Devices, systems, compositions, and methods for diagnosis of traumatic brain injury

Abstract

Provided herein are systems, devices and methods relating to the diagnosis of traumatic brain injury such as mild traumatic brain injury.

Description

DEVICES, SYSTEMS, COMPOSITIONS, AND METHODS FOR DIAGNOSIS OF TRAUMATIC BRAIN INJURYREEATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 561,162, filed on March 4, 2024; the entire contents of which are expressly incorporated herein by reference.SEQUENCE LISTING

[0002] The instant application contains a sequence listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 27, 2025, is named 13953 l-00120.xml and is 55,776 bytes in size.BACKGROUND

[0003] Concussions, often referred to as mild traumatic brain injuries (mTBIs), affect 69 million individuals worldwide. Traumatic Brain Injury (TBI) is a common occurrence in a variety of contexts including sports, military exercises, natural disasters and vehicular accidents. The direct costs associated with TBI treatment in the U.S. have been estimated to exceed $4 billion annually. Between 1.6-3.8 million sports or recreational activities-related concussions occur annually in the United States alone, and approximately half of these involve children and adolescents. A TBI is a sudden damage to the brain, and it occurs when an external force is strong enough to move the brain within the skull. Common causes include falls, traffic accidents, as well as head trauma associated with sports and military combat. The extent of the condition depends on the severity and location of the injury, and it can range from mild concussions to severe brain damage. Patients with mTBI can experience a variety of symptoms including loss of consciousness, memory loss, frustration, headaches, seizures, dizziness, fatigue, and emotional change.

[0004] Patients can appear normal following the injury, however, their condition may worsen as the brain undergoes a delayed trauma reducing the flow of oxygen-rich-blood (known as a secondary brain injury). In addition, studies have shown that there is an opening and damage of the blood brain barrier (BBB) as a result of mTBI that persists between 6-24 hours following the injury. This may significantly impact the quality of life of the patient and may lead to further long-term effects including neurodegenerative disease such as Alzheimer’ sand epilepsy. Early diagnosis of TBI reduces the risk of the secondary head injury and enables patients to be more aware of their injuries and seek proper care and immediate medical assistance.

[0005] When TBI patients report to hospitals, doctors often use computed tomography (CT) scan, Magnetic resonance imaging (MRI), Magnetic resonance spectroscopy (MRS), and the known Glasgow coma score (GCS; a 15 point test used to grade the patient’s level of consciousness) to diagnose TBI. These imaging tools are oftentimes used to diagnose moderate and severe TBI cases as their injuries are visible in patients. However, symptoms for mTBI cases may not be visible and patients may be released based on subjective questionnaire responses. Even if a CT scan is performed, mTBI is frequently invisible to imaging, leading to misdiagnosis and a lack of proper care. Since mTBI represent about 90% of all TBI cases, there is a need for accurate diagnosis.SUMMARY

[0006] Provided herein are systems, devices and methods that provide for the easy, immediate, accurate, and non-invasive diagnosis of traumatic brain injury such as mTBI. Unlike the existing test devices, such as Abbott i-STAT TBI Plasma, Alinity® i TBI or i- STAT TBI, the bioMerieux Banyan BTI, andt the Simoa® Neurology 2-Plex B assay, which require the patients to get to a health care facility and have the blood test drawn, the inventors of the present invention developed a lateral flow saliva test that can be run at the point of the injury, not requiring blood draw or a healthcare facility or practitioner. The device of the presented application is at least 90% accurate when obtained in less than 15 minutes, for example 5-10 minutes, and is planned to about <20% of cost of the serum tests.

[0007] In one aspect, provided are systems for diagnosing mild traumatic brain injury in a subject, the system comprising (a) a test device having a sample collector configured to receive a biological sample from the subject, and a reaction stage configured to receive the sample from the sample collector, the stage comprising a plurality of reaction zones where the sample is contacted with a detection reagent sensitive to a biomarker for traumatic brain injury to form a biomarker-detection reagent complex; and (c) a capture reagent conjugated to a visualization reagent in each reaction zone that generates a visible change when the capture reagent binds to the biomarker-detection reagent complex.

[0008] In another aspect, provided are methods for diagnosing mild traumatic brain injury in a subject, the method comprising: a. obtaining a biological sample from the subject; b. placing the sample in a sample collector that is configured to conduct the sample into areaction stage configured, the reaction stage comprising a plurality of reaction zones, each reaction zone comprising a combination of a detection reagent sensitive to a biomarker for traumatic brain injury, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarker-detection reagent complex, and when the capture reagent binds to the biomarkerdetection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones and then reading a set of visible signals from the plurality of reaction zones; and d. comparing the set of signals obtained with a reference to determine whether the subject has a) no traumatic brain injury, or b) mild traumatic brain injury.

[0009] In yet another aspect, provided are methods for diagnosing traumatic brain injury in a subject, the method comprising: a. obtaining a biological sample from the subject; b. placing the sample in a sample collector that is configured to conduct the sample into a reaction stage configured, the reaction stage comprising a plurality of reaction zones, each reaction zone comprising a combination of a detection reagent sensitive to a biomarker for traumatic brain injury, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarker-detection reagent complex, and when the capture reagent binds to the biomarker-detection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones and then reading a set of visible signals from the plurality of reaction zones; and d. comparing the set of signals obtained with a reference to determine whether the subject has a) no traumatic brain injury, b) mild traumatic brain injury, or c) moderate to severe traumatic brain injury.

[0010] In yet another aspect, provided are methods for detecting mild traumatic brain injury in a subject, comprising: a. obtaining a biological sample from the subject; b. placing the sample in a test device and allowing the sample to contact a plurality of reaction zones, each reaction zone containing a combination of a detection reagent sensitive to a biomarker for traumatic brain injury, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarker-detection reagent complex, and when the capture reagent binds to the biomarkerdetection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones; d. reading a set of visible signals from theplurality of reaction zones; and e. determining based on the set of visible signals whether the subject has a) no traumatic brain injury, or b) mild traumatic brain injury.

[0011] In yet another aspect, provided are methods for diagnosing traumatic brain injury in a subject, the method comprising: a. obtaining a biological sample from the subject; b. placing the sample in a sample collector that is configured to conduct the sample into a reaction stage configured, the reaction stage comprising a plurality of reaction zones, each reaction zone comprising a combination of a detection reagent sensitive to a biomarker for traumatic brain injury, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarker-detection reagent complex, and when the capture reagent binds to the biomarker-detection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones and then reading a set of visible signals from the plurality of reaction zones; and d. comparing the set of signals obtained with a reference to determine whether the subject has a) no traumatic brain injury, b) mild traumatic brain injury, or c) moderate to severe traumatic brain injury.

[0012] In yet another aspect, provided are methods for detecting mild traumatic brain injury in a subject, comprising: a. obtaining a biological sample from the subject; b. placing the sample in a test device and allowing the sample to contact a plurality of reaction zones, each reaction zone containing a combination of a detection reagent sensitive to a biomarker for traumatic brain injury, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarker-detection reagent; complex, and when the capture reagent binds to the biomarkerdetection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones; d. reading a set of visible signals from the plurality of reaction zones; and e. determining based on the set of visible signals whether the subject has a) no traumatic brain injury, or b) mild traumatic brain injury.

[0013] In yet another aspect, provided are methods for detecting traumatic brain injury in a subject, comprising: a. obtaining a biological sample from the subject; b. placing the sample in a test device and allowing the sample to contact a plurality of reaction zones, each reaction zone containing a combination of a detection reagent sensitive to a biomarker for traumatic brain injury, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarkerdetection reagent complex, and when the capture reagent binds to the biomarker-detectionreagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones; d. reading a set of visible signals from the plurality of reaction zones; and e. determining based on the set of visible signals whether the subject has a) no traumatic brain injury, b) mild traumatic brain injury, or c) moderate to severe traumatic brain injury.

[0014] In yet another aspect, provided are methods for treating mild traumatic brain injury in a subject, comprising: a. obtaining a biological sample from the subject; b. placing the sample in a test device and allowing the sample to contact a plurality of reaction zones, each reaction zone containing a combination of a detection reagent sensitive to a biomarker for traumatic brain injury mild traumatic brain injury / concussion, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarker-detection reagent complex, and when the capture reagent binds to the biomarker-detection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones; d. reading a set of visible signals from the plurality of reaction zones; e. determining based on the set of visible signals whether the subject has a) no traumatic brain injury, or b) mild traumatic brain injury; and f. administering an appropriate medical care for the subject determined to have mild traumatic brain injury.

[0015] In yet another aspect, provided are methods for treating traumatic brain injury in a subject, comprising: a. obtaining a biological sample from the subject; b. placing the sample in a test device and allowing the sample to contact a plurality of reaction zones, each reaction zone containing a combination of a detection reagent sensitive to a biomarker for traumatic brain injury mild traumatic brain injury / concussion, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarker-detection reagent complex, and when the capture reagent binds to the biomarker-detection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones; d. reading a set of visible signals from the plurality of reaction zones; e. determining based on the set of visible signals whether the subject has a) no traumatic brain injury, b) mild traumatic brain injury, or c) moderate to severe traumatic brain injury; and f. administering an appropriate medical care for the subject determined to have traumatic brain injury.

[0016] In yet another aspect, provided is an isolated antibody, or antigen-binding fragment thereof, that specifically binds to S100 Calcium Binding Protein B (SIOOB), wherein theantibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), (i) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:5, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO: 10; (ii) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO: 15, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:20; (iii) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:25, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:30; and (iv) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:35, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:40.

[0017] Further provided are alternative isolated nucleic acid molecules as well as isolated mRNAs, vectors, cells, compositions, and kits.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 depicts biogenesis and generation of a single stranded mature miRNA from stem- loop pri-miRNAs.

[0019] FIG. 2 is a table of 86 miRNAs expressed in saliva that are believed to be associated with mTBI.

[0020] FIG. 3 depicts different binding patterns between miRNA and their mRNAs.

[0021] FIGS. 4A-4C, are graphs showing expression levels of 46 miRNAs in brain tissue selected from the Table in FIG. 2.

[0022] FIG. 5A depicts an aspect of a mild traumatic brain injury detector.

[0023] FIG. 5B depicts the mild traumatic brain injury detector of FIG. 5 A.

[0024] FIG. 6A and 6B are internal schematic drawings of the mild traumatic brain injury detector of FIG. 5 A.

[0025] FIG. 7 depicts method steps for determining a mild traumatic brain injury, using the detector of FIGS. 5A-6.

[0026] FIG. 8A depicts an internal design of a lateral flow device for detecting mild traumatic brain injuries.

[0027] FIG. 8B depicts an alternative embodiment of the lateral flow device of FIG. 8A.

[0028] FIG. 8C depicts an alternative embodiment of the lateral flow device of FIG. 8A.

[0029] FIG. 8D depicts an internal design of a lateral flow device for detecting mild and severe traumatic brain injuries.

[0030] FIG. 8E depicts an alternative embodiment of the lateral flow device of FIG. 8A.

[0031] FIG. 8F depicts an internal design of the lateral flow device of FIG. 8C with a fluid sample deposited.

[0032] FIGS. 8G and 8H depict an internal design of the lateral flow device of FIG. 8C, where FIG. 8G displays a positive result and FIG. 8H displays a negative result.

[0033] FIGS. 9A and 9B depict an external design of the lateral flow device of FIGS. 8B and 8D having a cap.

[0034] FIG. 9C depicts the lateral flow device of FIGS. 9 A and 9B.

[0035] FIG. 10 shows 2 epitopes of the SIOOB protein.

[0036] FIG. 11 shows 3 epitopes of the SIOOB protein.

[0037] FIG. 12 shows a table of purified fusions products for antibody isolation.

[0038] FIG. 13 shows a table of purified fusions products for antibody isolation of FIG. 12, with 24 products exhibiting specific binding to the SIOOB protein.

[0039] FIG. 14 shows a table of fusion product pairs and their SIOOB detection ability at various concentrations.

[0040] FIG. 15 depicts a graphical user interface.

[0041] FIG. 16A-D depict the binding data of several subclones against human SIOOB as determined by ELISA.DETAILED DESCRIPTION

[0042] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition.

[0043] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural (i.e., one or more), unless otherwise indicated herein or clearly contradicted by context.

[0044] The terms “comprising, “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value recited or falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited.

[0045] The term “about” or “approximately” means within 5%, or more preferably within 1% or 2%, of a given value or range.

[0046] As used herein, the term “Traumatic Brain Injury” or TBI includes a condition in which an external force causes trauma or damage to the brain. Such external force may penetrate or not penetrate the skull. TBI can classified as being mild, moderate and severe. In some instances, the initial trauma or damage can result in expanding hematoma, subarachnoid hemorrhage, cerebral edema, raised intracranial pressure (ICP), and cerebral hypoxia, which can, in turn, lead to severe secondary events due to low cerebral blood flow (CBF).

[0047] Mild traumatic brain injury (mTBI), also known as concussion, accounts for approximately 80% of all TBI cases and may be characterized according to the Glasgow Coma Scale (GCS) with a GCS value of 13-15. See e.g., Teasdale G, Jennett B (July 1974). "Assessment of coma and impaired consciousness. A practical scale". The Lancet. 2 (7872): 81-4. It is also defined as a loss of consciousness from less than 20 min to 1 hour. Moderate TBI may be characterized as a GCS score of 9-12 and / or a loss of consciousness from 1 hour to 24 hours. Severe TBI may be characterized as a GCS score of 3-8 and / or a loss of consciousness of greater than 24 hours.

[0048] As used herein, “biological sample” refers to any biological sample obtained from or derived from a subject. In some aspects, the biological sample is a liquid biological sample.

[0049] The term “liquid sample” or “liquid biological sample,” as used herein, refers to a sample that is substantially in liquid form. In some aspects, a liquid sample is a body fluid. Exemplary body fluids may include, e.g., saliva, serum, plasma, whole blood, or cerebrospinal fluid (CSF). In some aspects, the biological sample is a saliva sample. In some aspects, the biological sample is a blood, serum, or plasma sample.

[0050] As used herein, “subject” and “human” are used synonymously.

[0051] As used herein, the term “antibody” is intended to include any polypeptide chaincontaining molecular structure with a specific shape that fits to and recognizes an epitope (e.g., an antigen), where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g. human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken, other avians, etc., are considered to be “antibodies.”

[0052] Examples thereof include chimeric antibodies, human antibodies and other nonhuman mammalian antibodies, humanized antibodies, single chain antibodies (such as scFvs), camelbodies, nanobodies, IgNAR (single-chain antibodies which may be derived from sharks, for example), small-modular immunopharmaceuticals (“SMIPs”), and antibody fragments such as Fabs, Fab', F(ab')2, and the like (See Streltsov et al., Protein Sci., 14(1 l):2901-9 (2005); Greenberg et al., Nature, 374(6518): 168-73 (1995); Nuttall et al., Mol. Immunol., 38(4):313-26 (2001); Hamers-Casterman et al., Nature, 363(6428):446-8 (1993); Gill et al., Curr. Opin. Biotechnol., (6):653-8 (2006)). The term “antibody,” as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies.

[0053] Antibodies consist of two identical light polypeptide chains of molecular weight approximately 23,000 daltons (the “light chain”), and two identical heavy chains of molecular weight 53,000-70,000 (the “heavy chain”). The four chains are joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” configuration. The “branch” portion of the “Y” configuration is designated the Fab region; the stem portion of the “Y” configuration is designated the FC region. The amino acid sequence orientation runs from the N-terminal end at the top of the “Y” configuration to the C-terminal end at the bottom of each chain. The N-terminal end possesses the variable region having specificity for the antigen that elicited it, and is approximately 100 amino acids in length, there being slight variations between light and heavy chain and from antibody to antibody.

[0054] The variable region is linked in each chain to a constant region that extends the remaining length of the chain and that within a particular class of antibody does not vary with the specificity of the antibody (i.e., the antigen eliciting it). There are five known major classes of constant regions that determine the class of the immunoglobulin molecule (IgG,IgM, IgA, IgD, and IgE corresponding to y, p, a, 5, and a (gamma, mu, alpha, delta, or epsilon) heavy chain constant regions). The constant region or class determines subsequent effector function of the antibody, including activation of complement (see Kabat, E. A., Structural Concepts in Immunology and Immunochemistry, 2nd Ed., p. 413-436, New York, NY: Holt, Rinehart, Winston (1976)), and other cellular responses (see Andrews et al., Clinical Immunology, pp. 1-18, W. B. Sanders, Philadelphia, PA (1980); Kohl et al., Immunology, 48:187 (1983)); while the variable region determines the antigen with which it will react. Light chains are classified as either K (kappa) or (lambda). Each heavy chain class can be prepared with either kappa or lambda light chain. The light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages when the immunoglobulins are generated either by hybridomas or by B-cells.

[0055] The expression “variable region” or “VR” refers to the domains within each pair of light and heavy chains in an antibody that are involved directly in binding the antibody to the antigen. Each heavy chain has at one end a variable region (VH) followed by a number of constant domains. Each light chain has a variable region (VL) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.

[0056] The expressions “complementarity-determining region,” “hypervariable region,” or “CDR” refer to one or more of the hyper- variable or complementarity-determining regions (“CDRs”) found in the variable regions of light or heavy chains of an antibody (See Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., Bethesda, MD: U.S. Dept, of Health and Human Services, Public Health Service, National Institutes of Health (1987)). These expressions include the hypervariable regions as defined by Kabat et al., (Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda, MD: U.S. Dept, of Health and Human Services, National Institutes of Health (1983)) or the hypervariable loops in 3-dimensional structures of antibodies (Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)). The CDRs in each chain are held in close proximity by framework regions (“FRs”) and, with the CDRs from the other chain, contribute to the formation of the antigen binding site. Within the CDRs there are select amino acids that have been described as the selectivity determining regions (“SDRs”) that represent the critical contact residues used by the CDR in the antibody- antigen interaction (see Kashmiri et al., Methods, 36(l):25-34 (2005)).

[0057] An “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds an antigen, e.g., SIOOB, is substantially free of antibodies that specifically bind antigens other than SIOOB). An isolated antibody that specifically binds SIOOB may, however, have cross -reactivity to other antigens, such as SIOOB molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0058] The expressions “framework region” or “FR” refer to one or more of the framework regions within the variable regions of the light and heavy chains of an antibody (See Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, Bethesda, MD: U.S. Dept, of Health and Human Services, Public Health Service, National Institutes of Health (1987)). These expressions include those amino acid sequence regions interposed between the CDRs within the variable regions of the light and heavy chains of an antibody.

[0059] The phrase “specifically binds to S100B” as used herein, refers to the ability of an anti- SIOOB antibody or antigen-binding fragment thereof to interact with SIOOB with a dissociated constant (KD) of, for example, about 1,000 nM or less, about 500 nM or less, about 200 nM or less, about 100 nM or less, about 75 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less, about 100 pM or less, about 10 pM nM or less, about 1 pM or less, or about 0.1 pM or less. In another aspect, the phrase “specifically binds to S100B”, as used herein, refers to the ability of an anti-SlOOB antibody or antigen-binding fragment thereof to interact with SIOOB with a KD of between about 0.1 pM to 1,000 nM, between about 1 pM to 500 nM, between about 10 pM to 100 nM, between about 0.1 nM to 50 nM, or between about 1 nM to 50 nM. In one aspect, KD is determined by surface plasmon resonance, ELISAs, radioimmunoassays, bio-layer interferometry (BLI), or by any other methods known in the art.I. Devices and Systems

[0060] Provided herein are devices and systems for diagnosing traumatic brain injury, e.g., mTBI / concussion, through analyzing biomarkers associated with mTBI in a biological sample, e.g., a saliva sample. The devices and systems are for Real-Time Concussion Detection (RTCD).

[0061] As shown in FIGS. 5A and 5B, two similar aspects of a mild traumatic brain injury detector are depicted. The mild traumatic brain injury detectors include a microfluidic device 500 to diagnose mTBI through analyzing biomarkers associated with mTBI found in a fluidsample (sf), for example saliva. A user / patient’s fluid sample is collected by depositing the fluid sample directly into a swab collector 504. At that time, the built-in technology initiates an internal mechanism in a chip to analyze the saliva. The device 500 can include a signal (not pictured), such as an LED, located on a side of the device 500. In an aspect, the signal stays active, i.e. on, until a predetermined volume of fluid sample, necessary to diagnose mTBI, is collected. Once the predetermined volume of fluid sample is collected, the signal will become inactive, i.e. off. The signal may output a constant signal or may oscillate on and off, i.e. blink. In alternative aspects, the signal remains inactive until the predetermined volume of the fluid sample is collected. Once the predetermined volume of fluid sample is collected, the signal is activated, for example a light is emitted.

[0062] A minimum volume of fluid sample is necessary for analysis of biomarkers. At minimum, 70uL of fluid should be introduced into the device 500 to ensure an accurate result. In an embodiment, a minimum lOOuL of fluid should be introduced into the device 500. In some embodiments, the device 500 may include a collection buffer (not shown). The collection buffer stabilizes low-volume samples. The collection buffer is a solution. The device 500 may include 30uL - 55uL of collection buffer. In this embodiment, the minimum fluid sample may include 30uL - 55uL, where the combination of the fluid sample and collection buffer provides a total fluid of 70uL - lOOuL. In an embodiment, the collection buffer comprises EDTA configured to inhibit enzymatic degradation, thereby maintaining the integrity of the fluid sample (sf), a pH control configured to preserve DNA and / or protein of the fluid sample, chelating metal ions configured to activate any degrading enzymes, and stabilizing agents configured to prevent denaturation or aggregation of the fluid sample. These elements of the collection buffer enable the collection buffer to stabilize the fluid sample. In an embodiment, the collection buffer comprises TSB / 4% EDTA, 2% Tween 20, 0.5% BSA and pH 8.2. Introduction of 0.5% BSA into the collection buffer reduces NSB at the test line without affecting the signal at 50 ng / mL SIOOB. Accordingly, the inclusion of 0.5% BSA is advantageous as it improves signal noise and hence the sensitivity of the assay.

