a biomarker, such as a biomarker of acquired brain injury (such as traumatic brain injury (TBI)), such as GFAP, one might expect that the ratio of GFAP sampled from venous blood to capillary blood might be time-dependent (i.e., or "dynamic"), and thus, being out of equilibrium means that the factor varies based on the time of sampling after the event. This means that the correlation may have a time-delay factor, and thus, for example, a longitudinal sampling design is needed to develop, for example, a "correlation table" that includes, for example, two factors ((magnitude of the ratio) and time) relative to the "event" and the amount in venous blood versus capillary blood.

[0194] In some aspects, the conversion factor (e.g., static correlation ratio, dynamic correlation ratio, or a combination of static and dynamic correlation ratios) can be selected based on: (a) the amount of GFAP in the sample; (b) the disease, disorder, condition, stage, or state 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) through (c).

[0195] For example, in some aspects, the conversion factor selected for GFAP can be the dynamic correlation ratio.

[0196] Alternatively, in other aspects, when the disease, disorder, condition, stage, or state associated with GFAP is ABI or TBI, the conversion factor selected is the dynamic correlation ratio.

[0197] In other aspects, 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.

[0198] Alternatively, in some aspects, the conversion factor for GFAP can be from about 1.2:1.0 (venous whole blood or plasma to capillary whole blood or plasma) to about 1.0:0.5 (venous whole blood or plasma to capillary whole blood or plasma). Alternatively, in some aspects, the conversion factor for GFAP is about 1.0:0.85 (venous whole blood or plasma to capillary whole blood or plasma). In other aspects, the conversion factor is about 1.0:0.8 (venous whole blood or plasma to capillary whole blood or plasma).

[0199] Once the conversion factor (e.g., static correlation ratio, dynamic correlation ratio, both static and dynamic correlation ratios) is selected, the process also involves normalizing the amount of GFAP in the capillary blood sample to the amount of GFAP in the 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 provide the normalized amount of GFAP in the sample.

[0200] In some aspects, the processing of the GFAP amount (e.g., the result) can be carried out by a processing system in conjunction with the computer process, the processing system including a computer processor and a non-transitory computer memory, which are configured to select a conversion factor to compare the GFAP amount in the sample with the GFAP amount in venous blood, and to normalize the GFAP amount in the sample with the GFAP amount in venous blood by applying the selected conversion factor to the GFAP amount in the sample.

[0201] Once the normalized GFAP amount in the capillary blood sample is obtained, this normalized result can be communicated (e.g., reported) for further analysis, interpretation, reading, processing, and / or display. The result can be communicated (e.g., reported) by a computer, in a document and / or spreadsheet, on a mobile device (e.g., a smart phone), on a website, in an email, or any combination thereof.

[0202] In some aspects, the result is communicated by, for example, displaying on an instrument. In other aspects, the result is displayed as indicating that the GFAP amount of the subject is elevated, not elevated, or that the determination for GFAP should be repeated.

[0203] As will be discussed in detail in Section 2(B), the systems and assays of the present disclosure utilize point-of-care devices. Point-of-care devices suitable for 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 include a user interface that can display assay results.

[0204] In some aspects, the results of GFAP (in some aspects, the normalized results) are communicated within about 4 minutes to about 40 minutes from the time of collecting the sample (such as from the time of injury or suspected injury). In other aspects, the results are communicated within about 4 minutes to about 30 minutes from the time of collecting the sample (e.g., the time of injury or suspected injury). In other aspects, the results are communicated within about 4 minutes to about 20 minutes from the time of collecting the sample (such as from the time of injury or suspected injury). In some aspects, the results are communicated within 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 12 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 of collecting the sample (such as from the time of injury or suspected injury).

[0205] In some aspects, the instrument contains software that performs one or more tasks, including carrying out the methods and algorithms described herein. In some aspects, the instrument contains software for automatically determining the next appropriate step in the methods and algorithms described herein. For example, the instrument can contain software for determining the amount or presence of an analyte of interest. The software can display this determination, such as on a graphical user interface.

[0206] In some aspects, the instrument stores software that instructs the processor to perform a given task. In some aspects, the software stores machine-readable instructions that instruct the processor to perform a given task. The machine-readable instructions can be one or more executable programs or a part of an executable program for a computer to execute. 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 disc, or memory associated with the processor. Alternatively, the entire program and / or a part thereof can alternatively be executed and / or embodied in firmware or dedicated hardware other than the processor. Additionally or alternatively, the process can be implemented by one or more hardware circuits (e.g., discrete and / or integrated analog and / or digital circuits, FPGA, ASIC, comparator, operational amplifier (op-amp), logic circuits, etc.) configured to perform the corresponding operations without executing software or firmware.

[0207] Machine-readable instructions can be stored in one or more of a compressed format, an encrypted format, a segmented format, a compiled format, an executable format, a packaged format, etc. The machine-readable instructions as described herein can be stored as data that can be used to create, manufacture, and / or generate machine-executable instructions (e.g., a portion of an instruction, code, a representation of code, etc.). For example, the machine-readable instructions can be segmented and stored on one or more storage devices and / or computing devices (e.g., servers). The machine-readable instructions may need to be installed, modified, adapted, updated, merged, supplemented, configured, decrypted, decompressed, unpacked, distributed, redistributed, compiled, etc. in order to be directly readable, interpretable, and / or executable by a computing device and / or other machine. For example, the machine-readable instructions can be stored in multiple parts, where the multiple parts are individually compressed, encrypted, and stored on separate computing devices, and where the parts form a set of executable instructions that implement a program such as that described herein when decrypted, decompressed, and combined.

[0208] In another embodiment, the machine-readable instructions can be stored in a state readable by a computer, but libraries (e.g., dynamic link libraries (DLLs)), software development kits (SDKs), application programming interfaces (APIs), etc. need to be added in order to execute the instructions on a particular computing device or other device. In another embodiment, it may be necessary to configure the machine-readable instructions (e.g., stored settings, data inputs, recorded network addresses, etc.) before the machine-readable instructions and / or the corresponding program can be executed in whole or in part. Accordingly, the disclosed machine-readable instructions and / or corresponding program are intended to cover such machine-readable instructions and / or programs regardless of the particular format or state of the machine-readable instructions and / or program when stored or otherwise placed or in transit.

[0209] 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 one of the following languages: C, C++, Java, C#, Perl, Python, JavaScript, HyperText Markup Language (HTML), Structured Query Language (SQL), Swift, etc.

[0210] Machine-readable instructions can be stored in a non-transitory computer and / or machine-readable medium (such as a hard disk drive, flash memory, read-only memory, optical disk, digital versatile disk, cache, random access memory, and / or any other storage device or storage disk for any duration (e.g., for an extended period of time, permanently, for a simple case, for temporary buffering, and / or for caching of information). As used herein, the term non-transitory computer-readable medium is expressly defined to include any type of computer-readable storage device and / or storage disk and to exclude propagated signals and to exclude transmission media).

[0211] In some aspects, the GFAP assays described herein are used in combination with assays for UCH-L1 that do not employ a cartridge including 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 further aspects, the reference level for UCH-L1 is about 400 pg / mL.

[0212] In some aspects, the reference level for UCH-L1 is from about 320 to about 400 pg / mL and the sample is obtained from the subject in about 24 hours or less. In other aspects, the reference level for UCH-L1 is about 360 pg / mL and the sample is obtained from the subject in about 24 hours or less. In other aspects, the reference level for UCH-L1 is about 400 pg / mL and the sample is obtained from the subject in about 24 hours or less.

[0213] In some aspects, the reference level for GFAP is from about 25 to about 40 pg / mL. In other aspects, the reference level for GFAP is about 30 pg / mL. In further aspects, the reference level for GFAP is about 35 pg / mL.

[0214] In some aspects, the reference level for GFAP is from about 25 to about 40 pg / mL and the sample is obtained from the subject in about 24 hours or less. In other aspects, the reference level for GFAP is about 30 pg / mL and the sample is obtained from the subject in about 24 hours or less. In other aspects, the reference level for GFAP is about 35 pg / mL and the sample is obtained from the subject in about 24 hours or less.

[0215] In some aspects, the reference level of UCH-L1 is about 360 pg / mL and the reference level of GFAP is about 30 pg / mL. In other aspects, the reference level of UCH-L1 is about 400 pg / mL and the reference level of GFAP is about 35 pg / mL. In additional aspects, the reference level of UCH-L1 is 360 pg / mL and the reference level of GFAP is about 30 pg / mL, and the sample is obtained from the subject in about 24 hours or less. In other aspects, the reference level of UCH-L1 is about 400 pg / mL and the reference level of GFAP is about 35 pg / mL, and the sample is obtained from the subject in about 24 hours or less.

[0216] In some aspects, 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 based on the assay results, determining whether the amounts (e.g., levels) of GFAP and UCH-L1 in the subject are elevated. In some aspects, the method includes determining whether the amounts of GFAP and UCH-L1 in the subject are elevated. In some aspects, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are elevated when the amount of GFAP in the sample is equal to or higher than 30 pg / mL and the level of UCH-L1 is below about 360 pg / mL and cannot be determined by or is not reported by the assay for UCH-L1. In some aspects, the method includes determining that the amounts of GFAP and UCH-L1 in a sample obtained from the subject are elevated when the level of GFAP is equal to or higher than about 30 pg / mL and the level of UCH-L1 is equal to or higher than about 360 pg / mL. In some aspects, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are elevated when the level of GFAP cannot be determined by or is not reported by the assay for GFAP and the level of UCH-L1 is equal to or higher than about 360 pg / mL.

[0217] In some aspects, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are not elevated. In some aspects, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are not elevated when the level of GFAP in the sample is below about 30 pg / mL and the level of UCH-L1 in the sample is below about 360 pg / mL.

[0218] In some aspects, the method includes determining that the assays for GFAP and UCH-L1 should be repeated. In some aspects, the method includes determining that the assays for GFAP and UCH-L1 or GFAP should be repeated when the amount of GFAP is below about 30 pg / mL and the level of UCH-L1 cannot be determined by the assay for UCH-L1 or is not reported by the assay for UCH-L1. In some aspects, the method includes determining that the assays for GFAP and UCH-L1 or GFAP should be repeated when the amount of GFAP cannot be determined by the assay or is not reported by the assay for GFAP and the level of UCH-L1 is below about 360 pg / mL. In some aspects, the method includes determining that the assays for GFAP and UCH-L1 or GFAP should be repeated when the amount of GFAP cannot be determined by the assay for GFAP or is not reported by the assay for GFAP and the level of UCH-L1 cannot be determined by the assay for UCH-L1 or is not reported by the assay for UCH-L1.

[0219] In some aspects, 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 based on the assay results, determining whether the amounts of GFAP and UCH-L1 in the subject are elevated. In some aspects, the method includes determining whether the amounts of GFAP and UCH-L1 in the subject are elevated. In some aspects, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are elevated when the amount of GFAP in the sample is equal to or higher than 35 pg / mL and the amount of UCH-L1 is below about 400 pg / mL and cannot be determined by the assay for UCH-L1 or is not reported by the assay for UCH-L1. In some aspects, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are elevated when the amount of GFAP is equal to or higher than about 35 pg / mL and the level of UCH-L1 is equal to or higher than about 400 pg / mL. In some aspects, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are elevated when the amount of GFAP cannot be determined by the assay for GFAP or is not reported by the assay for GFAP and the amount of UCH-L1 is equal to or higher than about 400 pg / mL.

[0220] In some aspects, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are not elevated. In some aspects, the method includes determining that the amounts of GFAP and UCH-L1 in the subject are not elevated when the amount of GFAP in the sample is below about 35 pg / mL and the amount of UCH-L1 in the sample is below about 400 pg / mL.

[0221] In some aspects, the method includes determining that the assays for GFAP and UCH-L1 or GFAP should be repeated. In some aspects, the method includes determining that the assays for GFAP and UCH-L1 or GFAP should be repeated when the amount of GFAP is below about 35 pg / mL and the amount of UCH-L1 cannot be determined by the assay for UCH-L1 or is not reported by the assay for UCH-L1. In some aspects, the method includes determining that the assays for GFAP and UCH-L1 or GFAP should be repeated when the amount of GFAP cannot be determined by the assay for GFAP or is not reported by the assay for GFAP and the amount of UCH-L1 is below about 400 pg / mL. In some aspects, the method includes determining that the assays for GFAP and UCH-L1 or GFAP should be repeated when the amount of GFAP cannot be determined by the assay for GFAP or is not reported by the assay for GFAP and the amount of UCH-L1 cannot be determined by the assay for UCH-L1 or is not reported by the assay for UCH-L1.

[0222] In some aspects, 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 a subject when the level of GFAP or GFAP and UCH-L1 in the subject is elevated. For example, in some aspects, the method further includes performing a head CT scan on a subject when the level of GFAP or GFAP and UCH-L1 in the subject is elevated. As another example, in some aspects, the method further includes performing an MRI procedure on a subject when the level of GFAP or GFAP and UCH-L1 in the subject is elevated. In some aspects, the method further includes performing a head CT scan and an MRI procedure on a subject when the level of GFAP or GFAP and UCH-L1 in the subject is elevated.

[0223] B. Point-of-care device including a magnetic immunosensor

[0224] As previously mentioned herein, the systems and assays of the present disclosure utilize point-of-care devices. Exemplary 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.

[0225] The point-of-care device used in the systems and assays of the present disclosure contains at least one cartridge, which includes at least one magnetic immunosensor or magnetic immunosensing device. In some aspects, the at least one magnetic immunosensor or magnetic immunosensing device includes a base sensor or sensing electrode. In some aspects, the base sensor or sensing electrode can be on a substantially planar chip (e.g., immunosensor or sensor chip), where the sensing electrode is in a conduit for receiving a biological sample (e.g., a blood sample such as a venous blood sample, a capillary blood sample, a finger stick blood sample, or a combination thereof) and is mixed with magnetosensitive beads that can be attracted by a magnet or responsive to a magnetic field.

[0226] A high-field magnet (e.g., a permanent magnet or an electromagnet) is positioned close to, incorporated in, or on the chip (e.g., on top of the chip or beneath or below the chip) to attract the magnetosensitive beads in the conduit substantially close to the sensing electrode. This magnetic region serves 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 washing of the electrode. As detailed 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 an analyte (e.g., GFAP) in the sample. In some aspects, the at least one first specific binding partner is an anti-GFAP antibody. In another aspect, the at least one first specific binding partner is an anti-UCH-L1 antibody.

[0227] In additional aspects, in addition to the magnetic immunosensor or magnetic immunosensing device, the cartridge further includes one or more regions that contain: (a) one or more beads (e.g., paramagnetic beads) coated with at least one first specific binding partner (e.g., an anti-GFAP antibody), the first specific binding partner specifically binding an analyte (e.g., GFAP), the beads being printed or dispensed on the cartridge and dried using conventional techniques known in the art; and (b) at least one second specific binding partner (e.g., an anti-GFAP antibody) labeled with a detectable label, which is printed or dispensed on the cartridge and dried using conventional techniques known in the art. The one or more beads containing at least one first specific binding partner (e.g., an anti-GFAP antibody) and the at least one second specific binding partner (e.g., an anti-GFAP antibody) labeled with a detectable label can be printed and dried in the same region on the cartridge, or alternatively, in regions adjacent to or near each other.

