WO2019169309A1 - Methods, apparatuses and kits for rapid testing of traumatic brain injuries - Google Patents

Abstract

The present disclosure relates to methods for diagnosing, assessing, and monitoring traumatic brain injuries, and devices (e.g., a test strip) for carrying out such methods.

Description

METHODS, APPARATUSES AND KITS FOR RAPID TESTING OF TRAUMATIC

BRAIN INJURIES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional

Application No. 62/637,477, filed on 2 March 2018, U.S. Provisional Application No. 62/645,288, filed on 20 March 2018, and U.S. Provisional Application No. 62/651,614, filed on 2 April 2018, each of which are herein incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

[0002] Concussions, mild traumatic brain injuries and other traumatic brain injuries affect approximately 3.8 million people in the United States alone (Rutland-Brown W, et al., (2006) J Head Trauma Rehabil, 2l(6):544-8). The current methods of detection for TBI include computerized tomography or magnetic resonance imagining scans of the brain, as well as cognitive evaluation by a trained neuropsychologist. There is need in the art for simple, definitive, and rapid detection of TBI, such as tests that can be administered non- invasively and not require processing in a laboratory.

[0003] Additionally, 15% of pediatric patients with concussion still show symptoms

3 months after the event (Eisenberg MA, et al. , (2013) Pediatrics, 132(8)8-17). It would be desirable to have a quick, low-cost, and non-invasive solution for detecting and monitoring TBI so that information could be used in the injury diagnosis and management of an individual’s ongoing treatment and lifestyle choices, as well as prognosticating and monitoring certain resulting short and long-term effects (cognitive, physiological and psychological) of TBI event and/or other non-TBI conditions that can affect cognitive functionality. Additionally, creating a simple way to compile test information and history from participating users in a database would increase the breadth and depth of knowledge relating to traumatic brain injuries as well as improve the accuracy of guidance regarding suggested medical attention, symptom prognostication and suggested lifestyle adjustments. SUMMARY OF THE INVENTION

[0004] The present invention aims to present new and definitive methods for the diagnosis of TBI, assessing injury severity and/or monitoring its progression or impact that is cheaper, faster and easier than current methods and less-invasive than traditional blood draws, along with a device for doing so. The present invention also provides an

accompanying database to compile such test result information in order to provide feedback to healthcare professionals and participating users regarding potential injury assessment, prognostication of symptoms and guidance for medical attention and lifestyle adjustments.

[0005] In one aspect of the invention, a method of diagnosing whether a subject is having or at risk of developing a traumatic brain injury (TBI) is provided, the method comprising the steps of: a) collecting a body fluid sample from the subject; b) detecting the presence of one or more biomarkers selected from the group consisting of S-100B, plasma- soluble prion protein, neutrophil gelatinase-associated lipocalin, occludin, C-C motif chemokine 11, phosphorylated heavy neurofilaments, neuron-specific enolase, glial fibrillary acidic protein, marinobufagenin, aII-spectrin N-terminal fragment, tumor necrosis factor receptor superfamily member 16, tau, A-tau, C-tau, and T-tau, and any fragment of at least five amino acids long of any thereof in the body fluid sample of the subject; and c) determining the subject as having or at risk of developing a TBI when the presence of one or more traumatic brain injury biomarkers is detected in the body fluid sample.

[0006] In another aspect of the invention, a method of assessing the severity of a traumatic brain injury in a subject is provided, the method comprising the steps of: a) collecting a body fluid sample from the subject; b) detecting the level of one or more biomarkers selected from the group consisting of S-100B, plasma-soluble prion protein, neutrophil gelatinase-associated lipocalin, occludin, C-C motif chemokine 11,

phosphorylated heavy neurofilaments, neuron-specific enolase, glial fibrillary acidic protein, marinobufagenin, aII-spectrin N-terminal fragment, tumor necrosis factor receptor superfamily member 16, tau, A-tau, C-tau, and T-tau, and any fragment of at least five amino acids long of any thereof in the body fluid sample of the subject; c) comparing the level of one or more traumatic brain injury biomarkers detected in the body fluid sample of the subject with the level of the corresponding biomarkers detected in a control sample or with a reference value; and d) determining the severity of the traumatic brain injury in the subject.

[0007] In another aspect, a method for monitoring the progression of a traumatic brain injury in a subject is provided, the method comprising: a) detecting at a first point in time the level of a biomarker in a body fluid sample from the subject, wherein the biomarker is one or more biomarkers selected from the group consisting of S-100B, plasma-soluble prion protein, neutrophil gelatinase-associated lipocalin, occludin, C-C motif chemokine 11, phosphorylated heavy neurofilaments, neuron-specific enolase, glial fibrillary acidic protein, marinobufagenin, aII-spectrin N-terminal fragment, tumor necrosis factor receptor superfamily member 16, tau, A-tau, C-tau, and T-tau, and any fragment of at least five amino acids long of any thereof; b) repeating step a) at a subsequent point in time; and c) comparing the level detected in steps a) and b), and therefrom monitoring the progression of the TBI in the subject. In some embodiments, the subject has undergone treatment between the first and the subsequence points in time.

[0008] Numerous embodiments are further provided that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in one embodiment, the one or more traumatic brain injury biomarkers comprise tumor necrosis factor receptor superfamily member 16. In another embodiment, the one or more traumatic brain injury biomarkers comprise neutrophil gelatinase- associated lipocalin. In still another embodiment, the one or more traumatic brain injury biomarkers comprise tumor necrosis factor receptor superfamily member 16 and neutrophil gelatinase-associated lipocalin.

[0009] In one embodiment, the presence and/or level of one or more biomarkers is detected by contacting the body fluid sample with an antibody against a biomarker and detecting the binding between the biomarker and the antibody. In another embodiment, the presence and/or level of one or more biomarkers is detected by Western-blot, ELISA (Enzyme-Linked Immunosorbent Assay), RIA (Radioimmunoassay), Competitive EIA (Competitive Enzyme Immunoassay), DAS-ELISA (Double Antibody Sandwich-ELISA), immunocytochemical or immunohistochemical techniques. In still another embodiment, the presence and/or level of one or more biomarkers is detected by exposing the body fluid sample to a test strip containing reactive agents against one or more biomarkers. In yet another embodiment, the presence and/or level of one or more traumatic brain injury biomarkers is detected by mass spectrometry, HPLC, or NMR. In another embodiment, the presence and/or level of one or more traumatic brain injury biomarkers is detected using more than one technique.

[0010] In any of the aforementioned embodiments, the body fluid sample is selected from the group consisting of blood, plasma, serum, urine, saliva, perspiration, and exhaled breath condensate.

[0011] In one embodiment, the method described herein further comprises storing the data of the subject into a database. In another embodiment, the data is collected with the mobile phone application described herein. In still another embodiment, the data in the database can be combined and analyzed to provide information which comprises injury screening, injury diagnosis, injury severity, prognostication of resulting symptoms, resulting physiological, psychological or cognitive impact, medical attention, activity abstinence, or suggested action or lifestyle adjustment.

[0012] In another aspect, a test strip for the detection of a traumatic brain injury in a subject is provided, the test strip comprising a substrate-based test strip containing one or more reactive agents against one or more biomarkers selected from the group consisting of S-100B, plasma-soluble prion protein, neutrophil gelatinase-associated lipocalin, occludin, C-C motif chemokine 11, phosphorylated heavy neurofilaments, neuron-specific enolase, glial fibrillary acidic protein, marinobufagenin, aII-spectrin N-terminal fragment, tumor necrosis factor receptor superfamily member 16, tau, A-tau, C-tau, and T-tau, and any fragment of at least five amino acids long of any thereof, and a means by which to detect the binding of said agents to said biomarkers.

[0013] In one embodiment, the one or more biomarkers comprise tumor necrosis factor receptor superfamily member 16. In another embodiment, the one or more traumatic brain injury biomarkers comprise neutrophil gelatinase-associated lipocalin. In still another embodiment, the one or more traumatic brain injury biomarkers comprise tumor necrosis factor receptor superfamily member 16 and neutrophil gelatinase-associated lipocalin. In yet another embodiment, the reactive agent is an antibody against the biomarker.

[0014] In one embodiment, the detection means further comprises a means to detect varying concentrations of said biomarkers. In another embodiment, the detection means comprises at least one human-discernible graphic or alphanumeric element. In still another embodiment, the detection means comprises at least one element that can be read or processed by machine. In yet another embodiment, the detection means or reactive agents are printed onto a transparent film, which is then impregnated onto the test strip.