[0063] The built-in technology further determines the concentrations of the biomarker associated with mTBI (i.e., miRNA). Upon completion of the test, a biomarker meter 505 can be attached to the user’s phone 506 camera to scan the device. The biomarker meter 505 can include a clip mechanism 507 to allow it to securely attach to the device 500 when collecting the results and the user’s phone 506 when scanning the results. The clip mechanism 507 may include an indent on a distal end of the clip mechanism 507 to providean improve grip surface. In another aspect, the clip mechanism 507 may include a textured surface on the distal end to provide an improve grip surface. The results may then be displayed on the phone screen in a mobile application. The report provides concentrations of the miRNA biomarker and determines whether the patient has TBI or not. With reference to FIGS 4A and 4B certain aspects may, for example, provide for the measuring the severity of TBI.

[0064] A schematic drawing of the mild traumatic brain injury detector of FIGS. 5 A and 5B is shown in FIGS. 6A-B illustrating the internal components and further illustrating the external components. The device 500 includes a fluid sample collector opening 501 having an opening in an exterior surface of the device 500, a screener 502, and a blinking signal on the side of the device 500. In an aspect, the microfluidic device 500 can include a second signal503, such as an LED, located on a side or top surface of the device 500. The signal stays inactive, i.e. off, until the test is complete. Once the test is complete the signal 503 may emit a certain color or blinking pattern to notify the user that the test is complete and the sample is ready for input into the mobile application. The device 500 may be re-used. In this aspect, the collector 504 is removeable and can be replaced with a new collector 504.

[0065] With continued reference to FIGS. 6A-B, the mechanisms and the processing steps of how the fluid sample is analyzed are shown. The fluid sample is collected into the collector504, for example a swab, and then the fluid sample migrates through a channel 2 into a chamber 3 via a mechanical pressure as shown in the diagram. Chamber 3 is designed to promote mobilization in a circular motion allowing it to work as a centrifuge and communicate with one or more chambers. Chamber 3 includes an open opening at an edge of the circumference of the chamber 3. This opening initially opened to collect the sample from chamber 2 then closed as the fluid sample is circulated in the centrifugal motion of chamber 3. The fluid sample undergoes auto-lysed, RNA extraction and isolation, RNA purification, and nCounter hybridization. This can take place in the chamber 3 or can take place in one or more chambers. In the aspect with one or more chambers, the opening in the chamber 3 is opened and mechanical pressure is used to push the sample to a chamber 4. In the chamber 4, the fluid sample is auto-lysed in a way that only the portion containing the RNA is pushed back to the chamber 3. Following that, the opening closes, the chamber 3 circulates, the opening re-opens and the chamber 3 delivers the fluid sample to a chamber 5 in which the RNA extraction and isolation process takes place. Then the extracted RNA returns to the chamber 3 where the opening is closed and the chamber 3 circulates and re-opens the openingto deliver the fluid sample to a chamber 6 to purify the RNA. Lastly, the chamber 3 picks up the purified RNA fluid sample from the chamber 6 closing it and delivering it to a chamber 7 where nCounter hybridization occurs. nCounter hybridization technique is based on color- coded fluorescent tags for each miRNA (Nanostring Technologies, Seattle, WA). In an alternative aspect, the chamber 3 includes a plurality of openings that correspond with the one or more chambers to allow the sample to transfer to and from the chamber 3. The size of chamber 7 may be configured (e.g., sized and dimensioned) to accommodate the purified RNA sample. However, dimensioning may vary depending on the dimensions and portability of the device 500. Once hybridization of the RNA is completed, the data can be read using a similar software to the nSolver and results on the screen and a report is generated based on expression levels of specific miRNA markers that if deregulated, indicates whether the patient has mTBI or not.

[0066] In some embodiments, the device 500 may include a filter component (not shown) configured to filter substances from the fluid sample. Substances in the fluid sample may interfere with the analysis and test performance. Accordingly, it is advantageous to include a filter component to remove substances in the fluid sample prior to analysis. The filter component may filter substances such as, but not limited to, food particles, blood, medications, and particle debris. The filter component is positioned downstream of the collector 504 and upstream of the chamber 3. The filter component may be positioned between the collector 504 and the channel 2, between the channel 2 and the chamber 3, or along the channel 2. In some embodiments, the device 500 may include a plurality of filter components positioned downstream of the collector 504 and upstream of the chamber 3.

[0067] The total RNA isolation and auto-lyzed process may be, for example, completed using the miRNeasy Mini Kit (Qiagen, USA Cat.No: 217004). Other viable kits that may be used to conduct RNA extraction and isolation are, for example, the following non-phenol ones: AxyPrep Multisource Total RNA Miniprep, EasySpin and Hlustra RNAspin Mini RNA Isolation. The Axyprep multisource kit produced the best results with high yield (see Tavares, L., Alves, P. M., Ferreira, R. B., & Santos, C. N. (2011). Comparison Of Different Methods For Dna-Free Rna Isolation From Sk-N-Mc Neuroblastoma. Bmc Research Notes, 4(1)). The miRNA sample preparation may be, for example, done using the miRNA human sample prep kit from nanostring (Cat No. Hu-MIRTAG-12) and hybridization may be, for example, done using the nCounter analysis system from nanostring (Nanostring Technologies, Seattle, WA). In order to conduct the RNA purification process, the following kits may, for example, beutilized: RNeasy Mini, RiboPure and Purelink extraction kits (see Rump, L. V., Asamoah, B., & Gonzalez-Escalona, N. (2010). Comparison Of Commercial Rim Extraction Kits For Preparation Of Dna-Free Total Rna From Salmonella Cells. Bmc Research Notes, 3(1). Various kits have been mentioned for use with the different types of processes. For the Autolyzed, isolation, and RNA purification processes the miRNeasy Mini Kit, may be used and provides a highly cost effective option.

[0068] While this aspect refers to targeting specific miRNAs expressed in patient fluid sample with mTBI, the device 500 may also be used to detect the expression levels of specific genes and / or proteins that are altered during mTBI. Certain proteins are overexpressed in patients who recently underwent a traumatic brain injury, with the following types present in greater abundance. These proteins include 1) markers of glial cell injury like glial fibrillary acidic protein, GFAP; 2) calcium binding protein B, SIOOB; 3) myelin basic protein (MBP), axonal and neuronal injury (ubiquitin carboxyl-terminal hydrolase-El (UCH- El)); 4) neuron-specific enolase (NSE); 5) Tau protein and phosphorylated-Tau [p-Tau]. Chemiluminescent-based EEISA may be used to measure the concentration of two proteins, GFAP and SIOOB, which has been shown to be able to predict the TBI-positive CT scan with a sensitivity of 97.5% (Pankratova et al. , 2021). SIOOB is a calcium-binding protein released into the blood from astroglial cells due to brain injury (Rodriguez et al. , 2012). SIOOB is a strong predictor of an unfavorable outcome with 100% discrimination in TBI patients (Jacobs et al. , 2010). Thus, SIOOB levels are sensitive and may act, for example, as an effective biomarker.

[0069] Other aspects have different applications. By changing some of the reagents and the algorithm providing instructions to the mild traumatic brain injury detector, different aspects may have the capabilities to detect and help solve other medical problems. This includes but is not limited to: Covid- 19, certain periodontal diseases using salivary macrophage inflammatory protein- 1 a, matrix metalloproteinase-8, interleukin (IL)-l PP, IL-6, prostaglandin E2 and tumor necrosis factor (TNF)-a, oral cancer which may be detected by deregulation of certain miRNA biomarkers including miR125a and miR200a, Sjogren’s syndrome which may be detected by change in levels of IL-4, IL-5 biomarkers, viral infections including hepatitis C using RNA antigen tests, diabetes mellitus where levels of a- 2-macroglobulin in saliva may serve as a potential diagnosis for this disease, pancreatic cancer using miRNA biomarkers, cardiovascular disease using a-2-HS-glycoprotein biomarker in saliva, breast cancer, lung cancer and prostate cancer.

[0070] In one aspect, the present invention provides a system for diagnosing a disease in a subject, the system comprising (a) a test device having a sample collector configured to receive a biological sample from the subject, and a reaction stage configured to receive the sample from the sample collector, the stage comprising a plurality of reaction zones where the sample is contacted with a detection reagent sensitive to a biomarker for the disease to form a biomarker-detection reagent complex; and (b) a capture reagent conjugated to a visualization reagent in each reaction zone that generates a visible change when the capture reagent binds to the biomarker-detection reagent complex.

[0071] In some embodiments, the plurality of reaction zones have different sensitivities to the biomarker and thereby provide a plurality of output signals that together can be interpreted to differentiate whether the subject has a) no disease, or b) disease, e.g., mild disease, or moderate to severe disease.

[0072] In some embodiments, the disease is selected from the group consisting of CO VID- 19, periodontal diseases, cancers (e.g., oral cancer, pancreatic cancer, breast cancer, lung cancer, prostate cancer, etc), or viral infections (e.g., hepatitis C), diabetes mellitus, Sjogren’s syndrome, cardiovascular diseases, or combination thereof.

[0073] In some embodiments, the biological sample is a body fluid. In some embodiments, the biological sample is a saliva sample. In some embodiments, the biological sample is a serum, sweat, urine, breath, chloride, sodium, lactate, glucose, cortisol, urea, and various proteins and peptides, plasma, or blood sample.

[0074] In some embodiments, the biomarker is a protein biomarker, a DNA biomarker, an RNA biomarker (e.g., a miRNA marker), nitric oxide (NO), or fractional exhaled nitric oxide (FeNO).

[0075] In some embodiments, the biomarker is selected from the group consisting of macrophage inflammatory protein- 1 a, matrix metalloproteinase- 8, interleukin (IL)-l PP, IL- 6, prostaglandin E2, tumor necrosis factor (TNF)-a, IL-4, IL-5, a-2-macroglobulin, a-2-HS- glycoprotein, miR125a and miR200a, or combination thereof.

[0076] In some embodiments, the system further comprises a mobile device-based application that is configured to provide diagnostic information based on the visible change generated by the reagents in the plurality of reaction zones.

[0077] Depending on the biomarker and reagent, performance of the analysis and test may be influenced by environmental factors. Environmental factors that may influence the testinclude, but are not limited to, humidity, temperature, and light exposure. Accordingly, it is advantageous to include an alert mechanism (not shown) in the device 500 to alert the user when an environmental factor is present that may influence the results. The alert mechanism may include a sensor configured to monitor humidity levels, the temperature of the device 500, and / or the amount of light exposure. The alert mechanism may further include an indicator component such as an LED. When an environmental factor is detected by the sensor of the alert mechanism, the LED may be activated. For example, the LED may be activated when the temperature of the device 500 exceeds a predetermined threshold. The LED may further be activated when the device 500 is exposed to light over a predetermined threshold. The predetermined threshold may include UV levels and / or time. In some embodiments, the device 500 may further include a stabilizing agent configured to mitigate biomarker degradation. Biomarker degradation may occur due to improper storage of the device 500. As such, it is advantageous to include a stabilizing agent. The stabilizing agent is configured to prevent denaturation or aggregation of the fluid sample.

[0078] With reference to FIG. 7, the method for fluid sample analysis includes collecting the fluid sample 601; using mechanical pressure, migrating the fluid sample into a centrifuge chamber 602; circulating the fluid sample in the centrifuge chamber 603; pushing the fluid sample by mechanical pressure into an auto-lyse chamber 604; the RNA portion of the saliva sample is returned to the centrifuge 605; circulating the RNA portion of the fluid sample into an RNA extraction and isolation chamber 606; extracting and isolating the RNA 607; returning the extracted RNA to the centrifuge chamber 608; circulating the extracted RNA 609; opening the gate to deliver the sample to a purification chamber 610; purifying the RNA 611; returning the purified RNA to the centrifuge 612; delivering the purified RNA to a hybridization chamber for nCounter hybridization 613; retrieving data from RNA hybridization 614; analyzing expression levels of specific miRNA markers for deregulation 615; creating a report on whether the patient has mTBI or not 616.

[0079] In a certain aspect, provided is a test device for the rapid detection and / or diagnosis of mild traumatic brain injury (e.g., concussion). In certain aspects, the test device is configured to be useful in non-clinical settings where rapid (i.e., real time) assessment of an injured person’s brain injury status (e.g., concussion) can be important in making appropriate care decisions.

[0080] In an alternative aspect, the traumatic brain injury detector is a lateral assay device that is a one-time use rapid test device. The lateral assay device can be dimensioned suchthat it can fit in a user’ s pocket. Such a test device provides numerous advantages over existing clinical diagnostic methods: for example, it can be can easily be kept with first aid supplies, provides rapid results, and is convenient use at or near a site where a head injury occurs (e.g., at a sporting event, accident site, or battlefield).

[0081] In certain aspects, the provided test device is configured to receive a sample of fluid obtained from an injured person and to rapidly provide a set of visible signals indicative of the brain injury status of the person based on detection of the presence and / or concentration of biomarkers in the fluid sample. In certain aspects, the test device comprises a sample collector configured to receive a small sample of bodily fluid from the injured person and a reaction stage configured to receive fluid from the sample collector (e.g., via lateral flow or capillary action). In certain aspects, the reaction stage comprises a plurality of reaction zones where the fluid sample is contacted with one or more detection reagents sensitive to at least one biomarker associated with a traumatic brain injury. In certain aspects, the reaction stage further comprises one or more visualization reagents that generate a visible signal indicating that the detection reagent(s) have reacted with one or more biomarkers for traumatic brain injury.

[0082] In certain aspects, the sample collector is configured to receive a defined volume of the bodily fluid from the injured person — for example, the sample collector can include a reservoir configured to hold a predetermined volume of fluid or may contain an absorptive element such as a porous membrane, sponge, or pad configured to absorb a predetermined volume of fluid. In certain aspects, the sample collector includes an absorptive pad on which the fluid sample is placed. In an aspect, the fluid sample includes, for example, saliva.

[0083] In certain aspects, the test device further comprises a reaction stage a5 where the fluid (e.g., saliva) from the injured person is contacted with one or more reagents configured to react with biomarkers in the sample and generate one or more visible signals which provide information about the brain injury status of the injured person. The reaction stage may be an integral part of the sample collector, or the reaction stage may be separate from the sample collector and coupled to it such that fluid placed in the sample collector flows from the sample collector into the reaction stage (e.g., by capillary flow, pumping, or gravity). In certain aspects, the reaction stage comprises an area that is wetted with the fluid sample and which contains two or more discrete visualization zones each comprising less than the entire area of the reaction stage. Each visualization zone is configured to provide a visible signal indicative of different characteristics of the fluid sample. For example, in certain aspects,separate visualization zones are configured to provide distinct visible signals that are indicative of different concentrations (or different concentration ranges) of a particular biomarker in the sample. In other aspects, separate visualization zones may be configured to provide visible signals that are indicative of the presence or absence of different biomarkers or to provide visible signals that are indicative of the presence of absence of different combinations of biomarkers in the sample. In certain aspects, the reaction stage may comprise separate visualization zones that provide information related both to the presence or absence of different biomarkers and to the concentration (or concentration range) of one or more biomarkers.

[0084] With reference to the aspect of FIGS. 8 A - 8C, an aspect of the lateral flow device 800A is shown. The device 800A includes a sample collection region al, the collection swab al may be an absorption pad, for example cotton or a similar material. The sample collection swab al is configured (e.g., shaped and dimensioned) to collect a patient fluid sample by, for example, a patient placing the sample collection swab al in their mouth and applying a suction force by, for example, sucking on the sample collection swab al. A patient may, for example, suck on the sample collection swab al for a period of approximately 10 seconds to 20 seconds to provide a saliva sample. Alternatively, the patient may suck on the sample collection swab al up to, but less than, one minute. By sucking on the sample collection swab al, suction is created and saliva may be transferred by a capillary tube a2 to a sample pad a3. The capillary tube a2, is configured (e.g., shaped and dimensioned) to detect and allow a range of volume of saliva, to transfer to the pad a3 in a sufficient quantity or volume required to perform the sample testing, but without overfilling the sample pad a3. In certain aspects, the lateral flow device 800 includes a signal, such as an LED al4 (FIG. 8C), that is connected to the capillary tube a2, to indicate that a sufficient volume of fluid has been obtained and that testing can be initiated. In an aspect, the signal stays active, i.e. on, until a predetermined volume of fluid sample, necessary to diagnose mTBI, is collected. The signal may output a constant signal or may oscillate on and off, i.e. blink. In alternative aspect, the signal remains inactive until the required volume of fluid sample is collected. Once the required volume of fluid sample is collected, the signal is activated, for example a light is emitted. The signal may output a constant signal or may oscillate on and off, i.e. blink.

[0085] A minimum volume of fluid sample is necessary for analysis of biomarkers. At minimum, 70uL of fluid should be introduced into the device 800A to ensure an accurate result. In an embodiment, a minimum lOOuL of fluid should be introduced into the device800A. In some embodiments, the device 800A may include a collection buffer (not shown). The collection buffer stabilizes low-volume samples. The collection buffer stabilizes low- volume samples. The collection buffer is a solution. The device 800A may include 30uL - 55uL of collection buffer. In this embodiment, the minimum fluid sample may include 30uL - 55uL, where the combination of the fluid sample and collection buffer provides a total fluid of 70uL - lOOuL. In an embodiment, the collection buffer comprises EDTA configured to inhibit enzymatic degradation, thereby maintaining the integrity of the fluid sample (sf), a pH control configured to preserve DNA and / or protein of the fluid sample, chelating metal ions configured to activate any degrading enzymes, and stabilizing agents configured to prevent denaturation or aggregation of the fluid sample. These elements of the collection buffer enable the collection buffer to stabilize the fluid sample. In an embodiment, the collection buffer comprises TSB / 4% EDTA, 2% Tween 20, 0.5% BSA and pH 8.2. Introduction of 0.5% BSA into the collection buffer reduces NSB at the test line without affecting the signal at 50 ng / mL SIOOB. Accordingly, the inclusion of 0.5% BSA is advantageous as it improves signal noise and hence the sensitivity of the assay.

[0086] Continuing through the capillary tube a2, the fluid sample may, for example, flow from the sample pad a3 to a conjugate pad a4 by capillary forces. The conjugate pad a4 is coated with a monoclonal antibody against SIOOB, and which will conjugate to any SIOOB protein (antigen) that is present in the sample fluid.

[0087] In some embodiments, the device 800A may include a filter component (not shown) configured to filter substances from the fluid sample. Substances in the fluid sample may interfere with the analysis and test performance. Accordingly, it is advantageous to include a filter component to remove substances in the fluid sample prior to analysis. The filter component may filter substances such as, but not limited to, food particles, blood, medications, and particle debris. The filter component is positioned downstream of the sample collection swab al and upstream of the sample pad a3. The filter component may be positioned between the sample collection swab al and the capillary tube a2, between the capillary tube a2 and the sample pad a3, or along the capillary tube a2. Alternatively, the filter component may be positioned between the sample pad a3 and the conjugate pad a4. In some embodiments the capillary tube a2 is configured to filter the sample fluid as it travels therethrough to the sample pad a3. In yet another embodiment, the device 800A may include a plurality of filter components positioned downstream of the sample collection swab al and upstream of the conjugate pad a4.

[0088] The antigen- antibody complex will flow from conjugate pad a4 through a nitrocellulose membrane a5 where at least one test line is present. A capture antibody conjugated to an enzyme such as, for example, horseradish peroxidase is present on the nitrocellulose membrane a5 and will attach to the antigen-detection antibody complex. In an aspect, the device 800A includes two test lines (FIG. 8B). A first line a7 is a control line to indicate that the device is working as expected. A second line a8 is the normal line, which is to indicate the normal levels of SIOOB in the saliva sample, in other words the test results would be negative for traumatic brain injury. In another aspect, the device 800A includes three test lines. The aspect includes a third line a9 in addition to the first line a7 and the second line a8. The third line a9 is mTBI line to indicate the elevated levels of SIOOB in the saliva sample.

[0089] In addition to the at least one testing line that is visible to the user / patient, the biomarker meter 505 can be used with the device 800A to transfer the results to the mobile application. Upon completion of the test, the biomarker meter 505 can be attached to the user’s phone 506 camera to scan the device 800A at area a6. The results may then be displayed on the phone screen in a mobile application. The report provides concentrations of the miRNA biomarker and determines whether the patient has TBI or not. With reference to FIGS 4A and 4B certain aspects may, for example, provide for the measuring the severity of TBI. The biomarker meter 505 can include a clip mechanism 507 to allow it to securely attach to the device 800 when collecting the results and the user’s phone 506 when scanning the results. The clip mechanism 507 may include an indent on a distal end of the clip mechanism 507 to provide an improve grip surface. In another aspect, the clip mechanism 507 may include a textured surface on the distal end to provide an improve grip surface.

[0090] As described above with reference to device 800A, depending on the biomarker and reagent, performance of the analysis and test may be influenced by environmental factors. Accordingly, it is advantageous to include an alert mechanism (not shown) in the device 800A to alert the user when an environmental factor is present that may influence the results. The alert mechanism may include a sensor configured to monitor humidity levels, the temperature of the device 800A, and / or the amount of light exposure. The alert mechanism may further include an indicator component such as an LED. When an environmental factor is detected by the sensor of the alert mechanism, the LED may be activated. For example, the LED may be activated when the temperature of the device 800A exceeds a predetermined threshold. The LED may further be activated when the device 800A is exposed to light overa predetermined threshold. The predetermined threshold may include UV levels and / or time. In some embodiments, the device 800A may further include a stabilizing agent configured to mitigate biomarker degradation. Biomarker degradation may occur due to improper storage of the device 800A. As such, it is advantageous to include a stabilizing agent. The stabilizing agent is configured to prevent denaturation or aggregation of the fluid sample.