[0228] In some aspects, during the determination, the printed beads containing at least one first specific binding partner (e.g., anti-GFAP antibody) and the printed at least one second specific binding partner labeled with a detectable label (e.g., anti-GFAP antibody) are reconstituted by wetting or dissolving the printed area with at least one diluent, buffer, or other liquid. Once reconstituted, a sample can be added and mixed with the beads and the detectable label. In other aspects, the sample can be used to reconstitute the printed beads and label, 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 at least one second specific binding partner labeled with a detectable label, a complex of the first specific binding partner-coated beads - analyte - detectable-labeled second specific binding partner is formed. Once formed, the beads containing the complex are captured and retained using a magnet contained in or on the cartridge, and the amount of analyte (e.g., GFAP) is determined using conventional techniques known in the art. In some aspects, the magnet can be positioned on top of the chip. In other aspects, the magnet can be positioned below or beneath the chip.

[0229] In addition, in some other aspects, a point-of-care device includes a reading device or reader and a single-use cartridge that contains a magnetic immunosensor or immunosensing device and all other assay components for analyzing an analyte in a biological sample.

[0230] (1) High-field magnet and magnetic layer

[0231] In some aspects, at least one magnetic immunosensor includes sensing electrodes on a substantially planar chip and has a high-field magnet, such as a permanent magnet or an electromagnet, positioned close to (e.g., beneath) the chip or associated with the chip. The magnetic immunosensor can provide a field greater than about 0.1 tesla and has an event horizon (e.g., a point of no return) that can effectively map beads in the region of the sensing electrodes within a range of about 0.05 mm to about 5 mm.

[0232] High-field magnets, such as permanent magnets or electromagnets, include any material that provides a high magnetic field (e.g., greater than about 0.1 tesla, greater than 0.4 tesla, or greater than 1 tesla). For example, the magnetic field can be measured as the remanent field on a substantially flat surface area of the magnet. Examples of materials that can be used include neodymium iron boron alloy (NdFeB) or Nd2Fe 14 B, but other materials can also be used. For example, high-field permanent magnets can include ferrite or alnico (AlNiCo) magnets, which typically exhibit fields of 0.1 to 1 tesla. Other high-field permanent magnets containing rare earth element alloys (e.g., neodymium alloys and samarium cobalt (SmCo) alloys) exhibit fields exceeding 1 tesla (e.g., greater than 1.2 tesla or greater than 1.4 tesla).

[0233] Rare earth magnets are generally brittle and also prone to corrosion. Therefore, these materials are often electroplated or coated to prevent cracking and fragmentation. In addition, the Curie point of rare earth magnets is substantially higher than the temperatures encountered in the assays described herein, and the assays can be performed at ambient temperature up to about 50°C. In some aspects, the assay is held 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.

[0234] In another aspect, the high-field magnet includes an electromagnet in which the magnetic field is generated by the flow of an electric current. The electric current can be provided by a reader in a point-of-care device in which a magnetic immunosensor or magnetic immunosensing device is inserted and is in electrical contact therewith.

[0235] The magnetic immunosensor or magnetic immunosensing device includes a sensing electrode on a substantially planar chip and a bulk high-field permanent magnet positioned adjacent to the electrode (e.g., below or on the opposite side of the chip). In some aspects, the bulk high-field permanent magnet is positioned within the housing of a point-of-care device (e.g., cut out or grooved in a plastic cartridge). In other aspects, the bulk high-field permanent magnet is positioned within the base of a plastic cartridge housing (e.g., non-coplanar with the sensing electrode). In other aspects, the magnet is positioned near or within the reading device or reader of a point-of-care device.

[0236] In some aspects, the bulk high-field permanent magnet is substantially cylindrical, having a diameter in the range of about 0.1 mm to about 5 mm and a length of about 0.1 mm to about 5 mm, and is positioned to create an event horizon in a conduit suitable for bead capture within a short period of time (e.g., 1 - 5 minutes). The conduit generally has a height of about 0.2 mm to about 5 mm and a width of about 0.2 mm to about 5 mm, and a uniform or non-uniform cross-sectional area. In other embodiments, the bulk magnet shape can be square, rectangular, oval, sheet-like, conical, spherical, sub-spherical, or other shapes.

[0237] On the other hand, a magnetic immunosensor or magnetic immunosensing device includes a sensing electrode on a substantially planar chip. The electrode is positioned in a conduit for receiving a sample that has been mixed with one or more first specific binding partners (e.g., antibodies) immobilized on magnetosensitive beads and one or more labeled second specific binding partners, as well as a magnetization layer (e.g., a microfabricated magnetic layer). The magnetization (or magnetic) layer can be on the chip (e.g., positioned above the chip, directly attached, coated, or patterned on any surface of the chip) or embedded in the chip (e.g., positioned within the chip, integral with the chip). This configuration attracts the magnetosensitive beads to be substantially close to the electrode and retains them substantially at the electrode during removal of unbound sample and washing of the electrode.

[0238] The magnetization layer can be formed from a mobile magnetic composition (e.g., a slurry) that contains a material capable of maintaining a high-field permanent magnetic field (e.g., an NdFeB alloy) in the form of particles in a fixed or supporting matrix (e.g., polyimide, polyvinyl alcohol (PVA), or a thermoplastic equivalent). This slurry is not limited by viscosity and can include any viscosity known to be suitable in the art. In some aspects, the viscosity of the mobile magnetic composition ranges from 0.3 to 300,000 CPS, such as 100 to 100,000 CPS or 1000 to 10,000 CPS. In another aspect, the average particle size of the magnetic particles in the slurry is from 0.01 μm to 100 μm, such as 0.1 μm to 10 μm or 3 μm to 7 μm.

[0239] In addition to polyimide, PVA, and thermoplastic polyimide, two-part chemically cured epoxy resins, kapton, etc. can also be used as the supporting matrix for fixing the magnetic particles to the wafer. The method of curing the matrix can be based on a photoinitiated, thermally initiated, or chemically initiated process. In certain embodiments, the fixing matrix contains other photoformable matrix materials.

[0240] As discussed above, the slurry can be applied to multiple locations in or on the immunosensing device (e.g., the front or back of a wafer or chip, an electrode, a housing, a reader, etc.). For example, in some aspects, the high-field permanent magnetic material is applied to the substantially planar chip in a patterned manner (e.g., using a mask). In certain aspects, the high-field permanent magnetic material is also applied to the microfabricated sensing electrode. In other aspects, the slurry is applied in a layer beneath the sensing electrode.

[0241] Before the application process, the slurry may or may not be magnetized. However, after the deposition step, the magnetic layer can be magnetized to provide directionality to the field.

[0242] (2) Sensing electrode

[0243] The sensing clicks can use any techniques suppressed in the art, such as those described in U.S. Patent Nos. 5,200,051 and 7,419,821, to be microfabricated (e.g., gold arrays for measuring current) on a substantially planar chip (e.g., a silicon wafer), the contents of which patents are incorporated herein by reference.

[0244] (3) Magnetosensitive beads

[0245] In some aspects, a biological sample (e.g., a blood sample) is mixed with magnetosensitive beads. The magnetosensitive beads can comprise any materials known in the art that are amenable to being moved by the magnets (e.g., permanent magnets or electromagnets) utilized in the devices of the present disclosure or in motion with the devices of the present disclosure. Thus, the terms "magnetic" and "magnetosensitive" with respect to the beads can be used interchangeably.

[0246] In some aspects, the beads include a magnetic core, which can be fully or partially coated with a coating material. The magnetic core can comprise ferromagnetic, paramagnetic, or superparamagnetic materials. In some aspects, the magnetosensitive beads comprise a ferrite magnetic core and an external polymer coating. However, the magnetic core can comprise one or more of the following: Fe, Co, Mn, Ni, metals containing one or more of these elements, ordered alloys of these elements, crystals containing these elements, magnetic oxide structures (such as ferrites), and combinations thereof. In other aspects, the magnetic core can comprise magnetite (Fe3O4), maghemite (γ-Fe2O3), or divalent metal ferrites provided by the formula Me 1-x OFe3+xO3, where Me is, for example, Cu, Fe, Ni, Co, Mn, Mg, or Zn, or combinations of these materials, and where x ranges from 0.01 to 99.

[0247] Suitable materials for the coating include synthetic and biological polymers, copolymers, and polymer blends, as well as inorganic materials. The polymer materials can include various combinations of polymers of acrylates, siloxanes, styrenes, acetates, alkylene glycols, olefins, alkylene oxides, parylene, lactic acid, and glycolic acid. The biological polymer materials include starches 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, carboxylates, sulfonates, phosphates, ferrites, phosphonates, and oxides of Group IV elements of the periodic table.

[0248] In other aspects, the magnetic beads include non-magnetic substrate beads, which are formed, for example, from materials selected from the group consisting of polystyrene, polyacrylic acid, and dextran, on which a magnetic coating is placed.

[0249] Taking into account the requirements for the dispersion of the magnetosensitive beads, any magnetosensitive beads of the correct size that can be positioned with the high-field magnet can be used. In some aspects, at least 50 wt% (e.g., at least 75 wt%) of the magnetosensitive beads remain on the electrode surface. In some aspects, the average particle size of the magnetosensitive beads can range from 0.01 μm to 20 μm, such as 0.1 μm to 10 μm, 0.1 μm to 5 μm, or 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., beads) as determined by methods well known in the art, such as the diameter of spherical particles. The particle size distribution of the magnetosensitive beads can be unimodal, but multimodal distributions can also be used. Although spherical magnetosensitive beads can be used, other bead shapes and structures, such as oval, sub-spherical, cylindrical, and other irregularly shaped particles, are also within the meaning of the terms "beads" and "particles" as used herein.

[0250] Commercial sources of magnetosensitive beads include Life Technologies TM of Invitrogen TM (Carlsbad, Calif., U.S.A.), Ademtech (Pessac, France), Chemicell GmbH (Berlin, Germany), Bangs Laboratories, Inc. TM (Fishers, Ind.) and Seradyn, Inc. (Indianapolis, Ind.). Many commercially available products incorporate surface functionalization, which can be used to immobilize antibodies (e.g., IgG) on the bead surface. Exemplary functionalizations include carboxyl, amino, or streptavidin-modified magnetosensitive beads.

[0251] In some aspects, the magnetosensitive beads are coated with an antibody, such as an anti-GFAP antibody. Coating the beads with an antibody immobilizes the antibody on the beads.

[0252] In certain aspects, the magnetosensitive beads are deposited as a suspension in a mixture of lactitol and DEAE-dextran (such as those supplied by Advanced Enzyme Technologies (Pontypool, Great Britain)) in a suitable area of the magnetic immunosensing device. Evaporation of the solvent (usually water) produces a vitreous deposit in which the beads are immobilized. Lactitol / DEAE-dextran localizes the beads in a mechanically and biochemically stable state within the device, but it also dissolves rapidly upon contact with the sample.

[0253] In various aspects, the beads are movable so as to be able to interact with the analyte. After binding to GFAP, the beads are concentrated at the electrode using magnetic force for measurement, thereby positioning the magnetosensitive beads at the electrode for measuring current for signal detection.

[0254] (4) Fabrication of the magnetic immunosensor or magnetic immunosensing device

[0255] The magnetic immunosensor or immunosensing device can be fabricated 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. Then a titanium / tungsten layer is sputtered onto the oxide layer, preferably having a thickness of about to about After that, a gold layer is sputtered, with a thickness of to Most preferably, the thickness is about Next, photoresist is spin-coated onto the wafer, dried, and baked. Then the surface is exposed using a contact mask, the latent image is developed, and the wafer is exposed to a gold etchant. The patterned gold layer is coated with a photo-definable polyimide, baked appropriately, exposed using a contact mask, developed, washed in an oxygen plasma, and preferably imidized at 350 °C for about 5 hours. This leaves a large number of electrode openings in the polyimide layer in the form of a square array. In some embodiments, the diameter of the square array is, for example, about 2 μm to about 100 μm, about 5 μm to about 15 μm, or about 7 μm, and the pitch 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, having a diameter of, for example, about 50 μm to about 1000 μm, about 100 μm to about 300 μm, or about 300 μm.

[0256] 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, a sensor is positioned within a conduit for receiving a sample, and a high-field magnet (e.g., a permanent magnet or an electromagnet) is positioned directly below the sensor, such as in its central region. In another aspect, the high-field magnet can be positioned above the sensor region of the conduit. These components can be in fixed positions within the instrument housing or adapted to an actuator capable of moving into and out of position relative to the immunosensor and the conduit. One or more high-field magnets can be used to attract magnetically sensitive beads (e.g., substantially near the sensor) within the conduit and retain them within the region of the sensor during removal of the sample and washing of the sensor to remove unbound or partially adsorbed reagents. As described above, the magnetic beads are coated with an antibody (e.g., an anti-GFAP antibody) to detect GFAP in the sample.

[0257] C. System

[0258] 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), which utilizes at least one magnetically sensitive bead. Additionally, the system utilizes a point-of-care device that includes at least one cartridge, the cartridge including at least one magnetic immunosensor or immunosensing device as described in Section 2(B). The point-of-care device determines the amount of GFAP in the sample by magnetically capturing and retaining beads containing the complex on at least one magnetic immunosensor and evaluating the signal from the complex, wherein the amount of the detectable signal from the detectable label indicates the amount of GFAP in the sample.

[0259] In another aspect, it has been found that when the system uses the GFAP assay described in Section 2A, the assay exhibits an increase in sensitivity of at least 5-fold compared to an assay in which the first specific binding partner is not immobilized on the magnetosensitive beads and the beads containing the complex are not magnetically captured and retained on at least one magnetic immunosensor in a cartridge contained in a point-of-care device. In some aspects, it has been found that the assay for measuring GFAP as described in Section 2A exhibits an increase in sensitivity of at least 6-fold, 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 compared to an assay in which the first specific binding partner is not immobilized on the magnetosensitive beads and the beads containing the complex are not magnetically captured and retained on at least one magnetic immunosensor in a cartridge contained in a point-of-care device.

[0260] 3. Plasma separation device

[0261] As previously described, in some aspects of the systems and assays described herein, a device that serves as 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 a top layer that is on the side of, above, or on top of the hydrophobic layer. The hydrophobic layer can include or be constructed of at least one hydrophobic material. The hydrophobic material can be a diaphragm, membrane, fabric, fiber, filter, microfilm, sieve, mesh, or any combination thereof. In one aspect, the hydrophobic layer is a diaphragm or a membrane. Hydrophobic diaphragms or membranes that can be used are known in the art. Specifically, diaphragms or membranes such as those available from Adhesive Research (Glen Rock, PA), 3M (Minneapolis, Minnesota), and / or Tesa SE (Norderstadt, Germany) can be used.