[0015] In one embodiment, the test strip further comprises a transparent protective coating over the strip. In another embodiment, the transparent protective coating is latex. In still another embodiment, the test strip further comprises a non-absorbent protective layer covering at least a part of one or more surfaces of the strip. In yet another embodiment, the detection means of the test strip is visible through the non-absorbent protective layer. In another embodiment, the non-absorbent protective layer is a plastic casing. In still another embodiment, said plastic casing further comprises a window for the viewing of the detection means of the test strip.

[0016] In still another aspect, a kit is provided, the kit comprising: the test strip described herein; instructions for the use of said test strip; and a cup for the collection of a fluid sample. In one embodiment, the said test strip is contained in a disposable plastic wrapper.

[0017] In yet another aspect, a mobile phone application capable of reading the test strip described herein, which can record, interpret, and/or distribute the results of said test strip.

[0018] In any of the aforementioned embodiments, the TBI is a brain injury selected from the group consisting of concussions, contusions, coup-contrecoup injuries, diffuse axonal injuries, penetrating injuries, skull fractures, and scalp wounds. In some

embodiments, the TBI is a mild, a moderate, or a severe TBI. [0019] In one aspect, the method of the invention is the detection of certain proteins in a body fluid sample from a patient having recently suffered an injury to the central nervous system in order to confirm TBI. Such proteins are considered biomarkers for TBI. These biomarkers are selected from the group consisting of S-100B, plasma-soluble prion protein, neutrophil gelatinase-associated lipocalin, occludin, C-C motif chemokine 11, phosphorylated heavy neurofilaments, neuron-specific enolase, glial fibrillary acidic protein, marinobufagenin, aII-spectrin N-terminal fragment, tumor necrosis factor receptor superfamily member 16, tau, A-tau, C-tau, and T-tau, and any fragment of at least five amino acids long of any thereof. In a preferred embodiment, the body fluid sample is urine.

[0020] In another aspect, the device of the invention is considered to be a substrate- based test strip (paper or otherwise) containing antibodies against one or more protein or peptide fragments known to be biomarkers for cognitive impairment and/or TBI, as well as a method for the use of said test strip in the diagnosis of TBI, or the assessment of treatment and recovery from TBI. In a preferred embodiment, the test strip is tested with a urine sample or saliva from a patient who may have recently suffered an TBI.

[0021] In a preferred embodiment, the test strip of the instant invention is an immunoassay designed for home use as follows: a patient suspected of suffering from a recent TBI applies a fluid sample to one end of the test strip. The fluid sample is drawn through the test strip via capillary action into a zone containing an antibody against a TBI biomarker. As the fluid enters this zone, if the TBI biomarker of interest is present, it will bind to the antibody present. The fluid is then drawn further into the test strip into a second zone, which contains a secondary antibody which will react to the presence of the anti-TBI biomarker antibody by producing a colored band on the test strip. If the fluid sample contains a TBI biomarker, it will carry the anti-TBI biomarker antibody into the second zone for the above reaction to occur. In a preferred embodiment, the first zone of the test strip also contains a control antibody, such as Immunoglobin G (or IgG), and a third zone beyond the second zone which contains an antibody against IgG, which also produces a colored band on the test strip when this reaction occurs as a means of positive control for the test. [0022] In an embodiment, the reaction zones and the control and test zones are on opposite sides of the substrate, this reducing physical distance between zones for more rapid testing (FIG. 10).

[0023] In an embodiment, the test strip contains an absorbent pad at the end of the strip designed to collect the fluid sample.

[0024] In an embodiment, the test strip is coated with a transparent protective coating to prevent contact and/or contamination with the test area. In a further embodiment, the transparent protective coating comprises latex (FIG. 7).

[0025] In an embodiment, the test strip is housed in a plastic casing, which allows the unit to be handheld and protects the strip from environmental contaminants. In a further embodiment, the plastic casing further contains a window that allows the colored bands to be viewed. In a further embodiment, the plastic casing further comprises a plastic cap which fits over the end of the strip where the fluid sample is deposited, again to prevent contact during handling (FIG. 9).

[0026] In an embodiment, the test strip is packaged as part of a kit, which additionally contains instructions for use of the test strip, as well as a cup for the collection of a fluid sample. Such a kit would be manufactured for home use at low cost, with results that are easily discernable to an average person without the need for lab-based testing for TBI.

[0027] The test strip of the instant invention is printed with columns containing antigens or other reactive agents against a cognitive impairment and/or TBI biomarker (FIG. 1A) (Creran B. et al. , ACS Appl. Mater. Interfaces (2014) 6(22): 19525-19530). When the column is exposed to the selected target biomarker from a fluid sample deposit, it produces a change in color of the strip if the concentration of the biomarker reaches a certain threshold (FIG. 1C). This change in color may rapidly assess the presence or absence of a cognitive impairment and/or TBI biomarker in the fluid sample without the need for drawing blood. In a preferred embodiment, the fluid sample is urine. [0028] In a preferred embodiment, the test strips of the instant invention contain antigens for the testing of more than one biomarker with the same fluid sample (FIG. 11 A). The use of multiple biomarkers associated with TBI would increase the accuracy of the test by ruling out certain false positives; for example, NGAL is a biomarker for kidney disease but is also upregulated in cerebral injury (Kim HJ et al ., (2018) JCI Insight, 3(l):97l05).

[0029] In a further embodiment, the columns of the test strip are printed with a tiled gradient of thresholds necessary to cause a change in color (FIG. 2A). This gradient allows the user to assess the approximate levels of each tested biomarker, rather than merely confirming its presence or absence in the fluid sample. In a further embodiment, the test strip can be read optically by a computer for the assessment of changes in biomarker levels over time to diagnose treatment efficacy and recovery from TBI.

[0030] In an embodiment, the reagents of the test strip are printed onto a transparent film, prior to the imprinting onto the substrate (FIG. 8).

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 illustrates an example of a binary dipstick or test strip method. The test strips FIG. 1 (A) each contain a reactive agent to detect biomarker proteins above a certain threshold concentration. These strips may be superimposed with ink FIG. 1 (B) to make the strips visible to the patient prior to fluid sample deposit. After sample deposit, the color of each column changes FIG. 1 (C) if the threshold presence of a biomarker protein is detected by the reactive agent.

[0032] FIG. 2 illustrates an example of a multiple threshold concentration detection strip and resultant output. FIG. 2 (A) shows how reactive agents may be printed onto each strip in a gradient; each column (a, b, c, d) is a different reactive agent, and each row (1, 2,

3...9) detects a different threshold of protein concentration. These strips may be superimposed with ink FIG. 2 (B) to make the strips visible to the patient prior to fluid sample deposit. After sample deposit, the color of each column changes FIG. 2 (C) in a gradient manner, allowing assessment of the concentration of each protein biomarker for each column, based upon the varying threshold levels of each protein detected. [0033] FIG. 3 illustrates an example of how the reagent solutions and other features are deposited to make a test strip. FIG. 3 (A) shows how reagent solutions are deposited in continuous lines along a substrate to form three zones: reaction, test, and control, with at least one solution per zone. FIG. 3 (B) shows the printing of additional information to the substrate prior to die-cutting into individual test strips FIG. 3 (C). FIG. 3 (D) shows an example of a finished test strip, prior to sample being deposited. The completed reaction produces colored lines across the test strip, validating the presence of a biomarker and the correct functioning of the strip via the control zone FIG. 3 (E).

[0034] FIG. 4 illustrates an example of how multiple reagent solutions may be used in the test zone. FIG. 4 (A) shows how reagent solutions are deposited in continuous lines along a substrate to form three zones: reaction, test, and control, with at least one solution per zone. The use of multiple lines of reagent in the test zone may be utilized to provide either binary detection of the same protein in different concentration levels, binary detection of multiple different proteins, each in a specific concentration, or some combination thereof. FIG. 4 (B) shows the printing of additional information to the substrate prior to die-cutting into individual test strips FIG. 4 (C). FIG. 4 (D) shows an example of a finished test strip, prior to sample being deposited. The completed reaction produces colored lines across the test strip, validating the presence of a biomarker and the correct functioning of the strip via the control zone FIG. 4 (E). The colored strip 1 indicates the correct functionality of the control reaction, while the multiple colored bars at the bottom of the strip 1 confirm increasingly higher levels of a protein, or multiple different proteins of a particular concentration for the purpose of diagnosing TBI with reduced potential for a false negative result.