[0091] With reference to FIGS. 8C and 8D, an alternative aspect of the lateral flow device 800B in FIG. 8A is shown. Similar to the aspect shown in FIGs. 8A and 8B, as described above, the device 800B includes a sample collection swab al, the collection swab al may be an absorption pad, for example cotton or a similar material. The sample collection swab al is configured (e.g., shaped and dimensioned) to collect a patient sample by, for example, a patient placing the sample collection swab al in their mouth and applying a suction force by for example sucking on the sample collection swab al. A patient may, for example, suck on the sample collection swab al for a period of approximately 10 seconds to 20 seconds to provide a saliva sample. Alternatively, the patient may suck on the sample collection swab al up to, but less than, one minute. By sucking on the sample collection swab al, suction is created and saliva may be transferred by capillary tube a2 to sample pad a3. The capillary tube a2, is configured (e.g., shaped and dimensioned) to detect and allow a range of volume of saliva to transfer to the pad a3 in a sufficient quantity or volume required to perform the sample testing, but without overfilling the sample pad a3. In certain aspects, the lateral flow device shown in FIG. 8B, a signal, such as an LED, (not pictured) may be connected to the capillary tube a2, to indicate that a sufficient volume of fluid has been obtained and that testing can be initiated. The signal stays active, i.e. one, until a predetermined amount of fluid sample, necessary to diagnose mTBI, is collected. The signal may output a constant signal or may oscillate on and off, i.e. blink. In alternative aspect, the signal remains inactive until the required volume of fluid sample is collected. Once the required volume of fluid sample is collected, the signal is activated, for example a light is emitted. The signal may output a constant signal or may oscillate on and off, i.e. blink.

[0092] A minimum volume of fluid sample is necessary for analysis of biomarkers. At minimum, 70uL of fluid should be introduced into the device 800B to ensure an accurate result. In an embodiment, a minimum lOOuL of fluid should be introduced into the device 800B. In some embodiments, the device 800B may include a collection buffer (not shown). The collection buffer stabilizes low-volume samples. The collection buffer stabilizes low- volume samples. The collection buffer is a solution. The device 800B may include 30uL -55uL of collection buffer. In this embodiment, the minimum fluid sample may include 30uL - 55uL, where the combination of the fluid sample and collection buffer provides a total fluid of 70uL - lOOuL. In an embodiment, the collection buffer comprises EDTA configured to inhibit enzymatic degradation, thereby maintaining the integrity of the fluid sample (sf), a pH control configured to preserve DNA and / or protein of the fluid sample, chelating metal ions configured to activate any degrading enzymes, and stabilizing agents configured to prevent denaturation or aggregation of the fluid sample. These elements of the collection buffer enable the collection buffer to stabilize the fluid sample. In an embodiment, the collection buffer comprises TSB / 4% EDTA, 2% Tween 20, 0.5% BSA and pH 8.2. Introduction of 0.5% BSA into the collection buffer reduces NSB at the test line without affecting the signal at 50 ng / mL SIOOB. Accordingly, the inclusion of 0.5% BSA is advantageous as it improves signal noise and hence the sensitivity of the assay.

[0093] The fluid sample may, for example, flow from the sample pad a3 to the conjugate pad a4. The conjugate pad a4 is coated with a monoclonal antibody against SIOOB, and which will conjugate to any SIOOB protein (antigen) that is present in the sample fluid.

[0094] In some embodiments, the device 800B may include a filter component (not shown) configured to filter substances from the fluid sample. Substances in the fluid sample may interfere with the analysis and test performance. Accordingly, it is advantageous to include a filter component to remove substances in the fluid sample prior to analysis. The filter component may filter substances such as, but not limited to, food particles, blood, medications, and particle debris. Similar to the filter component in device 800A, the filter component in 800B is positioned downstream of the sample collection swab al and upstream of the sample pad a3. The filter component may be positioned between the sample collection swab al and the capillary tube a2, between the capillary tube a2 and the sample pad a3, or along the capillary tube a2. Alternatively, the filter component may be positioned between the sample pad a3 and the conjugate pad a4. In some embodiments the capillary tube a2 is configured to filter the sample fluid as it travels therethrough to the sample pad a3. In yet another embodiment, the device 800A may include a plurality of filter components positioned downstream of the sample collection swab al and upstream of the conjugate pad a4.

[0095] The antigen- antibody complex will flow from conjugate pad a4 through a nitrocellulose membrane al2 where at least one test line is present. A capture antibody conjugated to an enzyme such as, for example, horseradish peroxidase is present on the nitrocellulose membrane al2 and will attach to the antigen-detection antibody complex. Acapture antibody conjugated to an enzyme such as, for example, horseradish peroxidase is present on the nitrocellulose membrane al2 and will attach to the antigen-detection antibody complex. In an aspect, the device 800B includes two test lines (FIG. 8D). A first line a7 is a control line (C) to indicate that the device is working as expected. A second line alO is an indicator for mild TBI (T), which is to indicate elevated levels of SIOOB in the saliva sample. In another aspect, the device 800B includes three test lines. The aspect includes a third line al 1 in addition to the first line a7 and the second line alO. The third line al 1 is an indicator for severe TBI (H), which is to indicate highly elevated levels of SIOOB in the saliva sample.

[0096] In addition to the at least one testing line that is visible to the user, the biomarker meter 505 can be used with the device 800B transfer the results to the mobile application. Upon completion of the test, the biomarker meter 505 can be attached to the user’s phone 506 camera to scan the device 800B at area a6. The results may then be displayed on the phone screen in a mobile application. The report provides concentrations of the miRNA biomarker and determines whether the patient has TBI or not. With reference to FIGS 4A and 4B certain aspects may, for example, provide for the measuring the severity of TBI. The biomarker meter 505 can include a clip mechanism 507 to allow it to securely attach to the device 800 when collecting the results and the user’s phone 506 when scanning the results. The clip mechanism 507 may include an indent on a distal end of the clip mechanism 507 to provide an improve grip surface. In another aspect, the clip mechanism 507 may include a textured surface on the distal end to provide an improve grip surface.

[0097] As described above with reference to device 800B, depending on the biomarker and reagent, performance of the analysis and test may be influenced by environmental factors. Accordingly, it is advantageous to include an alert mechanism (not shown) in the device 800B to alert the user when an environmental factor is present that may influence the results. The alert mechanism may include a sensor configured to monitor humidity levels, the temperature of the device 800B, and / or the amount of light exposure. The alert mechanism may further include an indicator component such as an LED. When an environmental factor is detected by the sensor of the alert mechanism, the LED may be activated. For example, the LED may be activated when the temperature of the device 800B exceeds a predetermined threshold. The LED may further be activated when the device 800B is exposed to light over a predetermined threshold. The predetermined threshold may include UV levels and / or time. In some embodiments, the device 800B may further include a stabilizing agent configured to mitigate biomarker degradation. Biomarker degradation may occur due to improper storageof the device 800B. As such, it is advantageous to include a stabilizing agent. The stabilizing agent is configured to prevent denaturation or aggregation of the fluid sample.

[0098] With reference to FIGS. 8 A - 8D, in other aspects each line may have a specific range and a cutoff of SIOOB concentration corresponding to the diagnoses level of the patient. In an aspect, the devices 800A, 800B can include a second signal (not pictured), such as an LED, located on a side or top surface of the device 800B, 800B. The signal stays inactive, i.e. on, until the test is complete. Once the test is complete the signal may emit a certain color or blinking pattern to notify the user that the test is complete and the sample is ready for input into the mobile application. In an alternative aspect, the devices 800A, 800B the sample collection swab al is not coupled to the devices 800A, 800B, and the devices can include a reservoir coupled to the capillary tube a2 to transfer to fluid sample to the pad a3 in a sufficient quantity required to perform the sample testing, but without overfilling the sample pad a3. The reservoir may include an absorbent material configured to collect and retain the fluid sample that will be flown through the devices 800A, 800B during testing. The user may use a sample collection swab that is separate from the devices by depositing the fluid sample on the sample collection swab and then placing it over and / or on the reservoir to transfer the fluid sample to the devices 800A, 800B for testing.

[0099] FIG. 8F depicts an internal design of the lateral flow device of FIG. 8C with a fluid sample deposited. FIGS. 8G and 8H depict an internal design of the lateral flow device of FIG. 8C, where FIG. 8G displays a positive result and FIG. 8H displays a negative result. In some embodiments the capillary tube a2 may have an upper portion having a diameter dimensioned larger than a lower portion. The capillary tube a2 may further comprise a middle portion having a diameter progressively decreasing from an end coupled to the upper portion to an end coupled to the lower portion. The middle portion may have substantially a funnel shape. The upper portion of the capillary tube a2 is coupled to the sample collection swab al and the lower portion is coupled to the pad a3. FIG. 8G illustrates the device displaying a control line a7 and a test line a8, indicating a positive result. FIG. 8H illustrates the device displaying only a control line a7, indicating a negative result.

[0100] With reference to FIGS. 8A-8D, in certain aspects, the lateral flow sample collector is configured to receive a volume of fluid less than about 1 mL. In certain aspects, the sample collector is configured to receive a volume of fluid less than about 0.5 mL. In certain aspects, the sample collector is configured to receive a volume of fluid less than about 0.25 mL. In certain aspects, the sample collector is configured to receive a volume of fluid between about0.05 mL and about 0.5 mL. inclusive. In certain aspects, the sample collector is configured to receive a volume of fluid between about 0.1 mL and about 0.25 mL, inclusive. In certain aspects, the sample collector is configured to receive a volume of fluid between about 0.05 mL and about 0.15 mL, inclusive. In certain aspects, the sample collector is configured to receive a volume of fluid between about 0.1 mL and about 0.15 mL, inclusive.

[0101] With reference to FIG. 9, the external design of the lateral flow devices shown in FIGs. 8A-8H is depicted. A removable cap Bl that is sterile and a housing B2 contain the lateral flow device of FIGs. 8A-8H within. Removing the cap Bl, exposes the swab al. A housing B2, contains the remainder of the lateral flow device. The housing B2 has a test result window B4, under which the nitrocellulose membrane (e.g., a5 or al2), to display the testing lines (e.g., a first line a7, a second line a8, and a third line a9 or a first line a8, a second line alO, and a third line all). As shown in FIG. 9C, the cap Bl being secured on the device, moving in direction X, after the fluid sample (sf) has been deposited.

[0102] The RTCD device consists of a removable flexible cap B 1 that is sterile and a housing B2 containing the lateral flow device of FIGs 8A-8H. Removing the cap Bl, exposes the B3 swab al which consist of al cotton tip, al4 the sample volume indicator, and a2 the buffer chamber connected to sf 1 capillary tube. The housing B2 has the RTCD test strip B5 at the bottom which consist of the test result window B4, under which the nitrocellulose membrane (e.g., a5 or al2), to display the testing lines (e.g., a first line a7, and a second line a8). As shown in FIG 9C, the cap Bl is secured on the device, moving in direction X to expose the swab Al B3. The fluid sample will be collected via absorbent cotton tip al until the sample volume indicator is active and turns red to indicate that a sufficient volume of fluid sample (sf) has been obtained and that testing can be initiated. The signal can stay active, i.e. on, until a predetermined volume of the fluid sample, necessary to diagnose mTBI, is collected. The signal may output a constant signal or may oscillate on and off, i.e. blink. In the alternative aspect, the signal remains inactive until the required volume of fluid sample is collected. Once the required volume of fluid sample is collected, the signal is activated, for example a light is emitted. The signal may output a constant signal or may oscillate on and off, i.e. blink. The sample fluid then moves through the capillary tube sfl towards the running buffer solution. Once the buffer solution combines with fluid sample sf, it will flow down the test strip via capillary action where antibodies against the SIOOB protein will detect the levels of the biomarker that will be shown in the test result window B4 (e.g., a first line a7, and a second line a8).

[0103] The test results window B4 and testing lines constitute visualization zones. The visualization zones can have a circular geometric shape or a non-circular geometric shape. For example, the test results window B4 shape can be circular, egg, oval, ovoid, or stadium. The test results window B4 is further dimensioned such that the testing lines are sufficiently visible and discernable, such that the testing lines separated sufficiently and a user of the device can apprehend the results based on the pattern of visible signals generated upon reaction with the fluid sample. Specific examples of suitable arrangements include but are not limited to configuring the visualization zones as lines (e.g., as a series of parallel lines), configuring the visualization zones as dots or configuring the visualization zones as a grid or matrix containing a plurality of defined shapes (e.g., squares or hexagons).

[0104] In an aspect, the device may include a plurality of reaction zones configured with a plurality of reagents. In certain aspects, the reaction stage comprises between 2 and 10 discrete visualization zones. In certain aspects, the reaction stage comprises between 3 and 8 discrete visualization zones. In certain aspects, the reaction stage comprises between 2 and 5 discrete visualization zones. In certain aspects, the reaction stage comprises between 3 and 8 discrete visualization zones. In certain aspects, the reaction stage comprises 3 discrete visualization zones. In certain aspects, the reaction stage comprises 4 discrete visualization zones. In certain aspects, the reaction stage comprises 5 discrete visualization zones. In certain aspects, the reaction stage comprises 6 discrete visualization zones.

[0105] In certain aspects, the reaction stage comprises a discrete reaction zone comprising a discrete structure or a portion of the reaction stage that is smaller than the total area of the reaction stage where the sample interacts with one or more reagents prior to entering a visualization zone. In such aspects, the reaction zone is configured to allow the sample to react with one or more detection reagents prior to entering visualization zones.

[0106] As mentioned above, the reaction zones each comprise one or more detection reagents that selectively react with one or more biomarkers associated with traumatic brain injury. In certain aspects, each reaction zone contains a detection reagent that selectively reacts with one or more protein (or peptide) biomarkers associated with brain injury. In certain aspects, a reaction zone in the provided device contains a reagent that selectively reacts with a protein biomarker for brain injury selected from the group consisting of: protein biomarkers associated with neuronal cell body injury (e.g., UCH-L1, NSE); protein biomarkers associated with astroglial injury (e.g., GFAP, SIOOB); protein biomarkers associated with neuronal cell death (e.g., all-spectrin breakdown products, including SBDP150 produced by 1calpain during necrosis, and SBDP120 produced by caspase-3 during apoptosis); protein biomarkers associated with axonal injury (e.g. NF proteins such as PNF-H); protein biomarkers associated with white matter injury (e.g. MBP); protein biomarkers associated with post-injury neurodegeneration (e.g., Tau and phospho-Tau); biomarkers indicative of post- injury autoimmune response (e.g., brain antigen-targeting autoantibodies); proteins involved in the breakdown of extracellular matrix in normal physiological processes (e.g., matrix metallopeptidase 9 (MMP9)); and any combination of two or more of these.

[0107] In certain aspects, the device is configured to react with one or more protein or peptide-based biomarkers present in the saliva of an injured person after a traumatic brain injury. In certain aspects, saliva-based biomarkers are further characterized in that the concentration of the biomarker in a patient’ s saliva varies within a short time after a brain injury. For example, in certain aspects, the reaction zone is configured to facilitate reaction(s) with one or more biomarkers whose concentrations in saliva vary within about 5 minutes, within about 10 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 45 minutes, within about 1 hour, or within about 1 to 6 hours after a brain injury. In certain aspects, the reaction zone is configured to facilitate reaction(s) with one or more biomarkers whose concentrations in saliva increase within a short time after a brain injury. In certain aspects, the reaction zone is configured to facilitate reaction(s) with one or more biomarkers whose concentrations in saliva decrease within a short time after a brain injury.

[0108] In certain aspects, the reaction stage comprises one or more reaction zones containing a reagent that selectively reacts with a protein or peptide biomarker that is associated with traumatic brain injury and which has a measurable concentration in saliva after a brain injury. In certain aspects, at least one reaction zone in the provided device contains a reagent that selectively reacts with a calcium binding protein present in the sample. In certain aspects, at least one reaction zone in the provided device contains a reagent that selectively reacts with a S100 calcium binding protein present in the sample. In certain aspects, at least one reaction zone in the provided device contains a reagent that selectively reacts with a SIOOB calcium binding protein present in the sample.

[0109] In certain aspects, a reagent in one or more reaction zone comprises an antibody (e.g., a monoclonal antibody). In certain aspects, a reagent in one or more reaction zone comprises an antibody that selectively binds to a protein biomarker for brain injury selected from the group consisting of: protein biomarkers associated with neuronal cell body injury (e.g.,UCH-L1, NSE); protein biomarkers associated with astroglial injury (e.g., GFAP, SIOOB); protein biomarkers associated with neuronal cell death (e.g., all-spectrin breakdown products); protein biomarkers associated with axonal injury (e.g., NF proteins); protein biomarkers associated with white matter injury (e.g. MBP); and protein biomarkers associated with post-injury neurodegeneration (e.g., Tau and phospho-Tau).

[0110] In certain aspects, at least one reaction zone in the provided device contains a monoclonal antibody that selectively reacts with the protein SIOOB.

[0111] In certain aspects, a provided device is configured to evaluate an injury victim’s saliva and the reaction zone contains a reagent that selectively reacts with a protein or peptide biomarker that is associated with traumatic brain injury and that is characterized in that it has a measurable concentration in saliva. In certain aspects, a reaction zone in the provided device contains a reagent that selectively reacts with the protein SIOOB in an injured person’s saliva. In certain aspects, a reaction zone in the provided device contains a monoclonal antibody that selectively reacts with the protein SIOOB.

[0112] In certain aspects, a provided device is characterized in that it has a plurality of reaction zones configured to detect the protein SIOOB. In certain aspects, two or more of the reaction zones reactive toward SIOOB are distinguished in that they have sensitivities to different concentrations of SIOOB protein (e.g., the zones are differentiated in that they provide distinct signals that are dependent upon a concentration of SIOOB in the sample). In certain aspects, the reaction stage of the device comprises two reaction zones reactive toward different concentrations of SIOOB. In certain aspects, the reaction stage of the device comprises three reaction zones reactive toward different concentrations of SIOOB. In certain aspects, the reaction stage of the device comprises four or more reaction zones reactive toward different concentrations of SIOOB.

[0113] In certain aspects, a provided device is characterized in that it comprises a plurality of reaction zones at least one of which is configured to react with the protein SIOOB. In certain aspects, the reaction stage comprises at least one reaction zone reactive toward SIOOB and at least one other reaction zone reactive toward a brain injury biomarker other than S 100B. In certain aspects, the reaction stage comprises at least one reaction zone reactive toward both SIOOB and at least one other brain injury biomarker other than SIOOB. In certain aspects, the reaction stage comprises at least one reaction zone reactive toward SIOOB and at least one reaction zone reactive toward a protein biomarker associated with neuronal cell body injury (e.g. UCH-Ei or NSE). In certain aspects, the reaction stage comprises at least one reactionzone reactive toward SIOOB and at least one reaction zone reactive toward a protein biomarker other than SIOOB associated with astroglial injury (e.g., GFAP). In certain aspects, the reaction stage comprises at least one reaction zone reactive toward SIOOB and at least one reaction zone reactive toward a protein biomarker associated with neuronal cell death (e.g., all-spectrin breakdown products). In certain aspects, the reaction stage comprises at least one reaction zone reactive toward SIOOB and at least one reaction zone reactive toward a protein biomarker associated with axonal injury (e.g., NF proteins). In certain aspects, the reaction stage comprises at least one reaction zone reactive toward SIOOB and at least one reaction zone reactive toward a protein biomarker associated with white matter injury (e.g., MBP). In certain aspects, the reaction stage comprises at least one reaction zone reactive toward SIOOB and at least one reaction zone reactive toward a protein biomarker associated with post-injury neurodegeneration (e.g. Tau or phospho-Tau).

[0114] In certain aspects, the reaction stage comprises at least one reaction zone reactive toward both SIOOB and a protein biomarker associated with neuronal cell death (e.g., all- spectrin breakdown products). In certain aspects, the reaction stage comprises at least one reaction zone reactive toward both SIOOB and a protein biomarker associated with axonal injury (e.g., NF proteins). In certain aspects, the reaction stage comprises at least one reaction zone reactive toward both SIOOB and a protein biomarker associated with white matter injury (e.g., MBP). In certain aspects, the reaction stage comprises at least one reaction zone reactive toward both SIOOB and a protein biomarker associated with post- injury neurodegeneration (e.g. Tau or phospho-Tau).

[0115] As noted above, the reaction zones are configured to detect one or more biomarkers for brain injury present in a fluid sample. In certain aspects, this detection is based on reaction of an antibody with the biomarker of interest. In certain aspects, one or more reaction zones comprise a monoclonal antibody that selectively reacts with a biomarker selected from the group consisting of: protein biomarkers associated with neuronal cell body injury (e.g. UCH-L1, NSE); protein biomarkers associated with astroglial injury (e.g., GFAP, SIOOB); protein biomarkers associated with neuronal cell death (e.g., all-spectrin breakdown products); protein biomarkers associated with axonal injury (e.g., NF proteins); protein biomarkers associated with white matter injury (e.g., MBP); and protein biomarkers associated with post-injury neurodegeneration (e.g. Tau and phospho-Tau). In certain aspects, one or more reaction zones comprise a polyclonal antibody that selectively reacts with a biomarker selected from the group consisting of: protein biomarkers associated with neuronalcell body injury (e.g. UCH-L1, NSE); protein biomarkers associated with astroglial injury (e.g., GFAP, SIOOB); protein biomarkers associated with neuronal cell death (e.g., al lspectrin breakdown products); protein biomarkers associated with axonal injury (e.g., NF proteins); protein biomarkers associated with white matter injury (e.g., MBP); and protein biomarkers associated with post-injury neurodegeneration (e.g. Tau and phospho-Tau). In certain aspects, such antibodies are conjugated with a detectable label to form a complex that can be detected, for example by interaction with a visualization reagent present in the reaction zone.

[0116] Specific examples were tested and are described in more detail below. Potential cross reactants may exist within the S100A family. In certain aspects, one or more reaction zones comprise a monoclonal or polyclonal antibody that selectively reacts with SIOOB but not with S100A.

[0117] In certain aspects, one or more reaction zones comprise a monoclonal antibody selective for SIOOB. In certain aspects, one or more reaction zones comprise a monoclonal antibody that is selective for the following epitopes of the SIOOB protein as shown in FIGS. 10 and 11: SEEEKAMVAEIDVFHQYSGR (SEQ ID NO: 41); GDGECDFQEFMAFVAMVTTA (SEQ ID NO: 42); KAMVALIDVFHQ (SEQ ID NO: 43); QEVVDKV (SEQ ID NO: 44); and MAFVAMVTTACHEF (SEQ ID NO: 45).