[0262] The hydrophobic layer includes at least one microchannel that has a first end and a second end and that defines a path for capillary fluid flow of a processed blood sample or blood product (e.g., plasma). In some aspects, at least one microchannel extends longitudinally from the first end along a portion of the hydrophobic layer to an opening at the second (e.g., opposite) end of the microchannel. In other aspects, 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 aspects, the microchannel contains a first opening that is connected to the first end of the microchannel. In these aspects, the processed blood or blood product (e.g., plasma) can flow from the first opening into the first end of the microchannel and then to a second opening at the second end of the microchannel.

[0263] The opening at the second end of the microchannel allows the treated blood or blood product (e.g., plasma) to flow out of the device. For example, if the treated fluid is blood, the plasma can flow from the first end to the second opening at the second (e.g., opposite) end through the capillary fluid end. A collection device or other means can be used to collect the plasma at the second (e.g., opposite) end, or if the device is operably connected, removably coupled, or in fluid communication with another device (such as a sample analysis cartridge (such as a microfluidic cartridge)), it allows for continued flow directly on or into the device for further processing and / or analysis.

[0264] The microchannel can have any length. In some aspects, the length of the microchannel is less than about 80 mm. In other aspects, the length of at least one microchannel is about 70 mm, the length of at least one microchannel is 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 aspects, the width of at least one microchannel is 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.

[0265] In addition, at least one microchannel can be located anywhere on the hydrophobic layer. For example, at least one microchannel can be located at the center of the hydrophobic layer, it can be slightly offset from the center of the hydrophobic layer, or it can be located at or near the side or edge of the hydrophobic layer.

[0266] In additional aspects, the thickness of the hydrophobic layer can be from about 50 to about 200 microns. In other aspects, the thickness of the hydrophobic layer can be from about 100 to about 200 microns. In some aspects, the thickness of the hydrophobic layer can be from about 100 to about 150 microns.

[0267] The device also includes a top layer that is lateral to, above, or on top of the hydrophobic layer. In some aspects, the top layer adheres to the hydrophobic layer. In other aspects, the surface of the top layer facing the hydrophobic layer comprises a material coated with a hydrophilic material or is coated with a hydrophilic material. The hydrophilic material can be an adhesive, a separator, a membrane, a fabric, a fiber, a filter, a micro-membrane, a sieve, a mesh, or any combination thereof. In other aspects, the entire top layer is made of or constructed from a hydrophilic material such as a separator, a membrane, a fabric, a fiber, a filter, a micro-membrane, a sieve, a mesh, or any combination thereof. For example, in one aspect, the top layer is a separator or a membrane. In some aspects, the separator or membrane is not constructed from a hydrophilic material but is coated with a hydrophilic material. The portion of the top layer coated with the hydrophilic material faces the hydrophobic layer. In another aspect, the entire separator or membrane is made of or constructed from a hydrophilic material. Hydrophilic separators or membranes that can be used are known in the art. Examples of hydrophilic membranes that can be used are the 9984 Diagnostic Microfluidic Surfactant Free Hydrophilic Film available from 3M (Minneapolis, MN), the Kemafoil H Hydrophiliccoated polyester film available from Coveme (S. Lazzaro di Savena, Italy), and the Tesa 62580 Hydrophiliccoated polyester film available from Tesa SE (Norderstadt, Germany). In additional aspects, the thickness of the top layer can be from about 50 to about 200 microns. In other aspects, the thickness of the top layer can be from about 100 to about 200 microns. In some aspects, the thickness of the top layer can be from about 100 to about 150 microns.

[0268] When a blood sample or blood product is placed on the top layer of the device, the blood sample or product flows through the top layer. As it does so, 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 hydrophilic material, allowing the plasma to continue flowing 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 of the channel to an opening at the second (e.g., opposite) end. A collection device or other means can be used to collect the plasma, or, if the device is operably connected, removably coupled, or in fluid communication with another device (such as a sample analysis cartridge (e.g., a microfluidic cartridge)), allowing the plasma to continue flowing directly on or into the device for further processing and analysis.

[0269] In another aspect, the device optionally includes a bottom layer. The bottom layer is on the side of or below or beneath the hydrophobic layer. In these aspects, the device includes at least three layers: a top layer, a hydrophobic layer, and a bottom layer. In some aspects, the bottom layer adheres to the hydrophobic layer. In additional aspects, the top layer and the bottom layer each adhere to the hydrophobic layer.

[0270] In further aspects, the surface of the bottom layer facing the hydrophobic layer comprises a material coated with a hydrophilic material or is coated with a hydrophilic material. The hydrophilic material can be an adhesive, a separator, a membrane, a fabric, a fiber, a filter, a micro-membrane, a sieve, a mesh, or any combination thereof. In other aspects, the entire bottom layer is made of or constructed from a hydrophilic material such as a separator, a membrane, a fabric, a fiber, a filter, a micro-membrane, a sieve, a mesh, or any combination thereof. For example, in one aspect, the bottom layer is a separator or a membrane. In some aspects, the separator or the membrane 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 separator or membrane is made of or constructed from a hydrophilic material. Hydrophilic separators or membranes that can be used are known in the art. Examples of hydrophilic membranes that can be used are the 9984 Diagnostic Microfluidic Surfactant Free Hydrophilic Film available from 3M (Minneapolis, MN), the Kemafoil H Hydrophiliccoated polyester film available from Coveme (S. Lazzaro di Savena, Italy), and the Tesa 62580 Hydrophiliccoated polyester film available from Tesa SE (Norderstadt, Germany).

[0271] In additional aspects, the thickness of the bottom layer can be from about 50 to about 200 microns. In other aspects, the thickness of the bottom layer can be from about 100 to about 200 microns. In some aspects, the thickness of the bottom layer can be from about 100 to about 150 microns.

[0272] In further aspects, the device can contain a protective film, which is on the side of or below or beneath the bottom layer. In some aspects, the protective film adheres to the bottom layer. The protective film protects the device from moisture and / or other contamination. The protective film can be a plastic film such as a self-adhesive plastic film, cardboard with an adhesive, a plastic sheet with an adhesive, or a combination thereof.

[0273] In other aspects, when the device includes a top layer, a hydrophobic layer, and a bottom layer, the combined thickness of the three layers is between about 100 and about 600 microns. In other aspects, the combined thickness of the three layers is between about 150 and about 600 microns. In other aspects, the combined thickness of the three layers is between about 200 and about 600 microns. In other aspects, the combined thickness of the three layers is between about 150 and about 500 microns. In additional aspects, the combined thickness of the three layers is between about 200 and about 500 microns.

[0274] In addition, in some aspects, when the device includes a top layer, a hydrophobic layer, and a bottom layer, the top layer and the bottom layer can be made of the same material. Alternatively, the top layer and the bottom layer can be made of different materials. For example, the top layer and the bottom layer can be made or constructed of the same hydrophilic material or different hydrophilic materials. Alternatively, the top layer and the bottom layer can be made or constructed of non-hydrophilic materials, but are coated with a hydrophilic material on the surface of the layer facing the hydrophobic layer. Additionally, either the top layer or the bottom layer can be made or constructed of a non-hydrophilic material and coated with a hydrophilic material on the surface facing the hydrophobic layer, and the other layer is made entirely of a hydrophilic material.

[0275] In some aspects, the top layer includes a sample inlet, and a blood sample or blood product is placed in the inlet to initiate sample processing through the device. 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 aspects, a hydrophobic transfer material (such as, for example, a transfer belt) can surround the sample inlet. The hydrophobic transfer material helps prevent the blood sample or blood product from being sucked away or removed from the sample inlet area.

[0276] When the top 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 can have a different shape. In some aspects, when the device includes two layers (e.g., a top layer with a sample inlet and a hydrophobic layer), one or more openings can be made in the hydrophobic layer. In other aspects, when the device includes three layers, one or more openings can be made in the hydrophobic layer, but not in the bottom layer. In other aspects, when the device includes 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 located directly below the sample inlet.

[0277] For example, Figure 8A device for plasma separation is shown, which comprises a hydrophilic top layer and a hydrophobic layer containing microchannels having a first end and a second end. The top layer adheres to the hydrophobic layer. The hydrophilic top 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, it flows from the opening into 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 allowed to flow directly onto or into a sample analysis cartridge.

[0278] Figure 9 Another embodiment of a device for plasma separation is shown. In this embodiment, the device includes a hydrophilic top layer, a hydrophobic layer, and a hydrophilic bottom layer. The hydrophilic top layer adheres to the hydrophobic layer, and the bottom layer adheres to the hydrophobic layer. The hydrophilic top layer includes a hydrophobic transfer band 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 the microchannel. When plasma reaches the first opening on the hydrophobic layer, it flows into 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 allowed to flow directly onto or into a sample analysis cartridge.

[0279] In a further aspect, the sample inlet may further include a separation diaphragm (such as a plasma separation diaphragm). In some aspects, the separation diaphragm is a glass fiber material, such as a membrane, film, fabric, fiber, filter, micro-membrane, sieve, mesh, or any combination thereof. Additionally, the separation diaphragm 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 diaphragm may have the same shape as the sample inlet. In other aspects, the separation diaphragm may have a different shape from the sample inlet.

[0280] The separation membrane can be made of any material known in the art that can be used to separate 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 captured in the pores and / or fibers of the membrane or material, allowing the plasma to continue flowing through the sample inlet and onto the first opening on the hydrophobic layer. The first opening on the hydrophobic layer is connected to a microchannel such that when the plasma reaches the first opening on the hydrophobic layer, it flows into the first end of the microchannel and continues flowing to the second (e.g., opposite) end of the microchannel at the second opening. Once the plasma reaches the second opening at the second end of the microchannel, the plasma can be collected using a collection device or other means, or, if the device is operably connected, removably coupled, or in fluid communication with another device (such as a sample analysis cartridge (e.g., a microfluidic cartridge)), it is allowed to continue flowing directly onto or into the device for further processing and analysis.

[0281] In some aspects, once the plasma reaches the first opening, the second opening, or both the first and second openings of the microchannel, a pump (such as a gas pump, a foam ring pump, a foil bubble cap pump, a membrane bubble cap pump, or any combination thereof) can be used to direct and / or disperse the plasma in the microchannel. In some aspects, a pump can be used to direct and / or disperse the plasma from the first end of the microchannel to the second end. In other aspects, a pump can be used to direct and / or disperse the plasma from the second end of the microchannel to another device (such as a sample analysis cartridge (e.g., a microfluidic cartridge)). The pump can be connected at any point on the microfluidic channel, such as at the first opening, along the side, near the second end, or any combination thereof.

[0282] In some aspects, the separation membrane can be positioned or placed above, below, or inside the sample inlet. For example, in some aspects, the separation membrane can be cut to a size larger than the sample inlet and simply placed on top of the sample inlet. Alternatively, the separation membrane can be cut to a size the same as or slightly smaller than the sample inlet and placed inside the inlet. Additionally, the separation membrane can be placed below the sample inlet and attached or adhered by any means known in the art, such as by an adhesive, glue, etc.

[0283] When the hydrophobic layer and / or the underlying layer contains one or more openings, the hydrophobic layer and / or the underlying layer may further contain one or more separation diaphragms positioned or placed above, below, or within the openings in the layer. The separation diaphragm may be constructed of the same material as the sample inlet or may be constructed of a different material. In another aspect, the separation diaphragm may be positioned or placed above, below, or within one or more openings. For example, in some aspects, the separation diaphragm may be cut to a size larger than the opening and simply placed on top of the opening. Alternatively, the separation diaphragm may be cut to a size the same as or slightly smaller than the opening and placed within the inlet. Additionally, the separation diaphragm may be placed below the opening and attached or adhered by any means known in the art such as by an adhesive, glue, etc.

[0284] In another aspect, one or more hydrophilic meshes or hydrophilic membranes may be on the sides of the separation diaphragm at the sample inlet and / or at one or more openings. For example, the hydrophilic mesh or hydrophilic membrane may be placed above or on top of the separation diaphragm to facilitate the dispersion of a blood sample or blood product into the device. Alternatively, the hydrophilic mesh or hydrophilic membrane may be located below or beneath the separation diaphragm to help facilitate the continued movement of the blood sample or blood product through to the hydrophobic layer and the first opening connected to the first end of the microchannel during processing.

[0285] In some aspects, the device further includes an upper substrate material that includes a sample inlet. The upper substrate material is on the side of the top layer or above or on top of it. In some aspects, the top layer adheres to the upper substrate. The upper substrate material may be a diaphragm, a membrane, a fabric, a fiber, a filter, a micro-membrane, a sieve, 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.

[0286] The upper substrate material includes a sample inlet into which a blood sample or blood product is placed to initiate sample processing. The sample inlet may have any shape. For example, the sample inlet may be circular, oval, rectangular, square, triangular, or any combination thereof. In some aspects, a hydrophobic transfer material (such as, for example, a transfer tape) may surround the sample inlet. The hydrophobic transfer material helps prevent the blood sample or blood product from being sucked away or removed from the sample inlet area.

[0287] In another aspect, the sample inlet may include a separation diaphragm. In some aspects, the separation diaphragm includes a fiberglass material, such as a diaphragm, a membrane, a fabric, a fiber, a filter, a micro-membrane, a sieve, a mesh, or any combination thereof. The separation diaphragm may be made of any material known in the art that can be used to separate a blood sample or blood product into its components (such as, for example, plasma). The function of the separation diaphragm in the upper substrate is the same as that of the separation diaphragm used in the sample inlet of the top layer.

[0288] In some aspects, the separation membrane can be positioned or placed above, below, or within the sample inlet. For example, in some aspects, the separation membrane can be cut to a size larger than 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 sample inlet and placed within the inlet. Additionally, the separation membrane can be placed below the sample inlet and attached or adhered by any means known in the art such as by an adhesive, glue, etc.

[0289] When there is an upper substrate and a sample inlet, one or more openings are made in each of the top layer and the hydrophobic layer or in each of the top 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 aspects, the shape of the opening is the same as that of the sample inlet. In other aspects, the shape of the opening is different from that of the sample inlet. In some aspects, when the device includes an upper substrate, one or more openings can be made in the top layer and the hydrophobic layer but not in the bottom layer. In other aspects, one or more openings can be made in the top layer, the hydrophobic layer, and the bottom layer. Each opening in one or more of the layers can be located directly below the sample inlet. One or more openings in each of the top layer, the hydrophobic layer, and / or the bottom layer can also include one or more separation membranes located above, below, or within the openings in the layer. The separation membrane can be constructed of the same material as the sample inlet or can be constructed of a different material. Additionally, one or more hydrophilic meshes or membranes can be on the sides of the separation membrane at the sample inlet and / or at one or more of the openings. For example, the hydrophilic mesh or hydrophilic membrane can be located above or below the separation membrane as described previously herein.