[0035] FIG. 5 illustrates an example of how many different type of information may be conferred on a single test strip, as printed in FIG. 4. In this example, the presence of the colored band 1 on the test strip indicates a successful control reaction; the presence of the colored strip 2 confirms the presence of one biomarker for TBI; the presence of the colored strip 4 confirms the presence of a second biomarker for TBI; and the presence of the colored strip 3 confirms the presence of a higher concentration of either of the two above biomarkers. This method produces additional diagnostic information to rule out false results.

[0036] FIG. 6 illustrates an example of a graded test strip to detect certain concentrations of a TBI biomarker for the purpose of diagnosing the severity of the TBI, or to monitor the progression of biomarker level during assessment, treatment, and recovery. FIG. 6 (A) shows how reagent solutions are deposited in continuous lines along a substrate to form three zones: reaction, test, and control, with at least one solution per zone. In the test zone, at least one line of reagent is utilized to detect a specific biomarker. The reagent produces a color change in response to the presence of the biomarker, with greater amount of biomarker producing a stronger color change. FIG. 6 (B) shows the printing of additional information to the substrate prior to die-cutting into individual test strips FIG. 6 (C). FIG. 6 (D) shows an example of a finished test strip, prior to sample being deposited. The completed reaction produces colored lines across the test strip, validating the presence and concentration of a biomarker and the correct functioning of the strip via the control zone FIG. 6 (E). In this example, the colored bar I confirms functionality of the control reaction. The presence of the colored bar II confirms the presence of a TBI biomarker, while the particular shade (#3) indicates the detected concentration. The presence of the colored band III confirms the presence of a second biomarker, while the particular shade (b) indicates the detected concentration. The colored bar III may also be used to re-confirm the biomarker from the colored band II, as above.

[0037] FIG. 7 illustrates an example of how the test strips of the invention may be coated in a transparent protective agent such as latex. After the substrate is imprinted with reagents and other additional information FIG. 7 (A), one end of the substrate is coated on at least one surface with a transparent protective agent such as latex, shown in light gray and in cross-section FIG. 7 (B). The substrate is then die-cut FIG. 7 (C) into individual test strips FIG. 7 (D). Results may appear as in FIG. 7 (E), with the colored circle in the middle confirming a successful control reaction, and one or more colored lines at the bottom confirming the presence of the TBI biomarker(s), and/or their concentrations.

[0038] FIG. 8 illustrates an example of how the reagents may be added to the test strip by imprinting onto a transparent film. Either before or after one reagent is deposited onto the substrate, along with any additional information added by ink-printing FIG. 8 (A), at least one reagent solution is deposited onto a plastic film, shown in light gray, which is then adhered to the substrate FIG. 8 (B). If the reagent is dry before contract with the substrate, the reagent will not absorb into the substrate. The resulting combination of film and substrate, shown in cross-section FIG. 8 (C), is the die-cut into individual test strips FIG. 8 (D)

[0039] FIG. 9 illustrates how the test strip of the invention may be further protected by a plastic cap to fit over the end of the test strip where the fluid sample is deposited, again to prevent contact during handling. After printing of reagents and other information, at least one end of the substrate is coated on at least one surface with a transparent material, preferably latex FIG. 9 (A). Plastic film, show in 1, is coated on one side with removable adhesive, shown in 2, and on the other side with a line of permanent adhesive, shown in brown FIG. 9 (B). The plastic film is then folded and adhered to the surface of the test strip ribbon, as shown in cross-section FIG. 9 (C). The substrate is then die-cut into individual test strips FIG. 9 (D). FIG. 9 (E) shows an example test strip prior to fluid deposit. FIG. 9 (F) shows how, after fluid sample is deposited on the test strip, the plastic film may be unfolded and extended around the end of the test strip and sealed, for the prevention of contact with the sample area during handling.

[0040] FIG. 10 illustrates an example of how the test strips of the invention may be assembled with the reaction zone on the opposite side of the strip as the control and test zones. The reagents of the reaction zone are deposited on the back side of the substrate FIG. 10 (A), while the reagents of the control zone and the test zone are deposited in lines on the front side of the substrate FIG. 10 (B). Either before or after reagents are deposited, additional information is printed onto the front side of the substrate FIG. 10 (C).

Transparent film, shown in light gray, is applied to the front side of the substrate FIG. 10 (D) to protect the integrity of the test and control zones from contamination, prior to die- cutting the substrate into individual test strips, with back side shown in FIG. 10 (E) and front side shown in FIG. 10 (F) and FIG. 10 (G), and cross-section shown in FIG. 10 (H). FIG. 10 (I) shows an example test strip prior to fluid sample deposit on the back side. FIG. 10 (J) shows an example test strip after fluid deposit, where the top line confirms functionality of the control reaction, and one or more lines below are used to diagnose TBI. [0041] FIG. 11 illustrates how reagent solutions may be printed onto the substrate in a tiled array. FIG. 11 (A) shows how reagent solutions are deposited in a line for the reaction and control zones, and an array for the test zone along the substrate. In the test zone, each“dot” of reagent solution in a given column will detect a higher concentration of a particular biomarker in the fluid sample. For example, dot A1 will only react to a very high biomarker concentration, and dot A5 will react to a lower concentration of the same biomarker. Each row of dots in the test zone will react to a different biomarker. After reagents are printed onto the substrate, the substrate is further ink-printed with additional information FIG. 11 (B). The substrate is then die-cut FIG. 11 (C) into individual test strips FIG. 11 (D). FIG. 11 (E) shows an example of a test’s results: the colored bar 1 on the left validates correct functioning of the control reaction, and the appearance of at least any one colored square in any column confirms the presence of the biomarker being tested in that row. The more colored squares in a column, the higher the detected concentration of that particular protein. This detection of multiple biomarkers at multiple levels would facilitate an understanding of the proteome of the fluid sample, allowing better

understanding of the TBI event, and allow for better management of recovery. A high result as shown in FIG. 11 (F) would confirm a high level of severity of TBI, while a low result as shown in FIG. 11 (G) may also confirm TBI, but a less severe incident.

[0042] FIG. 12 illustrates the method in which the use of multiple different biomarkers may be used to diagnose TBI with improved accuracy. These figures describe three groups of patients: A) patients with confirmed TBI, B) patients who sustained significant physical injury, but not diagnosed with TBI, and C) patients with no known injuries and no past diagnosis of TBI. FIG. 12 (A) compares four different biomarkers among the three populations of patients. In this example, detection of protein #3 confirms TBI. FIG. 12 (B) compares four different biomarkers among the three populations of patients, wherein the detection of all four biomarkers would confirm TBI with a statistical probability of 99.98%, and only a 0.02% chance of false positive. FIG. 12 (C) compares four different biomarkers among the three populations of patients, wherein all four biomarkers are also present at minimal levels in patient group C. In this example, the detection of all four biomarkers would confirm TBI with a statistical probability of 99.9725%, and only a 0.0275% chance of false positive. [0043] FIG. 13 is a table that shows the detection and protein identification probability via mass spectrometry for each sample.

[0044] FIG. 14 is a table that shows the detection and protein measurement (pg/ml) via ELISA for each sample.

[0045] FIG. 15 is a table that shows the sample number and protein detection by combining the mass spectrometry and ELISA data.

DETAILED DESCRIPTION OF THE INVENTION

[0046] Traumas to the central nervous system have been shown to increase levels of certain proteins or peptide fragments within blood serum, as a result of neuro-inflammation or disruption of the blood-brain barrier. As a result, these proteins may be used as biomarkers for cognitive impairment and inflammation within the brain. For example, high concentrations of S-100B protein are associated with inflammation within the brain (Korfias S, et al ., Curr. Medic. Chem. (2006), 13(30) 3719-3731, and Rodriguez R, et al ., Clin. Chim. Acta. (2012) 414:228-33). Neuro-specific enolase (NSE), glial fibrillary acidic protein (GFAP) and occludin (OCLN) are also examples of proteins whose upregulation and presence in blood serum are considered biomarkers for brain injury (Kawata K. et al., Neurosci Biobehav Rev. (2016), 68:460-473). Likewise, upregulating of neutrophil gelatinase-associated lipocalin (NGAL) has been seen in association with cerebral injury (Kim HJ et al., (2018) JCI Insight, 3(l):97l05) and tumor necrosis factor receptor superfamily member 16 (TNR16) is considered a biomarker of neuro-inflammation when associated with Schwann cell damage (Weiss T., et al., (2016) GLIA, 64(12):2133-2153 and US application 15/467,646). Detecting the presence of one or more of such trauma- related indicators of neuro-inflammation or blood-brain barrier disruption can be used to confirm TBI outside of other evaluation protocols. The use of such biomarkers in assessing TBI, as well as progress following treatment or rehabilitation, is an integral part of both diagnosis and ongoing treatment plans.