[0118] As noted above, the reaction zones can be configured to provide a visible change when a brain injury biomarker present in a fluid sample reacts with a detection reagent in the reaction zone. In certain aspects, this visible change is affected by combining a detection reagent such as a monoclonal antibody that is conjugated with a detectable label. In certain aspects, such conjugated labels are configured to react with a visualization reagent to generate a visible change when the antibody reacts with the target biomarker. Such systems are well known in the art and the skilled artisan can readily select and appropriate label, and a compatible visualization reagent. In certain aspects, different reaction zones employ different labels and / or different visualization reagents such that the signal provided by different reaction zones is visually distinct (e.g., provides a signal of a different color). In certain aspects, the detection reagent comprises horseradish peroxidase. See Michigan Diagnostics, https: / / michigandiagnostics.squarespace.com / use-of-horseradish-peroxidase-and-alkali ne- phosphatase-in-chemil umi nescence-the-pros-and-cons.

[0119] FIG. 15 illustrates a graphical user interface (GUI) 1600 including user prompts to read the test result of the later flow assay device as well as to monitor the patient’s healthstatus and perform the Glasgow Coma Scale test [GCS] in accordance with various aspects taught herein. The GUI 1600 can be deployed on an external device such as a mobile device. The GUI 1600 includes static elements that provide information to the user and actuatable elements that can be actuated by a user to perform certain actions. Actuation of the actuatable elements can occur when the user clicks the element with a mouse, inputs information with a keyboard, or gestures using one or more touches on a touchscreen. The actuatable elements can include Capture Test Result 1602, View Reports 1604, Email Report 1606, and Perform GCS Test 1608. Static elements can include test result information. The test result information includes qualitative and quantitative measurements of the isolated antibody levels in the fluid sample. The information displayed in these static elements can be retrieved from a database by the application running the GUI 4100 or can be pushed from a central server (e.g., from a physician’s office) to the external device and populated into the appropriate fields in the application.

[0120] Actuatable elements can be dynamically displayed in the GUI 1600 when certain conditions are met. For example, the Capture Test Result 1602 element can appear on the GUI 1600 in an actuatable state or a deactivated state depending upon whether patient status requirements have been met. If patient status requirements have been met, the Capture Test Result 1602 element is depicted in an actuatable state that can be actuated by the user to initiate a reading of the test results. If the application determines that patient status requirements have not been met, the Capture Test Result 1602 element is depicted in a deactivated state that will not respond to user input. The deactivated state can be maintained until the patient status requirements are resolved. Upon activation of the Capure Test Result 1602 element, the patient and / or user may be asked a series of questions, for example which of the two lines appeared on the device; the date and time the test was taken; and who administered the test. Following input of the information, a result report based on the results is generated.

[0121] In some aspects, the use of the mobile application running on the external device can be restricted through the application until patient status requirements are met. For example, use of the mobile application can be restricted subject to account creation by the patient and / or user. The GUI 1600 can prompt the user that the account creation requirement has not been met using an account creation element (not pictured) of the GUI 1600. Actuation of the account creation element prompt can take a user to further information or explanation as to what steps should be taken to resolve this patient status requirement. These operations canbe incorporated into a downloadable software application that includes all operational modules, or any periodic updates thereto, to perform the mobile application as generally described herein.Although the static and actuatable elements are shown on GUI 1600 as visual elements, the skilled person would appreciate that other forms for the elements are possible to increase accessibility such as replacing and / or augmenting the visual static or actuatable elements with auditory prompts or prompts to receive speech from the user.

[0096] The GUI 1600 can include additional actuatable elements and static elements. These elements can include Real-time Monitoring, enabling real-time monitoring of vital signs or health parameters, providing users with instant feedback on their health status; Data Analytics and Insights, implementing advanced data analytics algorithms to analyze the collected health data over time, providing users with insights into trends, patterns, and potential health risks; Predictive Analysis, utilizing machine learning algorithms to predict potential health issues based on historical data and provide personalized recommendations for preventive measures or interventions; Telemedicine Integration, incorporating telemedicine features, allowing users to consult with healthcare professionals remotely, share their health data, and receive personalized advice or prescriptions; Medication Management, including features for medication management such as reminders for medication intake, tracking of medication adherence, and interactions with other medications or food; Secure Data Storage and Sharing, enabling secure sharing of user data with healthcare providers or family members; Customize User Profiles, providing customizable profiles with users’ health goals, preferences, and medical history, enabling personalized recommendations and tailored user experiences; Health Challenges, allowing users to identify health challenges, goals, and rewards to motivate users to adopt healthy behaviors and stay engaged with content provided on the GUI 1600; Emergency Response Integration, integrating emergency response features, such as SOS alerts with GPS location tracking, quick access to emergency contacts, and instructions for basic first aid procedures; Community and Support Groups, a community platform where users can connect with others facing similar health challenges, share experiences, and provide mutual support; Accessibility Features, ensuring the GUI 1600 is accessible to users with disabilities by incorporating features such as voice commands, screen reader compatibility, and adjustable font sizes; Integration with Electronic Health Records (EHR), enable seamless integration with electronic health record systems used by healthcare providers, allowing for easy sharing of health data and facilitating better coordination of care;Al-Powered Symptom Checker, a symptom checker powered by artificial intelligence to help users assess their symptoms, provide initial recommendations, and guide them on whether they need to seek medical attention.

[0097] In an embodiment, the GUI 1600 is compatible with wearable devices. Users’ wearable health devices such as smartwatches or fitness trackers can be paired with the GUI 1600 to gather additional health data and provide a comprehensive view of the user's health.II. Methods for Detecting or Diagnosing Traumatic Brain Injury

[0098] Provided herein are methods for diagnosing mild traumatic brain injury in a subject, the method comprising: a. obtaining a biological sample from the subject; b. placing the sample in a sample collector that is configured to conduct the sample into a reaction stage configured, the reaction stage comprising a plurality of reaction zones, each reaction zone comprising a combination of a detection reagent sensitive to a biomarker for traumatic brain injury, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarker-detection reagent complex, and when the capture reagent binds to the biomarker-detection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones and then reading a set of visible signals from the plurality of reaction zones; d. comparing the set of signals obtained with a reference to determine whether the subject has a) no traumatic brain injury, or b) mild traumatic brain injury.

[0099] In some aspects, provided are methods for diagnosing traumatic brain injury in a subject, the method comprising: a. obtaining a biological sample from the subject; b. placing the sample in a sample collector that is configured to conduct the sample into a reaction stage configured, the reaction stage comprising a plurality of reaction zones, each reaction zone comprising a combination of a detection reagent sensitive to a biomarker for traumatic brain injury, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarker-detection reagent complex, and when the capture reagent binds to the biomarker-detection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones and then reading a set of visible signals from the plurality of reaction zones; d. comparing the set of signals obtained with a reference to determine whether the subject has a) no traumatic brain injury, b) mild traumatic brain injury, or c) moderate to severe traumatic brain injury.

[0100] In some aspects, the sample collector and the plurality of reaction zones are linked to each other such that capillary flow draws the sample from the sample collector into the plurality of reaction zones.

[0101] In some aspects, the sample collector and the plurality of reaction zones are configured as a lateral flow device.

[0102] In some aspects, the sample collector and the plurality of reaction zone are integrated into a disposable single use test device.

[0103] In some aspects, provided are methods for detecting mild traumatic brain injury in a subject, comprising: a. obtaining a biological sample from the subject; b. placing the sample in a test device and allowing the sample to contact a plurality of reaction zones, each reaction zone containing a combination of a detection reagent sensitive to a biomarker for traumatic brain injury, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarkerdetection reagent complex, and when the capture reagent binds to the biomarker-detection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones; d. reading a set of visible signals from the plurality of reaction zones; and e. determining based on the set of visible signals whether the subject has a) no traumatic brain injury, or b) mild traumatic brain injury.

[0104] In some aspects, provided are methods for detecting traumatic brain injury in a subject, comprising: a. obtaining a biological sample from the subject; b. placing the sample in a test device and allowing the sample to contact a plurality of reaction zones, each reaction zone containing a combination of a detection reagent sensitive to a biomarker for traumatic brain injury, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarkerdetection reagent complex, and when the capture reagent binds to the biomarker-detection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones; and d. reading a set of visible signals from the plurality of reaction zones; and e. determining based on the set of visible signals whether the subject has a) no traumatic brain injury, b) mild traumatic brain injury, or c) moderate to severe traumatic brain injury.

[0105] In some aspects provided are methods for treating mild traumatic brain injury in a subject, comprising: a. obtaining a biological sample from the subject; b. placing the sample in a test device and allowing the sample to contact a plurality of reaction zones, each reactionzone containing a combination of a detection reagent sensitive to a biomarker for traumatic brain injury mild traumatic brain injury / concussion, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarker-detection reagent complex, and when the capture reagent binds to the biomarker-detection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones; d. reading a set of visible signals from the plurality of reaction zones; e. determining based on the set of visible signals whether the subject has a) no traumatic brain injury, or b) mild traumatic brain injury; and f. administering an appropriate medical care for the subject determined to have mild traumatic brain injury.

[0106] In some aspects provided are methods for treating traumatic brain injury in a subject, comprising: a. obtaining a biological sample from the subject; b. placing the sample in a test device and allowing the sample to contact a plurality of reaction zones, each reaction zone containing a combination of a detection reagent sensitive to a biomarker for traumatic brain injury mild traumatic brain injury / concussion, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarker-detection reagent complex, and when the capture reagent binds to the biomarker-detection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones; d. reading a set of visible signals from the plurality of reaction zones; e. determining based on the set of visible signals whether the subject has a) no traumatic brain injury, b) mild traumatic brain injury, or c) moderate to severe traumatic brain injury; and f. administering an appropriate medical care for the subject determined to have traumatic brain injury.

[0107] In some aspects, the biological sample is a liquid biological sample. In some aspects, a liquid sample is a body fluid. Exemplary body fluids may include, e.g., saliva, serum, plasma, whole blood, or cerebrospinal fluid (CSF). In some aspects, the biological sample is a saliva sample. In some aspects, the biological sample is a blood, serum, or plasma sample. In some aspects, the biological sample is a saliva sample. In some aspects, the biological sample is a blood sample. In some aspects, the biological sample is a plasma sample. In some aspects, the biological sample is a serum sample. In some embodiments, the biological sample is obtained within about 24 hours of injury suspected to be traumatic brain injury.

[0108] In some aspects, the subject is a human. In some aspects, the subject is about 5-75 years of age. In some aspects, the subject has a Glasgow Coma Scale (GCS) of 13-15. Insome aspects, the subject does not have a history of or does not currently have a major psychiatric disorder selected from the group consisting of schizophrenia, active psychosis, and uncontrolled bipolar disorder. In some aspects, the subject is an adult, i.e., 18 years of age or older, and has a body mass index of >17 or < 36. In some aspects, the subject is a child, i.e., under 18 years of age, e.g., 5-17 years of age, and has a body mass index in the 5th -85th percentile.

[0109] In some aspects, the bio marker for traumatic brain injury is a protein biomarker, a DNA biomarker, or an RNA biomarker.

[0110] In some aspects, a protein biomarker is selected from the group consisting of: protein biomarkers associated with neuronal cell body injury (e.g., UCH-L1, NSE); protein biomarkers associated with astroglial injury (e.g., GFAP, SIOOB); protein biomarkers associated with neuronal cell death (e.g., all-spectrin breakdown products, including SBDP150 produced by calpain during necrosis, and SBDP120 produced by caspase-3 during apoptosis); protein biomarkers associated with axonal injury (e.g. NF proteins such as PNF- H); protein biomarkers associated with white matter injury (e.g. MBP); protein biomarkers associated with post-injury neurodegeneration (e.g., Tau and phospho-Tau); biomarkers indicative of post-injury autoimmune response (e.g., brain antigen-targeting autoantibodies); proteins involved in the breakdown of extracellular matrix in normal physiological processes (e.g., matrix metallopeptidase 9 (MMP9)); and any combination of two or more of these.

[0111] In some aspects, the protein biomarker is S100 Calcium Binding Protein B (S100B).

[0112] In some aspects, the detection reagent and / or the capture reagent is an antibody, or antigen-binding fragment thereof, that selectively binds to the biomarker.

[0113] In some aspects, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VE), wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:5, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO: 10.

[0114] In some aspects, the antibody, or antigen-binding fragment thereof, comprises a VH and a VL, wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO: 15, and wherein the VL comprises an amino acid sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:20.

[0115] In some aspects, the antibody, or antigen-binding fragment thereof, comprises a VH and a VL, wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:25, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:30.

[0116] In some aspects, the antibody, or antigen-binding fragment thereof, comprises a VH and a VL, wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:35, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:40.

[0117] In some aspects, the antibody, or antigen-binding fragment thereof, comprises a VH and a VL, wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO:1, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:2, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:3, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:6, a VL CDR2 domain comprising the amino acid sequence of YAS, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:8.

[0118] In some aspects, the antibody, or antigen-binding fragment thereof, comprises a VH and a VL, wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO: 11, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO: 12, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO: 13, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 domain comprising the amino acid sequence of YTS, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO: 18.

[0119] In some aspects, the antibody, or antigen-binding fragment thereof, comprises a VH and a VL, wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO:21, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:22, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:23, and wherein the VL comprises a VL CDR1 domain comprising the amino acidsequence of SEQ ID NO:26, a VL CDR2 domain comprising the amino acid sequence of YAS, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:28.

[0120] In some aspects, the antibody, or antigen-binding fragment thereof, comprises a VH and a VL, wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO:31, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:32, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:33, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:36, a VL CDR2 domain comprising the amino acid sequence of YAS, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:38.

[0121] In some aspects, the visualization reagent is a combination of a peroxidase enzyme and a chromogenic substrate for the enzyme.III. Anti-SIOOB Antibodies and Uses for Detecting or Diagnosing Traumatic Brain Injury

[0122] Provided herein are antibodies and antigen-binding fragments thereof that specifically bind SIOOB, methods of making the antibodies or antigen binding fragments thereof, and methods of using such antibodies to, for example, detect human SIOOB in a biological sample, e.g., a saliva or blood sample, for the diagnosis of traumatic brain injury, and other neurological disorders.

[0123] As used herein, the term “S100B” or “S100 Calcium Binding Protein B” refers to a member of the S100 family of proteins containing 2 EF-hand calcium-binding motifs. S100 proteins are localized in the cytoplasm and / or nucleus of a wide range of cells, and involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. Chromosomal rearrangements and altered expression of SIOOB gene have been implicated in several neurological, neoplastic, and other types of diseases, including Alzheimer's disease, Down's syndrome, epilepsy, amyotrophic lateral sclerosis, melanoma, and type I diabetes.

[0124] SIOOB is also known to be overexpressed in patients who recently underwent a traumatic brain injury. Under physiological conditions, SIOOB mainly produced by astrocytes do not cross the blood-brain barrier (BBB), and the concentration of SIOOB in the cerebrospinal fluid is reported to be about 100-fold higher than in serum (Petzold A., et al. Brain Res. Bull. 2003;61:281-285). After brain insults, SIOOB released from damaged glialcells can diffuse into the bloodstream. Thus, SIOOB is a strong predictor of an unfavorable outcome in TBI patients.

[0125] An exemplary sequence for SIOOB is a human SIOOB, the amino acid sequence of which may be found in for example, GenBank Accession No. NP_006263.1.

[0126] The term “SIOOB” includes a wild type, a variant or an isoform of SIOOB protein or a fragment or domain thereof. In some aspects, the variant forms of SIOOB include those SIOOB mutants with one or more substitutions. The term “SIOOB” also encompasses SIOOB protein or a fragment thereof coupled to, for example, a mouse or human Fc, a signal peptide sequence, and / or a protein tag.

[0127] Provided herein are antibodies, or antigen-binding fragments thereof, that bind SIOOB, which optionally may be affinity-matured. Other antibodies or antigen-binding fragments thereof that bind SIOOB, including those having different CDRs, and epitopic specificity may be obtained using the disclosure of the present specification, and using methods that are generally known in the art. In some aspects, the antibody, or antigen-binding fragment thereof, according to the disclosure comprises one or more CDRs, a VL chain and / or VH chain of the anti-SlOOB antibodies, or antigen-binding fragments thereof, described herein.

[0128] Exemplary anti-SlOOB antibodies, or antigen-binding fragments thereof, according to the disclosure, and the specific CDRs thereof are identified in this section. For convenience, the exemplified antibody, or antigen-binding fragment thereof, and corresponding sequences are separately identified by a specific nomenclature as shown in Tables 1 and 2.A. Anti-SlOOB Antibody Polypeptide Sequences and Nucleic Acid Sequences

[0129] Anti-SlOOB antibodies, and antigen-binding fragments thereof, provided herein include any one of the antibodies disclosed in Tables 1 and 2, and antigen-binding fragments thereof. Any Fc variant may be used in combination with any of the variable sequences disclosed herein.

[0130] Tables 1 and 2 show (i) the amino acid sequences of the VH, VH CDR1, VH CDR2, VH CDR3, VL, VL CDR1, VL CDR2, and VL CDR3, and (ii) the DNA sequences of the VH and VL chains for the antibodies.Table 1. Heavy Chain and Light Chain CDR SequencesTable 2. VH and VL sequences

[0131] In one aspect, provided are anti-SlOOB antibodies, or antigen-binding fragments thereof, comprising (i) a VH CDR that is same as the VH CDR3 of, (ii) a VH CDR3 and VL CDR3, both of which as same as both of the VH CDR3 and the VL CDR3 of, (iii) at least 1, 2, 3, 4, 5, or 6 CDRs that are same as the corresponding CDR(s) of, or (iv) 6 CDRs that are all the same as the 6 CDRs of the antibody of the disclosure, e.g., AVS-5955, AVS-5956, AVS-5957, or AVS-5958.

[0132] In some aspects, provided herein are anti-SlOOB antibodies, or antigen-binding fragments thereof, wherein (a) the VH comprises an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino acid sequence of the VH of, and (b) the VL comprises an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino acid sequence of the VL of the antibody of the disclosure, e.g., AVS-5955, AVS-5956, AVS-5957, or AVS-5958.

[0133] In further aspects, provided herein are anti-SlOOB antibodies, or antigen-binding fragments thereof, comprising (i) a VH CDR that is same as the VH CDR3 of, (ii) a VH CDR3 and VL CDR3, both of which as same as both of the VH CDR3 and the VL CDR3 of, (iii) at least 1, 2, 3, 4, 5, or 6 CDRs that are same as the corresponding CDR(s) of, or (iv) 6 CDRs that are all the same as the 6 CDRs of the antibody of the disclosure, e.g., AVS-5955, AVS-5956, AVS-5957, or AVS-5958.

[0134] In further aspects, provided herein are anti-SlOOB antibodies, or antigen-binding fragments thereof, comprising one of the CDR requirements (i)-(iv) of the immediately above paragraph, further wherein (a) the VH comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the VH of, and (b) the VL comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the VL of the antibody of the disclosure, e.g., AVS-5955, AVS-5956, AVS-5957, or AVS-5958.

[0135] In other aspects, the anti-SlOOB antibodies, or antigen-binding fragments thereof, comprise, or alternatively consist of, combinations of one or more of the CDRs, the VH and VL sequences, and the heavy chain and light chain sequences set forth above, including all of them, or sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0136] In a further aspect, the anti-SlOOB antibodies, or antigen-binding fragments thereof, comprise, or alternatively consist of, Fab fragments having binding specificity for SIOOB. The Fab fragment preferably includes the VH and the VL sequence of the antibody of the disclosure, e.g., AVS-5955, AVS-5956, AVS-5957, or AVS-5958, or sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. This aspect further includes Fabs containing additions, deletions, and variants of such VH and VL sequence while retaining binding specificity for SIOOB.

[0137] In some aspects, Fab fragments may be produced by enzymatic digestion e.g., papain) of the parent full antibody. In another aspect, anti-SlOOB antibodies such as the antibody of the disclosure, e.g., AVS-5955, AVS-5956, AVS-5957, or AVS-5958, and Fab fragments thereof may be produced via expression in mammalian cells, such as CHO, NS0, or HEK 293 cells, fungal, insect, or microbial systems, such as yeast cells.

[0138] In additional aspects, provided herein are polynucleotides encoding antibody polypeptides having binding specificity to SIOOB, including the VH and VL of the antibodyof the disclosure, e.g. AVS-5955, AVS-5956, AVS-5957, or AVS-5958, as well as fragments, and combinations of one or more of the CDRs, the VH and VL sequences, and the heavy chain and light chain sequences set forth above, including all of them, or sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0139] In other aspects, provided herein are isolated anti-SlOOB antibodies and antigen binding fragments comprising (i) a VH which is same as the VH of the antibody of the disclosure, e.g., AVS-5955, AVS-5956, AVS-5957, or AVS-5958; or (ii) a VL which is same as the VL of the antibody of the disclosure, e.g., AVS-5955, AVS-5956, AVS-5957, or AVS- 5958, or a variant thereof, wherein optionally one or more of the framework region residues (“FR residues”) and / or CDR residues in said VH or VL polypeptide has been substituted with another amino acid residue resulting in an anti-SlOOB antibody that binds, e.g., specifically binds, SIOOB.

[0140] Provided herein are also humanized, primatized and other chimeric forms of these antibodies. The chimeric and humanized antibodies may include an Fc derived from IgGl, IgG2, IgG3, or IgG4 constant regions.

[0141] In some aspects, provided herein are a nucleic acid molecule, e.g., a DNA or an mRNA molecule, encoding any of the antibodies or antigen binding fragments thereof disclosed herein.

[0142] In some aspects, provided herein are vectors comprising a nucleic acid molecule encoding an anti-SlOOB antibody or antigen binding fragment thereof as disclosed herein. In some aspects, provided herein are host cells comprising a nucleic acid molecule encoding an anti-SlOOB antibody or antigen binding fragment thereof as disclosed herein.