[0290] In another aspect, the top layer, the hydrophobic layer, the bottom layer, the hydrophilic mesh or hydrophilic membrane, the separation membrane, or any combination thereof is ubiquitous for any analyte or is specific for an analyte or a class of analytes.

[0291] In another aspect, the device further includes at least one aggregating agent that aggregates red blood cells to form red blood cell aggregates, improves separation, and produces cleaner plasma. In some aspects, in some embodiments, the aggregating agent is coated on or incorporated into one or more of the top layer, the hydrophobic layer, the bottom layer, the upper substrate, the separation membrane, the hydrophilic mesh or hydrophilic membrane, 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.

[0292] In another aspect, the top layer, hydrophobic layer, bottom layer, upper substrate, separation diaphragm, hydrophilic mesh or hydrophilic membrane, or any combination thereof is coated with a coating, such as a surfactant, hydrophilic coating, or any combination thereof, to improve or increase the rate or speed at which plasma and / or serum separates from whole blood when passing through any layer, substrate, diaphragm, mesh, membrane, or any combination thereof.

[0293] The plasma generated using the devices disclosed herein may not be pure plasma, but rather plasma depleted of one or more blood components (e.g., red blood cells, white blood cells, platelets, and combinations thereof). In some aspects, the plasma contains about 5 volume % or less 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 other aspects, the plasma contains about 3% or less by volume of red blood cells. In other aspects, the plasma contains about 2% or less by volume of red blood cells. In additional aspects, the plasma contains about 1% or less by volume of red blood cells.

[0294] Another aspect relates to an apparatus. In one aspect, the apparatus includes the devices previously described herein and at least one sample analysis cartridge (e.g., a microfluidic cartridge) having a sample application area for applying a test sample. In one aspect, the device is configured to have a sample analysis cartridge such that the microchannel is operably connected, removably coupled, or in fluid communication with the sample application area. Specifically, as Figure 11 shown, the second end of the microchannel can be operably connected, removably coupled, or in fluid communication with the sample application area of the sample analysis cartridge. In another aspect, the device is included or incorporated as part of a clip, such as a moby clip, extended moby clip, etc., and is configured to have a sample analysis cartridge, coupled or integrated therewith, such that the microchannel is operably connected, removably coupled, or in fluid communication with the sample application area.

[0295] In another aspect, the plasma separation device includes a pre-evacuated container or tube having an inlet end and an outlet end, each end being closed. There is a pressure differential between the inlet end and the outlet end of the container or tube. The inlet end of the container or tube includes a cap or septum that can be pierced by a needle or blood collection needle assembly used to obtain a whole blood sample from a subject. The outlet end includes a serum receiving chamber (e.g., a filtrate container) that receives the serum and / or plasma (e.g., filtrate) generated by the device.

[0296] The inlet end of the container or tube contains a cap or septum adapted to be pierced by a needle or standard blood collection needle assembly, the inlet end defining a first end of a blood receiving chamber that freely accepts a whole blood sample from the needle or blood collection needle assembly for filtration.

[0297] In some aspects, the filter component is adjacent to the second end of the blood receiving chamber. The filter component captures the cellular components of blood (e.g., red blood cells) and allows the serum and / or plasma components to pass through the component to the outlet end of the serum receiving chamber. In some aspects, the filter component covers the entire cross-sectional area of the container or tube. In some aspects, the filter component allows 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 to pass through, and functions similar 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 component faster than larger-sized molecules (e.g., red blood cells). The filter component can be made of any material or combination of materials, and the filter component can be used to separate blood components based on size and allow the serum and / or plasma components to pass through the component and flow towards the serum receiving chamber. For example, in some aspects, the filter component includes one or more microfiber diaphragms and / or fiberglass filter materials (e.g., such as low-density fiber filter materials). For example, Micro-Strand glass microfibers from Johns Manville (Fruita, CO) can be used. In some aspects, highly hydrophilic, highly porous materials can be used in the filter component, such as materials available from Porex Filtration Group (South Chesterfield, VA), such as POR 410 or POR 4711. In additional aspects, the filter component terminates and is retained and supported near the middle of the container or tube by a screen member or other perforated material.

[0298] Optionally, in some aspects, a flow regulator can be placed adjacent to the lid or septum and can be used to regulate the flow rate of blood towards the filter component. In other aspects, the flow regulator can be included as part of the blood collection needle assembly.

[0299] Since the container or tube is pre-vacuumed, there is a pressure difference in the area around the filter component. The pressure at the top of the container or tube in the blood receiving chamber is higher than the pressure at the bottom of the container in the serum receiving chamber. Due to this pressure difference, the whole blood sample in the blood receiving chamber moves to and through the filter component, where the larger red blood cells are captured and entangled in the filter component, and the serum and / or plasma (e.g., filtrate) move through the filter component at a much faster rate and reach the serum receiving chamber, where they are collected.

[0300] The serum containment chamber has a hollow space sized to contain the serum and / or plasma produced by the filter assembly. The serum containment chamber is removably attached (e.g., detachable) to a container or tube and is capable of being detached from the container or tube to enable further processing and / or analysis of the serum or plasma. For example, the serum containment chamber can be detached from the container or tube by twisting or sliding the serum containment chamber in a clockwise or counterclockwise motion to break the vacuum. Once the serum containment chamber is detached from the container or tube, the serum or plasma sample can be further processed and / or analyzed on another device and / or apparatus. For example, the serum or plasma sample in the serum containment chamber can be transferred to another container or device (e.g., a cartridge) for further processing and / or analysis.

[0301] In other aspects, plasma separation can be performed in a container or tube that is not pre-evacuated. In such aspects, plasma separation can be performed using any suitable means, including, for example, any means employing size exclusion chromatography that separates components in the blood by differences in their size as they pass through a material (e.g., a filter assembly) contained within the container or tube. For example, any material (e.g., glass or porous beads, membranes, one or more filters, glass or other fibrous materials, or any combination thereof) that preferentially helps slow the movement of red blood cells relative to plasma such that plasma can be recovered can be employed. Such methods include using methods other than a pressure differential, such as gravity feed separation can be employed without using any pressure differential.

[0302] Examples of pre-evacuated containers or tubes having the components described above that can be used as plasma separation devices in the methods described in the present disclosure include those described in U.S. Patent No. 9,427,707, the content of which is incorporated herein by reference.

[0303] In other aspects, the plasma separation device described above can be used in conjunction with capillary blood samples that are (1) collected in a decentralized setting; (2) without the use of a syringe, standard needle, or combination thereof; (3) by a user untrained in collecting blood samples from a subject; (4) by a robot; (5) by a blood collection device self-administered or administered by another person; or (6) any combination thereof.

[0304] In another aspect, the plasma separation device described above can be used in combination with a blood sample obtained from a subject collected using a syringe, a standard needle, or a combination thereof. Such a sample can be collected in a decentralized or centralized setting (e.g., in a traditional medical setting such as a hospital, a doctor's office, an independent laboratory, etc.) by a user trained in blood collection or a combination thereof. In some aspects, the blood sample obtained from a subject collected using a syringe, a standard needle, or a combination thereof is a venous blood sample. In other aspects, the blood sample obtained from a subject collected using 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 using a syringe, a standard needle, or a combination thereof (and optionally, in a centralized setting and / or by a user trained in collecting a blood sample from a subject), and processed using the plasma separation device described above, followed by determination of GFAP.

[0305] 4. Microsampling System for Determining GFAP Amount Using Capillary Blood Sample

[0306] Referring Figure 2 , a microsampling system 10 for determining the amount of GFAP is provided. The amount of GFAP can be used to assist in the diagnosis and / or evaluation of a subject who has suffered or may have suffered a head injury. In one aspect, the microsampling system 10 includes a microsampling device 14, a reaction vessel 22, and an instrument, such as instrument 26 (e.g., a point-of-care device). It should be noted that although Figure 2 the shape of the reaction vessel 22 shown in

[0307] is rectangular, the shape is not important. For example, in some aspects, the reaction vessel 22 can be tubular. In another aspect, the reaction vessel 22 can be a microfluidic cartridge. Figure 2 ) When a plasma separation device 18 is present, the microsampling device 14 collects a capillary blood sample from the subject, and the plasma separation device 18 produces a processed capillary blood sample (e.g., serum or plasma) from the capillary blood sample.

[0308] In another aspect, the system can further include a transfer tube 78 ( Figure 7 ). The transfer tube 78 can include a cap or plug 79. The transfer tube 78 also has a hole 80. The hole 80 allows the reaction tube to receive the capillary blood sample or the processed capillary blood sample.

[0309] In some aspects, the reaction vessel 22 or the transfer tube 78 receives the processed capillary blood sample from the plasma separation device 18.

[0310] The instrument 26 analyzes the reaction vessel 22 to determine the amount of GFAP of the subject. In some aspects, 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 smart phone), on a website, in an email, or any combination thereof.

[0311] In some aspects, the communicated amount of GFAP can be displayed on an instrument such as 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 of the system 10 are described in further detail with respect to Figures 3A - 3E and Figure 4 A-4C herein.

[0312] Reference Figure 3A , the microsampling device 14 is coupled to the subject 30 and draws a capillary blood sample from the subject 30. In the illustrated embodiment, the microsampling device 14 includes a housing 34 and a reservoir 38 coupled to the housing 34. In the illustrated embodiment, the capillary blood sample is collected in the reservoir 38. In other aspects, the reservoir 38 is removably coupled to the housing 34. For example, once the capillary blood sample is collected, the reservoir 38 can be separated from the housing 34. In other aspects, the reservoir 38 is the reaction vessel 22. In these aspects, when the reservoir is the reaction vessel, the container can be removed from the microsampling device 14 and directly inserted into the instrument 26.

[0313] The microsampling device 14 also includes a microneedle, lancet, micro-lancet, blade, micro-blade, micro-screw, 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 actuate the microneedle or similar component into the skin of the subject to initiate drawing a capillary blood sample from the subject 30.

[0314] Reference Figure 3B , the system 10 can further include a lid 42 coupled to the reservoir 38. In some embodiments, the lid 42 is attached to the reservoir after the reservoir 38 is removed from the housing 34. In various aspects, the lid 42 seals the capillary blood sample within the container 38. For example, a threaded configuration can couple the lid 42 to the reservoir 38. In some embodiments, the lid 42 is part of the interface between the reservoir 38 and the plasma separation device 18.

[0315] Reference Figure 3C, the plasma separation device 18 includes an inlet 46 that receives a capillary blood sample from the micro-sampling 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 a capillary blood sample from the reservoir 38. In some aspects, the plasma separation device 18 is integrated within the reservoir 38 (e.g., it is not intended for the user to remove the plasma separation device 18 from the reservoir 38).

[0316] In some other aspects, the plasma separation device 18 is formed separately from the reaction vessel 22 or the transfer tube 78 (i.e., the plasma separation device 18 is movable and can be separated from the reaction vessel 22 or the transfer tube 78). In other aspects, the plasma separation device 18 is integrated with the reaction vessel 22 or the transfer tube 78. For example, in some aspects, the plasma separation device 18 is integrated within the housing 54 of the reaction vessel 22 (i.e., it is not intended for the user to remove the plasma separation device 18 from the reaction vessel 22 or the transfer tube 78).

[0317] The plasma separation device 18 includes a filter, a diaphragm, synthetic paper, or any combination thereof. In one aspect, the plasma separation device 18 is removably coupled to the reaction vessel 22 or the transfer tube 78 using 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 reaction vessel 22 or the transfer tube 78 to hold the plasma separation device 18 in place once the plasma separation device 18 is installed on the reaction vessel 22 or the transfer tube 78. The outlet 50 of the plasma separation device 18 is positioned in fluid communication with a hole 62 in the reaction vessel 22 or a hole 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 hole 62 of the reaction vessel 22 or the hole 80 of the transfer tube 78.

[0318] In additional aspects, the reservoir 38 is removably coupled to the plasma separation device 18 using a removable coupling 66 ( Figure 3C ). In some aspects, after the plasma separation device 18 is coupled to the reaction vessel 22 or the transfer tube 78, the reservoir 38 is coupled to the plasma separation device 18. In additional aspects, the lid 42 is removed before the reservoir 38 is coupled to the plasma separation device 18. In some aspects, the lid 42 remains in place when the reservoir 38 is coupled to the plasma separation device 18. In some aspects, when the reservoir 38 is coupled to the plasma separation device 18, the lid 42 is pierced, allowing the capillary blood sample to flow into the plasma separation device 18.

[0319] In additional aspects, the plasma separation device 18 is removably coupled to the reaction vessel 22 or the transfer tube 78 and the reservoir 38. Refer to Figure 3D, the plasma separation device 18 is located between the reservoir 38 and the reaction vessel 22. In other aspects, the plasma separation device 18 is located between the reservoir 38 and the transfer tube 78. In some aspects, the user or operator squeezes the reservoir 38 to force the capillary blood sample through the plasma separation device 18 and into the reaction vessel 22 or the transfer tube 78. In other aspects, the reservoir 38 includes a plunger to force the capillary blood sample through the plasma separation device 18 and into the reaction vessel 22 or the transfer tube 78. In other embodiments, the capillary blood sample is gravity-fed through the plasma separation device 18 and into the reaction vessel 22 or the transfer tube 78.

[0320] Reference Figure 3D , the reaction vessel 22 is in some aspects a microfluidic cartridge. The processed capillary blood sample flows from the plasma separation device 18 to the reaction vessel 22, where analysis is performed. In some aspects, the assay is for GFAP.

[0321] In additional aspects, reference Figure 4 , the plasma separation device 18 is placed in fluid communication with the aperture 62 at any point along the reaction vessel 22 (e.g., microfluidic cartridge). In some aspects, the plasma separation device 18 is placed in fluid communication with the aperture 62 at one end, side, or middle of the reaction vessel 22 (e.g., microfluidic cartridge). In some aspects, as Figure 5 shown, the plasma separation device 18 is placed in fluid communication with the aperture 62 at an angle at one end or side of the reaction vessel 22 (e.g., microfluidic cartridge), the angle being such as 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, forming an L or J shape). In other aspects, as Figure 6 shown, the plasma separation device 18 is placed in fluid communication with at least one aperture 62 at one end or side of the reaction vessel 22 (e.g., microfluidic cartridge).

[0322] In one aspect, reference Figure 3E , in the case of loading the processed capillary blood sample into the reaction vessel 22, the reaction vessel 22 is inserted into the instrument 26. In the illustrated embodiment, the reaction vessel 22 is inserted into the bottom 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 as 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 with visual, auditory, or tactile feedback.

[0323] In some aspects, results are communicated within about 4 minutes from when the sample is collected (e.g., from when an injury or suspected injury occurs). In some aspects, results are communicated within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes from when the sample is collected (e.g., from when an injury or suspected injury occurs). In some aspects, results are communicated within a range of about 4 minutes to about 40 minutes from when the sample is collected (such as, from when an injury or suspected injury occurs). In some aspects, results are communicated within a range of about 4 minutes to about 30 minutes from when the sample is collected (such as, from when an injury or suspected injury occurs). In some aspects, results are communicated within a range of about 4 minutes to about 20 minutes from when the sample is collected (such as, from when an injury or suspected injury occurs).