IB [0047] While traditional tests for such biomarkers involve the use of enzyme-linked immunosorbent assays (ELISA) or high-performance liquid chromatography (HPLC) on a patient’s blood sample, many of these biomarkers are also present in other fluids, including saliva (Salivary Biomarkers in Pediatric Traumatic Brain Injury, ClinicalTrials.gov, https://clinicaltrials.gov/ct2/show/NCT02609568 (last visited Feb 27, 2018), perspiration (Kawata K. et al ., Neurosci Biobehav Rev. (2016), 68:460-473), and urine (Rodriguez R, el al., Clin. Chim. Acta. (2012) 414:228-33). Biomarkers present in blood serum may be filtered out by the kidneys and then excreted with urine. While protein detection in urine and saliva is possible and has become increasingly accurate and sensitive, blood has historically been (and continues to be) considered by the medical community the body fluid used for absolute, confirmatory testing. However, greater accuracy in diagnostic methods using urine as a fluid sample for detecting the presence of a protein have made this a promising technique. Home pregnancy tests, which detect human chorionic gonadotropin in urine at concentrations as low as 10 mlU/ml, are touted as being highly accurate.

Significant technological advances continue to improve the cost, speed and accuracy of protein detection in other body fluids, like exhaled breath condensate, as well.

[0048] The present disclosure provides a simple and quick test to detect protein presence and protein level changes which would indicate, detect or confirm a TBI event, as well as monitor the resulting physiological impact on cognitive functioning over time.

[0049] The term“brain injury” refers to a condition that results in central nervous system damage, irrespective of its pathophysiological basis. Among the most frequent origins of a“brain injury” are stroke and traumatic brain injury (TBI). The term“brain injury” also refers to, e.g., subclinical brain injury, spinal cord injury, and anoxic-ischemic brain injury. In preferred embodiments, the brain injury described herein refers to a traumatic brain injury or TBI.

[0050] The term“TBI” or“traumatic brain injury” refers to a damage which directly or indirectly affects the normal functioning of the brain, skull, or scalp. In specific embodiments, TBI is caused by an external force that causes brain to move inside the skull or damages the skull, which in turn damages the brain. The injury may and may affect just one functional area of the brain, various areas, or all areas of the brain. The non-limiting examples of TBI include, for example, concussions, contusions, coup-contrecoup injuries, diffuse axonal injuries, penetrating injuries, skull fractures, scalp wounds, etc. The severity of brain damage can vary with the type of brain injury. For example, TBI can be a mild TBI, a moderate TBI, or a severe TBI. The methods and compositions of the present disclosure can be used for any brain injury. In preferred embodiments, the methods and compositions of the present disclosure are used for TBI.

[0051] As used herein, the term“biomarker” refers to a molecule that is associated either quantitatively or qualitatively with a biological change. Examples of biomarkers include polypeptides, proteins or fragments of a polypeptide or protein; and

polynucleotides, such as a gene product, RNA or RNA fragment; and other body metabolites. In certain embodiments, a“biomarker” means a compound that is differentially present (i.e., increased or decreased) in a biological sample from a subject or a group of subjects having a first phenotype (e.g., having a disease or condition) as compared to a biological sample from a subject or group of subjects having a second phenotype (e.g., not having the disease or condition or having a less severe version of the disease or condition). A biomarker may be differentially present at any level, but is generally present at a level that is increased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100%, by at least 110%, by at least 120%, by at least 130%, by at least 140%, by at least 150%, or more; or is generally present at a level that is decreased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by 100% (i.e., absent). A biomarker is preferably differentially present at a level that is statistically significant (e.g., a p-value less than 0.05 and/or a q-value of less than 0.10 as determined using, for example, either Welch's T-test or Wilcoxon's rank-sum Test).

[0052] A biological sample can be obtained from a subject by conventional techniques. For example, CSF can be obtained by lumbar puncture. Blood can be obtained by venipuncture, while plasma and serum can be obtained by fractionating whole blood according to known methods. Surgical techniques for obtaining solid tissue samples are well known in the art. For example, methods for obtaining a nervous system tissue sample are described in standard neuro-surgery texts such as Atlas of Neurosurgery: Basic

Approaches to Cranial and Vascular Procedures, by F. Meyer, Churchill Livingstone, 1999; Stereotactic and Image Directed Surgery of Brain Tumors, 1 st ed., by David G. T. Thomas, W B Saunders Co., 1993; and Cranial Microsurgery: Approaches and Techniques, by L. N. Sekhar and E. De Oliveira, lst ed., Thieme Medical Publishing, 1999. Methods for obtaining and analyzing brain tissue are also described in Belay et al, (2001) Arch. Neurol. 58: 1673-1678; and Seijo et al, (2000) J. Clin. Microbiol. 38: 3892-3895.

[0053] Any animal that expresses the neural proteins, such as for example, those listed herein, can be used as a subject from which a biological sample is obtained.

Preferably, the subject is a mammal, such as for example, a human, dog, cat, horse, cow, pig, sheep, goat, primate, rat, mouse and other vertebrates such as fish, birds and reptiles. More preferably, the subject is a human. Particularly preferred are subjects suspected of having or at risk for developing traumatic brain injuries, such as victims of brain injury caused by traumatic insults ( e.g ., gunshots wounds, automobile accidents, sports accidents, shaken baby syndrome).

[0054] The biomarkers of the invention can be detected in a sample by any means.

Methods for detecting the biomarkers are described in detail in the Examples which follow. For example, immunoassays, include but are not limited to competitive and non

competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays,

immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, fluorescent immunoassays and the like. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in

Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety).

[0055] The biomarkers of the present invention may also be detected by mass spectrometry, a method that employs a mass spectrometer to detect gas phase ions.

Examples of mass spectrometers are time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer, hybrids or combinations of the foregoing, and the like. In one embodiment, mass spectrometry can be combined with another appropriate method(s) as may be contemplated by one of ordinary skill in the art. In another embodiment, the mass spectrometric technique is multiple reaction monitoring (MRM) or quantitative MRM.

[0056] The biomarkers of the present invention may also be detected by means of an electrochemicaluminescent assay developed by Meso Scale Discovery (Gaithersrburg, MD). Electrochemiluminescence detection uses labels that emit light when

electrochemically stimulated. Background signals are minimal because the stimulation mechanism (electricity) is decoupled from the signal (light). Labels are stable, non radioactive and offer a choice of convenient coupling chemistries. They emit light at -620 nm, eliminating problems with color quenching. See U.S. Patents No. 7,497,997; No. 7,491,540; No. 7,288,410; No. 7,036,946; No. 7,052,861; No. 6,977,722; No. 6,919,173; No. 6,673,533; No. 6,413,783; No. 6,362,011; No. 6,319,670; No. 6,207,369; No.

6,140,045; No. 6,090,545; and No. 5,866,434. See also U.S. Patent Applications

Publication No. 2009/0170121; No. 2009/006339; No. 2009/0065357; No. 2006/0172340; No. 2006/0019319; No. 2005/0142033; No. 2005/0052646; No. 2004/0022677; No.

2003/0124572; No. 2003/0113713; No. 2003/0003460; No. 2002/0137234; No.

2002/0086335; and No. 2001/0021534.

[0057] The biomarkers of the present invention can be detected by other suitable methods, see. e.g., U.S. Patent Applications Publication No. 2016/0178643.