[0143] In some aspects, provided herein are pharmaceutical or diagnostic compositions comprising at least one antibody or antigen binding fragment thereof as disclosed herein.

[0144] In some aspects, provided herein are an antibody or antigen binding fragment thereof that selectively binds to SIOOB, wherein the antibody or antigen binding fragment thereof binds to SIOOB with a Ko of less than or equal to 5xl0'5M, 10'5M, 5xl0'6M, 10'6M, 5xl0'7M, IO’7M, 5xl0'8M, 10’8M, 5xl0’9M, 10’9M, 5xl0’10M, IO’10M, 5xl01M, 10’11M, 5xl0'12M, 10'12M, 5xl0'13M, or 10'13M; preferably, with a KD of less than or equal to 5xl0'10M, 10'10M, 5xl0-11M, 10'11M, 5xl0'12M, or 10'12M; more preferably, with a KD that is less than about 100 pM, less than about 50 pM, less than about 40 pM, less than about 25 pM, less than about 1 pM, between about 10 pM and about 100 pM, between about 1 pM and about 100 pM, or between about 1 pM and about 10 pM.

[0145] The antibodies and antigen binding fragments thereof may be modified post- translationally to add effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.

[0146] Antibodies and antigen binding fragments thereof may also be chemically modified to provide additional advantages such as increased solubility, stability and circulating time (in vivo half-life) of the polypeptide, or decreased immunogenicity. The chemical moieties for derivatization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, and the like. The antibodies and fragments thereof may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three, or more attached chemical moieties.

[0147] Regarding detectable moieties, further exemplary enzymes include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta-galactosidase, and luciferase. Further exemplary fluorescent materials include, but are not limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin, and dansyl chloride. Further exemplary chemiluminescent moieties include, but are not limited to, luminol. Further exemplary bioluminescent materials include, but are not limited to, luciferin and aequorin. Further exemplary chemiluminescent moieties include, but are not limited to, luminol. Further exemplary bioluminescent materials include, but are not limited to, luciferin and aequorin. Further exemplary radioactive materials include, but are not limited to, Iodine 125 (125I), Carbon 14 (14C), Sulfur 35 (35S), Tritium (3H) and Phosphorus 32 (32P).

[0148] Methods are known in the art for conjugating an antibody or antigen binding fragment thereof to a detectable moiety and the like, such as for example those methods described by Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J., Histochem. and Cytochem., 30:407 (1982).

[0149] Aspects described herein further include variants and equivalents that are substantially homologous to the antibodies, antibody fragments, diabodies, SMIPs, camelbodies, nanobodies, IgNAR, polypeptides, variable regions, and CDRs set forth herein. These may contain, e.g., conservative substitution mutations, (i.e., the substitution of one or more amino acids by similar amino acids). For example, conservative substitution refers to thesubstitution of an amino acid with another within the same general class, e.g., one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid by another neutral amino acid. The intent of a conservative amino acid substitution is well known in the art.

[0150] In other aspects, provided herein are polypeptide sequences having at least 90% or greater sequence homology to any one or more of the polypeptide sequences of antigen binding fragments, variable regions and CDRs set forth herein. More preferably, the disclosure contemplates polypeptide sequences having at least 95% or greater sequence homology, even more preferably at least 98% or greater sequence homology, and still more preferably at least 99% or greater sequence homology to any one or more of the polypeptide sequences of antigen binding fragments, variable regions, and CDRs set forth herein.

[0151] Methods for determining homology between nucleic acid and amino acid sequences are well known to those of ordinary skill in the art.

[0152] Provided herein are anti-SlOOB antibodies comprising any of the polypeptide or polynucleotide sequences described herein substituted for any of the other polynucleotide sequences described herein. For example, without limitation thereto, provided herein are antibodies comprising the combination of any of the VL and VH sequences described herein, and further provided herein are antibodies resulting from substitution of any of the CDR sequences described herein for any of the other CDR sequences described herein.

[0153] In other aspects, provided herein are these polynucleotides incorporated into an expression vector for expression in mammalian cells such as CHO, NS0, or HEK-293 cells, or in fungal, insect, or microbial systems such as yeast cells. In one aspect of the disclosure described herein, Fab fragments can be produced by enzymatic digestion e.g., papain) of the antibody of the disclosure, e.g., AVS-5955, AVS-5956, AVS-5957, or AVS-5958; following expression of the full-length polynucleotides in a suitable host. In another aspect, anti-SlOOB antibodies, such as the antibody of the disclosure, e.g., AVS-5955, AVS-5956, AVS-5957, or AVS-5958, or Fab fragments thereof, can be produced via expression of the polynucleotides encoding AVS-5955, AVS-5956, AVS-5957, or AVS-5958, in mammalian cells such as CHO, NS0, or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells.

[0154] Provided herein are vectors comprising the polynucleotide sequences encoding the variable heavy and light chain polypeptide sequences, as well as the individual CDRs (hypervariable regions), as set forth herein, as well as host cells comprising said vectorsequences. In some aspects, the host cells are mammalian cells, such as CHO cells. In other aspects, the host cells are yeast cells.B. Methods of Producing Antibodies and Antigen-Binding Fragments Thereof

[0155] Provided herein are methods for producing anti-SlOOB antibodies and fragments thereof. Methods of producing antibodies are well known to those of ordinary skill in the art. For example, methods of producing chimeric antibodies are now well known in the art (See, for example, U.S. Patent No. 4,816,567 to Cabilly et al. Morrison et al., Proc. Natl. Acad. Sci. U.S.A., 81:8651-55 (1984); Neuberger et al., Nature, 314:268-270 (1985); Boulianne, G.L. et al., Nature, 312:643-46 (1984), the disclosures of each of which are herein incorporated by reference in their entireties).

[0156] For example, antibodies or antigen-binding fragments thereof may be produced by genetic engineering. In this technique, as with other methods, antibody-producing cells are sensitized to the desired antigen or immunogen. The messenger RNA isolated from antibody producing cells is used as a template to make cDNA using PCR amplification. A library of vectors, each containing one heavy chain gene and one light chain gene retaining the initial antigen specificity, is produced by insertion of appropriate sections of the amplified immunoglobulin cDNA into the expression vectors. A combinatorial library is constructed by combining the heavy chain gene library with the light chain gene library. This results in a library of clones that co-express a heavy and light chain (resembling the Fab fragment or antigen -binding fragment of an antibody molecule). The vectors that carry these genes are cotransfected into a host cell. When antibody gene synthesis is induced in the transfected host, the heavy and light chain proteins self-assemble to produce active antibodies that can be detected by screening with the antigen or immunogen.

[0157] Antibody coding sequences of interest include those encoded by native sequences, as well as nucleic acids that, by virtue of the degeneracy of the genetic code, are not identical in sequence to the disclosed nucleic acids, and variants thereof. Variant polypeptides can include amino acid (“aa”) substitutions, additions, or deletions. The amino acid substitutions can be conservative amino acid substitutions or substitutions to eliminate non-essential amino acids, such as to alter a glycosylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function. Variants can be designed so as to retain or have enhanced biological activity of a particular region of the protein (e.g., a functional domain, catalytic amino acid residues, etc). Variants also include fragments of the polypeptides disclosed herein, particularly biologically active fragmentsand / or fragments corresponding to functional domains. Techniques for in vitro mutagenesis of cloned genes are known. Also included in the subject disclosure are polypeptides that have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.

[0158] Chimeric antibodies may be made by recombinant means by combining the VL and VH regions, obtained from antibody producing cells of one species with the constant light and heavy chain regions from another. Typically, chimeric antibodies utilize rodent or rabbit variable regions and human constant regions, in order to produce an antibody with predominantly human domains. The production of such chimeric antibodies is well known in the art, and may be achieved by standard means (as described, e.g., in U.S. Patent No. 5,624,659, incorporated herein by reference in its entirety). It is further contemplated that the human constant regions of chimeric antibodies of the disclosure may be selected from IgGl, IgG2, IgG3, and IgG4 constant regions.

[0159] Humanized antibodies are engineered to contain even more human-like immunoglobulin domains, and incorporate only the complementarity determining regions of the animal-derived antibody. This is accomplished by carefully examining the sequence of the hyper- variable loops of the variable regions of the monoclonal antibody and fitting them to the structure of the human antibody chains. Although facially complex, the process is straightforward in practice. See, e.g., U.S. Patent No. 6,187,287, incorporated fully herein by reference.

[0160] Antibody polypeptides of the disclosure having SIOOB binding specificity may also be produced by constructing, using conventional techniques well known to those of ordinary skill in the art, an expression vector containing a promoter (optionally as a component of a eukaryotic or prokaryotic operon) and a DNA sequence encoding an antibody heavy chain in which the DNA sequence encoding the CDRs required for antibody specificity is derived from a non-human cell source, e.g., a rabbit or rodent B-cell source, while the DNA sequence encoding the remaining parts of the antibody chain is derived from a human cell source.

[0161] A second expression vector is produced using the same conventional means well known to those of ordinary skill in the art, said expression vector containing a promoter (optionally as a component of a eukaryotic or prokaryotic operon) and a DNA sequence encoding an antibody light chain in which the DNA sequence encoding the CDRs required for antibody specificity is derived from a non-human cell source, e.g., a rabbit or rodent B-cell source, while the DNA sequence encoding the remaining parts of the antibody chain is derived from a human cell source.

[0162] The expression vectors are transfected into a host cell by convention techniques well known to those of ordinary skill in the art to produce a transfected host cell, said transfected host cell cultured by conventional techniques well known to those of ordinary skill in the art to produce said antibody polypeptides.

[0163] The host cell may be co-transfected with the two expression vectors described above, the first expression vector containing DNA encoding a promoter (optionally as a component of a eukaryotic or prokaryotic operon) and a light chain-derived polypeptide and the second vector containing DNA encoding a promoter (optionally as a component of a eukaryotic or prokaryotic operon) and a heavy chain-derived polypeptide. The two vectors contain different selectable markers, but preferably achieve substantially equal expression of the heavy and light chain polypeptides. Alternatively, a single vector may be used, the vector including DNA encoding both the heavy and light chain polypeptides. The coding sequences for the heavy and light chains may comprise cDNA, genomic DNA, or both.

[0164] The host cells used to express the antibody polypeptides may be either a bacterial cell such as E. coli, or a eukaryotic cell such as P. pastoris. In one aspect, a mammalian cell of a well-defined type for this purpose, such as a myeloma cell, a CHO cell line, a NSO cell line, or a HEK293 cell line may be used.

[0165] The general methods by which the vectors may be constructed, transfection methods required to produce the host cell and culturing methods required to produce the antibody polypeptides from said host cells all include conventional techniques. Although preferably the cell line used to produce the antibody is a mammalian cell line, any other suitable cell line, such as a bacterial cell line such as an E. co / z-derived bacterial strain, or a yeast cell line, may alternatively be used.

[0166] Similarly, once produced the antibody polypeptides may be purified according to standard procedures in the art, such as for example cross-flow filtration, ammonium sulphate precipitation, affinity column chromatography, hydrophobic interaction chromatography (“HIC”), and the like.

[0167] The antibody polypeptides described herein may also be used for the design and synthesis of either peptide or non-peptide mimetics that would be useful for the same therapeutic applications as the antibody polypeptides of the disclosure (See, for example,Saragobi et al., Science, 253:792-795 (1991), the contents of which are herein incorporated by reference in its entirety).

[0168] In another aspect, provided herein are methods for humanizing antibody heavy and light chains which bind to SIOOB. Exemplary methods for humanizing antibody heavy and light chains that may be applied to anti-SlOOB antibodies are identified herein and are conventional in the art.

[0169] In some aspects, provided herein are methods for producing an antibody, or antigenbinding fragment thereof, that specifically binds to SIOOB, the methods comprise expressing the antibody, or antigen-binding fragment thereof, in a recombinant cell, and isolating the antibody, or antigen-binding fragment thereof, from the cell. In some aspects, the methods further comprise preparing the antibody, or antigen-binding fragment thereof, isolated from the cell into a test device. In other aspects, the methods further comprise formulating the antibody, or antigen-binding fragment thereof, isolated from the cell into a pharmaceutical composition.C. Methods of Detecting or Diagnosing Disease or Disorders

[0170] Provided herein are methods for diagnosing an SlOOB-associated disease or disorder based on the level of SIOOB protein. Exemplary SlOOB-associated diseases or disorder include, but are not limited to, traumatic brain injury, Alzheimer's disease, strokes, epilepsy, amyotrophic lateral sclerosis, melanoma, Down's syndrome, type I diabetes, or any other neurological diseases.

[0171] In some aspects, provided herein are methods for diagnosing traumatic brain injury (TBI) in a subject. The methods comprise obtaining a biological sample from the subject; contacting the sample with an anti-SlOOB antibody, or antigen-binding fragment thereof, as disclosed herein, measuring the level of SIOOB in the sample; and determining based on the level of SIOOB whether the subject has traumatic brain injury.

[0172] In some aspects, the traumatic brain injury is a mild traumatic brain injury. In some aspects, the traumatic brain injury is a moderate to severe traumatic brain injury.

[0173] In some aspects, provided herein are methods for diagnosing an SlOOB-associated disease in a subject. The methods comprise obtaining a biological sample from the subject; contacting the sample with an anti-SlOOB antibody, or antigen-binding fragment thereof, as disclosed herein, measuring the level of SIOOB in the sample; and determining based on the level of SIOOB whether the subject has an SlOOB-associated disease.

[0174] In some aspects, the SlOOB-associated disease is selected from the group consisting of traumatic brain injury, Alzheimer's disease, strokes, epilepsy, amyotrophic lateral sclerosis, melanoma, Down's syndrome, or type I diabetes.

[0175] In some aspects, provided herein are methods for detecting or measuring the level of SIOOB in a subject. The methods comprise obtaining a biological sample from the subject, and contacting the sample with an antibody, or antigen-binding fragment thereof, as disclosed herein, thereby detecting or measuring the level of SIOOB in the subject.

[0176] The anti-SlOOB antibody, or antigen-binding fragment thereof, can be used for detecting SIOOB in any samples, or any biological samples obtained from or derived from a subject.

[0177] In some aspects, the biological sample is a liquid biological sample. In some aspects, a liquid biological sample is a body fluid. In some aspects, the biological sample is a saliva sample. In some aspects, the biological sample is a blood sample. In some aspects, the biological sample is a plasma sample. In some aspects, the biological sample is a serum sample.

[0178] In some aspects, the subject is a human.D. Compositions and Kits

[0179] Provided herein are compositions and kits comprising an antibody, or antigen-binding fragment thereof, of the disclosure, as described herein, or an isolated nucleic acid molecule, e.g., an isolated mRNA molecule, encoding the antibody or antigen-binding fragment thereof, or a vector comprising the nucleic acid molecule, and a package insert with instructions to perform any of the methods described herein.

[0180] In some aspects, the kits include instructions for using the kit. The instructions will generally include information about the use of the kit for diagnosing an SlOOB-associated disease or disorder, e.g., traumatic brain injury. In other aspects, the instructions include at least one of the following: precautions; warnings; clinical studies; and / or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. In a further aspect, a kit can comprise instructions in the form of a label or separate insert (package insert) for suitable operational parameters.

[0181] In some aspects, the kit includes a composition including an antibody, or antigenbinding fragment thereof, or an isolated nucleic acid molecule, e.g., an isolated mRNAmolecule, encoding the antibody or antigen-binding fragment thereof, and a package insert with instructions to perform any of the methods described herein.

[0182] The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein.

[0183] In some aspects, the kit can comprise one or more containers with appropriate positive and negative controls or control samples, to be used as standard(s) for detection, calibration, or normalization.

[0184] The kit can further comprise a second container comprising a pharmaceutically- acceptable buffer, such as (sterile) phosphate-buffered saline, Ringer's solution, or dextrose solution; and other suitable additives such as penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients, as described herein. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, and package inserts with instructions for use. The kit can further include a delivery device, such as needles, syringes, pumps, and package inserts with instructions for use.EXAMPLESExample 1. Antibody Isolation

[0185] This example described the isolation of anti-SlOOB antibody for use as a detection and capture reagent in the device / system.

[0186] The suitability of the purified fusion products as capture and detection reagents for immunoassays were characterized. Purified fusion products from hybridoma fusion SIOOB- AJ-1.2. All fusion products were biotinylated in MBS’ MultiPure process for use in Sandwich ELISA, with estimated molar substitution rates of 1.8 to 4.1. Additional Reagents consisted of: Plate coat buffers dPBS, pH 7.6 and Carbonate / Bicarbonate buffer pH 9.6; Blocking / Diluent Buffer lx PBS-Casein (from 5x PBS-Casein Block / Diluent, Surmodics Cat# PBSC); SIOOB Calcium-binding protein, ProSpec Cat# pro-2312, lot# 622PS100B; S100A1 R&D Systems Cat# 9705-S1, lot# QDH0321121 (Cross-reactant); S100A4 R&D Systems Cat# 4137-S4, lot# QEG0321102 (Cross -reactant); S100A6 ProSpec Cat# pro-148, lot# 622PS100A6 (Cross-reactant); S100A13 R&D Systems Cat# 4327-SA, lot# QHQ0212081 (Cross -reactant); S100A Mixture 1:1: 1:1 mixture of SIOO Al, A4, A6 andA13, negative screen; ELISA microwell plates Greiner MicroIon 600 Hi-bind, 96 well flatbottom Wash Buffer PBS- 0.05% Tween 20 Goat anti-Mouse IgG Fc fragment-HRP Jackson Immunoresearch Cat# 115-035-164, 1:10,000 dilution in Block / Diluent; Streptavidin-HRP Jackson Immunoresearch Cat# 016-030-084, 1:10,000 dilution in TMB Substrate, Surmodics Cat# TMBW; and Stop solution IN HCI. With reference to FIG. 12, a listing of the purified fusions products is shown.

[0187] EEISA assays were incubated at controlled room temperature. The incubation steps, except substrate, were run on an orbital plate shaker (0.2 micron orbit) at 350 rpm. An Indirect ELISA test was performed to ensure that the candidate Fusion Products retained specific binding profiles post purification: e.g., strong binding of 1 pg / mL antibody on an SIOOB -coated plate (1 pg / mL), and a lack of binding to plates coated with 2 pg / mL of 5100A Mix. The remaining specific fusion products were run on an Isoelectric focusing (IEF) gel to visualize their isoform profiles for purposes of detecting obvious polyclonals, optimizing plate coating buffers and identifying possibly redundant samples.

[0188] With reference to FIG. 13, six fusion products (02F12, 04C04, 06G06, 11D05, 11 H03 and 12D05) no longer exhibit specific binding to the SIOOB protein. Of the remaining 24 candidates, none display any binding to the S100A Mix coated on the plate. The remaining 24 fusion products were run on IEF gels. Two of the fusion products displayed multiple dominant isoform groupings on the IEF gels, indicating definite polyclonality (06C03 and 11E11). These were removed from Sandwich ELISA testing, as the presence of multiple positive clones could confound results in the assay. However, six other fusion products had dominant plus faint groups of isoforms. These may be polyclonal but moved on to further testing.

[0189] Matched pair screening of MultiPure Fusion Products with 0 and 40 ng / mL SIOOB sample, was conducted using the Sandwich ELISA protocol. Twenty-two fusion products were coated on plates at 2 pg / mL in pl- appropriate coating buffer. Coating on 384- well plates occurred overnight at 4°C, followed by a 2x wash and PBS-Casein Block. PBS-Casein Blocking Buffer also served as diluent for all samples and reagents. The same twenty-two biotinylated fusion products were tested as detectors in the Sandwich ELISA, with SIOOB sample prepared in the PBS-Casein block at 0 and 40ng / mL. Samples were incubated on the coated plates for 45 minutes. Biotinylated antibodies were added at 0.5 pg / mL after samples were washed out, for 30 minutes. Following a wash, Streptavidin-HRP detection reagent was incubated for 30 minutes. After a final wash, TMB Substrate incubated for four minutes, andcolor development was stopped with an equal volume of IN HC1. Absorbance values were measured at 450 nm with subtraction of a 650 nm reference reading.

[0190] Selections of top pairs were re-tested with multiple sample concentrations to determine relative sensitivity. The assay protocol was the same used in the initial sandwich ELISA but utilized 96-well rather than 384-well plates. The S100A Mix sample was tested with all these pairs at 1 pg / mL. The results are shown in FIG. 14. None of the pairings detected the 1 pg / mL 5100A mix sample and all were specific for SIOOB. The combinations in the top half of the table demonstrate SIOOB detection down to the 0.78 ng / mL sample with reasonable linearity of dilution. The combinations in the bottom half of the table demonstrate SIOOB detection as measured by their absorbance values of the 25ng / mL sample, but linearity of detection was not maintained throughout the dilution series.

[0191] Seven monoclonal antibodies were selected for further subcloning: 11C03, 02H09, 02C11, 07F04, 12C03, 03A07, 10B06, 07F04, and 12C03. The following monoclonal antibodies have been identified as capture antibodies: 11C03, 02H09, 02C11, 07F04, and 12C03. The following antibodies have been identified as antigen detection antibodies: 03A07, 10B06, 07F04, and 12C03. The following antibodies have been identified as both capture antibodies and antigen detection antibodies: 07F04 and 12C03. Referring to the lateral flow device of FIGS. 8 A and 8B, one or more of the identified capture antibodies may be conjugated to an enzyme (e.g., horseradish peroxidase) in the nitrocellulose membrane a5. One or more of the detection antibodies identified may attach to the capture antibodies to form test lines (e.g., test lines a5 or test lines al2).

[0192] Each parental line (fusion products) will be subcloned by limiting dilution. The top three subclones from each will be selected for cryo-preservation (2 vials hybridoma cells each) and scaled up (15 ml).Example 2. Subcloning, Sequencing and Purification of Monoclonal Antibodies

[0193] Fusion products were subcloned via limiting dilution in order to obtain a monoclonal culture of cells producing your antibody of interest. The subcloning procedure includes a scoring of each and every well to identify the presence of a single colony of cells or multiple colonies of cells and then screened for the production of specific antibody.