[0324] Advantageously, a portion of system 10 can be used in a decentralized environment. In other words, system 10 is portable. Advantageously, system 10 or a portion thereof can be reused for multiple samples and / or subjects. In some embodiments, the microsampling device 14, the plasma separation device 18, and the reaction vessel 22 or transfer tube 78 are single-use components (i.e., can be used for a single test on a single subject); while the instrument 26 can be reused for analyzing multiple reaction vessels 22.

[0325] 5. Treatment and Monitoring of Subjects

[0326] Subjects identified in the above methods can be treated or monitored. In some embodiments, the method further includes treating a subject (such as a human subject) with a treatment for an acquired brain injury or traumatic brain injury (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 head injury. For example, for a subject with a mild TBI, treatment can include one or more periods of rest, abstaining from physical exercise (such as sports), avoiding light or wearing sunglasses in the sun, medications to relieve headaches or migraines, anti-nausea medications, etc. Treatment of patients with moderate, severe, or moderate-to-severe TBI may include administering one or more suitable medications (such as diuretics, anti-seizure medications, medications to sedate and induce a drug-induced coma, or other pharmaceutical or biopharmaceutical medications (known or developed in the future for the treatment of TBI), one or more surgical procedures (such as hematoma removal, skull fracture repair, decompressive craniectomy, etc.), protecting the airway, and one or more therapies (such as one or more rehabilitations, cognitive behavioral therapy, anger management, psychological counseling, etc.). In some embodiments, the method further includes monitoring a subject (such as a human subject). In some embodiments, a subject can be monitored using a CT scan or an MRI procedure.

[0327] 6. Methods for Measuring the Level of GFAP

[0328] In the above method, 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, protein immunostaining, electrophoretic analysis, protein analysis, competitive binding analysis, functional protein analysis, or chromatographic or spectroscopic methods, such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). Additionally, the assay can be performed in a clinical chemistry format known to those skilled in the art.

[0329] In some embodiments, measuring the level of GFAP includes contacting a sample with a first specific binding member and a second specific binding member. In some embodiments, the first specific binding member is a capture antibody and the second specific binding member is a detection antibody. In some embodiments, measuring the level of GFAP includes contacting the sample simultaneously or sequentially in any order with: (1) a capture antibody (e.g., a GFAP capture antibody) that binds to an epitope on GFAP or a GFAP fragment to form a capture antibody-GFAP antigen complex (e.g., a GFAP capture antibody-GFAP antigen complex), and (2) a detection antibody (e.g., a GFAP detection antibody) that includes a detectable label and binds to an epitope on GFAP that is not bound by the capture antibody to form a GFAP antigen-detection antibody complex (e.g., a GFAP antigen-GFAP detection antibody complex), such that a capture antibody-GFAP antigen-detection antibody complex (e.g., a GFAP capture antibody-GFAP antigen-GFAP detection antibody complex) is formed, and measuring the amount or concentration of GFAP in the sample based on a signal generated by the detectable label in the capture antibody-GFAP antigen-detection antibody complex.

[0330] In some embodiments, the first specific binding member is immobilized on a solid support. In some embodiments, the second specific binding partner is immobilized on a solid support. In some embodiments, the first specific binding partner is a GFAP antibody as described below.

[0331] 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 liter, 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.

[0332] Some instruments other than point-of-care devices (such as Abbott Laboratories instruments and other core laboratory instruments) may be able to measure GFAP levels in a sample that are higher than or greater than 25,000 pg / mL.

[0333] Other detection methods include using nanopore devices or nano-well devices or can be adapted for use on nanopore devices or nano-well devices. Examples of nanopore devices are described in International Patent Publication No. WO 2016 / 161402, which is incorporated herein by reference in its entirety. Examples of nano-well devices are described in International Patent Publication No. WO 2016 / 161400, which is incorporated herein by reference in its entirety.

[0334] 7. GFAP Antibody

[0335] The methods described herein can use isolated antibodies that specifically bind to glial fibrillary acidic protein (“GFAP”) (or fragments thereof), referred to as “GFAP antibodies”. GFAP antibodies can be used to assess 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 its amount.

[0336] a. Glial Fibrillary Acidic Protein (GFAP)

[0337] Glial fibrillary acidic protein (GFAP) is a 50 kDa cytoplasmic filamentous protein that forms part of the cytoskeleton in astrocytes and has been shown to be the most specific marker for astrocyte-derived cells. The GFAP protein is encoded by the human GFAP gene. GFAP is the major intermediate filament in mature astrocytes. In the central rod domain of the molecule, GFAP has considerable structural homology with other intermediate filaments. GFAP is involved in the movement and shape of astrocytes by providing structural stability to astrocytic processes. Glial fibrillary acidic protein and its breakdown products (GFAP-BDP) are brain-specific proteins released into the blood as part of the pathophysiological response following traumatic brain injury (TBI). After injury to the human CNS by trauma, genetic disorders, or chemicals, astrocytes proliferate and show extensive hypertrophy of the cell body and processes, and GFAP is significantly upregulated. In contrast, GFAP production gradually decreases as astrocytic malignancies increase. GFAP can also be detected in Schwann cells, enteric glial cells, salivary gland tumors, metastatic renal cell carcinoma, epiglottic cartilage, pituitary cells, immature oligodendrocytes, papillary meningiomas, and mammary myoepithelial cells.

[0338] Human GFAP can have the following amino acid sequence:

[0339] MERRRITSAARRSYVSSGEMMVGGLAPGRRLGPGTRLSLARMPPPLPTRVDFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTKLADVYQAELRELRLRLDQLTANSARLEVERDNLAQDLATVRQKLQDETNLRLEAENNLAAYRQEADEATLARLDLERKIESLEEEIRFLRKIHEEEVRELQEQLARQQVHVELDVAKPDLTAALKEIRTQYEAMASSNMHEAEEWYRSKFADLTDAAARNAELLRQAKHEANDYRRQLQSLTCDLESLRGTNESLERQMREQEERHVREAASYQEALARLEEEGQSLKDEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKESKQEHKDVM (SEQ ID NO:2).

[0340] Human GFAP can be a fragment or variant of SEQ ID NO:2. The length of the fragment 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. The fragment can contain many consecutive amino acids from SEQ ID NO:2. The human GFAP fragment or variant of SEQ ID NO:2 can be a GFAP breakdown product (BDP). GFAP BDP can be 38 kDa, 42 kDa (weak 41 kDa), 47 kDa (weak 45 kDa), 25 kDa (weak 23 kDa), 19 kDa, or 20 kDa. In some embodiments, the human GFAP fragment or variant can be a GFAP BDP containing 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.

[0341] b. GFAP recognition antibody

[0342] An antibody is an antibody that binds to GFAP, its fragment, an epitope of GFAP, or its variant. The antibody can be a fragment or a variant or derivative of an anti-GFAP antibody. 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. Antibody fragments or derivatives can include F(ab')2, Fv, or scFv fragments. Antibody derivatives can be produced by mimotopes. In addition, techniques described for producing single-chain antibodies can be applicable to produce single-chain antibodies.

[0343] The anti-GFAP antibody can be a chimeric anti-GFAP or a humanized anti-GFAP antibody. In one embodiment, both the humanized antibody and the chimeric antibody are monovalent. In one embodiment, both the humanized antibody and the chimeric antibody comprise a single Fab region linked to an Fc region.

[0344] Human antibodies can be derived from phage display technology or transgenic mice expressing human immunoglobulin genes. Human antibodies can be generated as a result of an immune response in a human body and isolated. See, for example, Funaro et al., BMC Biotechnology, 2008(8):85. Thus, the antibody can be a product of the human rather than the animal repertoire. Since it is of human origin, the risk of autoantigenic reactions can be reduced. Alternatively, standard yeast display libraries and display techniques can be used to select and isolate human anti-GFAP antibodies. For example, a library of initial human single-chain variable fragments (scFv) can be used to select human anti-GFAP antibodies. Transgenic animals can be used to express human antibodies.

[0345] A humanized antibody can be an antibody molecule from an antibody of a non-human species that binds a desired antigen with one or more complementarity-determining regions (CDRs) from a non-human species and framework regions from a human immunoglobulin molecule.

[0346] The antibody differs from known antibodies in that it has a biological function different from those known in the art.

[0347] (1) Epitope

[0348] The antibody can immunospecifically bind to GFAP (SEQ ID NO:2), a fragment thereof, or a variant thereof. The antibody can immunospecifically recognize and bind at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, or at least ten amino acids within an epitope region. The antibody can immunospecifically recognize and bind an epitope having at least three contiguous amino acids, at least four contiguous amino acids, at least five contiguous amino acids, at least six contiguous amino acids, at least seven contiguous amino acids, at least eight contiguous amino acids, at least nine contiguous amino acids, or at least ten contiguous amino acids of the epitope region.

[0349] c. Antibody preparation / production

[0350] Antibodies can be prepared by any of a variety of techniques, including those well known to those skilled in the art. Generally, antibodies can be produced by cell culture techniques, including by conventional techniques, or by transfecting antibody genes, heavy and / or light chains, into a suitable bacterial or mammalian cell host to produce monoclonal antibodies for antibody production, where the antibodies can be recombinant. The term "transfection" in its various forms is intended to encompass a wide variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although it is possible to express antibodies in prokaryotic or eukaryotic host cells, it is preferred to express antibodies in eukaryotic cells and most preferably in mammalian host cells because such eukaryotic cells (and especially mammalian cells) are more likely to assemble and secrete correctly folded and immunologically active antibodies than prokaryotic cells.

[0351] Exemplary mammalian host cells for expressing recombinant antibodies include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), which are used with a DHFR selectable marker, such as described in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982); NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a time sufficient to allow the antibody to be expressed in the host cell or more preferably secreted into the culture medium in which the host cell is growing. The antibody can be recovered from the culture medium using standard protein purification methods.

[0352] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be understood that variations of the above processes can be performed. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of the light and / or heavy chains of the antibody. Recombinant DNA techniques can also be used to remove some or all of the DNA encoding portions of one or both of the light or heavy chains that are not necessary for binding to the antigen of interest. The antibodies also encompass molecules expressed from such truncated DNA molecules. In addition, bispecific antibodies can be produced by cross-linking an antibody with a second antibody using standard chemical cross-linking methods, where one heavy chain and one light chain are the antibody (i.e., bind to human GFAP) and the other heavy chain and the other light chain are specific for an antigen other than human GFAP.

[0353] In a preferred system for the recombinant expression of an antibody or an antigen-binding portion thereof, recombinant expression vectors encoding 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 to drive high-level transcription of the genes. The recombinant expression vector also carries the DHFR gene, which permits selection / amplification using methotrexate to select CHO cells that have been transfected with the vector. The selected transformed host cells are cultured to express the antibody heavy chain and light chain, and the intact antibody is recovered from the culture medium. The recombinant expression vectors are prepared using standard molecular biology techniques, the host cells are transfected, the transformants are selected, the host cells are cultured, and the antibody is recovered from the culture medium. Further, the method for synthesizing a recombinant antibody can be by culturing the host cells in a suitable medium until the recombinant antibody is synthesized. The method can also include isolating the recombinant antibody from the culture medium.

[0354] Methods for preparing monoclonal antibodies include preparing an immortal cell line capable of producing an antibody with the desired specificity. Such cell lines can be generated from spleen cells obtained from an immunized animal. The animal can be immunized with GFAP or a fragment and / or variant thereof. The peptide used to immunize the animal can contain amino acids encoding the human Fc (e.g., the crystallizable fragment) or the tail region of a human antibody. The spleen cells can then be immortalized by fusion, for example, with a myeloma cell fusion partner. A variety of fusion techniques can be employed. For example, spleen cells and myeloma cells can be mixed with a non-ionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells but not myeloma cells. One such technique is selection using hypoxanthine, aminopterin, thymidine (HAT). Another technique involves electrofusion. After a sufficient time (usually about 1 to 2 weeks), colonies of hybrids are observed. Individual colonies are selected and their culture supernatants are tested for binding activity to the polypeptide. Hybridomas with high reactivity and specificity can be used.

[0355] Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. Additionally, various techniques can be employed to increase the yield, such as injecting the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host (such as a mouse). The monoclonal antibody can then be harvested from the ascites or blood. Contaminants can be removed from the antibody by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. Affinity chromatography is an example of a method that can be used to purify the antibody.

[0356] The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (F(ab) fragments) each contain a covalently linked heterodimer with a complete antigen-binding site. Pepsin is capable of cleaving IgG molecules to provide multiple fragments that include two antigen-binding sites, including the F(ab')2 fragment.

[0357] Fv fragments can be generated by the preferential proteolytic cleavage of IgM and occasionally the proteolytic cleavage of IgG or IgA immunoglobulin molecules. Fv fragments can be derived using recombinant techniques. Fv fragments include non-covalently linked VH:VL heterodimers that contain an antigen-binding site that retains many of the antigen recognition and binding capabilities of the native antibody molecule.

[0358] An antibody, antibody fragment or derivative can contain a set of heavy chain complementarity determining regions ("CDRs") and a set of light chain complementarity determining regions ("CDRs") that are respectively interposed between sets of heavy chain frameworks ("FRs") and light chain frameworks ("FRs"), the framework sets providing support for the CDRs and defining the spatial relationship of the CDRs relative to each other. The set of CDRs can include the three hypervariable regions of the heavy or light chain V region.

[0359] Other suitable methods for generating or isolating antibodies with the required specificity can be used, including but not limited to methods for selecting recombinant antibodies from peptide or protein libraries (such as but not limited to phage, ribosome, oligonucleotide, RNA, cDNA, yeast, etc. display libraries); for example, as can be obtained from various commercial suppliers such as Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid / Planegg, Del.), Biovation (Aberdeen, Scotland, UK), BioInvent (Lund, Sweden) using methods known in the art. See U.S. Patent Nos. 4,704,692, 5,723,323, 5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862. Alternative methods rely on immunizing transgenic animals capable of generating a human antibody repertoire (e.g., SCID mice, Nguyen et al. (1997) Microbiol. Immunol. 41:901-907; Sandhu et al. (1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol. 93:154-161), as known in the art and / or as described herein. Such techniques include but are not limited to ribosome display (Hanes et al. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998) Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibody production techniques (e.g., 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); B cell selection (Steenbakkers et al. (1994) Molec. Biol. Reports 19:125-134 (1994)).

[0360] Affinity matured antibodies can be generated by any of a number of methods known in the art. For example, see Marks et al., BioTechnology, 10:779-783 (1992) which describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described in Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-3319 (1995); Hawkins et al., J. Mol. Biol., 226:889-896 (1992). Selective mutagenesis at selected mutagenic positions and at contacting or hypermutable positions with activity enhancing amino acid residues is described in U.S. Patent No. 6,914,128 B1.