[0058] The biomarkers are differentially present in control sample (e.g., healthy or non-brain injury) sample and brain injury sample, and, therefore, are useful in aiding in the determination of brain injury status. In certain embodiments, the biomarkers are measured in a patient sample using the methods described herein and compared, for example, to predefined biomarker levels and correlated to brain injury status. In particular

embodiments, the measurement(s) may then be compared with a relevant diagnostic amount(s), cut-off(s), or multivariate model scores that distinguish a positive brain injury status from a negative brain injury status. The diagnostic amount(s) represents a measured amount of a biomarker(s) above which or below which a patient is classified as having a particular brain injury status. For example, if the biomarker(s) is/are up-regulated compared to normal during brain injury, then a measured amount(s) above the diagnostic cutoff(s) provides a diagnosis of brain injury. Alternatively, if the biomarker(s) is/are down- regulated during brain injury, then a measured amount(s) at or below the diagnostic cutoff(s) provides a diagnosis of non-brain injury. As is well understood in the art, by adjusting the particular diagnostic cut-off(s) used in an assay, one can increase sensitivity or specificity of the diagnostic assay depending on the preference of the diagnostician. In particular embodiments, the particular diagnostic cut-off can be determined, for example, by measuring the amount of biomarkers in a statistically significant number of samples from patients with the different brain injury statuses, and drawing the cut-off to suit the desired levels of specificity and sensitivity.

[0059] Indeed, as the skilled artisan will appreciate there are many ways to use the measurements of two or more biomarkers in order to improve the diagnostic question under investigation. In a quite simple, but nonetheless often effective approach, a positive result is assumed if a sample is positive for at least one of the markers investigated.

[0060] Furthermore, in certain embodiments, the values measured for markers of a biomarker panel are mathematically combined and the combined value is correlated to the underlying diagnostic question. Biomarker values may be combined by any appropriate state of the art mathematical method. Well-known mathematical methods for correlating a marker combination to a disease status employ methods like discriminant analysis (DA) (e.g., linear-, quadratic-, regularized-DA), Discriminant Functional Analysis (DFA), Kernel Methods (e.g., SVM), Multidimensional Scaling (MDS), Nonparametric Methods (e.g., k- Nearest-Neighbor Classifiers), PLS (Partial Least Squares), Tree-Based Methods (e.g., Logic Regression, CART, Random Forest Methods, Boosting/Bagging Methods),

Generalized Linear Models (e.g., Logistic Regression), Principal Components based Methods (e.g., SIMCA),

[0061] Generalized Additive Models, Fuzzy Logic based Methods, Neural

Networks and Genetic Algorithms based Methods. The skilled artisan will have no problem in selecting an appropriate method to evaluate a biomarker combination of the present invention. In one embodiment, the method used in a correlating a biomarker combination of the present invention, e.g. to diagnose brain injury, is selected from DA (e.g., Linear-, Quadratic-, Regularized Discriminant Analysis), DFA, Kernel Methods (e.g., SVM), MDS, Nonparametric Methods (e.g., k-Nearest-Neighbor Classifiers), PLS (Partial Least

Squares), Tree-Based Methods (e.g., Logic Regression, CART, Random Forest Methods, Boosting Methods), or Generalized Linear Models (e.g., Logistic Regression), and Principal Components Analysis. Details relating to these statistical methods are found in the following references: Ruczinski et al.,l2 J. OF COMPUTATIONAL AND GRAPHICAL STATISTICS 475-511 (2003); Friedman, J. FL, 84 J. OF THE AMERICAN

STATISTICAL ASSOCIATION 165-75 (1989); Hastie, Trevor, Tibshirani, Robert, Friedman, Jerome, The Elements of Statistical Learning, Springer Series in Statistics (2001); Breiman, L., Friedman, J. FL, Olshen, R. A., Stone, C. J. Classification and regression trees, California: Wadsworth (1984); Breiman, L., 45 MACHINE LEARNING 5-32 (2001); Pepe, M. S., The Statistical Evaluation of Medical Tests for Classification and Prediction, Oxford Statistical Science Series, 28 (2003); and Duda, R. O., Hart, P. E., Stork, D. G., Pattern Classification, Wiley Interscience, 2nd Edition (2001).

[0062] In a specific embodiment, the present invention provides methods for determining the risk of developing brain injury (e.g., TBI) in a patient. Biomarker percentages, amounts or patterns are characteristic of various risk states, e.g., high, medium or low. The risk of developing brain injury is determined by measuring the relevant biomarkers and then either submitting them to a classification algorithm or comparing them with a reference amount, i.e., a predefined level or pattern of biomarkers that is associated with the particular risk level.

[0063] In another embodiment, the present invention provides methods for determining the severity of brain injury in a patient. Each grade or stage of brain injury likely has a characteristic level of a biomarker or relative levels of a set of biomarkers (a pattern). The severity of brain injury is determined by measuring the relevant biomarkers and then either submitting them to a classification algorithm or comparing them with a reference amount, i.e., a predefined level or pattern of biomarkers that is associated with the particular stage. [0064] In one embodiment, the present invention provides methods for determining the course of brain injury in a patient, brain injury course refers to changes in brain injury status over time, including brain injury progression (worsening) and brain injury regression (improvement). Over time, the amount or relative amount (e.g., the pattern) of the biomarkers changes. For example, biomarker "X" may be increased with brain injury (e.g., TBI), while biomarker "Y" may be decreased with brain injury (e.g., TBI). Therefore, the trend of these biomarkers, either increased or decreased over time toward brain injury or non-brain injury indicates the course of the condition. Accordingly, this method involves measuring the level of one or more biomarkers in a patient at least two different time points, e.g., a first time and a second time, and comparing the change, if any. The course of brain injury is determined based on these comparisons.

[0065] In certain embodiments of the methods of qualifying brain injury status, the methods further comprise managing patient treatment based on the status. Such

management includes the actions of the physician or clinician subsequent to determining brain injury status. For example, if a physician makes a diagnosis of brain injury, then a certain regime of monitoring would follow. An assessment of the course of brain injury using the methods of the present invention may then require a certain brain injury therapy regimen. Alternatively, a diagnosis of non-brain injury might be followed with further testing to determine a specific disease that the patient might be suffering from. Also, further tests may be called for if the diagnostic test gives an inconclusive result on brain injury status.

[0066] In another embodiment, the present invention provides methods for determining the therapeutic efficacy of a pharmaceutical drug. These methods are useful in performing clinical trials of the drug, as well as monitoring the progress of a patient on the drug.

[0067] Therapy or clinical trials involve administering the drug in a particular regimen. The regimen may involve a single dose of the drug or multiple doses of the drug over time. The doctor or clinical researcher monitors the effect of the drug on the patient or subject over the course of administration. If the drug has a pharmacological impact on the condition, the amounts or relative amounts (e.g., the pattern or profile) of one or more of the biomarkers of the present invention may change toward a non-brain injury profile. Therefore, one can follow the course of one or more biomarkers in the patient during the course of treatment. Accordingly, this method involves measuring one or more biomarkers in a patient receiving drug therapy, and correlating the biomarker levels with the brain injury status of the patient (e.g., by comparison to predefined levels of the biomarkers that correspond to different brain injury statuses). One embodiment of this method involves determining the levels of one or more biomarkers at at least two different time points during a course of drug therapy, e.g., a first time and a second time, and comparing the change in levels of the biomarkers, if any. For example, the levels of one or more biomarkers can be measured before and after drug administration or at two different time points during drug administration. The effect of therapy is determined based on these comparisons. If a treatment is effective, then the one or more biomarkers will trend toward normal, while if treatment is ineffective, the one or more biomarkers will trend toward brain injury indications.

[0068] In another aspect, the present invention provides kits for qualifying brain injury status, which kits are used to detect the biomarkers described herein. In a specific embodiment, the kit is provided as an ELISA kit comprising antibodies to the biomarkers of the present invention including, but not limited to, S-100B, plasma-soluble prion protein, neutrophil gelatinase-associated lipocalin, occludin, C-C motif chemokine 11,

phosphorylated heavy neurofilaments, neuron-specific enolase, glial fibrillary acidic protein, marinobufagenin, aII-spectrin N-terminal fragment, tumor necrosis factor receptor superfamily member 16, tau, A-tau, C-tau, and T-tau, and any fragment of at least five amino acids long of any thereof.

[0069] The ELISA kit may comprise a solid support, such as a chip, microtiter plate

(e.g., a 96-well plate), bead, or resin having biomarker capture reagents attached thereon. The kit may further comprise a means for detecting the biomarkers, such as antibodies, and a secondary antibody-signal complex such as horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody and tetramethyl benzidine (TMB) as a substrate for HRP.

