Method and device for detection of a biomarker

Abstract

A method of detecting a biomarker in a biological sample from a mammal is provided. The method comprises the steps of: (a) combining the biological sample with a conjugate to form a mixture, wherein the conjugate comprises a detectable label linked to a detection antibody that binds to an antigen on the biomarker, and wherein the conjugate binds with the biomarker to form a detectable complex; (b) contacting the mixture with a capture antibody that binds to the biomarker; and (c) detecting the presence of the biomarker in the sample when the detectable complex binds to the capture antibody and the metal particulate label in the detectable complex is detected. The method is useful to detect biomarkers of brain injury.

Description

METHOD AND DEVICE FOR DETECTION OF A BIOMARKERField of the Invention

[0001] The present invention generally relates to the diagnostic methods, and in particular, relates to methods and devices for detecting biomarkers useful for diagnosis of a medical condition.Background

[0002] Approximately 1 in 10 people who play a contact sport will sustain a concussion every year. While some athletes promptly acknowledge and seek treatment for their concussion symptoms, a significant number of concussions remain undetected. This is often attributed to the current reliance on inaccurate testing methods, underscoring the need for a precise and affordable objective concussion screening method. Concussions are defined by the Center for Disease Control and Prevention as “a type of traumatic brain injury - or TBI - caused by a bump, blow, or jolt to the head or by a hit to the body that causes the head and brain to move rapidly back and forth. This sudden movement can cause the brain to bounce around and twist in the skull, creating chemical changes in the brain and sometimes stretching and damaging brain cells’". Neurological injuries, including traumatic brain injuries, are frequently linked to cerebrovascular dysfunction and alterations in blood-brain barrier function. A compromised blood-brain barrier may play a role in the development of brain diseases by allowing the entry of cells and molecules that are typically confined to the periphery, making them detectable in bodily fluids. If a concussion goes untreated, re-injury of the brain may occur. Re-injury of the brain before it has fully recovered greatly increases the risk of chronic symptoms, permanent damage, and postconcussion syndrome. In the realm of sports, the challenge arises as athletes, keen to resume playing, may attempt to conceal the symptoms of their concussions.

[0003] Reducing the risk of undetected concussions in sports and beyond is crucial, given that the incidence is estimated to be around 50%. This is particularly significant as second impact syndrome can have devastating effects. While the origin of a concussion is broadly comprehended, the origin of second impact syndrome is not as thoroughly grasped. The widely acknowledged cause is linked to experiencing a second concussion before the brain has the opportunity to fully recover from the initial injury. Following a second impact, the individual will swiftly experience impaired cognitive function and loss of consciousness within seconds to minutes, leading to severe neurological damage. Although rare, second impact syndrome can result in fatalities. The mortality rate in confirmed cases of secondary impact syndrome exceeds 50%, with the likelihood of permanent disability approaching 100%.Moreover, about 20% of individuals with untreated concussions will endure post-concussion syndrome, characterized by persistent symptoms lasting for months to years after the initial injury.

[0004] Mild traumatic brain injuries, or concussions, are estimated to account for up to 85% of head injuries. Current diagnostic approaches rely on computed tomography (CT scan) or in-hospital observation. However, these methods are both time-consuming and expensive, rendering them inaccessible to a significant portion of the global population. CT scans, in particular, involve potential exposure to harmful radiation, with over 90% of brain scans yielding negative results for traumatic brain injury.

[0005] Other methods of concussion screening can be separated into two categories: either accurate and time consuming, or inaccurate and quick to administer. None of the numerous tools designed for the diagnosis and management of concussions, such as SCAT5 and ImPACT, singularly demonstrate complete effectiveness. Consequently, enhancing sensitivity and specificity involves employing a combination of various tools, yet the most effective combination remains undetermined. In general, the challenge in diagnosing concussions persists due to the absence of a universal definition and the lack of an objective measurement tool.