[0361] Antibody variants can also be prepared by delivering a polynucleotide encoding the antibody to a suitable host, such as a transgenic animal or mammal, such as a goat, cow, horse, sheep, etc. which produces such antibodies in its milk. These methods are known in the art and are described, for example, in U.S. Patent Nos. 5,827,690, 5,849,992, 4,873,316, 5,849,992, 5,994,616, 5,565,362 and 5,304,489.

[0362] Antibody variants can also be prepared by delivering polynucleotides to provide transgenic plants and cultured plant cells (such as but not limited to tobacco, corn, and duckweed), which produce such antibodies, specific portions, or variants in plant parts or cells cultured therefrom. For example, Cramer et al. (1999) Curr. Top. Microbiol. Immunol. 240:95-118 and the references cited therein describe, for example, the use of inducible promoters to produce transgenic tobacco leaves expressing large amounts of recombinant proteins. Transgenic corn has been used to express mammalian proteins at commercial production levels, and their biological activities are the same as those produced in other recombinant systems or purified from natural sources. See, for example, Hood et al., Adv. Exp. Med. Biol. (1999) 464:127-147 and the references cited therein. Antibody variants, including antibody fragments such as single-chain antibodies (scFv), have also been produced in large quantities from transgenic plant seeds (including tobacco seeds and potato tubers). See, for example, Conrad et al. (1998) Plant Mol. Biol. 38:101-109 and the references cited therein. Therefore, transgenic plants can also be used to produce antibodies according to known methods.

[0363] Antibody derivatives can be produced, for example, by adding foreign sequences to modify immunogenicity or to reduce, enhance, or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life, or any other suitable characteristic. Generally, 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.

[0364] Small antibody fragments can be diabodies having two antigen-binding sites, where the fragment contains a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH VL). See, for example, EP 404,097; WO93 / 11161; and Hollinger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. By using a linker that is too short to allow pairing between the two domains of the same chain, the domains are forced to pair with the complementary domains of the other chain and generate two antigen-binding sites. See also U.S. Patent No. 6,632,926 to Chen et al., which is incorporated herein by reference in its entirety and discloses antibody variants having one or more amino acids inserted into the hypervariable regions of the parental antibody and having a binding affinity for the target antigen that is at least about two-fold stronger than the binding affinity of the parental antibody for the antigen.

[0365] The antibody can be a linear antibody. Procedures for preparing linear antibodies are known in the art and are described in Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, these antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1), which form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0366] Antibodies can be recovered and purified from recombinant cell cultures by known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification.

[0367] Detectably labeled antibodies may be useful. Methods for conjugating antibodies to such reagents are known in the art. For illustrative purposes only, antibodies can be labeled with a detectable moiety such as a radioactive atom, a chromophore, or a fluorophore, etc. Such labeled antibodies can be used in diagnostic techniques in vivo or in isolated test samples. They can be linked to cytokines, ligands, and another antibody. Suitable reagents for conjugating with antibodies to achieve anti-tumor effects include cytokines such as interleukin 2 (IL-2) and tumor necrosis factor (TNF); photosensitizers for photodynamic therapy, including aluminum(III) phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine; radionuclides such as iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), technetium-99m (99mTc), rhenium-186 (186Re), and rhenium-188 (188Re); antibiotics such as adriamycin, doxorubicin, daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacteria, plant, and other toxins such as diphtheria toxin, Pseudomonas exotoxin A, staphylococcal enterotoxin A, Abrin-A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-α toxin, cytotoxins of Naja naja atra (cobra), and bryodin (phytotoxin); 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 ribonuclease; tyrosine kinase inhibitors; ly207702 (difluoropurine nucleoside); liposomes containing anti-cystic drugs (such as antisense oligonucleotides, plasmids encoding toxins, methotrexate, etc.); and other antibodies or antibody fragments such as F(ab).

[0368] Antibody production by using hybridoma technology, selection lymphocyte antibody method (SLAM), transgenic animals, and recombinant antibody libraries is described in more detail below.

[0369] (1) Monoclonal anti-GFAP antibody using hybridoma technology

[0370] A variety of techniques known in the art can be used to prepare monoclonal antibodies, including using hybridoma, recombinant, and phage display techniques or combinations thereof. For example, monoclonal antibodies can be generated using hybridoma technology, which includes those known in the art and taught, for example, in the following: Harlow et al., Antibodies: A Laboratory Manual, 2nd edition, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988); Hammerling et al., In Monoclonal Antibodies and T-Cell Hybridomas, (Elsevier, N.Y., 1981). It 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 the antibody is produced.

[0371] Methods for generating monoclonal antibodies and the antibodies generated by such methods can include culturing hybridoma cells that secrete the antibodies of the present disclosure, wherein the hybridomas are preferably generated 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 generated by the fusion for hybridoma clones that secrete antibodies capable of binding the polypeptides 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 to stimulate an immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immunostimulating complex). Such adjuvants can protect the polypeptide from rapid dispersion by sequestering the polypeptide in a local depot, or they can contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. Preferably, if the polypeptide is being administered, the immunization protocol will involve two or more administrations of the polypeptide, spread over several weeks; however, a single administration of the polypeptide can also be used.

[0372] After immunizing an animal with GFAP antigen, antibodies and / or antibody-producing cells can be obtained from the animal. By bleeding or sacrificing the animal, serum containing anti-GFAP antibodies is obtained from 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 antibody can be purified from the serum. The serum or immunoglobulin obtained in this way is polyclonal and thus has a range of heterogeneity.

[0373] Once an immune response is detected, for example, antibodies specific for the antigen GFAP are detected in rat serum, the rat spleen is harvested and the spleen cells are isolated. The spleen cells are then fused with any suitable myeloma cells (e.g., cells from the cell line SP20 available from the American Type Culture Collection (ATCC, Manassas, Va., US)) by well-known techniques. Hybridomas are selected and cloned by limiting dilution. The ability of the hybridoma clones to secrete antibodies that can bind to GFAP is then determined by methods known in the art. Ascites generally containing a high level of antibodies can be produced by immunizing rats with positive hybridoma clones.

[0374] In another embodiment, immortalized hybridomas that produce antibodies can be prepared from immunized animals. After immunization, the animals are sacrificed and the splenic B cells are fused with immortalized myeloma cells, as is well known in the art. See, e.g., Harlow and Lane, supra. In a preferred embodiment, the myeloma cells do not secrete immunoglobulin polypeptides (non-secreting cell lines). After fusion and antibiotic selection, the hybridomas are screened using GFAP, or a portion thereof, or cells expressing GFAP. In a preferred embodiment, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), preferably ELISA, is used for the initial screening. Examples of ELISA screening are provided in PCT Publication No. WO 00 / 37504.

[0375] Hybridomas that produce anti-GFAP antibodies are selected, cloned, and further screened for their desired characteristics, including robust hybridoma growth, high antibody production, and the desired antibody characteristics. Hybridomas can be cultured and amplified in vivo in syngeneic animals, in animals lacking an immune system (e.g., nude mice), or in vitro in cell culture. Methods for selecting, cloning, and amplifying hybridomas are well known to those of ordinary skill in the art.

[0376] In a preferred embodiment, the hybridomas are rat hybridomas. In another embodiment, the hybridomas are produced in non-human, non-rat species such as mice, sheep, pigs, goats, cattle, or horses. In another preferred embodiment, the hybridomas are human hybridomas, in which human non-secreting myelomas are fused with human cells expressing anti-GFAP antibodies.

[0377] 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 generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce two identical Fab fragments) or pepsin (to produce F(ab')2 fragments). The F(ab')2 fragment of an IgG molecule retains the two antigen-binding sites of the larger ("parent") IgG molecule, which includes two light chains (containing variable and constant light chain regions), the CH1 domain of the heavy chain, and the disulfide-bond-forming hinge region of the parent IgG molecule. Thus, the F(ab')2 fragment is still able to crosslink antigen molecules like the parent IgG molecule.

[0378] (2) Anti-GFAP monoclonal antibodies using SLAM

[0379] In another aspect of the present disclosure, recombinant antibodies are generated from single, isolated lymphocytes using a method known in the art as the selection of lymphocytes antibody method (SLAM), as described in U.S. Patent No. 5,627,052; PCT Publication No. WO 92 / 02551; and Babcock et al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996). In this method, single cells secreting an antibody of interest are screened using an antigen-specific hemolytic plaque assay, for example, lymphocytes derived from any immunized animal, where the antigen GFAP, a subunit of GFAP, or a fragment thereof is conjugated to sheep red blood cells using a linker such as biotin, and used to identify single cells secreting an antibody specific for GFAP. After identifying the antibody-secreting cells of interest, the heavy and light chain variable region cDNAs are rescued from the cells by reverse transcriptase-PCR (RT-PCR), and these variable regions can then be expressed in mammalian host cells such as COS or CHO cells in the context of an appropriate immunoglobulin constant region (e.g., a human constant region). Host cells transfected with the amplified immunoglobulin sequences (derived from in vivo-selected lymphocytes) can then be further analyzed and selected in vitro, for example, by panning the transfected cells to isolate cells expressing an antibody against GFAP. The amplified immunoglobulin sequences can be further manipulated in vitro, such as by in vitro affinity maturation methods. See, for example, PCT Publication No. WO 97 / 29131 and PCT Publication No. WO 00 / 56772.

[0380] (3) Anti-GFAP monoclonal antibodies using transgenic animals

[0381] In another embodiment of the present disclosure, antibodies are produced by immunizing a non-human animal comprising some or all of the human immunoglobulin loci with a GFAP antigen. In one embodiment, the non-human animal is a transgenic mouse, an engineered mouse strain that contains a large fragment of the human immunoglobulin locus and lacks murine antibody production. See, e.g., Green et al., Nature Genetics, 7:13-21 (1994) and U.S. Patent Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364. See also PCT Publication Nos. WO 91 / 10741, WO 94 / 02602, WO 96 / 34096, WO 96 / 33735, WO 98 / 16654, WO 98 / 24893, WO 98 / 50433, WO 99 / 45031, WO 99 / 53049, WO 00 / 09560 and WO 00 / 37504. The transgenic mouse produces an adult-like, fully human antibody repertoire and generates antigen-specific human monoclonal antibodies. The transgenic mouse contains approximately 80% of the human antibody repertoire by introduction of megabase-sized, germline configuration YAC fragments of the human heavy chain locus and kappa 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 which are incorporated herein by reference.

[0382] (4) Anti-GFAP monoclonal antibodies using recombinant antibody libraries

[0383] The antibodies of the present disclosure can also be prepared using in vitro methods, where antibody libraries are 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 those described in the following documents: for example, U.S. Patent No. 5,223,409 (Ladner et al.); PCT Publication No. WO 92 / 18619 (Kang et al.); PCT Publication No. WO 91 / 17271 (Dower et al.); PCT Publication No. WO 92 / 20791 (Winter et al.); PCT Publication No. WO 92 / 15679 (Markland et al.); PCT Publication No. WO 93 / 01288 (Breitling et al.); PCT Publication No. WO92 / 01047 (McCafferty et al.); PCT Publication No. WO 92 / 09690 (Garrard et al.); Fuchs et al., Bio / Technology, 9:1369-1372 (1991); Hay et al., Hum. Antibod. Hybridomas, 3:81-85 (1992); Huse et al., Science, 246:1275-1281 (1989); McCafferty et al., Nature, 348:552-554 (1990); Griffiths et al., EMBO J., 12:725-734 (1993); Hawkins et al., J. Mol. Biol., 226:889-896 (1992); Clackson et al., Nature, 352:624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992); Garrard et al., Bio / Technology, 9:1373-1377 (1991); Hoogenboom et al., Nucl. Acids Res., 19:4133-4137 (1991); Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991); U.S. Patent Application Publication No. 2003 / 0186374; and PCT Publication No. WO 97 / 29131, the content of each of which is incorporated herein by reference.

[0384] A recombinant antibody library can be from a subject immunized with GFAP or a portion of GFAP. Alternatively, a recombinant antibody library can be from a naïve subject, i.e., a human not immunized with GFAP, such as a human antibody library from a human subject not immunized with human GFAP. The antibodies of the present disclosure are selected by screening a recombinant antibody library with a peptide comprising human GFAP, thereby selecting those antibodies that recognize GFAP. Methods for performing such screening and selection are well known in the art, such as those described in the references of the previous paragraph. To select the antibodies of the present disclosure that have a specific binding affinity for GFAP, such as those that dissociate from human GFAP at a specific K off rate constant, surface plasmon resonance methods known in the art can be used to select antibodies having the desired K off rate constant. To select the antibodies of the present disclosure that have a specific neutralizing activity against GFAP, such as those having a specific IC 50 , standard methods known in the art for assessing inhibition of GFAP activity can be used.

[0385] In one aspect, the present disclosure relates to an isolated antibody that binds human GFAP, or an antigen-binding portion thereof. Preferably, the antibody is a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody.

[0386] For example, various phage display methods known in the art can also be used to generate antibodies. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. Such phages can be used to display antigen-binding domains expressed from combinatorial libraries or combinatorial antibody libraries (e.g., human or murine). Phages expressing antigen-binding domains that bind to an antigen of interest can be selected or identified with the antigen, e.g., using a labeled antigen or an antigen bound or captured to a solid surface or bead. The phages used in these methods are typically filamentous phages, which include fd and M13 binding domains, and are expressed by phages having a recombinant fusion to the phage gene III or gene VIII protein of a Fab, Fv, or disulfide-stabilized Fv antibody domain. Examples of phage display methods that can be used to prepare antibodies include the methods disclosed in the following references: Brinkmann et al., J. Immunol. Methods, 182:41-50 (1995); Ames et al., J. Immunol. Methods, 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic et al., Gene, 187:9-18 (1997); Burton et al., Advances in Immunology, 57:191-280 (1994); PCT Publication No. WO 92 / 01047; PCT Publication No. WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; and U.S. Patent Nos. 5,698,426, 5,223,40, 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.

[0387] As described in the above references, after phage selection, the antibody-encoding regions can be isolated from the phage and used to generate full antibodies, including human antibodies or any other desired antigen-binding fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, as detailed below. For example, methods known in the art can also be used to employ techniques for the recombinant production of Fab, Fab', and F(ab')2 fragments, such as those disclosed in the following documents: PCT Publication No. WO 92 / 22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); Sawai et al., Am. J. Reprod. Immunol., 34:26-34 (1995); and Better et al., Science, 240:1041-1043 (1988). Examples of techniques that can be used to generate 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).