[0194] After the selected fusion products from the primary screen (and subsequent re-screen) were cryopreserved and supernatants harvested. The harvested supernatants were re-screened toconfirm specific antibody production, and / or test against other reagents / cross -reactants to further characterize the lead fusion products.

[0195] FIG. 16A-D depict the binding data of several subclones against human SIOOB as determined by ELISA. Selected antibodies (z.e., AVS5955 (03A07-01G09-02E11), AVS5956 (07F04-01F11-02C11), AVS5957 (10B06-02F06-02D05), and AVS5958 (11C03-01C08- 02C12)) have been identified as antigen detection and / or capture antibodies.

[0196] The antibodies were cloned using rapid amplification of cDNA ends (RACE) methods. After sequencing 96 cloned PCR fragments, antibody sequence analysis identified 1 heavy chain and 1 light chain for each hybridoma sample.

[0197] The antibody variable region DNA fragments were then cloned into a TOPO vector. PCR fragments amplified from the TOPO clones were recovered after gel electrophoresis analysis and sequenced. CDR analysis was performed. The protein and DNA sequences for each antibody are shown in Tables 1 and 2.

[0198] Recombinant whole IgG (with human Fc backbone) or scFv-Fc protein are generated in CHO cells, and antigen binding assay is performed in vitro in a 96-well ELISA format to confirm specificity for the SIOOB antigen.

[0199] Antibodies AVS5955 (03A07-01G09-02E11), AVS5956 (07F04-01F11-02C11), AVS5957 (10B06-02F06-02D05) and AVS5958 (11C03-01C08-02C12) were separately purified using Protein A affinity chromatography (IC / P0036). About IL supernatant was loaded onto the column for purification. The binding buffer was 3.0 M NaCl in 0.1 M citrate / phosphate buffer (pH 9.0), and the elution buffer was 0.1 M citrate / phosphate buffer with 0.15M NaCl (pH 3.0). Antibody was eluted along a linear gradient. The elution pH is about 3.0.

[0200] For AVS5955 (03A07-01G09-02E11), about 35.7 mg of purified antibody was collected in 21 mL of 0.15 PBS (Dulbecco’s CMF) with a final concentration of 1.7 mg / mL.

[0201] For AVS5956 (07F04-01F11-02C11), about 36.1 mg of purified antibody was collected in 19 mL of 0.15 PBS (Dulbecco’s CMF) with a final concentration of 1.9 mg / mL.

[0202] For AVS5957 (10B06-02F06-02D05), about 25.0 mg of purified antibody was collected in 12.5 mL of 0.15 PBS (Dulbecco’s CMF) with a final concentration of 2.0 mg / mL.

[0203] For AVS5958 (11C03-01C08-02C12), about 32.4 mg of purified antibody was collected in 18 mL of 0.15 PBS (Dulbecco’s CMF) with a final concentration of 1.8 mg / mL.

[0204] Table 2 provides the summary of cell lines producing the SIOOB monoclonal antibodies.

[0205] Table 2. Summary of Cell LinesExample 3. Performance of the Conan MedTech Real-Time Saliva S100B Concussion Detection Device for Evaluation of Mild Traumatic Brain Injury

[0206] This examples provides a clinical study of the performance of the Conan MedTech Real-Time Saliva SIOOB concussion detection device for the evaluation of mild traumatic brain injury, also known as concussion.

[0207] This is a prospective, multi-center, double-blind, open-label study. Adults and children aged 5 and older who sustain a possible or probable head and / or body injury of sufficient impact to raise concern for concussion, after informed consent, have a saliva sample taken and applied to the device. This collection takes place in <24 hours, preferably as soon as possible at the point of care. A Glasgow Coma Scale (GCS) is administered prior to saliva sample and must be 13-15 inclusive.

[0208] The patient sucks the sample collection filter for about 10-20 seconds, allowing the saliva to be transferred to the collection pad. There is a blinking signal on the side to indicate when sufficient sample has been collected. After sufficient saliva has been collected, it goes to the sample pad so that capillary action can begin by allowing the sample to flow through the conjugate pad. There it captures the antibody for the SIOOB biomarker causing the control and possibly the indicator line to be visualized present based upon the level of the biomarker. This result can then be captured and shared using the app with the parent or health care practitioner.

[0209] The device identifies two lines on the lateral flow device strip for evaluation. A control line indicates that the device is functioning properly, and if there is detection of elevated SIOOB, a second line appears indicating possible TBI which requires the patient to seek further evaluation.

[0210] Study participants are not informed of the result but are referred to a study site (a “site”) within 24 hours, where they are evaluated by a Neurologist / concussion specialist and receive standard of care. Adults aged 18 and over have blood drawn for the Abbott i-Stat test.

[0211] The study participant follow-up with a blinded site investigator Neurologist at day 7- 10, who assess for evidence of concussion using patient history, general and neurological examinations, and the use of the Balance Error Scoring System (BESS), the King-Devick Test, and the Post-Concussion Symptom Score (PCSS).

[0212] Approximately 10% of the cohort are randomly selected to participate in the brain imaging sub-study (qDTI of the brain and CT scan of the head). This reduces the number of participants who receive CT radiation, especially in pediatrics, as CT is generally normal in mTBI. The imaging ideally occurs within 2 weeks (+ / - 2 weeks) of the injury. The sub-study cohort read and sign a separate radiological informed consent form (r-ICF).

[0213] A statistical analysis plan (SAP) is created to evaluate powering for both the pediatric and adult populations studied. In the adult population studied, powering includes comparison to the Abbott i-Stat test. In adults and children, powering includes comparison to clinical and (in a sub-set radiological) gold standards.

[0214] The primary objective is to compare the ability of the Conan Medtech Real-Time Concussion Detection Device (“device”) to accurately detect a concussion (sensitivity, specificity, negative and positive predictive values) immediately (within 24 hours of injury) to examination by a board-certified Neurologist using patient history, examination and validated concussion clinical tests (herein referred to a “clinical gold standard”) at day 7-10 post-injury in adults and children age five and older.

[0215] Secondary objectives of the study include: comparing the device’s ability to accurately diagnose concussion to the Abbott i-Stat biomarker blood test (herein referred to as “biomarker gold standard”) in adults 18 years of age and older, and comparing (in a substudy of approximately 10% of total sample size) the device’s ability to accurately diagnose concussion to quantitative diffusion tensor imaging MRI (qDTI) brain imaging (herein referred to as “radiologic gold standard”) in adults and children aged 5 and older. All participants offered a qDTI also receive a head CT scan.

[0216] These outcomes can be prespecified by time of obtaining the results and ages in categories. Sensitivity, specificity, and positive / negative predictive values can be calculated from these outcomes.Inclusion criteria:

[0217] Patients are eligible to be included in the study if all of the following criteria apply:1. Written informed consent must be obtained from the subject, or in the case of a minor, a parent, in accordance with requirements of the study site’s IRB or ethics committee, prior to initiation of any protocol- specified procedures.2. Participant / parent must be able to read English.3. Male or female, 5 to 75 years of age inclusive.4. GCS of 13-15 inclusive.Exclusion criteria:

[0218] Patents are excluded from the study if any of the following criteria apply:1. GCS less than 132. Patients with clinically significant neurological illness, that, in the opinion of the Investigator, may have the potential of confounding test results.3. Patients with a history of or currently having major psychiatric disorders including schizophrenia, active psychosis, or uncontrolled bipolar disorder. Major depressive disorder and generalized anxiety disorder which, in the investigator’s opinion are stable and well- controlled, will be allowed.4. Patients with hematologic or solid malignancy diagnosis within 5 years prior to screening with the exception of basal cell carcinoma and squamous cell carcinoma if they have been cancer free prior to screening.5. Adult body mass index of < 17 or >36. In children, BMI must be in the 5th-85th percentile.6. Patients who within the past 3 years have a history of or have been treated for alcohol or drug abuse.7. Women who are pregnant or breastfeeding.Study Design and Visit Schedule

[0219] Participants with suspected concussion are identified primarily through four avenues:

[0220] A non-medical facility (NMF). This includes schools (elementary through university), sporting venues (such as an ice-skating rink, gymnastics facility), community sports teams (e.g. Pop Warner football). After administration of GCS, Participating NMFs perform the device saliva test on any athlete suspected of having a concussion. Informed consent is either previously obtained (for example, an entire football team signing consent to have the test performed at the beginning of the season), or at the time of suspected concussion. The injured participant (or their parent, as applicable) are not informed of the test result, but are referred to a local trial site as soon as possible (within 24 hours) for Visit 1 (see below). Device test result and signed informed consent are sent to site. Professional athletes and military personnel who sustain possible concussion are treated as referral from an NMF, unless a licensed medical profession is present and able to perform device test and GCS, in which case such participants are treated as per ER / HF.

[0221] Emergency Department or walk-in acute care medical facility (ER). Participant, after providing informed consent, and following administration of GCS with results 13-15, provide saliva for test device analysis. Results are not provided to participant. The ER providestandard care, and refer to local trial site for appointment in 7-10 days. The ER forward to the trial site the GCS score and test result, signed informed consent, and medical record of visit.

[0222] Healthcare Facility (HCF), such as a pediatric, internal medicine, neurology, or family practice office. Procedure is the same as for the ER.

[0223] Study or Trial site. If a participant presents immediately after injury (within 24 hours), VI takes place, and participant are scheduled for follow-up in 7-10 days.

[0224] VI: 0-24 hours after qualifying event

[0225] After the qualifying event, at the first site visit (VI), the following items must be completed and reviewed:• Explain the purpose of the study to the prospective participants.• Obtain written informed consent.• Document medical history and collect demographic information.• Record concomitant medications (con meds) that the patient is taking.• Perform a physical examination (including height, weight, and vital signs).• Perform Glasgow Coma Scale (GCS)• Review inclusion / exclusion criteria.• Obtain saliva sample (if not already performed)• Perform Abbott i-Stat blood draw• Assign Next Visit Date

[0226] VI: 7-10 days ( + / - 2 days) after qualifying event• Perform physical and neurological exam• Record concomitant medications (con meds) that the patient is taking• Perform BESS, King-Devick, and PCSS• Blinded Neurologist will review findings and determine whether participant sustained a concussion or not• Determine if participant has been randomly assigned to radiology sub-study, and if so schedule head CT and brain qfMRI at local facility• Assign next visit date (V2 for sub-study participants and / or V3 for all participants)

[0227] V2: Radiology Sub-study, 7-28 days after qualifying event

[0228] Participant will report to local imaging facility for studies. Results, including images and written report, will be transmitted to study site.

[0229] V3: End of study (telephone visit) 28-40 days after qualifying event

[0230] Collect AEs, if any

[0231] Con meds

[0232] Record whether participant has any symptoms of concussio

[0233] Recruitment Plan

[0234] Thirty (30) research sites conduct the study: 25 private Neurology research centers across the United States, 3 academic research centers, and 2 military sites to enroll 1,800 participants (approximately 60 participants per site), stratified by race and 3 age groups: age 5-11, age 12-17, and age 18 and up. Enrollment period is 12 months but may be extended as necessary. It is anticipated that each site get l / 3rdof participants (n=20). These participants come directly to the site within 24 hours of a suspected concussion (qualifying event) and receive the Conan Medtech saliva test onsite. The remaining 2 / 3rd(n=40) are referred to the Study or Trial site from local schools (grades 1-12, public and private), universities, emergency rooms / medical walk-in centers, and local sports leagues. It is anticipated the majority of these have the Conan Medtech saliva test performed immediately after qualifying event at the NMR (for example, on the sideline of a football game), in which case the participant are directed to the local Study or Trial site within 24 hours of the qualifying event, and the test results are transmitted to the site.

[0235] Statistical Analysis

[0236] The statistical analysis plan (SAP) are finalized prior to the database lock of the primary analysis, and it includes a more technical and detailed description of the statistical analyses described in this section. This section is a summary of the planned statistical analyses of the most important endpoints including primary and key secondary endpoints. Other endpoints as well as demographic, baseline characteristics and disposition are also included.

[0237] The primary analysis is estimating the diagnostic test characteristics [sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy] of Conan MedTech SIOOB against the clinical gold standard (physician evaluation). Point estimates with 95% CI (Clopper- Pearson) will be derived based on the following definitions:

[0238] Cl . ... . d — — _ T T & + clSensitivity = — Specificity = - PPV = - NPV = - Accuracy = - a+c b + d a+b c+d a+b+c+d

[0239] The secondary analyses involve comparing the sensitivity, specificity, PPV, and NPV of Conan MedTech SIOOB to the biomarker gold standard (Abbott i-Stat blood test) and also comparing it to the radiological gold standard (qDTI). The test characteristics are computed for the biomarker and radiological gold standards as described above. To compare sensitivity and specificity the nonparametric McNemar test for paired nominal data is used. To compare PPV and NPV (which unlike sensitivity and specificity will have different denominators for each test) the nonparametric permutation test is used.

[0240] Subgroup analyses are performed by splitting the sample into two age groups: 1) pediatric, defined as ages 5-17; and 2) adults, defined as age 18 and older. The primary and secondary analyses described above are run for each group separately.

[0241] Risk Management for Clinical Study

[0242] Risk Case 1: Data Entry Errors• Description: There is a risk that clinical sites will record incorrect or missing data, resulting in inaccurate study outcomes and protocol violations.• Severity (S): 6 (Moderate; impacts data integrity but can be corrected)• Occurrence (O): 7 (Likely; human errors are common in manual data entry)• Detectability (D): 8 (Low Detectability; errors may go unnoticed before data validation)• RPN Calculation: S x O x D = 6 x 7 x 8 = 336• Risk Level: High (Risk / Benefit Analysis Required.)• Mitigation Measures:• Prevention: a. Implement real-time error detection in the Electronic Data Capture (EDC) system to prevent incorrect data submission. b. Automated flagging of inconsistent or missing entries.• Detection: a. Perform weekly data audits at all clinical sites. b. Assign data monitors to review and validate all critical variables.• Correction: a. Establish a correction log process for investigators to amend errors.b. Provide mandatory refresher training for site staff every 3 months.

[0243] Risk Case 2: Participant Dropout• Description: Participants may withdraw owing to a lack of participation, unclear study objectives, or schedule conflicts, resulting in insufficient data collection.• Severity (S): 8 (Very High; reduces study power and affects statistical significance)• Occurrence (O): 6 (Dropouts occur occasionally but not constantly).• Detectability (D): 7 (Moderate; may only be detected after missing follow-ups)• RPN Calculation: S x O x D = 8 x 6 x 7 = 336• Risk Level: High (Risk / Benefit Analysis Required.)• Mitigation Measures:• Prevention: a. Develop personalized participant engagement strategies (e.g., periodic updates, newsletters). b. Offer financial compensation and travel reimbursements to improve retention.• Detection: a. Use automated alerts in EDC to flag participants at risk of dropping out (e.g., missed visits, lack of responses).• Correction: a. Re-engagement protocol: Reach out within 48 hours after a missed visit to reschedule. b. Conduct exit interviews to understand reasons for withdrawal and adjust study procedures accordingly.

[0244] Risk Case 3: Regulatory Non-Compliance• Description: The study may not fully comply with FDA criteria, resulting in regulatory delays or rejection.• Severity (S): 9 (Severe with Warning ; could prevent study approval)• Occurrence (O): 4 (Low; with proper compliance strategy)• Detectability (D): 6 (Moderate Detectability; misalignment may not be detected until review)• RPN Calculation: S x O x D = 9 x 4 x 6 = 216Risk Level: High (Risk / Benefit Analysis Required.)Mitigation Measures:• Prevention: a. Align protocol with FDA regulatory frameworks before submission. b. Schedule pre- submission meetings with regulatory agencies to address compliance concerns.• Detection: a. Conduct internal regulatory audits at key study milestones. b. Ensure all study documents are pre -reviewed by regulatory experts.• Correction: a. Establish a regulatory task force to quickly implement changes if non- compliance is detected. b. If deviations occur, file Corrective and Preventive Actions (CAPAs) within 7 days.

[0245] Risk Case 4: Imaging Sub-Study delays• Description: Delays in qDTI imaging data submission may impair study timeframes and secondary outcomes.• Severity (S): 7 (High; affects secondary outcomes)• Occurrence (O): 5 (Delays happen occasionally but can be managed.; due to logistical challenges)• Detectability (D): 6 (Moderate; delays can be detected through tracking)• RPN Calculation: S x O x D = 7 x 5 x 6 = 210• Risk Level: High (Requires risk / benefit analysis per)• Mitigation Measures:• Prevention: a. Establish partnerships with multiple imaging centers to reduce dependency on a single site. b. Ensure MRI appointments are pre-booked as part of participant scheduling.• Detection: a. Implement real-time tracking of imaging completion rates in the study database. b. Create a weekly reporting system for imaging sites to monitor backlog.• Correction: a. Allocate backup imaging slots at secondary locations for urgent cases.b. Develop a remote imaging review process to expedite analysis.

[0246] Risk Case 5: Low Pediatric Recruitment• Description: Low enrollment in pediatrics may reduce the statistical power of subgroup analysis.• Severity (S): 8 (Very High; affects subgroup analysis)• Occurrence (O): 6 (Pediatric recruitment is challenging but manageable.)• Detectability (D): 7 (Moderate; only visible after enrollment analysis)• RPN Calculation: S x O x D = 8 x 6 x 7 = 336• Risk Level: High (Risk / Benefit Analysis Required.)• Mitigation Measures:• Prevention: a. Partner with schools, pediatric hospitals, and sports leagues for recruitment. b. Offer parental education sessions to address concerns.• Detection: a. Monitor recruitment progress weekly and adjust outreach strategies accordingly.• Correction: a. Expand eligibility criteria slightly to include borderline age groups. b. Extend recruitment timelines if necessary.Example 4. Risk Assessment and Management

[0247] Risk Assessment for Lateral Flow Assay

[0248] Risk Case 1: Inaccurate Test Results (False Positives / Negatives)• Description: The LFA may yield false positive or false negative results, leading to misdiagnosis and improper medical decisions.• Severity (S): 9 (High; incorrect diagnosis could affect patient treatment)• Occurrence (O): 6 (Moderate; influenced by test sensitivity and specificity)• Detectability (D): 7 (Moderate; errors may only be detected through clinical validation)• RPN Calculation: 9 x 6 x 7 = 378• Risk Level: High• Mitigation Measures: Optimize antibody specificity, conduct rigorous clinical validation, and implement confirmatory testing recommendations.

[0249] Risk Case 2: Insufficient Sample Volume for Detection• Description: Inadequate saliva sample may lead to inaccurate or invalid results.• Severity (S): 7 (Moderate; test may fail, requiring retesting)• Occurrence (O): 5 (Low; mitigated by clear user instructions)• Detectability (D): 6 (Moderate; detected through invalid test warnings)• RPN Calculation: 7 x 5 x 6 = 210• Risk Level: Medium• Mitigation Measures: Design collection buffer to stabilize low-volume samples, add fail-safe indicators, and provide user training.

[0250] Risk Case 3: Degradation of Biomarkers in Storage• Description: The target biomarker may degrade due to improper storage, leading to test inaccuracies.• Severity (S): 8 (High; affects accuracy of results)• Occurrence (O): 5 (Low; controlled through storage guidelines)• Detectability (D): 7 (Moderate; detected upon performance testing)• RPN Calculation: 8 x 5 x 7 = 280• Risk Level: High• Mitigation Measures: Establish strict temperature controls, use stabilizing agents, and perform periodic quality checks.

[0251] Risk Case 4; Interference from Saliva Components• Description: Substances in saliva (food particles, blood, medications) may interfere with test performance.• Severity (S): 7 (Moderate; could lead to inaccurate results)• Occurrence (O): 6 (Moderate; variable based on user)• Detectability (D): 7 (Moderate; may only be noticed in QC checks)• RPN Calculation: 7 x 6 x 7 = 294• Risk Level: High• Mitigation Measures: Include filtration step in sample collection, instruct users to rinse mouth before testing.

[0252] Risk Case 5; Difficulty in Interpretation of ResultsDescription: Users may misread faint test lines, leading to confusion.Severity (S): 6 (Moderate; misinterpretation affects decision-making)• Occurrence (O): 5 (Low; can be minimized with clear indicators)• Detectability (D): 6 (Moderate; depends on visibility of results)• RPN Calculation: 6 x 5 x 6 = 180• Risk Level: Medium• Mitigation Measures: Implement digital reader option, enhance color contrast of test lines, provide clear visual instructions.

[0253] Risk Case 6: Cross-Reactivity with Other Biomarkers• Description: The test may detect non-target biomarkers, reducing specificity.• Severity (S): 8 (High; leads to false positives)• Occurrence (O): 5 (Low; can be addressed in assay design)• Detectability (D): 6 (Moderate; detected in validation studies)• RPN Calculation: 8 x 5 x 6 = 240• Risk Level: High• Mitigation Measures: Improve antibody specificity, test cross-reactivity during validation.

[0254] Risk Case 7; Environmental Sensitivity of the Test• Description: Humidity, temperature, or light exposure may impact test performance.• Severity (S): 7 (Moderate; may cause false results)• Occurrence (O): 6 (Moderate; depends on storage conditions)• Detectability (D): 6 (Moderate; can be identified in stability testing)• RPN Calculation: 7 x 6 x 6 = 252• Risk Level: High• Mitigation Measures: Develop robust packaging, test environmental stability, include storage guidelines.

[0255] Risk Case 8: User Error in Sample Collection• Description: Users may collect saliva improperly, affecting test accuracy.• Severity (S): 6 (Moderate; incorrect results could lead to retesting)• Occurrence (O): 7 (High; varies by user proficiency)• Detectability (D): 5 (Moderate; may not be detected until analysis)• RPN Calculation: 6 x 7 x 5 = 210• Risk Level: Medium• Mitigation Measures: Provide clear instructions, develop easy-to-use collection devices, conduct usability testing.