[0006] The SCAT-5 (Sport Concussion Assessment Tool) has long been the gold standard for concussion assessment, however, it was designed as a sideline tool and its utility can be seen to decrease after a few days post-concussion. It also does not prompt evaluation of all the phenotypes of concussion. More recently, the Concussion Office Based Rehabilitation Assessment (COBRA) was created to assess the majority of potential manifestations of concussion in the office setting a day or two after an injury has been sustained. The COBRA utilizes the eight phenotypes of concussion as a guide to assess each of the potential biopsychosocial features that can be associated with these injuries and can be used to guide evidence-based treatments. However, further work is required to determine the diagnostic utility of the COBRA.

[0007] Lateral flow assays find extensive application in point-of-care diagnostics owing to their simplicity, speed, and cost-effectiveness. Employing immunoassay technology, these tests facilitate the efficient identification and measurement of a target substance, such as a biomarker, in liquid samples, encompassing biological specimens like urine, saliva, serum, cerebrospinal fluid, and other bodily fluids. Testing can be performed using a variety of devices and accompanying equipment, either administered by a healthcare professional or self-administered by a patient. Traditional lateral flow assays, thoughwidely used, do not possess the necessary sensitivity and specificity to consistently identify specific biomarkers, especially those found in low concentrations within intricate biological samples.

[0008] In view of the foregoing, it would be desirable to develop a novel diagnostic method that is convenient, efficient and / or cost-effective which may be useful to diagnose medical conditions such as traumatic brain injury or disease.Summary of the Invention

[0009] A novel method for the detection of a protein biomarker in a biological sample has now been developed which is straightforward, and characterized by its simplicity, cost-effectiveness, accuracy, efficiency, and remarkable specificity and sensitivity. This method, along with a device designed for its execution, involves a lateral flow assay tailored to detect the biomarker in a biological sample obtained from a patient.

[0010] Thus, in one aspect, a method of detecting a target biomarker in a biological sample is provided. The method comprises the steps of (a) combining the sample with a conjugate comprising a detectable particulate metal label covalently linked to a detection antibody that binds to an antigen on the target biomarker to form a mixture, wherein the detection antibody of the conjugate binds with the target biomarker to form a detectable complex; (b) contacting the detectable complex with a capture antibody that binds to the target biomarker; and (c) detecting the presence of the target biomarker in the sample when the detectable complex binds to the capture antibody and the label in the detectable complex is detected.

[0011] In another aspect, a method of detecting SIOOB biomarker in a biological sample comprising the steps of (a) combining the biological sample with a conjugate to form a mixture, wherein the conjugate comprises a detectable label linked to a detection antibody that binds to an antigen on the biomarker, and wherein the conjugate binds with the biomarker to form a detectable complex; (b) contacting the mixture with a capture antibody that binds to the biomarker; and (c) detecting the presence of the biomarker in the sample when the detectable complex binds to the capture antibody and the label in the detectable complex is detected.

[0012] In another aspect, a test strip is provided for the detection of a target biomarker in a biological sample. The test strip comprises, a sample pad; a conjugate release pad comprising a particulate metal label / detection antibody conjugate; a detection zone comprising a test line withimmobilized capture antibody and a control line with immobilized detection antibody; and an absorbent pad.

[0013] A kit is also provided comprising the test strip and other components useful to conduct a method of detecting a target biomarker in a biological sample.

[0014] These and other aspects of the invention are described herein by reference to the detailed description, figures, and Example.Brief Description of the Figures

[0015] FIG. 1 illustrates the amino acid sequence of A) human and B) mouse SIOOB;

[0016] FIG. 2 illustrates a sample lateral flow strip in accordance with an embodiment of the invention.

[0017] FIG. 3 illustrate sensitivity results of a lateral flow strip in accordance with an embodiment of the invention.