[0388] As an alternative to screening recombinant antibody libraries by phage display, other methods known in the art for screening large combinatorial libraries can be applied to identify the antibodies of the present disclosure. One type of alternative expression system is a system in which a recombinant antibody library is expressed as an RNA-protein fusion, as described in PCT Publication No. WO 98 / 31700 (Szostak and Roberts) and Roberts and Szostak, Proc. Natl. Acad. Sci. USA, 94:12297-12302 (1997). In this system, the covalent fusion is between the mRNA and the peptide or protein it encodes, generated by in vitro translation of synthetic mRNA carrying puromycin (a peptidyl acceptor antibiotic) at its 3' end. Thus, specific mRNAs can be enriched from a complex mixture of mRNAs (e.g., a recombinant library) based on the properties of the encoded peptide or protein (e.g., an antibody) or a portion thereof, such as the binding of the antibody or a portion thereof to a bispecific antigen. The nucleic acid sequences encoding the antibody or a portion thereof recovered from screening such libraries can be expressed by recombinant means (e.g., in mammalian host cells) as described above, and furthermore, can be further affinity matured by additional rounds of mRNA-peptide fusion screening in which mutations have been introduced into the initially selected sequences or by other in vitro affinity maturation methods for recombinant antibodies, as described above. A preferred example of this method is the PROfusion display technology.

[0389] In another method, yeast display methods known in the art can also be used to generate antibodies. In yeast display methods, antibody domains are tethered to the yeast cell wall using genetic methods and displayed on the yeast surface. Such yeast can be used to display antigen-binding domains expressed from a library or a combinatorial antibody library (e.g., human or murine). Examples of yeast display methods that can be used to prepare antibodies include the methods disclosed in U.S. Patent No. 6,699,658 (Wittrup et al.), which is incorporated herein by reference.

[0390] d. Generation of recombinant GFAP antibodies

[0391] Antibodies can be produced by any of a variety of techniques known in the art. For example, expression from host cells, where one or more expression vectors encoding the heavy and light chains are transfected into the host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although it is possible to express the disclosed antibodies in prokaryotic or eukaryotic host cells, it is preferred to express the antibodies in eukaryotic cells and most preferably in mammalian host cells because such eukaryotic cells (and especially mammalian cells) are more likely to assemble and secrete correctly folded and immunologically active antibodies compared to prokaryotic cells.

[0392] Exemplary mammalian host cells for expressing the disclosed recombinant antibodies include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), which are used with a DHFR selection marker, such as described in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982); NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a time sufficient to allow expression of the antibody in the host cell or more preferably to secrete the antibody into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0393] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be understood that variations of the above procedures can be performed. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of the light and / or heavy chains of the disclosed antibodies. Recombinant DNA techniques can also be used to remove some or all of the DNA encoding portions of one or both of the light or heavy chains that are not necessary for binding to the antigen of interest. The antibodies of the present disclosure also encompass molecules expressed from such truncated DNA molecules. In addition, bispecific antibodies can be produced by cross-linking the disclosed antibodies with a second antibody using standard chemical cross-linking methods, where one heavy chain and one light chain are the disclosed antibody (i.e., bind to human GFAP) and the other heavy chain and the other light chain are specific for an antigen other than human GFAP.

[0394] In one preferred system for recombinantly expressing an antibody of the present disclosure, or an antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. In the recombinant expression vector, the antibody heavy chain and light chain genes are each operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high-level transcription of the genes. The recombinant expression vector also carries the DHFR gene, which allows selection / amplification using methotrexate to select CHO cells that have been transfected with the vector. The selected transformant host cells are cultured to express the antibody heavy chain and light chain, and the intact antibody is recovered from the culture medium. The recombinant expression vector is prepared using standard molecular biology techniques, the host cells are transfected, the transformants are selected, the host cells are cultured, and the antibody is recovered from the culture medium. Further, the present disclosure provides a method for synthesizing a recombinant antibody of the present disclosure, the method being carried out by culturing the host cells of the present disclosure in a suitable medium until the recombinant antibody of the present disclosure is synthesized. The method may also include isolating the recombinant antibody from the culture medium.

[0395] (1) Humanized antibody

[0396] 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 having substantially the amino acid sequence of a human antibody and complementarity-determining regions (CDRs) having substantially the amino acid sequence of a non-human antibody. A humanized antibody can be derived from a non-human species antibody that binds to the desired antigen, which has one or more complementarity-determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.

[0397] 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 and usually both variable domains substantially in their entirety (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., the donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. According to one aspect, a humanized antibody also comprises at least a portion of the immunoglobulin constant region (Fc) (usually the constant region of a human immunoglobulin). In some embodiments, a humanized antibody contains a light chain as well as at least the variable domain of the heavy chain. The antibody may 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 the heavy chain.

[0398] The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, as well as any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4. The humanized antibody can contain sequences 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 functions.

[0399] The framework regions and CDR regions of the humanized antibody need not precisely correspond to the parental sequences. For example, the donor antibody CDR or consensus framework can be mutagenized by substituting, inserting, or / or deleting at least one amino acid residue such that the CDR or framework residue at that site does not correspond to the donor antibody or consensus framework. However, in one embodiment, such mutations will not be extensive. Typically, at least 90%, at least 95%, at least 98%, or at least 99% of the residues of the humanized antibody will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework regions in consensus immunoglobulin sequences. 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 related family of immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, 1987)). In an immunoglobulin family, each position in the consensus sequence is occupied by the amino acid that occurs most frequently in that position in the family. If two amino acids occur equally frequently, then either may be included in the consensus sequence.

[0400] Humanized antibodies can be designed to minimize unwanted immune responses to rodent anti - human antibodies, which limits the duration and effectiveness of the therapeutic applications of those moieties in human recipients. Humanized antibodies can have one or more amino acid residues introduced therein from non - human sources. These non - human residues are often referred to as "import" residues and are typically taken from variable domains. Humanization can be performed by substituting the hypervariable region sequences for the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies in which substantially less than the complete human variable domains have been replaced by the corresponding sequences from non - human species. See, e.g., U.S. Patent No. 4,816,567, the contents of which are incorporated herein by reference. A humanized antibody can be a human antibody in which some hypervariable region residues and possibly some FR residues are replaced by residues at similar sites in a rodent antibody. Any known method can be used for humanizing or engineering the antibodies of the present disclosure, such as, but not limited to, the methods 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.

[0401] Humanized antibodies can retain high affinity for GFAP and other favorable biological properties. Three - dimensional models of the parent and humanized sequences can be used to prepare humanized antibodies by analytical methods of the parent sequence and the products of various conceptual humanizations. Three - dimensional immunoglobulin models are generally available. Computer programs that illustrate and display the possible three - dimensional conformational structures of selected candidate immunoglobulin sequences are available. Examination of these displays allows analysis of the possible role of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and incorporated from the recipient and import sequences to achieve the desired antibody characteristics, such as increased affinity for GFAP. Generally speaking, hypervariable region residues are directly and most substantially involved in affecting antigen binding.

[0402] As an alternative to humanization, human antibodies (also referred to herein as "fully human antibodies") can be generated. For example, it is possible to isolate human antibodies from libraries via PROfusion and / or yeast - related technologies. Transgenic animals (e.g., mice) can also be produced that are capable of generating a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production after immunization. For example, in chimeric and germline - mutated mice, the antibody heavy - chain joining region (J H)Homozygous deletion of the gene results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies upon antigen challenge. Humanized or fully human antibodies can be prepared according to the methods described in U.S. Patent Nos. 5,770,429, 5,833,985, 5,837,243, 5,922,845, 6,017,517, 6,096,311, 6,111,166, 6,270,765, 6,303,755, 6,365,116, 6,410,690, 6,682,928, and 6,984,720, the contents of each of which are incorporated herein by reference.

[0403] e. anti-GFAP antibody

[0404] Anti-GFAP antibodies can be generated using the techniques described above and using conventional techniques known in the art. In some embodiments, the anti-GFAP antibody can be an unconjugated GFAP antibody, such as a GFAP antibody 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); EMDMillipore (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); Sino Biological Inc. (Catalog No.: 100140-R012-50). The anti-GFAP antibody can be conjugated to a fluorophore, such as a conjugated GFAP antibody 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).

[0405] Alternatively, the antibodies described in WO 2018 / 067474, WO2018 / 081649, U.S. Patent No. 11,078,298, U.S. 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) may also be used, the contents of which are incorporated herein by reference.

[0406] 8. A method for measuring the level of an analyte other than GFAP (e.g., UCH-L1)

[0407] In the methods described above, the magnetic point-of-care assay for GFAP can be combined with other assays (i.e., non-magnetic point-of-care assays) for other analytes, including, for example, UCH-L1. The level of UCH-L1 can be measured by any means, such as antibody-dependent methods, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, protein immunostaining, electrophoretic analysis, protein analysis, competitive binding analysis, functional protein analysis, or chromatographic or spectroscopic methods, such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS). Additionally, the assays can be performed in a clinical chemistry format known to those of skill in the art. For example, such methods for measuring UCH-L1 are described, for example, in 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.

[0408] In some embodiments, measuring the level of UCH-L1 includes contacting a sample with a first specific binding member and a second specific binding member. In some embodiments, the first specific binding member is a capture antibody and the second specific binding member is a detection antibody. In some embodiments, measuring the level of UCH-L1 includes contacting the sample simultaneously or sequentially in any order with: (1) a capture antibody (e.g., a UCH-L1 capture antibody) that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form a capture antibody-UCH-L1 antigen complex (e.g., a UCH-L1 capture antibody-UCH-L1 antigen complex), and (2) a detection antibody (e.g., a UCH-L1 detection antibody) that includes a detectable label and binds to an epitope on UCH-L1 that is not bound by the capture antibody to form a UCH-L1 antigen-detection antibody complex (e.g., a UCH-L1 antigen-UCH-L1 detection antibody complex), such that a capture antibody-UCH-L1 antigen-detection antibody complex (e.g., a UCH-L1 capture antibody-UCH-L1 antigen-UCH-L1 detection antibody complex) is formed; and measuring the amount or concentration of UCH-L1 in the sample based on the signal generated by the detectable label in the capture antibody-UCH-L1 antigen-detection antibody complex.

[0409] In some embodiments, the first specific binding member is immobilized on a solid support. In some embodiments, the second specific binding partner is immobilized on a solid support. In some embodiments, the first specific binding partner is a UCH-L1 antibody as described below.

[0410] 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 liter, 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.

[0411] 9. UCH-L1 Antibody

[0412] The methods described herein can use isolated antibodies that specifically bind to ubiquitin carboxyl-terminal hydrolase L1 (“UCH-L1”) (or fragments thereof), referred to as “UCH-L1 antibodies”. UCH-L1 antibodies can be used to assess UCH-L1 status as a measure of traumatic brain injury, detect the presence of UCH-L1 in a sample, quantify the amount of UCH-L1 present in a sample, or detect the presence of UCH-L1 in a sample and quantify its amount.

[0413] a. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1)

[0414] 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 that hydrolyzes small C-terminal adducts of ubiquitin to generate ubiquitin monomers. The expression of UCH-L1 is highly specific for neurons and for cells of the diffuse neuroendocrine system and their tumors. It is present in large amounts in all neurons (1-2% of total brain protein), and is particularly expressed in neurons and testis / ovary. The catalytic triad of UCH-L1 contains cysteine at position 90, aspartic acid at position 176, and histidine at position 161, which are responsible for its hydrolase activity.

[0415] Human UCH-L1 can have the following amino acid sequence:

[0416] MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFPLTAQHENFRKKQIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGSVLKQFLSETEKMSPEDRAKCFEKNEAIQAAHDAVAQEGQCRVDDKVNFHFILFNNVDGHLYELDGRMPFPVNHGASSEDTLLKDAAKVCREFTEREQGEVRFSAVALCKAA (SEQ ID NO:1).

[0417] Human UCH-L1 can be a fragment or variant of SEQ ID NO:1. The length of a fragment 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 175 amino acids, or between 175 and 225 amino acids. The fragment can contain a number of contiguous amino acids from SEQ ID NO:1.

[0418] b. UCH-L1 recognizing antibody

[0419] The antibody is an antibody that binds to UCH-L1, a fragment thereof, an epitope of UCH-L1, or a variant thereof. The antibody can be a fragment, variant, or derivative of an anti-UCH-L1 antibody. 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. Antibody fragments or derivatives can include F(ab')2, Fv, or scFv fragments. Antibody derivatives can be produced by mimotopes. In addition, techniques described for producing single-chain antibodies can be applicable to producing single-chain antibodies.

[0420] 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 comprise a single Fab region linked to an Fc region.

[0421] Human antibodies can be derived from phage display technology or transgenic mice expressing human immunoglobulin genes. Human antibodies can be generated as a result of an immune response in a human body and isolated. See, for example, Funaro et al., BMC Biotechnology, 2008(8):85. Thus, the antibody can be a product of a human rather than an animal repertoire. Since it is of human origin, the risk of autoantigenic reactions can be reduced. Alternatively, standard yeast display libraries and display techniques can be used to select and isolate human anti-UCH-L1 antibodies. For example, an original human single-chain variable fragment (scFv) library can be used to select human anti-UCH-L1 antibodies. Transgenic animals can be used to express human antibodies.

[0422] A humanized antibody can be an antibody molecule from an antibody of a non-human species that binds a desired antigen having one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule.

[0423] The antibody is distinguished from known antibodies in that it has a biological function different from those known in the art.

[0424] (1) Epitope

[0425] The antibody can immunospecifically bind to UCH-L1 (SEQ ID NO: 1), its fragment or its variant. The antibody can immunospecifically recognize and bind to at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids or at least ten amino acids within the epitope region. The antibody can immunospecifically recognize and bind to an epitope having at least three consecutive amino acids, at least four consecutive amino acids, at least five consecutive amino acids, at least six consecutive amino acids, at least seven consecutive amino acids, at least eight consecutive amino acids, at least nine consecutive amino acids or at least ten consecutive amino acids in the epitope region.

[0426] c. Antibody preparation and production and anti-UCH-L1 antibody

[0427] Anti-UCH-L1 antibodies can be generated or prepared using conventional 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 which are incorporated herein by reference. In some embodiments, the anti-UCH-L1 antibody can be an unconjugated UCH-L1 antibody, such as UCH-L1 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); Thermo Fisher 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 available from BioVision (Catalog No.: 6960-25) or Aviva Systems Biology (Catalog No.: OAAF01904-FITC).

[0428] 10. Other factors

[0429] The methods of diagnosis, prognosis, and / or assessment as described above can further include diagnosing, prognosticating, and assessing using other factors. In some embodiments, the Glasgow Coma Scale can be used to diagnose traumatic brain injury. Other tests, scales, or indices can be used alone or in combination with the Glasgow Coma Scale. One example is the Ranchos Los Amigos Scale. The Ranchos Los Amigos Scale measures the levels of consciousness, cognition, behavior, and interaction with the environment. The Ranchos Los Amigos Scale 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, which is a self - report scale for measuring the severity of post - concussion symptoms after TBI. Patients are asked to rate the severity of each of 16 symptoms (such as headache, dizziness, nausea, vomiting) experienced in the past 24 hours. In each case, the symptoms are compared to the severity before the injury (pre - onset). These symptoms are reported on a scale of 0 to 4: not experienced, no longer a problem, mild problem, moderate problem, and severe problem.