[0070] The kit for qualifying brain injury status may be provided as an immuno- chromatography strip comprising a membrane on which the antibodies are immobilized, and a means for detecting, e.g., gold particle bound antibodies, where the membrane, includes NC membrane and PVDF membrane. The kit may comprise a plastic plate on which a sample application pad, gold particle bound antibodies temporally immobilized on a glass fiber filter, a nitrocellulose membrane on which antibody bands and a secondary antibody band are immobilized and an absorbent pad are positioned in a serial manner, so as to keep continuous capillary flow of blood serum or urine.

[0071] In certain embodiments, a patient can be diagnosed by adding blood or urine from the patient to the kit and detecting the relevant biomarkers conjugated with antibodies, specifically, by a method which comprises the steps of: (i) collecting blood or urine from the patient; (ii) separating blood serum from the patient's blood if the blood sample is used; (iii) adding the blood serum or urine from patient to a diagnostic kit; and, (iv) detecting the biomarkers conjugated with antibodies. In this method, the antibodies are brought into contact with the patient's blood or urine. If the biomarkers are present in the sample, the antibodies will bind to the sample, or a portion thereof. In other kit and diagnostic embodiments, blood or urine need not be collected from the patient (i.e., it is already collected). Moreover, in other embodiments, the sample may comprise a tissue sample or a clinical sample.

[0072] The kit can also comprise a washing solution or instructions for making a washing solution, in which the combination of the capture reagents and the washing solution allows capture of the biomarkers on the solid support for subsequent detection by, e.g., antibodies or mass spectrometry. In a further embodiment, a kit can comprise instructions for suitable operational parameters in the form of a label or separate insert. For example, the instructions may inform a consumer about how to collect the sample, how to wash the probe or the particular biomarkers to be detected, etc. In yet another embodiment, the kit can comprise one or more containers with biomarker samples, to be used as standard(s) for calibration.

[0073] Various methodologies of the instant invention include a step that involves comparing a value, level, feature, characteristic, property, etc. to a“suitable control,” referred to interchangeably herein as an“appropriate control,” a“control sample,” a “reference” or simply a“control.” A“suitable control,”“appropriate control,”“control sample,”“reference” or a“control” is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. A“reference level” of a biomarker means a level of the biomarker that is indicative of a particular disease state, phenotype, or lack thereof, as well as combinations of disease states, phenotypes, or lack thereof. A“positive” reference level of a biomarker means a level that is indicative of a particular disease state or phenotype. A“negative” reference level of a biomarker means a level that is indicative of a lack of a particular disease state or phenotype. For example, a“brain injury-positive reference level” of a biomarker means a level of a biomarker that is indicative of a positive diagnosis of brain injury in a subject, and a“brain injury-negative reference level” of a biomarker means a level of a biomarker that is indicative of a negative diagnosis of brain injury in a subject. A“reference level” of a biomarker may be an absolute or relative amount or concentration of the biomarker, a presence or absence of the biomarker, a range of amount or concentration of the biomarker, a minimum and/or maximum amount or concentration of the biomarker, a mean amount or concentration of the biomarker, and/or a median amount or concentration of the biomarker; and, in addition,“reference levels” of combinations of biomarkers may also be ratios of absolute or relative amounts or concentrations of two or more biomarkers with respect to each other. Appropriate positive and negative reference levels of biomarkers for a particular disease state, phenotype, or lack thereof may be determined by measuring levels of desired biomarkers in one or more appropriate subjects, and such reference levels may be tailored to specific populations of subjects (e.g., a reference level may be age-matched so that comparisons may be made between biomarker levels in samples from subjects of a certain age and reference levels for a particular disease state, phenotype, or lack thereof in a certain age group). Such reference levels may also be tailored to specific techniques that are used to measure levels of biomarkers in biological samples (e.g., LC-MS, GC-MS, ELISA, PCR, etc.), where the levels of biomarkers may differ based on the specific technique that is used.

[0074] In one embodiment, a“suitable control” or“appropriate control” is a value, level, feature, characteristic, property, etc., determined in a cell, organ, or patient, e.g., a control or normal cell, organ, or patient, exhibiting, for example, normal traits. For example, the biomarkers of the present invention may be assayed for levels/ratios in a sample from an unaffected individual (UI) or a normal control individual (NC) (both terms are used interchangeably herein). For example, a“suitable control” or“appropriate control” can be a value, level, feature, characteristic, property, ratio, etc. determined prior to performing a therapy (e.g., brain injury treatment) on a patient or a value, level, feature, characteristic, property, ratio, etc. determined prior to injury (e.g., a baseline test). In yet another embodiment, a transcription rate, mRNA level, translation rate, protein level/ratio, biological activity, cellular characteristic or property, genotype, phenotype, etc., can be determined prior to, during, or after administering a therapy into a cell, organ, or patient. In a further embodiment, a“suitable control” or“appropriate control” is a predefined value, level, feature, characteristic, property, ratio, etc. A“suitable control” can be a profile or pattern of levels/ratios of one or more biomarkers of the present invention that correlates to brain injury, to which a patient sample can be compared. The patient sample can also be compared to a negative control, i.e., a profile that correlates to not having brain injury.

[0075] In one aspect, the test strip of the invention is intended for the rapid diagnosis of TBI by the detection of a known TBI biomarker in a fluid sample, preferably blood or urine. These biomarkers are typically associated neuro-inflammation or the disruption of the blood-brain barrier. The efficacy of the invention can be monitored by comparing fluid samples from the following three groups:

A) Patients with confirmed TBI

B) Patients who sustained significant physical injury, but not diagnosed with TBI

C) Patients with no known injuries and no past diagnosis of TBI

[0076] The comparison of biomarkers among these three groups can determine the statistical probability of TBI with sufficient (99%+) accuracy. For example (FIG. 12A), a biomarker that is present in the samples of group A, but not groups B or C, in a patient’s sample would confirm TBI. The use of multiple biomarkers may further increase the accuracy of diagnosis, as the probability of each biomarker being present in a TBI patient is multiplicative.

[0077] As further example (FIG. 12C), using the above groups, a sample taken from a patient in group A may produce 100% detection of four TBI biomarkers, whereas a sample taken from a patient in group B may produce 20% detection of biomarker #1, 10% detection of biomarker #2, 5% detection of biomarker #3, and 20% detection of biomarker #4. For the purposes of diagnosis, assume a sample taken from a patient in group C produces 5% detection of biomarker #1, 3% detection of biomarker #2, 1% detection of biomarker #3, and 5% detection of biomarker #4. This would give an indication of TBI with 99.97% accuracy, based upon the calculation of positive predicative value (Suojanen JN, (1999) N Engl J Med, 341 : 131).

[0078] Each test strip of the invention may be marked with multiple reagents in the test zone, allowing for the detection of multiple different proteins, or one protein in varying concentrations, or a combination thereof (FIG. 4). This use of more detailed information of the proteome in the fluid sample will reduce the potential of false readings. The test strip may also include a graded result, which measures the concentration of a TBI biomarker in the fluid sample for the purpose of diagnosing the severity of the TBI, or to monitor the progression of biomarker level during assessment, treatment, and recovery (FIG. 6 and FIG. 10). Higher concentrations of biomarker in the sample produce a different color.

EXEMPLIFICATION

[0079] This invention is further illustrated by the following examples, which should not be construed as limiting.

Example 1:

Background:

[0080] Neuro-inflammatory response proteins do not exist in elevated levels within the body (brain, blood stream, urine, saliva, exhaled breath condensate, cutaneous secretions etc.) without an underlying causal factor. There are neuro-inflammatory proteins whose elevated levels are seen as biomarkers of certain conditions within the body and/or have also been noted in association with other injuries and conditions. For example, NGAL is seen as a biomarker for kidney disease, but is also seen up-regulated in cerebral diseases (Kim et al. (2018) JCI Insight 3 : e97105).