[0018] FIG. 4 graphically illustrate the results of image J software to determine the relative test line intensity at various concentrations of SIOOB.Detailed Description of the Invention

[0019] A method of detecting a target biomarker in a biological sample from a mammal is provided. The method comprises the steps of: (a) combining the sample with a conjugate comprising a detectable label linked to a detection antibody to form a mixture, wherein the detection antibody binds to an antigen on the biomarker, and the conjugate binds with the biomarker to form a detectable complex; (b) contacting the mixture with a capture antibody that binds to the detectable complex; and (c) detecting the presence of the biomarker in the sample when the detectable complex binds to the capture antibody and the detectable label in the detectable complex is detected.

[0020] The present method is useful to detect biomarkers of associated with brain injury such as traumatic brain injury (TBI), including, but not limited to, mild TBI such as concussion as well as moderate to severe TBI, and brain injury resulting from disease. Exemplary biomarkers include, but are not limited to, the calcium-binding protein SIOOB, ubiquitin carboxy-terminal hydrolase LI (UCH-L1), glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), tau proteins, a-II spectrin N- terminal fragment (SNTF), and neuron specific enolase (NSE).

[0021] To perform the present method, a biological sample is collected from a mammal. The term “biological sample” is used herein to refer to blood, including serum and plasma, saliva, urine, cerebrospinal fluid, amniotic fluid and other bodily fluids. The selected biological sample may be collected using protocols well-established in the art.

[0022] In one embodiment, the biological sample is a saliva sample, including a whole saliva sample. The term “whole saliva” refers to saliva containing fluid from the salivary glands, e.g. water, proteins, electrolytes and small organic molecules, as well as oral microbiota, blood and blood derivatives from intraoral bleeding, epithelial lining cells, other fluids, e.g. bronchial and nasal secretions, and extrinsic substances (such as food debris, toothpaste, oral rinse). Saliva sample collection methods include, but are not limited to, collection of saliva using an absorbent material such as swabs, cotton, pads, gauze or sponge such as hydrocellulose sponge. The material may be placed in the mouth, such as under the tongue of the mammal, for a period of time to absorb saliva, or the material may be chewed to collect saliva. Saliva is then removed from the material by squeezing into a sample container. This process may have to be repeated a number times to collect a sufficient amount of saliva sample for testing. Saliva may also be collected by passive drooling or active spitting into a sample container. Saliva is ideally collected at least 15 minutes after eating, drinking, using tobacco products, or chewing gum. Generally, the amount of saliva for use in the present method is at least about 0.1 ml, for example, 0.08- 1 ml. The term “mammal” is used herein to refer to both human and non-human mammals including domestic and undomesticated animals.

[0023] Once the biological sample is collected, it may be treated and / or purified to remove materials therefrom that may interfere with detection of the biomarker, and / or decrease the accuracy of the present method. Various chemical and physical purification methods are known in the art including, but not limited to, absorption, adsorption, chromatography, distillation, extraction, ion exchange, filtration, complex formation, crystallization, drying, and the like.

[0024] Following purification, if conducted, the sample is contacted with a conjugate comprising a first detection antibody linked to a labelling agent under conditions which permit binding of the conjugate to a target biomarker that may be present in the sample. The biological sample may be used as is, or alternatively, the sample may be combined with a suitable buffer solution, for example, a phosphate-based buffer solution (e.g. PBS, pH 7.4). Binding of the conjugate to the biomarker in the sample, via binding of the detection antibody to an antigenic site on the biomarker, effectively results inlabelling of the biomarker for subsequent detection. Thus, binding of the conjugate to the biomarker forms a detectable complex.

[0025] As used herein, the term “detection antibody” refers to an antibody that selectively binds to an antigen of the target biomarker. The term “antibody” is used herein to refer to monoclonal or polyclonal antibodies, or antigen-binding fragments thereof, e.g. an antibody fragment that retains specific binding affinity for an antigen of the target biomarker. As one skilled in the art will appreciate, detection antibodies that bind target antigens may be commercially available, or alternatively, may be raised using techniques conventional in the art.

[0026] In one embodiment, the detection antibody selectively binds to a biomarker that is detectable in saliva.