[0256] Risk Case 9: Regulatory Non-Compliance• Description: Failure to meet FDA and ISO regulatory requirements may delay approval.• Severity (S): 9 (High; non-compliance could prevent market entry)• Occurrence (O): 4 (Low; mitigated through compliance checks)• Detectability (D): 6 (Moderate; may be detected in audits)• RPN Calculation: 9 x 4 x 6 = 216• Risk Level: High• Mitigation Measures: Align with FDA and ISO guidelines, conduct internal audits, consult regulatory experts.

[0257] Risk Case 10: Delays in Manufacturing and Supply Chain Issues• Description: Disruptions in reagent supply or production could lead to test shortages.• Severity (S): 8 (High; affects availability of tests)• Occurrence (O): 5 (Moderate; depends on supply chain stability)• Detectability (D): 6 (Moderate; may only be noticed in production)• RPN Calculation: 8 x 5 x 6 = 240• Risk Level: High• Mitigation Measures: Establish multiple supplier agreements, maintain stock of critical reagents, implement contingency plans.

[0258] Risk Management for Lateral Flow Test

[0259] This risk management analysis provides a comprehensive assessment of potential risks associated with Lateral Flow Assay for Concussion Detection.

[0260] Risk Case 1: The patient does not provide an adequate saliva sample.• Description: Primarily, the test may produce inaccurate results or become invalid, causing potential misdiagnosis or the need for retesting. This can delay diagnosis and treatment, potentially allowing a medical condition to progress unchecked. Additionally, it can lead to increased anxiety for the patient due to uncertainty or the inconvenience of repeated testing.Severity (S): 7 (Moderate; test may fail, requiring retesting)Occurrence (O): 5 (Low; mitigated by clear user instructions)Detectability (D): 6 (Moderate; detected through invalid test warnings)• RPN Calculation: 7 x 5 x 6 = 210• Risk Level: Medium• Mitigation Measures: Ensuring proper collection techniques and clear instructions can help mitigate these risks.1. **Clear Instructions**: Provide the patient with clear, detailed instructions on how to properly collect the saliva sample. Use both written and visual aids, such as illustrations or instructional videos via our mobile app.2. **Demonstration**: demonstrate the sample collection process through a video demonstration to ensure patient understanding via our mobile app.3. **Pre-collection Preparation**: Advise patients to avoid eating, drinking colorful beverages, chewing gum, or brushing teeth for a certain period before sample collection to prevent contamination or insufficient sample volume.4. **Use of Collection Aids**: Provide devices that facilitate easier saliva collection to help ensure adequate sample volume.5. **Reinforce the Importance**: Educate patients on the importance of providing an adequate sample and how it affects the accuracy of their test results.6. **Feedback and Support**: Offer a channel for patients to ask questions or express concerns about the sample collection process.7. **Pilot Test**: Conduct a user experience and human factor study or dry run to ensure the patient is comfortable and capable of providing an adequate sample.

[0261] Risk Case 2; Patient receives false negative• Description: Despite having a concussion, the test result incorrectly indicates a negative result, meaning the device fails to detect the presence of the analyte that it's designed to measure. This false negative may lead to delayed diagnosis and inappropriate medical decisions. The patient may not seek further medical evaluation or treatment based on the erroneous assurance that they are free of the condition.• Severity (S): 9 (High; incorrect diagnosis could affect patient treatment)• Occurrence (O): 6 (Moderate; influenced by test sensitivity and specificity)• Detectability (D): 7 (Moderate; errors may only be detected through clinical validation)• RPN Calculation: 9 x 6 x 7 = 378• Risk Level: High• Mitigation Measures: Device sensitivity and specificity improvement, quality control and testing, use instructions and education, providing extra tests if the first one has a manufacturing issue and encouraging follow-up testing. Statistical analysis and postmarketing surveillance by employing statistical analyses to monitor false negative rates and identify patterns or factors contributing to these errors. Conduct post-marketing surveillance to gather real-world performance data and adjust recommendations accordingly.

[0262] Risk Case 3: Patient receives false positive• Description: In the risk case of a patient receiving a false positive result from a salivabased lateral flow device (LFD), the test incorrectly indicates the presence of a disease or condition when it is not actually present. This situation can lead to several potential consequences, including unnecessary stress and anxiety for the patient, unwarranted medical treatments or procedures, and potential resource wastage in healthcare settings. Additionally, false positives may overload healthcare systems with individuals seeking further confirmatory testing, which can divert resources from those who truly need them.• Severity (S): 9 (High; incorrect diagnosis could affect patient treatment)• Occurrence (O): 6 (Moderate; influenced by test sensitivity and specificity)• Detectability (D): 7 (Moderate; errors may only be detected through clinical validation)• RPN Calculation: 9 x 6 x 7 = 378• Risk Level: High• Mitigation Measures:1. **Quality Control and Manufacturing Standards**: Ensure that the devices are produced under strict quality control standards to minimize defects and ensure consistent performance.2. **Testing Protocols**: Implement standardized testing protocols that include clear instructions on how to properly collect and process saliva samples, reducing the chance of user error.3. * Confirmatory Testing**: Establish guidelines for follow-up confirmatory testing, such as scheduling an appointment with the doctor as soon as possible,for cases where a positive result is received, to verify the presence of the condition or disease.4. **Training and Education**: Provide a thorough mobile app to the patient with clear, detailed instructions on the correct use and interpretation of saliva LFDs.5. **Regulatory Approvals**: Ensure that the device meets all necessary regulatory approvals and standards, indicating that it has been validated for accuracy and reliability.6. Providing extra tests if the first one has a manufacturing issue and encouraging follow-up testing.

[0263] Risk Case 4: Test administered to patient with moderate or severe TBI• Description: When administering a saliva lateral flow device (LFD) designed for detecting mild concussions, there is a risk associated with testing patients who have moderate or severe concussions. This can present several challenges and potential issues: Inappropriate test application, resource misallocation, patient safety concerns.• Severity (S): ): 8 (High; leads to false positives)• Occurrence (O): 5 (Low; can be addressed in assay design)• Detectability (D): 6 (Moderate; detected in validation studies)• RPN Calculation: 8 x 5 x 6 = 240• Risk Level: High• Mitigation Measures: To address this risk, protocols should ensure that the saliva LFD is only administered under appropriate indications, and complementary or alternative diagnostic methods are used for cases suspected to be moderate or severe. Moreover, inform patients and their caregivers about the purpose and limitations of the test, ensuring they understand that additional evaluation may be necessary. Develop a feedback loop where outcomes of the testing are reviewed to improve protocols and diagnostic accuracy, allowing for continuous improvement based on real-world use.

[0264] Risk Case 5; Control line is not present• Description: The absence of a control line typically indicates a problem with the test (manufacturing issue). A control line is a fundamental aspect of such devices as it verifies that the test is functioning properly and that the sample has been correctly introduced. In this risk case, the test is rendered invalid due to the absence of the control line, leading tothe potential for: invalid test results, misinterpretation and delayed diagnosis, increased anxiety and confusion, resource wastage.• Severity (S): 6 (Moderate; misinterpretation affects decision-making)• Occurrence (O): 5 (Low; can be minimized with clear indicators)• Detectability (D): 6 (Moderate; depends on visibility of results)• RPN Calculation: 6 x 5 x 6 = 180• Risk Level: Medium• Mitigation Measures: To address these issues, the proper use instructions of the device should emphasize checking for the presence of the control line and actions to undertake if it is absent. Responding swiftly to this issue by performing a repeat test can help to ensure accurate diagnosis and patient care.

[0265] Risk Case 6: Device is exposed to precipitation prior to or during use• Description: Exposing the device to moisture or precipitation, such as humidity, poses several risks: Compromised Test Accuracy: Moisture may alter the reagents or the flow of the test sample across the device, leading to unreliable results either through faint, false, or missing lines. Invalid Test Results: Incursion of buffer can disrupt the test’s chemistry, possibly resulting in no visible control or test lines, indicating a malfunction of the device. Sample Contamination: precipitation can dilute the saliva sample or introduce contaminants, affecting the test's specificity and sensitivity, and thus compromising its ability to correctly diagnose a concussion. Potential for Misdiagnosis: If results are accepted from a compromised device, there may be serious consequences such as misdiagnosis or delayed treatment.• Severity (S): 8 (High; affects accuracy of results)• Occurrence (O): 5 (Low; controlled through storage guidelines)• Detectability (D): 7 (Moderate; detected upon performance testing)• RPN Calculation: 8 x 5 x 7 = 280• Risk Level: High• Mitigation Measures: To mitigate these risks, it is crucial to ensure that instructions for the LFD emphasize keeping the device dry at all times until use, and suggest conducting it in a dry environment. Prompt retesting in optimal conditions should be the standard procedure if the device has been exposed to precipitation.

[0266] Risk Case 7; Positive line is faint or not fully present• Description: A faint or incomplete positive line poses specific risks that can affect result interpretation: Result Ambiguity: A faint or incomplete positive line can create uncertainty about whether the test result is indeed positive or negative, leading to ambiguous interpretations. Potential for False Negatives: Misinterpreting a faint positive line as negative may result in failure to identify a concussion, missing vital opportunities for early intervention and treatment. User Misinterpretation: Users, especially those without medical training, may struggle to accurately interpret faint lines, leading to varied and unreliable test outcomes. Need for Repeated Testing: Ambiguity resulting from a faint line may necessitate retesting, using up resources and possibly delaying diagnosis and care. Consequences for Clinical Decision-Making: The clarity of test results directly impacts healthcare decisions. Inaccurate interpretation may affect treatment strategies and patient management.• Severity (S): 6 (Moderate; misinterpretation affects decision-making)• Occurrence (O): 5 (Low; can be minimized with clear indicators)• Detectability (D): 6 (Moderate; depends on visibility of results)• RPN Calculation: 6 x 5 x 6 = 180• Risk Level: Medium• Mitigation Measures: To mitigate these risks, manufacturers should provide clear guidelines on interpreting faint lines, advising on test repetition when results are unclear, and emphasizing proper test protocol execution, including handling, timing, and lighting conditions. Additionally, there should be clear instructions for users to seek medical advice if test results are ambiguous, ensuring proper evaluation and care. Providing extra tests if the first one has a manufacturing issue and encouraging follow-up testing.

[0267] Risk Case 8: Patient’s saliva contains blood or other contaminant(s)• Description: The presence of blood or other contaminants in the saliva sample presents distinct risks: Interference with Test Accuracy: Blood or other contaminants can interfere with the reagents and detection processes of the LFD, potentially leading to inaccurate results. False Positives or Negatives: Contaminants may cause misleading test outcomes, such as false positives, where a concussion is incorrectly indicated, or false negatives, where a concussion is missed. Difficulty in Visual Interpretation: Contamination may alter the appearance of test lines, making them difficult to interpret or leading to misinterpretation by users. Compromised Clinical Decisions: Inaccurate results due to contamination can affect clinical decisions regarding the diagnosis andmanagement of a concussion, possibly putting the patient at risk if necessary interventions are delayed.• Severity (S): 7 (Moderate; could lead to inaccurate results)• Occurrence (O): 6 (Moderate; variable based on user)• Detectability (D): 7 (Moderate; may only be noticed in QC checks)• RPN Calculation: 7 x 6 x 7 = 294• Risk Level: High• Mitigation Measures: To address these risks, it is important to ensure: Proper Sample Collection Instruction: Users should receive detailed guidance on avoiding contamination during sample collection. Guidelines for Sample Quality: Instructions should specify how to verify that samples are free of blood or visible contaminants before use. Contingency Procedures: If contamination is suspected, users should be advised on steps to take, such as obtaining a new sample or seeking medical review. Rinsing their mouth with water before collecting the saliva sample will help to reduce this risk.

[0268] Risk Case 9: Are risks different based upon age group (explore all)• Description: When utilizing a saliva lateral flow device for diagnosing mild concussions, risks may vary across different age groups due to factors like physiological differences and variations in usage ability. Here's an overview:1. **Pediatrics (Children)**:• **Sample Collection Challenges**: Younger children may have difficulty providing an adequate saliva sample, affecting test accuracy.• **Inability to Follow Instructions**: Children might not follow sampling instructions carefully, leading to sample contamination or insufficient sample volume.• **Higher Parental / Carer Involvement**: Testing often requires supervision and assistance from adults, potentially leading to operator error if instructions are unclear.2. **Adults**:• **Consistent Ability to Provide Samples**: Usually better equipped to provide adequate saliva samples following instructions, leading to more reliable results.• **Risk Awareness and Compliance**: Adults often have higher awareness of testing importance and hence comply better with protocols.3. **Elderly**:• **Dry Mouth or Medication Effects**: Common conditions or medications in older adults can lead to dry mouth, impacting the ability to provide a sufficient sample.• **Cognitive Decline**: Seniors with cognitive impairments may struggle with test instruction comprehension and adherence, potentially compromising test results.• **Pre-existing Conditions**: Conditions common in older age might affect test validity or require adjustments in interpretation.• Severity (S): 9 (High; incorrect diagnosis could affect patient treatment)• Occurrence (O): 6 (Moderate; influenced by test sensitivity and specificity)• Detectability (D): ): 7 (Moderate; errors may only be detected through clinical validation)• RPN Calculation: 9 x 6 x 7 = 378• Risk Level: High• Mitigation Measures: Mitigating Risks Across Age Groups:1. Tailored Instructions: Provide age- appropriate and clear instructions via our mobile app, with visual aids for children and simplified guidelines for the elderly.2. Parental / Carer Guidance for Children and Seniors: Ensure caregivers understand the process to assist those incapable of independently completing the test.3. Consideration of Age-Specific Physiological Factors: Develop guidelines recognizing variable saliva composition or health conditions across age groups that could influence results.4. Clear Usage Protocols: Emphasize the importance of following testing protocols strictly across all age groups to ensure consistent reliability and accuracy of results.

[0269] Risk Case 10: Device is exposed to extreme heat or cold prior to or during use• Description: The effectiveness and accuracy of a saliva lateral flow device can be compromised if the device is exposed to extreme temperatures.1. Extreme Heat Exposure will lead to the following: a. Degradation of Reagents: High temperatures may cause the chemical reagents in the test to degrade or alter, potentially leading to false negatives or positives. b. Adhesive Failures: Heat could weaken any adhesives used in the device, leading to leakage or malfunction during the test. c. Test Component Damage: Plastic or other materials that make up the device might warp or become misshapen, affecting its functionality.2. Extreme Cold Exposure will lead to the following: a. Reagent Freezing: Certain reagents might freeze at low temperatures, which could impact the chemical reactions required for an accurate result. b. Delayed Reaction Times: Cold conditions can slow down chemical processes, causing extended wait times or unreliable readings. c. Brittle Materials: Some parts of the device might become brittle in cold temperatures, increasing the risk of physical damage.• Severity (S): 7 (Moderate; may cause false results)• Occurrence (O): 6 (Moderate; depends on storage conditions)• Detectability (D): 6 (Moderate; can be identified in stability testing)• RPN Calculation: 7 x 6 x 6 = 252• Risk Level: High• Mitigation Measures:1. Storage Guidelines: Clearly indicate the appropriate storage temperatures on packaging and in instructions to avoid exposure to extreme conditions.2. User Instructions: Provide guidance to users on acclimatizing the device to room temperature before use if it has been exposed to extreme temperatures.3. Temperature Monitoring: Consider incorporating temperature indicators on device packaging to alert users if the product may have exceeded safe temperature thresholds during storage or transport.

[0270] Risk Case 11: Delays in Manufacturing and Supply Chain Issues• Description: Disruptions in reagent supply or production could lead to test shortages.• Severity (S): 8 (High; affects availability of tests)• Occurrence (O): 5 (Moderate; depends on supply chain stability)• Detectability (D): 6 (Moderate; may only be noticed in production)• RPN Calculation: 8 x 5 x 6 = 240• Risk Level: High• Mitigation Measures: Establish multiple supplier agreements, maintain stock of critical reagents, implement contingency plans.

[0271] Risk Case 12; User Error in Sample Collection• Description: Users may collect saliva improperly, affecting test accuracy.• Severity (S): 6 (Moderate; incorrect results could lead to retesting)• Occurrence (O): 7 (High; varies by user proficiency)• Detectability (D): 5 (Moderate; may not be detected until analysis)• RPN Calculation: 6 x 7 x 5 = 210• Risk Level: Medium• Mitigation Measures: Provide clear instructions, develop easy-to-use collection devices, conduct usability testing.

[0272] Risk Case 13: Environmental Sensitivity of the Test• Description: Humidity, temperature, or light exposure may impact test performance.• Severity (S): 7 (Moderate; may cause false results)• Occurrence (O): 6 (Moderate; depends on storage conditions)• Detectability (D): 6 (Moderate; can be identified in stability testing)• RPN Calculation: 7 x 6 x 6 = 252• Risk Level: High• Mitigation Measures: Develop robust packaging, test environmental stability, include storage guidelines.

[0273] Risk Case 14: Cross-Reactivity with Other Biomarkers• Description: The test may detect non-target biomarkers, reducing specificity.• Severity (S): 8 (High; leads to false positives)• Occurrence (O): 5 (Low; can be addressed in assay design)• Detectability (D): 6 (Moderate; detected in validation studies)• RPN Calculation: 8 x 5 x 6 = 240Risk Level: High• Mitigation Measures: Improve antibody specificity, test cross-reactivity during validation.

[0274] Risk Case 15: Difficulty in Interpretation of Results• Description: Users may misread faint test lines, leading to confusion.• Severity (S): 6 (Moderate; misinterpretation affects decision-making)• Occurrence (O): 5 (Low; can be minimized with clear indicators)• Detectability (D): 6 (Moderate; depends on visibility of results)• RPN Calculation: 6 x 5 x 6 = 180• Risk Level: Medium• Mitigation Measures: Implement digital reader option, enhance color contrast of test lines, provide clear visual instructions.

[0275] Risk Case 16: Interference from Saliva Components• Description: Substances in saliva (food particles, blood, medications) may interfere with test performance.• Severity (S): 7 (Moderate; could lead to inaccurate results)• Occurrence (O): 6 (Moderate; variable based on user)• Detectability (D): 7 (Moderate; may only be noticed in QC checks)• RPN Calculation: 7 x 6 x 7 = 294• Risk Level: High• Mitigation Measures: Include filtration step in sample collection, instruct users to rinse mouth before testing.

[0276] Risk Case 17: Degradation of Biomarkers in Storage• Description: The target biomarker may degrade due to improper storage, leading to test inaccuracies.• Severity (S): 8 (High; affects accuracy of results)• Occurrence (O): 5 (Low; controlled through storage guidelines)• Detectability (D): 7 (Moderate; detected upon performance testing)• RPN Calculation: 8 x 5 x 7 = 280• Risk Level: High• Mitigation Measures: Establish strict temperature controls, use stabilizing agents, and perform periodic quality checks.

[0277] Risk Case 18: Insufficient Sample Volume for Detection• Description: Inadequate saliva sample may lead to inaccurate or invalid results.• Severity (S): 7 (Moderate; test may fail, requiring retesting)• Occurrence (O): 5 (Low; mitigated by clear user instructions)• Detectability (D): 6 (Moderate; detected through invalid test warnings)• RPN Calculation: 7 x 5 x 6 = 210• Risk Level: Medium• Mitigation Measures: Design collection buffer to stabilize low-volume samples, add fail-safe indicators, and provide user training.

[0278] Risk Case 20; Inaccurate Test Results (False Positives / Negatives)• Description: The LFA may yield false positive or false negative results, leading to misdiagnosis and improper medical decisions.• Severity (S): 9 (High; incorrect diagnosis could affect patient treatment)• Occurrence (O): 6 (Moderate; influenced by test sensitivity and specificity)• Detectability (D): 7 (Moderate; errors may only be detected through clinical validation)• RPN Calculation: 9 x 6 x 7 = 378• Risk Level: High• Mitigation Measures: Optimize antibody specificity, conduct rigorous clinical validation, and implement confirmatory testing recommendations.

[0279] Example 5. Step-by-Step Instructions for Using the Concussion Test

[0280] This examples provides a method of use of the Conan MedTech Real-Time Saliva SIOOB concussion detection device for the evaluation of mild traumatic brain injury, also known as concussion.

[0281] Step 1: Setup the Device and Mobile Application. Unbox the Kit: The kit contains two units of the test strips, each in a separate sterile foil pouch. Download the Mobile App: Scan the barcode on the kit using the phone camera to download the Conan MedTech app from the Apple or Google Play store. First-Time Setup: Open the app and create an account by entering basic information such as your name, age, and any relevant medical history. If the user is underage, they will be prompted to have a caregiver or administrator assist you with the setup.

[0282] Step 2: Collect the Saliva Sample. Peel and Open the Foil Pouch: Remove the test device from the foil pouch and uncover the flexible cap, place the round cotton tip inside themouth. Collect the Saliva: The device has a sample collector area (an round absorbent cotton pad) at the top. Place this area in mouth and suck at the tip to collect the saliva until the color on the indicator changes to red, which shows enough saliva has been collected. Next put the flexible cap on and gently squeeze the round absorbent cotton pad , this pushes the clear saliva through the tube towards the buffer solution. Saliva Mixed with Buffer Solution: The saliva-buffer solution will flow down the test strip via capillary action to the conjugate pad, where antibodies against the SIOOB protein will detect levels of the biomarker.

[0283] Step 3: Test Results. Wait for the Results: Once the saliva sample has moved through the test strip, wait for 5-10 minutes for the results to be displayed on the test which appear as colored lines. Positive Result: If a line appears in both the control and test lines, the result indicates a suspected concussion (mTBI). Negative Result: If only the control line appears, the result is considered normal, indicating no concussion.

[0284] Step 4: Capture Test Results via the Mobile App. Scan the Test Strip: To document your results, it is recommended that you use the Conan mobile app within 5-10 minutes after completing the RTCD test. Open the app and capture a clear image of the test strip, Ensure the red lines on the test strip are centered, clearly visible, sharply focused, and captured in a well-lit environment free of shadows or glare. If the app does not recognize the test results, it will prompt you to retake the picture.