Hypothesis:

[0081] In an otherwise healthy individual, the presence (or elevated level) of certain neuro-inflammatory protein(s) associated with cerebral dysfunction should be consistent with the expression of symptoms from mTBETB I/concussion. [0082] Additionally, in an otherwise healthy individual, the presence (or elevated level) of specific neuro-inflammatory protein(s) whose presence outside the brain would only result from a blood-brain barrier disruption should be consistent with that same individual exhibiting the symptoms of a traumatic brain injury (TBI or mTBI) and, thus, be a biomarker for concussion assessment and/or diagnosis. For example, it is known that Schwann cells play a crucial role in nerve repair and regeneration (Kim et al. (2017) Stem Cell Reports 8: 1714-1726). Particularly, Schwann cells are critical to blood brain barrier repair by remyelinating lesions (Felts PA and Smith KJ (1996) Neuroscience 75:643-655) and TNR16 expression in Schwann cells is critically important for this remyelination process (Tomita et al. (2007) Glia 55: 1199-1208). It is also known that concussions cause a disruption in blood brain barrier integrity (Sahyouni et al. (2017) Journal of Concussion 1 : 1-15; Johnson et al. (2018) Acta Neuropathol 135:711-726). So, in an otherwise healthy individual a detected presence of TNR16 in the brain would necessarily correspond to the Schwann cell activity following a cerebral trauma. Because a concussion/mTBI causes a disruption in blood brain barrier integrity, that corresponding TNR16 response would then be detectable in body fluid outside the brain. The presence of TNR16 outside the brain should, therefore, be consistent with other symptoms of TBI/mTB I/concussion.

Proteins:

[0083] Based on their specific neuro-inflammatory functionality and/or previously observed correlations with cerebral dysfunction, a list of fourteen potential proteins were targeted for analysis: S-100B protein, Neuro-specific enolase (NSE), glial fibrillary acidic protein (GFAP), occludin (OCLN), phosphorylated heavy neurofilaments (NFH), neutrophil gelatinase-associated lipocalin (NGAL) (aka LCN-2), tumor necrosis factor receptor superfamily member 16 (TNFR16) (aka p75NTR), marinobufagenin (MBG), plasma-soluble prion protein (PrPC), aII-spectrin N-terminal fragment (SNTF), C-C motif chemokine 11 (CCL11), A-tau, C-tau and T-tau.

Sample Universe:

[0084] Urine samples were taken during hospital emergency room admission from donors who recently experienced a significant physical trauma that resulted in either the symptoms of an mTBI/concussion or a long bone fracture with no symptoms of a concussion. Test Protocol:

[0085] Initial analysis was done utilizing mass spectrometry, followed-up with

ELISA testing.

Sample blindness:

[0086] Actions were taken to reduce sample-specific analytic bias by A) de- identifying the individual donor information and donor group status (i.e., Symptomatic or Asymptomatic) of each sample, and then B) removing a small number of randomly selected samples from the sample universe prior to testing.

Sample collection:

[0087] Samples were taken from of 72 donors (55 symptomatic, 17 asymptomatic) from which 2 were chosen at random and removed prior to testing.

Mass spectrometer Testing:

[0088] Sample preparation was done at Western Michigan School of Medicine utilizing Filter Aided Sample Preparation (FASP) technique (Wisniewski et al. (2009) Nat. Methods 6:359-362) and sent to University of Arkansas for Medical Sciences for mass spectrometry testing on an Orbitrap Lumos Mass Spectrometer with Nano LC Proteomics Workflow. Data analysis was done using Mascot algorithmic proteomic search engine and MaxQuant analytic software program with detection confirmation based on top three precursor intensity with 95%+ protein identification probability.

[0089] During mass spectrometer testing, a noticeable amount of background interference was noted and was expected to have impacted protein detection confirmation. The mass spectrometry test results showed no discernable detection for twelve of the target proteins and no immediately observable correlation between detections of the other two proteins: NGAL and TNR16 (FIG. 13).

[0090] In an attempt to look further for any detection correlation between the two detected proteins, the analysis universe was expanded to include those proteins which had been detected in approximately 15 samples or more. From this effort, a potential correlation of non-detection was observed across four additional proteins and one of the target proteins (TNR16) (FIG. 13), but further work would be required to determine any statistical significance.

ELISA Testing:

[0091] The following ELISA kits were utilized: LS-F39777-1 Human NGFR /

CD271 / TNR16 ELISA Kit (Sandwich ELISA kit from LifeSpan BioSciences, Inc.) and LS-F2720-1 Human LCN2 / Lipocalin 2 / NGAL ELISA Kit (Sandwich ELISA from LifeSpan BioSciences, Inc.). Sample preparation and testing were conducted at Western Michigan University School of Medicine in accordance with kit manufacturer instructions.

[0092] The samples were tested utilizing ELISA to cross-confirm the mass spectrometry detection of NGAL and TNR16 (FIG. 14). The initial ELISA testing results showed a significantly higher detection rate than mass spectrometry for NGAL, but no discernable detection of TNR16 in any of the samples because of high background interference. The NGAL testing would remain a cross-confirmation control group while in an attempt to improve ELISA TNR16 detection, dilution ratios of 1 : 1 and 1 :3 were employed but resulted in very little improvement. Further dilution experimentation determined that a 1 :8 dilution ratio was required to relieve the suppression with TNR16, but introduced a significant potential for insufficient sample volume in the assay. Test results for TNR16 detection at this 1 :8 dilution ratio, not surprisingly, yielded a TNR16 detection rate that was lower than with mass spectrometry.

Binary Output:

[0093] To better understand the observed detections across the different test techniques and proteins, results are shown in binary format in one chart (FIG. 15).

Summary of Conditions:

[0094] Known: the original sample universe was 72: 55 mTBI symptomatic, 17 mTBI asymptomatic; of the 72, 2 were removed and the remaining 70 samples were utilized for the mass spectrometry and ELISA testing; since the 70 samples were from the original sample set of 72, the only possible donor group ratios are: 53/17, 54/16 and 55/15. Unknown: which specific samples were from which donor group. Quantitative Observations:

[0095] There were no samples exhibiting the same multi-protein profile as detected by mass spectrometry ( i.e no duplicate samples were observed). In 21 of the 23 NGAL detections by mass spectrometry, NGAL was also detected by ELISA testing. Combining the mass spectrometry and ELISA results for TNR16 yielded a detection breakdown of 55 positive/ 15 negative, which is one of the three possible donor group ratios.

Qualitative Assessments:

[0096] Combined mass spec/ELISA results indicate a potential correlation of non detection* between TNR16 and 4 other proteins of associative functionality: TNR16:

Neuroinflammatory response protein, seen in blood-brain-barrier dysfunction; BSG: Protein that promotes inflammation, required for transmembrane movement; IGFBP3: Transport protein whose increased production is influenced by TNR-a; CD320: Transmembrane protein receptor; EPHB3: Transmembrane protein (*except for detection conflicts occurring in sample #16 and #70).

[0097] Background interference was a consistent footnote to confirmation of detection in both the mass spectrometry and ELISA testing of TNR16. The high dilution rate employed to reduce background interference in ELISA testing of TNR16 significantly lowered detectable protein concentration levels in the samples. The result was an increased difficulty in protein-specific detection, but a reduction in detection inaccuracy (i.e., false positive detection). The near-perfect cross-confirmation of‘shotgun’ mass spectrometry NGAL detection by the control group ELISA testing for NGAL, further substantiates the mass spectrometry detection accuracy (i.e., true-positive detection). Combining the mass spectrometry and ELISA results provides strong support of TNR16 presence in those samples in which it was detected and even more so its absence in the samples in which it was not.

Predictive Probabilities:

[0098] Based on the conditions, observations and resulting assessments, the predictive probability chart (Table 1) below are initially formulated with on Q Assumption: there were no False-Positive TNR16 detections in either the mass spectrometer or ELISA tests. Table 1

[0099] Based on the Assumption, the Positive Predictive Value is 100% with a

Sensitivity of detection ranging between 100% and 78.5%.

Implications of Deviation:

[0100] Deviating from the initial Assumption by introducing a False-Positive (B) values increases the range of possible predictive values. Further, adjusting the ratios of A and B across the total range of possible C values book-ends the range of the resulting possible predictive values (Table 2).

Table 2

[0101] Even adjusting the False-Positive ratio up to 9%, Positive-Predictive Value always remains above 90% and Sensitivity of detection drops only to about 77%.

[0102] Within the universe of sample donors who all experienced significant physical trauma, there are significant differences in the multi-protein detection profiles among all the individual samples. The ratio of combined TNR16 detection aligns exactly with the known ratio of donors within that universe exhibiting symptoms of mTBI. A significant variation in the detection accuracy still results in high predictive values for TNR16 detection and mTBI. In conclusion, a concussed person who exhibits the symptoms of mTBI has necessarily suffered a blood-brain-barrier disruption and TNR16 is detected outside the brain. The absence of TNR16 detection outside the brain indicates blood- barrier-integrity and necessarily ruling-out the potential of mTBI. Supported by the test results and a high positive predictive value, TNR16 is, thus, a biomarker for concussion assessment: detecting its absence for screening-out mTBI and its detected presence

(whether binary, in certain concentrations or in concert with other proteins) for diagnosing mTBI, injury severity and prognostication of symptoms.