[0027] In a particular embodiment, the detection antibody binds to a concussion biomarker that is detectable in saliva, for example, SIOOB. The SIOOB biomarker is glial-specific and is secreted by astrocytes or injured cells and enters the extracellular space or bloodstream. Serum levels of SIOOB increase in patients during the acute phase of brain damage. The term “S100B” encompasses mammalian SI 00 calcium-binding protein B, including both human and non-human SIOOB orthologs, as well as functionally equivalent variants thereof. Human SIOOB has the amino acid sequence of NCBI Ref. Sequence NP 006263 as shown in Fig. 1A, and mouse SIOOB has the amino acid sequence of NCBI Ref. Sequence NP 033141 as shown in Fig. IB. The term “functionally equivalent” as it relates to variants of SIOOB refers to protein variants which may include one or more amino acid substitutions, deletions or insertions while retaining all or a portion of the native activity of the protein, namely expression during the acute phase of brain damage.

[0028] Antibodies suitable to detect SIOOB in a biological sample bind to an antigenic region on the SIOOB. Such antigenic regions include, but are not limited to, full-length SIOOB (amino acids 1-92), a terminal region of SIOOB such as amino acids 31-92, or an internal region of SIOOB such as amino acids 27-56. As set out above, SIOOB antibodies may be raised using techniques conventional in the art. Antibodies suitable for detecting SIOOB in a biological sample are also readily available from various commercial sources. Notable suppliers of (anti) SIOOB antibodies include Invitrogen, Sigma Aldrich, Abeam, Cell Signaling Technologies and Proteintech. The antibodies may be polyclonal or monoclonal, and may be derived from a variety of sources including mammals such as mouse, rat, rabbit, goat, non- human primates, and genetically modified sources. Preferred antibodies for use in the present methods are monoclonal antibodies.

[0029] The detection antibody is combined with a detectable labelling agent to form a conjugate that is detectable. Any suitable detectable labelling agent may be used including, but not limited to, reporter enzymes such as horseradish peroxidase, fluorophores, luminescent labels (e.g. RuBPY), biotin, radioactive isotopes, electrochemical labels, magnetic beads, DNA / RNA tags, dye-based labels, polymer-based labels, particulate metals and others.

[0030] In one embodiment, the labelling agent comprises a particulate metal. Exemplary metals for use as a labelling agent include transition metals such as, but not limited to, gold, silver, platinum, iron copper, selenium, chromium, vanadium, titanium, manganese or an alloy thereof. In an embodiment, the particulate metal comprises nanoparticles of about 1 to 150 nm in size which may comprise various geometries including spherical and nanoshell structures, e.g. , or non-spherical shapes such as rods, cubes, tubes, fibers, prisms and the like. Such particulate metal labels are detectable based on their ability to absorb / scatter light at particular wavelengths. In one embodiment, gold nanoparticles (AuNPs) are used as the labelling agent. Gold particles with a diameter in the range of about 1 to 100 nm, preferably 1-50 nm, such as 10-30 nm, are generally utilized for labelling. AuNPs are detectable based on their ability to absorb / scatter light at particular wavelengths depending on their size. For example, particles of 10-20 nm or 20-30 nm in size are detectable by appearance of a colorimetric signal in the visible wavelength range. In other embodiments, metal nanoshell structures are utilized which comprise a dielectric core nanoparticle such as silica surrounded by an ultrathin metal shell (e.g. gold or silver). Preferred nanoshells for use are 50-150 nm in size, preferably 100-150 nm.

[0031] Gold nanoparticles (AuNPs) may be prepared by the reduction of chloroauric acid. There are a number of methods that may be used to prepare colloidal AuNPs. The appropriate method is selected based on a number of factors including the desired nanoparticle size. Reducing agents that may be used include, but are not limited to, citrate, tannic acid, borohydrate, hydroquinone, acetyl acetonate and hydroxyl radicals, optionally together with a suitable stabilizing agent such as citrate or tetraoctylammonium bromide. In a preferred embodiment, the AuNPs are prepared by the Turkevich method in which chloroauric acid is treated with sodium citrate solution to produce nanoparticles in the size range of about 20-30 nm.