[0285] Step 5: View and Store Results. View Results: The mobile app will generate a result report based on the test. The app provides guidance for understanding the results, such as whether further medical attention is required. Save the Report: Test results are stored in the app's report section, where you can view previous results and track your concussion history. It is a HIPAA compliance device which ensures the privacy and security of your health information.

[0286] Step 6: Share Results. Email the Report: If you need to share the test results with a physician, the app allows you to email the report securely to your healthcare provider.

Claims

CLAIMSWhat is claimed is:

1. A system for diagnosing mild traumatic brain injury / concussion in a subject, the system comprising(a) a test device having a sample collector configured to receive a biological sample from the subject, and a reaction stage configured to receive the sample from the sample collector, the stage comprising a plurality of reaction zones where the sample is contacted with a detection reagent sensitive to a biomarker for traumatic brain injury to form a biomarker-detection reagent complex; and(b) a capture reagent conjugated to a visualization reagent in each reaction zone that generates a visible change when the capture reagent binds to the biomarker-detection reagent complex.

2. The system of claim 1, wherein the plurality of reaction zones have different sensitivities to the biomarker and thereby provide a plurality of output signals that together can be interpreted to differentiate whether the subject has a) no traumatic brain injury, or b) mild traumatic brain injury; or can be interpreted to differentiate whether the subject has a) no traumatic brain injury, b) mild traumatic brain injury, or c) moderate to severe traumatic brain injury.

3. The system of claim 1, further comprising a mobile device-based application that is configured to provide diagnostic information based on the visible change generated by the reagents in the plurality of reaction zones.

4. The system of claim 1, wherein the biological sample is a body fluid.

5. The system of claim 1, wherein the biological sample is a saliva sample.

6. The system of claim 1, wherein the biological sample is a serum, sweat, urine, breath, chloride, sodium, lactate, glucose, cortisol, urea, and various proteins and peptides, plasma, or blood sample.

7. The system of claim 1, wherein the biomarker for mild traumatic brain injury is a protein biomarker, a DNA biomarker, an RNA biomarker, a miRNA marker, nitric oxide(NO), or fractional exhaled nitric oxide (FeNO).

8. The system of claim 7, wherein the protein biomarker is SI 00 Calcium Binding Protein B (SIOOB).

9. The system of claim 1, wherein the detection reagent and / or the capture reagent is an antibody, or antigen-binding fragment thereof, that selectively binds to the biomarker.

10. The system of claim 9, wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL),(i) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:5, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO: 10;(ii) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO: 15, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:20;(iii) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:25, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:30; and(iv) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:35, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:40.

11. The system of claim 10, wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL),(i) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:2, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:3, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:6, a VL CDR2 domain comprising the amino acid sequence of YAS, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:8;(ii) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO: 11, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO: 12, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO: 13, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 domain comprising the amino acid sequence of YTS, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO: 18;(iii) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO:21, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:22, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:23, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:26, a VL CDR2 domain comprising the amino acid sequence of YAS, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:28; or(iv) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO:31, a VH CDR2 domain comprising the amino acid sequence ofSEQ ID NO:32, and a VH CDR3 domain comprising the amino acid sequence of SEQ IDNO:33, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:36, a VL CDR2 domain comprising the amino acid sequence of YAS, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:38.

12. The system of claim 1, wherein the visualization reagent is a combination of a peroxidase enzyme and a chromogenic substrate for the enzyme.

13. The system of claim 1, wherein the sample collector and the plurality of reaction zones are linked to each other such that capillary flow draws the sample from the sample collector into the plurality of reaction zones.

14. The system of claim 13, wherein the test device is a lateral flow device.

15. The system of claim 14, wherein the lateral flow device is a disposable single use test device.

16. The system of claim 14, the lateral flow device further comprising: a. a housing; b. an absorption pad; c. a sample pad; d. a conjugate pad; e. a nitrocellulose membrane; f. at least one capillary tube; g. a sample collector cotton; h. an indicator; and i. a buffer chamber.

17. The system of claim 16, wherein the sample collector, the cotton, the indicator, the buffer chamber, the absorption pad; a sample pad; the conjugate pad, and the nitrocellulose membrane are linked to each other via the at least one capillary tube such thatcapillary flow draws the biological sample through each of the sample collector, the absorption pad; a sample pad; the conjugate pad, and the nitrocellulose membrane.

18. The system of claim 16, wherein the biological sample is deposited on the sample collector.

19. The system of claim 14, the lateral flow device further comprising a visualization window positioned on a top surface of the lateral flow device.

20. The system of claim 14, the lateral flow device further comprising a first signal, located on a side edge of the lateral flow device or the sample collector, the first signal activates or deactivates in response to a predetermined volume of the biological sample being collected.

21. The system of claim 14, the lateral flow device further comprising a collection buffer configured to stabilize low-volume fluid samples. .

22. The system of claim 14, the lateral flow device further comprising a second signal located on a side edge or a top surface of the lateral flow device, the second signal activates or deactivates in response to a predetermined volume of the biological sample being collected.

23. The system of claim 14, the lateral flow device further comprising a filter component configured to filter substances from the fluid sample..

24. The system of claim 13, wherein the sample collector and the plurality of reaction zones are configured as a microfluidic device.

25. The system of claim 24, wherein the microfluidic device is a re-useable test device.

26. The system of claim 24, the microfluidic device further comprising a. a sample collection opening;b. a capillary tube; c. a porous member located within the sample collection opening; and d. at least one chamber in fluid communication with the porous member via the capillary tube.

27. The system of claim 26, wherein the at least one chamber is a centrifuge chamber.

28. The system of claim 26, wherein the at least one chamber includes a first chamber, a second chamber, a third chamber, a fourth chamber, and a fifth chamber in fluid communication with each chamber.

29. The system of claim 24, the microfluidic device further comprising a first signal, located on a side edge of the microfluidic device, the first signal activates or deactivates in response to a predetermined volume of the biological sample being collected.

30. The system of claim 24, the microfluidic device further comprising: a collection buffer configured to stabilize low-volume fluid samples; and a filter component configured to filter substances from the fluid sample.

31. The system of claim 24, the microfluidic device further comprising a second signal located on a side edge or a top surface of the microfluidic device, the second signal activates or deactivates in response to a predetermined volume of the biological sample being collected.

32. The system of claim 14 or 24, the device further comprising an environmental indicator configured to monitor the light exposure, humidity, and / or temperature of the device..

33. A method for diagnosing mild traumatic brain injury in a subject, the method comprising: a. obtaining a biological sample from the subject;b. placing the sample in a sample collector that is configured to conduct the sample into a reaction stage configured, the reaction stage comprising a plurality of reaction zones, each reaction zone comprising a combination of a detection reagent sensitive to a biomarker for traumatic brain injury, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarker-detection reagent complex, and when the capture reagent binds to the biomarker-detection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones and then reading a set of visible signals from the plurality of reaction zones; and d. comparing the set of signals obtained with a reference to determine whether the subject has a) no traumatic brain injury, or b) mild traumatic brain injury.

34. A method for diagnosing traumatic brain injury in a subject, the method comprising: a. obtaining a biological sample from the subject; b. placing the sample in a sample collector that is configured to conduct the sample into a reaction stage configured, the reaction stage comprising a plurality of reaction zones, each reaction zone comprising a combination of a detection reagent sensitive to a biomarker for traumatic brain injury, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarker-detection reagent complex, and when the capture reagent binds to the biomarker-detection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones and then reading a set of visible signals from the plurality of reaction zones; and d. comparing the set of signals obtained with a reference to determine whether the subject has a) no traumatic brain injury, b) mild traumatic brain injury, or c) moderate to severe traumatic brain injury.

35. A method for detecting mild traumatic brain injury in a subject, comprising: a. obtaining a biological sample from the subject;b. placing the sample in a test device and allowing the sample to contact a plurality of reaction zones, each reaction zone containing a combination of a detection reagent sensitive to a biomarker for traumatic brain injury, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarker-detection reagent complex, and when the capture reagent binds to the biomarker-detection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones; d. reading a set of visible signals from the plurality of reaction zones; and e. determining based on the set of visible signals whether the subject has a) no traumatic brain injury, or b) mild traumatic brain injury.

36. A method for detecting traumatic brain injury in a subject, comprising: a. obtaining a biological sample from the subject; b. placing the sample in a test device and allowing the sample to contact a plurality of reaction zones, each reaction zone containing a combination of a detection reagent sensitive to a biomarker for traumatic brain injury, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarker-detection reagent complex, and when the capture reagent binds to the biomarker-detection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones; d. reading a set of visible signals from the plurality of reaction zones; and e. determining based on the set of visible signals whether the subject has a) no traumatic brain injury, b) mild traumatic brain injury, or c) moderate to severe traumatic brain injury.

37. A method for treating mild traumatic brain injury in a subject, comprising: a. obtaining a biological sample from the subject; b. placing the sample in a test device and allowing the sample to contact a plurality of reaction zones, each reaction zone containing a combination of a detection reagent sensitive to a biomarker for traumatic brain injury mild traumatic brain injury / concussion, and a capture reagent conjugated to a visualization reagent that generates avisible signal when the detection reagent binds to the biomarker to form a biomarkerdetection reagent complex, and when the capture reagent binds to the biomarker-detection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones; d. reading a set of visible signals from the plurality of reaction zones; e. determining based on the set of visible signals whether the subject has a) no traumatic brain injury, or b) mild traumatic brain injury; and f. administering an appropriate medical care for the subject determined to have mild traumatic brain injury.

38. A method for treating traumatic brain injury in a subject, comprising: a. obtaining a biological sample from the subject; b. placing the sample in a test device and allowing the sample to contact a plurality of reaction zones, each reaction zone containing a combination of a detection reagent sensitive to a biomarker for traumatic brain injury mild traumatic brain injury / concussion, and a capture reagent conjugated to a visualization reagent that generates a visible signal when the detection reagent binds to the biomarker to form a biomarkerdetection reagent complex, and when the capture reagent binds to the biomarker-detection reagent complex; c. waiting a predetermined interval for the sample to react with the reagents in the plurality of reaction zones; d. reading a set of visible signals from the plurality of reaction zones; e. determining based on the set of visible signals whether the subject has a) no traumatic brain injury, b) mild traumatic brain injury, or c) moderate to severe traumatic brain injury; and f. administering an appropriate medical care for the subject determined to have traumatic brain injury.

39. The method of any one of claims 33-38, wherein the biological sample is a body fluid.

40. The method of any one of claims 33-38, wherein the biological sample is a saliva sample.

41. The method of any one of claims 33-38, wherein the biological sample is a serum, plasma, or blood sample.

42. The method of any one of claims 33-38, wherein the biological sample is obtained within about 24 hours of injury suspected to be traumatic brain injury.

43. The method of any one of claims 33-38, wherein the biomarker for traumatic brain injury is a protein biomarker, a DNA biomarker, or an RNA biomarker.

44. The method of claim 43, wherein the protein biomarker is S100 Calcium Binding Protein B (S100B).

45. The method of any one of claims 33-38, wherein the detection reagent and / or the capture reagent is an antibody, or antigen-binding fragment thereof, that selectively binds to the biomarker.

46. The method of claim 45, wherein the antibody, or antigen -binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL),(i) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:5, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO: 10;(ii) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO: 15, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:20;(iii) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:25, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:30; and(iv) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:35, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:40.

47. The method of claim 45, wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL),(i) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:2, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:3, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:6, a VL CDR2 domain comprising the amino acid sequence of YAS, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:8;(ii) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO: 11, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO: 12, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO: 13, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 domain comprising the amino acid sequence of YTS, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO: 18;(iii) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO:21, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:22, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:23, andwherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:26, a VL CDR2 domain comprising the amino acid sequence of YAS, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:28; or(iv) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO:31, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:32, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:33, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:36, a VL CDR2 domain comprising the amino acid sequence of YAS, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:38.

48. The method of any one of claims 33-38, wherein the visualization reagent is a combination of a peroxidase enzyme and a chromogenic substrate for the enzyme.

49. The method of any one of claims 33-38, wherein the subject is a human subject.

50. The method of any one of claims 33-38 and 49, wherein the subject is about 5- 75 years of age.

51. The method of any one of claims 33-38, 49 and 50, wherein the subject has a Glasgow Coma Scale (GCS) of 13-15.

52. The method of any one of claims 33-38, and 49-51, wherein the subject does not have a history of or does not currently have a major psychiatric disorder selected from the group consisting of schizophrenia, active psychosis, and uncontrolled bipolar disorder.

53. The method of any one of claims 33-38, and 49-52, wherein the subject is 18 years of age or older, and has a body mass index of >17 or < 36.

54. The method of any one of claims 33-38, and 49-52, wherein the subject is 5-17 years of age, and has a body mass index in the 5th-85thpercentile.

55. An isolated antibody, or antigen -binding fragment thereof, that specifically binds to S100 Calcium Binding Protein B (SIOOB), wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL),(i) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:5, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO: 10;(ii) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO: 15, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:20;(iii) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:25, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:30; and(iv) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:35, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:40.

56. The isolated antibody, or antigen-binding fragment thereof, of claim 55, wherein the VH comprises a VH complementarity determining region (CDR) 1 domain comprising an amino acid sequence of SEQ ID NO:1, a VH CDR2 domain comprising anamino acid sequence of SEQ ID NO:2, and a VH CDR3 domain comprising an amino acid sequence of SEQ ID NO:3, and wherein the VL comprises a VL CDR1 domain comprising an amino acid sequence of SEQ ID NO:6, a VL CDR2 domain comprising an amino acid sequence of YAS, and a VL CDR3 domain comprising an amino acid sequence of SEQ ID NO:8.

57. The isolated antibody, or antigen-binding fragment thereof, of claim 55, wherein the VH comprises a VH CDR1 domain comprising an amino acid sequence of SEQ ID NO: 11, a VH CDR2 domain comprising an amino acid sequence of SEQ ID NO: 12, and a VH CDR3 domain comprising an amino acid sequence of SEQ ID NO: 13, and wherein the VL comprises a VL CDR1 domain comprising an amino acid sequence of SEQ ID NO: 16, a VL CDR2 domain comprising an amino acid sequence of YTS, and a VL CDR3 domain comprising an amino acid sequence of SEQ ID NO: 18.

58. The isolated antibody, or antigen-binding fragment thereof, of claim 55, wherein the VH comprises a VH CDR1 domain comprising an amino acid sequence of SEQ ID NO:21, a VH CDR2 domain comprising an amino acid sequence of SEQ ID NO:22, and a VH CDR3 domain comprising an amino acid sequence of SEQ ID NO:23, and wherein the VL comprises a VL CDR1 domain comprising an amino acid sequence of SEQ ID NO:26, a VL CDR2 domain comprising an amino acid sequence of YAS, and a VL CDR3 domain comprising an amino acid sequence of SEQ ID NO:28.

59. The isolated antibody, or antigen-binding fragment thereof, of claim 55, wherein the VH comprises a VH CDR1 domain comprising an amino acid sequence of SEQ ID NO:31, a VH CDR2 domain comprising an amino acid sequence of SEQ ID NO:32, and a VH CDR3 domain comprising an amino acid sequence of SEQ ID NO:33, and wherein the VL comprises a VL CDR1 domain comprising an amino acid sequence of SEQ ID NO:36, a VL CDR2 domain comprising an amino acid sequence of YAS, and a VL CDR3 domain comprising an amino acid sequence of SEQ ID NO:38.

60. An isolated antibody, or antigen-binding fragment thereof, that binds to S100 Calcium Binding Protein B (SIOOB), wherein said antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL),(i) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:2, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:3, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:6, a VL CDR2 domain comprising the amino acid sequence of YAS, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:8;(ii) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO: 11, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO: 12, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO: 13, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 domain comprising the amino acid sequence of YTS, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO: 18;(iii) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO:21, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:22, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:23, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:26, a VL CDR2 domain comprising the amino acid sequence of YAS, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:28; or(iv) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:32, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:33, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:36, a VL CDR2 domain comprising the amino acid sequence of YAS, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:38.

61. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 55, 56, and 60, wherein the VH comprises the amino acid sequence of SEQ ID NO:5, and wherein the VL comprises the amino acid sequence of SEQ ID NO: 10.

62. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 55, 57, or 60, wherein the VH comprises the amino acid sequence of SEQ ID NO: 15, and wherein the VL comprises the amino acid sequence of SEQ ID NO:20.

63. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 55, 58, or 60, wherein the VH comprises the amino acid sequence of SEQ ID NO:25, and wherein the VL comprises the amino acid sequence of SEQ ID NO:30.

64. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 55, 59, or 60, wherein the VH comprises the amino acid sequence of SEQ ID NO:35, and wherein the VL comprises the amino acid sequence of SEQ ID NO:40.

65. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 55-64, wherein the antibody, or antigen-binding fragment thereof, is a mouse, human, humanized, primatized, chimeric, bispecific, or multispecific antibody, or antigen-binding fragment thereof.

66. An isolated nucleic acid molecule encoding the VH of the antibody, or antigen-binding fragment thereof, of any one of claims 55-65; the VL of the antibody, or antigen-binding fragment thereof, of any one of claims 55-65, or the VH and the VL of the antibody, or antigen-binding fragment thereof, of any one of claims 55-65.

67. An isolated mRNA molecule encoding the VH of the antibody, or antigenbinding fragment thereof, of any one of claims 55-65; the VL of the antibody, or antigenbinding fragment thereof, of any one of claims 55-65, or the VH and the VL of the antibody, or antigen-binding fragment thereof, of any one of claims 55-65.

68. A vector comprising the isolated nucleic acid molecule of claim 66, or the isolated mRNA molecule of claim 67.

69. A cell expressing the vector of claim 68.

70. A composition comprising the antibody, or antigen-binding fragment thereof, of any one of claims 55-65, the isolated nucleic acid molecule of claim 66, the isolated mRNA molecule of claim 67, or the vector of claim 68.

71. A pharmaceutical composition comprising the antibody, or antigen-binding fragment thereof, of any one of claims 55-65, the isolated nucleic acid molecule of claim 66, the isolated mRNA molecule of claim 67, or the vector of claim 68, and a pharmaceutically acceptable carrier.

72. A kit comprising the antibody, or antigen-binding fragment thereof, of any one of claims 55-65, the isolated nucleic acid molecule of claim 66, the isolated mRNA molecule of claim 67, or the vector of claim 68, and instructions for use.

73. A method for diagnosing traumatic brain injury in a subject, the method comprising: a. obtaining a biological sample from the subject; b. contacting the sample with the antibody, or antigen-binding fragment thereof, of any one of claims 55-65, c. measuring the level of SIOOB; and d. determining based on the level of SIOOB whether the subject has traumatic brain injury.

74. The method of claim 73, wherein the traumatic brain injury is a mild traumatic brain injury, or a moderate to severe traumatic brain injury.

75. A method for detecting or measuring the level of SIOOB in a subject, comprising: a. obtaining a biological sample from the subject; and b. contacting the sample with the antibody, or antigen-binding fragment thereof, of any one of claims 55-65, thereby detecting or measuring the level of SIOOB in the subject.

76. The method of claim 73 or 75, wherein the biological sample is a body fluid.

77. The method of claim 73 or 75, wherein the biological sample is a saliva sample.

78. The method of claim 73 or 75, wherein the biological sample is a serum, plasma, or blood sample.

79. The method of claim 73 or 75, wherein the subject is a human subject.

80. A method of producing the antibody, or antigen-binding fragment thereof, of any one of claims 55-65, the method comprising expressing the antibody, or antigen-binding fragment thereof, in a recombinant cell, and isolating the antibody, or antigen-binding fragment thereof, from the cell.

81. The method of claim 80, further comprising preparing the antibody, or antigen-binding fragment thereof, isolated from the cell into a test device.

82. The method of claim 80, further comprising formulating the antibody, or antigen-binding fragment thereof, isolated from the cell into a pharmaceutical composition.

83. A method for the treatment of mild traumatic brain injury in a patient, the method comprising: obtaining a sample of saliva; placing the sample in a sample collector; providing mechanical pressure to move the saliva sample to a centrifugal chamber; activating the centrifugal chamber; providing an opening to a second chamber; providing mechanical pressure to move the saliva sample to the second chamber; auto-lasing the sample to separate portions containing RNA from the saliva sample; returning the portions containing RNA into the centrifugal chamber; activating the centrifugal chamber; providing an opening to a third chamber; providing mechanical pressure to move the portions containing RNA to the third chamber; isolating and extracting the RNA; returning the RNA to the centrifugal chamber; activating the centrifugal chamber; providing an opening to a fourth chamber; providing mechanical pressure to move the RNA to the fourth chamber; purifying the RNA;returning the purified RNA to the centrifugal chamber; providing mechanical pressure to move the saliva sample to a fifth chamber; conducting ncounter hybridization on the purified RNA; collecting data on the purified RNA; and displaying data.

84. A method for the treatment of mild traumatic brain injury in a patient, the method comprising: removing a cap from a lateral flow device; obtaining a sample of saliva; placing the sample in a sample collector until a sample volume indicator is activated; providing a capillary tube for the sample to travel through to combine with a buffer solution; flowing the combined sample and buffer solution to a test strip via capillary action; detecting levels of a biomarker; displaying reaction results in a test window formed in the lateral flow device.

85. A method for the treatment of mild traumatic brain injury in a patient, the method comprising: removing a cap from a lateral flow device to expose a sample collector area; inserting the sample collector area into a user’s mouth; obtaining a sample of saliva from the user’s mouth; placing the cap on the lateral flow device to enclose the sample collector area; applying pressure to the cap pushing saliva through a tube to combine with a buffer solution; flowing the combined sample and buffer solution to a test strip via capillary action; detecting levels of a biomarker; displaying reaction results in a test window formed in the lateral flow device; capturing an image of the lateral flow device on a mobile; and display a results report on a mobile device.

86. The system of claim 7, wherein the protein biomarker is selected from the group consisting of UCH-L1, GFAP & BDP, T- Tau & P-Tau, NSE, MAP2, MAP, SBP120, SBDP150, pNF-H, and Anti-GFAP AutoAb.

Images