[0430] 11. Sample

[0431] In some embodiments, a sample is obtained after a subject (such as a human subject) has suffered a head injury caused by blunt impact, one or more falls, explosion or shock wave, or other types of blunt force trauma resulting from physical shaking, external mechanical force, or other forces that cause closed or open head trauma. In some embodiments, a 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, paint, glue, gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organometals (such as methylmercury, tetraethyllead, and organotin), and / or one or more abused drugs. In some embodiments, the sample is obtained from a subject (such as a human subject) suffering from an autoimmune disease, metabolic disorder, brain tumor, hypoxia, viral infection (e.g., SARS - CoV - 2), fungal infection, bacterial infection, meningitis, hydrocephalus, or any combination thereof.

[0432] In another embodiment, the sample used in the methods described herein can also be used to determine whether a subject has a TBI (such as mild TBI, moderate TBI, severe TBI, or moderate-to-severe TBI) or is at risk of developing it by determining the levels of UCH-L1 and / or GFAP in the subject using the anti-UCH-L1 and / or anti-GFAP antibodies or antibody fragments described below. Thus, in certain embodiments, the present disclosure also provides a method for determining whether a subject having a traumatic brain injury as described herein and known in the art or at risk of a traumatic brain injury as described herein and known in the art is a candidate for therapy or treatment. Generally, the subject is at least one of the following: (i) has experienced a head injury; (ii) has ingested and / or been exposed to one or more chemicals and / or toxins; (iii) has an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection (such as SARS-CoV-2), a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof; or (iv) any combination of (i)-(iii); or, has actually been diagnosed with a TBI or is at risk of a TBI (such as, a subject having an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection (such as SARS-CoV-2), a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof) and / or exhibits 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.

[0433] c. Control

[0434] It may be desirable to include a control sample. The control sample can be analyzed simultaneously with the sample from the subject as described above. The results obtained from the subject sample can be compared with the results obtained from the control sample. A standard curve can be provided, and the assay results of the sample can be compared thereto. If a fluorescent label is used, such a standard curve presents the marker level as a function of the assay unit, i.e., the fluorescence signal intensity. Using samples taken from multiple donors, a reference level for GFAP in normal healthy tissue can be provided, as well as a standard curve for the "at risk" level of GFAP in tissue obtained from donors who may have one or more of the characteristics set forth above.

[0435] Accordingly, in view of the foregoing, a method for determining the presence, amount, or concentration of GFAP in a test sample is provided. The method includes assaying for GFAP in the test sample by an immunoassay that employs, for example, at least one capture antibody that binds to an epitope on GFAP and at least one detection antibody that binds to an epitope on GFAP that is different from the capture antibody and optionally includes a detectable label, and includes comparing a signal generated by the detectable label, which is a direct or indirect indication of the presence, amount, or concentration of GFAP in the test sample, with a generated signal that is a direct or indirect indication of the presence, amount, or concentration of GFAP in a calibrator. The calibrator is optionally, and preferably, part of a series of calibrators, where each calibrator differs from the other calibrators in the series by GFAP concentration.

[0436] The present disclosure has multiple aspects, which are illustrated by the following non-limiting examples.

[0437] Examples

[0438] Example 1

[0439] Figure 1 Demonstration of a GFAP assay using paramagnetic microparticles (e.g., magnetosensitive beads) that are magnetically captured and retained on a magnet (e.g., a magnetic immunosensor). Monoclonal antibody pairs are used, such as 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 in-house at Abbott Laboratories (Abbott Park, IL). A biotin linker is conjugated to antibody A using conventional techniques known in the art. Antibody A containing the biotin linker is also coated (e.g., immobilized) on the paramagnetic particles using conventional techniques known in the art. The paramagnetic particles used are Dynabeads TM MyOne TM Streptavidin T1, which are uniform and superparamagnetic beads that have a recombinant streptavidin monolayer covalently coupled to the surface of the beads. The paramagnetic particles coated with antibody A are printed and dried on a sensor chip in a cartridge for use in a point-of-care device. Antibody B is labeled with a detectable label (e.g., alkaline phosphatase) using conventional techniques known in the art. At least one antibody B labeled with the detectable label is printed and dried on the same sensor chip that contains the printed and dried paramagnetic particles coated with antibody A.

[0440] Fingerstick blood samples (whole blood) were obtained from normal subjects. The samples were inserted into a cartridge for use with a point-of-care device. Printed paramagnetic particles coated with antibody A and biotin linker and antibody B labeled with a detectable label were reconstituted and mixed with the whole blood samples. A complex comprising paramagnetic particles coated with antibody A - GFAP - antibody B labeled with a detectable label was formed. Magnets contained on the sensor chip (i.e., under the chip, which together constitute a magnetic immunosensor) captured and retained the complex, and the amount of GFAP was determined using an i-STAT Alinity instrument (or i-STAT1 instrument). The techniques described in U.S. Patent Nos. 9,233,370, 9,958,440, 10,145,843 and International Patent Publications WO18107016, WO18107015, WO18107007, WO18107009, WO18107012, WO18107013, WO21211331 and WO21211332 were also employed, the contents of which are incorporated herein by reference. The total assay time was approximately 10 minutes. The limit of quantification for the magnetic GFAP assay was ≤10 pg / mL. In contrast, the limit of quantification for the commercially available TBI plasma test (Abbott Laboratories) used on the Alinity instrument was approximately 23 pg / mL.

[0441] Example 2

[0442] Figure 10 Devices are shown that can be used as plasma separation devices in the methods and systems described herein. The device includes a hydrophilic top layer, a hydrophobic layer having microchannels (which have a first end and a second end), a hydrophilic bottom layer, and a protective film. The hydrophilic top layer adheres to the hydrophobic layer, and the protective film adheres to the hydrophilic bottom layer, and the hydrophilic bottom layer adheres to the hydrophobic layer. The top layer includes a sample inlet. The sample inlet is surrounded by a circular hydrophobic transfer band. On top of the hydrophobic transfer band is a plasma separation membrane (PSM), the top and bottom surfaces of which are flanked by hydrophilic mesh sheets. The hydrophilic mesh sheet located above or on top of the PSM helps to disperse the sample. The hydrophilic mesh sheet located below or beneath the PSM helps to process the blood sample or blood product into the hydrophobic layer. A restraint ring that helps to contain the sample encompasses the PSM, the hydrophilic mesh sheets, and the transfer band. The hydrophobic layer contains an opening (e.g., a first opening) directly below the sample inlet. The opening in the hydrophobic layer connects to the first end of the microchannel. Plasma flows from the opening at the first end (e.g., the first opening) to a second opening at the second (e.g., opposite) end. The device also contains an air pump for circulating air within the device.

[0443] Example 3

[0444] ​Blood samples were obtained from 6 donors on 4 different test days. Each blood sample was manipulated to provide a number of different samples. Specifically, the samples:

[0445] 1. Were tested without any manipulation.

[0446] 2. Were boosted with native GFAP antigen.

[0447] 3. Were spiked with recombinant GFAP antigen.

[0448] The samples were tested at nominal hematocrit (packed cell volume (PCV) of 37 - 45%) and modified hematocrits (low: 30, 33% PCV; high: 44, 50, 52, 54, and 55% PCV).

[0449] The samples were aspirated into 6 mL K2 - EDTA tubes and then plasma was separated by:

[0450] 1. Centrifugation; or

[0451] 2. Using a pre - evacuated tube having an inlet end and an outlet end. The inlet end of the tube has a blood - receiving chamber and the outlet end has a serum - receiving chamber. The tube also has a filter assembly between the blood - receiving chamber at the inlet end and the serum - receiving 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.

[0452] Once the plasma was obtained, it was placed in the sample wells of a cartridge and tested in a point - of - care device (such as a device from Abbott Laboratories (Abbott Park, IL)). device).

[0453] The results confirmed that both native and recombinant GFAP can be detected by a point - of - care device using plasma generated using a pre - evacuated tube.

[0454] Example 4

[0455] This study aimed to compare GFAP levels in capillary and venous blood samples obtained from normal subjects. Five (5) subjects were enrolled in this study. Each subject underwent two (2) blood draws. The first blood draw was a venous blood draw performed by a phlebotomist using conventional venous blood collection techniques known in the art. The second blood draw was a capillary blood draw and was performed using a TAP II sample collection device commercially available from YourBio Health (Medford, MA), in accordance with the manufacturer's instructions. The TAP II sample collection device is described in WO 2020 / 223710, the content of which is incorporated herein by reference.

[0456] A portion of each of the venous and capillary samples obtained from each subject was centrifuged to obtain plasma. For each subject, the venous whole blood and plasma samples and the capillary whole blood and plasma samples were tested.

[0457] Using the magnetic GFAP assay described in Example 1 and Figure 1 shown in, the venous whole blood and plasma samples and the capillary whole blood and plasma samples of each of five (5) subjects were tested.

[0458] As Figure 12 shown, the readings of GFAP levels in the venous whole blood and plasma samples were similar to those in the capillary whole blood and plasma samples (e.g., exhibiting a correlation of about 1.0 to about 0.85 (1.0:0.85) between venous whole blood and plasma and capillary whole blood and plasma).

[0459] It will be apparent to those skilled in the art that other suitable modifications and variations of the disclosed methods herein are readily applicable and understood and may be made using suitable equivalents without departing from the scope of the disclosure or the aspects and embodiments disclosed herein. The disclosure has now been described in detail and will be more clearly understood by reference to the following examples, which are intended to illustrate only some aspects and embodiments of the disclosure and should not be considered as limiting the scope of the disclosure. All journal references, U.S. patents, and published disclosures mentioned herein are hereby incorporated by reference in their entirety.

[0460] The disclosure has several aspects, which are illustrated by the non-limiting examples described herein.

[0461] It should be understood that the foregoing detailed description and the accompanying examples are illustrative only and should not be considered as limiting the scope of the disclosure, which is defined only by the appended claims and their equivalents.

[0462] Various changes and modificat...

Claims

1. An assay for measuring the amount of glial fibrillary acidic protein (GFAP) in a biological sample obtained from a subject, comprising: Perform a determination for GFAP, wherein the determination comprises: (a) contacting the sample with a cartridge comprising at least one magnetic immunosensor and (i) at least one first specific binding partner printed on the cartridge, comprising at least one anti-GFAP antibody that specifically binds GFAP in the sample, wherein the at least one first specific binding partner is immobilized on at least one magnetically sensitive bead; and (ii) at least one second specific binding partner printed on the cartridge, comprising a detectable label, thereby generating one or more complexes comprising the first specific binding partner-GFAP-second specific binding partner; (b) using the at least one magnetic immunosensor to magnetically capture and retain the beads containing the complex; and (c) evaluating the signal from the complex, wherein the amount of the detectable signal from the detectable label indicates the amount of GFAP in the sample, wherein the cartridge is used in a point-of-care device, and furthermore, wherein the determination exhibits at least a 5-fold increase in sensitivity compared to a determination in which the beads containing the complex are not magnetically captured and retained on at least one magnetic immunosensor in a cartridge in which the first specific binding partner is not immobilized on a magnetically sensitive bead and which is not used in a point-of-care device.

2. The assay according to claim 1, wherein the assay further comprises a step of washing unbound sample that is not magnetically captured and retained on the at least one magnetic immunosensor.

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

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

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

6. The assay according to any one of claims 1-5, wherein the assay exhibits 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 in which the first specific binding partner is not immobilized on the magnetosensitive beads and the beads containing the complex are not magnetically captured and retained on at least one magnetic immunosensor in a cartridge contained in a point-of-care device.

7. The assay according to any one of claims 1-6, wherein the assay is used to assist in diagnosing and evaluating a subject who has suffered or may have suffered a head injury.

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

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

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

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

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

13. The determination according to claim 12, wherein the sample is processed by plasmapheresis.

14. The determination according to claim 13, wherein the sample is processed using a plasmapheresis device.

15. The determination according to claim 14, wherein the plasmapheresis device: (a) is incorporated into or operably connected to a point-of-care device; or (b) is separate from the point-of-care device.

16. The determination according to any one of claims 5-15, wherein the amount of GFAP is communicated by display on the device.

17. A system, comprising: A determination for glial fibrillary acidic protein (GFAP), wherein the determination 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, comprising at least one anti-GFAP antibody that specifically binds GFAP in the sample, wherein the at least one first specific binding partner is immobilized on at least one magnetically sensitive bead; and (ii) at least one second specific binding partner printed on the cartridge, comprising a detectable label, thereby generating one or more complexes comprising the first specific binding partner-GFAP-second specific binding partner; A point-of-care device comprising the cartridge, wherein the device (a) determines the amount of GFAP in a sample obtained from the subject by magnetically capturing and retaining the beads containing the complex on the at least one magnetic immunosensor; and (b) evaluates the signal from the complex, wherein the amount of the detectable signal from the detectable label indicates the amount of GFAP in the sample, wherein the determination exhibits at least a 5-fold increase in sensitivity compared to a determination in which the beads containing the complex are not magnetically captured and retained on at least one magnetic immunosensor in a cartridge contained in a point-of-care device.

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-field magnetic particles.

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

21. The system according to any one of claims 17-20, wherein the determination exhibits an increase in sensitivity 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 compared to a determination in which the first specific binding partner is not immobilized on the magnetic beads and the beads containing the complex are not magnetically captured and retained on at least one of the magnetic immunosensors in a cartridge contained in the point-of-care device.

22. The system according to any one of claims 17-21, wherein the determination is used to assist in diagnosing and evaluating a subject who has suffered or may have suffered a head injury.

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

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

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

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

27. The system according to any one of claims 17 - 26, wherein the sample is processed prior to performing 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: (a) is incorporated into or operably connected to a point - of - care device; or (b) is separate from the point - of - care device.

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

32. A cartridge, comprising: a. A magnet; b. A region comprising printed paramagnetic particles coated with an anti-GFAP antibody; and c. A region comprising a plurality of printed, detectably labeled anti-GFAP antibodies, wherein the cartridge is used in a point-of-care device.

33. The cartridge according to claim 32, wherein the region containing the plurality of detectably - labeled anti - GFAP antibodies is the same as the region containing the printed paramagnetic particles coated with the anti - GFAP antibody.

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.

35. The assay according to claim 16, wherein prior to displaying the amount of GFAP on the device, the assay further comprises: a. Determine the amount of GFAP in the capillary blood sample; b. Select a conversion factor to compare the amount of GFAP in the sample with the amount of GFAP in venous blood, where the conversion factor is a static correlation ratio, a dynamic ratio, or a combination thereof; and c. Standardize the amount of GFAP in the sample using the amount of GFAP from venous blood by applying the conversion factor selected in step b) to the amount of GFAP in the sample.

36. The assay according to claim 35, wherein the normalized amount of GFAP is displayed by the device.

37. The assay according to claim 35 or claim 36, wherein the conversion factor is from about 1.2:1.0 to about 1.0:0.

5.

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

Images