[0032] The detection antibody conjugate is formed by combining the labelling agent such as a metal particulate label with the selected detection antibody. The conjugate may be prepared by physical absorption of the detection antibody directly onto the surface of the labelling agent. In this method, the labelling agent is introduced into the detection antibody solution, and the labelling agent adheres to thedetection antibody through non-covalent interactions, such as electrostatic forces or van der Waals interactions. Alternatively, the conjugate may be prepared by a covalent conjugation method in which the detection antibody and / or the labelling agent are functionalized to promote a strong stable binding reaction between the labelling agent and the detection antibody. This functionalization may involve modifying the surface of the labelling agent with chemical groups, such as thiol groups, amino groups, or carboxyl groups, that provide reactive sites for conjugation with the detection antibody.

[0033] Following the formation of the detection antibody conjugate, a second capture antibody is employed to capture the detectable complex formed through the binding of the biomarker in the sample to the detection antibody conjugate. The second capture antibody binds to the biomarker within the complex at an antigenic site that is either the same as or different from the antigen-binding site of the first detection antibody. The second capture antibody typically differs from the first antibody in its specificity or affinity for the biomarker. When the second antibody binds to a different antigenic site on the biomarker to the first antibody, a sandwich complex is formed, comprising detectable antibodybiomarker-capture antibody. Detection of this sandwich complex is achieved by detection of the labelling agent, e.g. metal nanoparticles or nanoshells, bound to the detection antibody. In cases where the second capture antibody binds to the same antigenic site as the first antibody, a competitive binding reaction occurs, leading to the displacement of the first detection antibody by the second capture antibody. In one embodiment, the detection and capture antibodies are each monoclonal antibodies. In another embodiment, the detection and capture antibodies are both monoclonal antibodies which bind to different antigenic sites on the biomarker. In a further embodiment, the detection and capture antibodies are monoclonal antibodies from different sources, e.g. different mammalian sources such as a monoclonal rabbit detection antibody and a monoclonal mouse capture antibody, or other combinations of mammalian detection and capture monoclonal antibodies.

[0034] The presence of a biomarker such as SIOOB in a biological sample such as saliva is detected as follows. Biomarker in the sample is first exposed to detection antibody conjugate to which it binds to form a detectable complex. The detectable complex comprising biomarker bound to the detection antibody conjugate is then exposed to capture antibody which binds to the biomarker at a site distinct from the site at which the detection antibody binds. The capture antibody is generally immobilized, and binding of detectable complex comprising the biomarker is then also immobilized on binding to the capture antibody. Binding of the detectable complex to the capture antibody is detected by detection of the metal particulate label via a colorimetric signal. The intensity of the signal isgenerally indicative of the concentration of the biomarker in the sample. Thus, the stronger the signal, the greater the concentration of biomarker in the biological sample.

[0035] In one embodiment, the presence of SIOOB in a saliva sample is detected. A saliva sample is obtained from a patient and reacted with a detection antibody conjugate comprising an SIOOB antibody conjugated to metal nanoparticles or nanoshells. If SIOOB is present in the sample, an SIOOB detectable complex is formed by binding of the SIOOB to the detection antibody conjugate. The sample is then contacted with an immobilized SIOOB capture antibody. As set out above, the capture antibody may be the same as or different from the SIOOB detection antibody, and each may be monoclonal antibodies from different sources. The capture antibody will immobilize any SlOOB-containing detectable complex in the sample. Immobilized SlOOB-containing detectable complex is detected by detection of the metal nanoparticles or nanoshells in the conjugate.

[0036] The present method of detecting a target biomarker may be used in any suitable assay format.

[0037] In one embodiment, the method is applied to a lateral flow assay in which the biological sample is applied to a lateral flow test strip. The lateral flow test strip generally comprises a series of overlapping membranes that may be mounted on a backing card or other support for stability. The sample is applied at one end of the strip, onto a sample pad and migrates along the strip as a result of capillary forces from the sample pad to a conjugate release pad and then a detection zone.