INCORPORATION BY REFERENCE

[0103] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0104] Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the World Wide Web and/or the National Center for Biotechnology Information (NCBI) on the World Wide Web.

EQUIVALENTS

[0105] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. A method of diagnosing whether a subject is having or at risk of developing a traumatic brain injury (TBI), comprising the steps of:

a) collecting a body fluid sample from the subject;

b) detecting the presence of one or more biomarkers selected from the group consisting of S-100B, plasma-soluble prion protein, neutrophil gelatinase-associated lipocalin, occludin, C-C motif chemokine 11, phosphorylated heavy neurofilaments, neuron-specific enolase, glial fibrillary acidic protein, marinobufagenin, aII-spectrin N- terminal fragment, tumor necrosis factor receptor superfamily member 16, tau, A-tau, C- tau, and T-tau, and any fragment of at least five amino acids long of any thereof in the body fluid sample of the subject; and

c) determining the subject as having or at risk of developing a TBI when the presence of one or more traumatic brain injury biomarkers is detected in the body fluid sample.

2. A method of assessing the severity of a traumatic brain injury in a subject, comprising the steps of:

a) collecting a body fluid sample from the subject;

b) detecting the level of one or more biomarkers selected from the group consisting of S-100B, plasma-soluble prion protein, neutrophil gelatinase-associated lipocalin, occludin, C-C motif chemokine 11, phosphorylated heavy neurofilaments, neuron-specific enolase, glial fibrillary acidic protein, marinobufagenin, aII-spectrin N-terminal fragment, tumor necrosis factor receptor superfamily member 16, tau, A-tau, C-tau, and T-tau, and any fragment of at least five amino acids long of any thereof in the body fluid sample of the subject;

c) comparing the level of one or more traumatic brain injury biomarkers detected in the body fluid sample of the subject with the level of the corresponding biomarkers detected in a control sample or with a reference value; and

d) determining the severity of the traumatic brain injury in the subject.

3. A method for monitoring the progression of a traumatic brain injury in a subject, the method comprising:

a) detecting at a first point in time the level of a biomarker in a body fluid sample from the subject, wherein the biomarker is one or more biomarkers selected from the group consisting of S-100B, plasma-soluble prion protein, neutrophil gelatinase-associated lipocalin, occludin, C-C motif chemokine 11, phosphorylated heavy neurofilaments, neuron-specific enolase, glial fibrillary acidic protein, marinobufagenin, aII-spectrin N- terminal fragment, tumor necrosis factor receptor superfamily member 16, tau, A-tau, C- tau, and T-tau, and any fragment of at least five amino acids long of any thereof;

b) repeating step a) at a subsequent point in time; and

c) comparing the level detected in steps a) and b), and therefrom monitoring the progression of the TBI in the subject.

4. The method of claim 3, wherein the subject has undergone treatment between the first and the subsequence points in time.

5. The method according to any one of claims 1-4, wherein the one or more traumatic brain injury biomarkers comprise tumor necrosis factor receptor superfamily member 16.

6. The method according to any one of claims 1-5, wherein the one or more traumatic brain injury biomarkers comprise neutrophil gelatinase-associated lipocalin.

7. The method according to any one of claims 1-6, wherein the one or more traumatic brain injury biomarkers comprise tumor necrosis factor receptor superfamily member 16 and neutrophil gelatinase-associated lipocalin.

8. The method according to any one of claims 1-7, wherein the presence and/or level of one or more biomarkers is detected by contacting the body fluid sample with an antibody against a biomarker and detecting the binding between the biomarker and the antibody.

9. The method according to any one of claims 1-8, wherein the presence and/or level of one or more biomarkers is detected by Western-blot, ELISA (Enzyme-Linked Immunosorbent Assay), RIA (Radioimmunoassay), Competitive EIA (Competitive Enzyme Immunoassay), DAS-ELISA (Double Antibody Sandwich-ELISA), immunocytochemical or immunohistochemical techniques.

10. The method according to any one of claims 1-9, wherein the presence and/or level of one or more biomarkers is detected by exposing the body fluid sample to a test strip containing reactive agents against one or more biomarkers.

11. The method according to any one of claims 1-10, wherein the presence and/or level of one or more traumatic brain injury biomarkers is detected by mass spectrometry, HPLC, or NMR.

12. The method according to any one of claims 1-11, wherein the presence and/or level of one or more traumatic brain injury biomarkers is detected using more than one technique.

13. The method according to any one of claims 1-12, wherein the body fluid sample is selected from the group consisting of blood, plasma, serum, urine, saliva, perspiration, and exhaled breath condensate.

14. A test strip for the detection of a traumatic brain injury in a subject, comprising a substrate-based test strip containing one or more reactive agents against one or more biomarkers selected from the group consisting of S-100B, plasma-soluble prion protein, neutrophil gelatinase-associated lipocalin, occludin, C-C motif chemokine 11, phosphorylated heavy neurofilaments, neuron-specific enolase, glial fibrillary acidic protein, marinobufagenin, aII-spectrin N-terminal fragment, tumor necrosis factor receptor superfamily member 16, tau, A-tau, C-tau, and T-tau, and any fragment of at least five amino acids long of any thereof, and a means by which to detect the binding of said agents to said biomarkers.

15. The test strip according to claim 14, wherein the one or more biomarkers comprise tumor necrosis factor receptor superfamily member 16.

16. The test strip according to claim 14 or 15, wherein the one or more traumatic brain injury biomarkers comprise neutrophil gelatinase-associated lipocalin.

17. The test strip according to any one of claims 14-16, wherein the one or more traumatic brain injury biomarkers comprise tumor necrosis factor receptor superfamily member 16 and neutrophil gelatinase-associated lipocalin.

18. The test strip according to any one of claims 14-17, wherein the reactive agent is an antibody against the biomarker.

19. The test strip according to any one of claims 14-18, wherein the detection means further comprises a means to detect varying concentrations of said biomarkers.

20. The test strip according to any one of claims 14-19, wherein the detection means comprises at least one human-discernible graphic or alphanumeric element.

21. The test strip according to any one of claims 14-20, wherein the detection means comprises at least one element that can be read or processed by machine.

22. The test strip according to any one of claims 14-21, wherein the detection means or reactive agents are printed onto a transparent film, which is then impregnated onto the test strip.

23. The test strip according to any one of claims 14-22, further comprising a transparent protective coating over the strip.

24. The test strip according to any one of claims 14-23, wherein the transparent protective coating is latex.

25. The test strip according to any one of claims 14-24, further comprising a non-absorbent protective layer covering at least a part of one or more surfaces of the strip.

26. The test strip according to any one of claims 14-25, wherein the detection means of the test strip is visible through the non-absorbent protective layer.

27. The test strip according to any one of claims 14-26, wherein the non absorbent protective layer is a plastic casing.

28. The test strip according to any one of claims 14-27, wherein said plastic casing further comprises a window for the viewing of the detection means of the test strip.

29. A kit comprising:

the test strip according to any one of claims 14-28;

instructions for the use of said test strip; and

a cup for the collection of a fluid sample.

30. The kit according to claim 29, wherein said test strip is contained in a disposable plastic wrapper.

31. A mobile phone application capable of reading the test strip according to any one of claims 14-28, which can record, interpret, and/or distribute the results of said test strip.

32. The method of any one of claims 1-13, further comprising storing the data of the subject into a database.

33. The method of any one of claims 1-13 and claim 32, wherein the data is collected with the mobile phone application according to claim 31.

34. The method of any one of claims 1-13 and claims 32-33, wherein data in the database can be combined and analyzed to provide information which comprises injury screening, injury diagnosis, injury severity, prognostication of resulting symptoms, resulting physiological, psychological or cognitive impact, medical attention, activity abstinence, or suggested action or lifestyle adjustment.

35. The method, test strip, kit or mobile phone application according to any one of claims 1-34, the TBI is a brain injury selected from the group consisting of concussions, contusions, coup-contrecoup injuries, diffuse axonal injuries, penetrating injuries, skull fractures, and scalp wounds.

36. The method, test strip, kit or mobile phone application according to any one of claims 1-35, the TBI is a mild, a moderate, or a severe TBI.