[0038] The function of the sample pad is to receive the sample. The sample pad may be designed to pretreat the sample, e.g. to separate sample components, remove interferents, adjust the pH, and the like. For example, the sample pad may incorporate additives which include, but not limited to, surfactants such as non-ionic surfactants, phosphate-buffered saline (PBS), Tween 20, bovine serum albumin (BSA), triton X-100, ethylenediaminetetraacetic acid (EDTA), trehalose, sodium azide, and silica gel. The sample pad is made of materials that do not adversely affect the sample, for example, the sample pad may be made of cellulose, nylon, polyvinylidene difluoride (PVDF), and / or glass fiber. In one embodiment, the sample pad is adapted to receive a saliva sample.

[0039] The conjugate pad comprises the detection antibody conjugate and is made of materials that do not affect the stability of the conjugate, the ability of the conjugate to react with the sample, nor the release of the conjugate from the pad. Materials such as glass fiber, cellulose, nylon and polyesters are used to make the conjugate pad. Conjugate is dispensed onto the conjugate pad using methods knownin the art including, for example, manual deposition by pipette, dipping of the conjugate pad into a solution containing conjugate particles, striping by a reagent dispensing printer and aerosolization by a reagent dispensing printer. The reagent printer is known to “stripe” reagent across the membrane at a constant rate. In this regard, reagent is loaded into the reagent printer at a concentration range of 0.25 - 3.0 mg / ml. Reagent is then dispensed through a printer tip that has a gauge size from 0.2 - 1 ,2mm. The printer tip lightly drags along the surface of the membrane at a constant speed, yielding a reagent dispensing rate between 0.5-3.0 ul / cm.

[0040] In one embodiment, to preserve the activity of the detection antibody conjugate on the conjugate pad, a solution comprising one or more sugars such as glucose, sucrose, lactose and / or trehalose is added. When dried, the sucrose molecules crystalize and preserve the detection antibody conjugate. Note that the conjugate particle preservative may be dispensed onto the conjugate pad before or after the conjugate particles, or may be combined with the conjugate particles in a solution to be dispensed onto the conjugate pad. When sample flows onto the conjugate pad, the sugar crystals dissolve and the detection antibody conjugate is released to flow with the sample when bound to biomarker within the sample.

[0041] The detection zone comprises a porous membrane, for example, composed of nitrocellulose having various pore sizes to yield various flow rates such as 75 sec / 4cm, 135 s / 4cm, 180s / 4cm, 240s / 4cm, etc. The membrane has a test line comprising immobilized capture antibody and a control line with immobilized biomarker or a control protein. The capture antibody reacts with biomarker-containing detectable complex, if biomarker is present in the sample, to immobilize the detectable complex at the test line. The presence of the biomarker in the sample is then detected at the test line by detecting a color change associated with the presence of the detectable label (such as a metal particulate label) within the immobilized detectable complex. The control line confirms proper working of the assay, e.g. proper fluid flow from the sample pad to the conjugate pad and along the detection zone. Immobilized on the control line is a protein that binds to the detection antibody in excess detection antibody conjugate released from the conjugate pad. The protein bound at the control line may be control biomarker (SIOOB), or a control immunoglobulin-binding protein that binds to antibodies, such as a bacterial immunoglobulin-binding protein, e.g. Protein A or Protein G. Binding of the conjugate at the control line is detected by detection of the colour change associated with the label (e.g. gold nanoparticles) in the conjugate.

[0042] For the purpose of dispensing capture antibodies onto nitrocellulose membranes in the detection zone, preservation additives may be utilized to promote even distribution of the antibodies, block non-specific binding and preserve the stability of the antibodies in dried storage. These preservation additives are often added in small amounts to the antibody buffer and include, but are not limited to, BSA (0.01-0.1%), histidine (20-40mM), methanol (0.5-5%), ethanol (0.5-5%), polyethylene glycol (molecular weight 20,000g / mol) (0.01-1%) and glycerol (0.5-5%). For example, BSA is added to the antibody buffer to reduce aggregation of the antibodies and block non-specific binding.

[0043] In an embodiment, the lateral flow test strip may also comprise a sensitivity pad assembled between the conjugate pad and the detection zone to enhance binding between biomarker present in the sample and the detection antibody conjugate on the conjugate pad. As the sample flows from the conjugate pad to the sensitivity pad, the contact time between the biomarker in the sample and the detection antibody conjugate increases thereby improving binding efficiency. This results in greater yields of biomarker-containing detectable complexes to be captured in the detection zone, resulting in an increased sensitivity of the assay. The sensitivity pad is made of materials similar to that of the conjugate pad, e.g. a cellulose fiber pad with a suitable thickness (e.g. 0.6-lmm). A cellulose fiber pad is more compact than other materials such as glass fiber, resulting in a slower flow rate and allowing more contact time between the biomarker and the detection antibody conjugate.

[0044] In order to prevent non-specific binding at the test line, and thereby enhance sensitivity and specificity of the assay, the membrane of the detection zone may be coated with blocking agent which serves to saturate and block available binding sites, preventing nonspecific interactions with proteins, antibodies, or other molecules. Common blocking agents include, but are not limited to, bovine serum albumin (BSA), skim milk, and gelatin. Other pads of the strip may also be coated with blocking agent, if desired.

[0045] The strip additionally comprises an absorption pad, made of materials similar to those in the sample and / or conjugate pads. The absorption pad is located at the end of the detection zone and functions to absorb unbound materials that migrate the length of the strip.

[0046] The lateral flow test strip, comprising sample pad, conjugate pad, optional sensitivity pad, and detection zone, will be a length that permits the flow of a given volume, e.g. 0.08-1 ml, of sample and conjugate the full length of the detection zone to ensure detection of target antibody in the sample at the test line and unbound conjugate at the control line. Strip length may be in the range of about 2-8 cm,such as 3-7 cm. The target flow rate to achieve detection of the biomarker using the lateral flow test strip is in the range of about 1-5 millimeters per minute (mm / min).

[0047] The strip may be provided in a housing to support the strip, provide windows to view portions of the strip with labelling, provide protective features to the user to prevent contact with the sample applied to the strip, and / or provide aesthetics. For example, the housing may be designed to include a sample window to receive the sample, a window showing the results of the test, a flip cover and the like. The housing may be made of any suitable material such as a synthetic or semi-synthetic polymer, including thermoplastic materials such as polyethylene, polypropylene, polystyrene, polyvinylchloride or polylactic acid.

[0048] In another embodiment, the method of detecting a biomarker such as SIOOB in a sample is conducted in a liquid reagent comprising a detection-antibody conjugate, for example, a gold nanoparticle-antibody conjugate. The reagent is prepared by admixing an alkaline solution with a solution of the labelling agent to achieve a solution having a pH in the range of about 8.5-9.5, e.g. about 9.0. An alkaline solution such as, but not limited to, potassium carbonate, sodium carbonate or sodium bicarbonate may be utilized. SIOOB monoclonal antibody is then added to the alkaline labelling agent solution, mixed and then incubated at room temperature for 30 minutes. To mitigate non-specific binding of other proteins or components to the SIOOB antibody, a blocking agent such as, but not limited to, bovine serum albumin (BSA), casein, non-fat dry milk (NFDM), gelatin, or other commercially available blocking buffer, is added to the labelled antibody solution in an amount sufficient to prevent non-specific binding. A surfactant such as Tween-20 may also be added to prevent non-specific binding. The reagent is used to detect SIOOB in a sample. A sample, such as a saliva sample, is added to the reagent and allowed to incubate for a sufficient period of time to permit binding of any SIOOB in the sample to the antibody conjugate. Binding of the SIOOB to the antibody conjugate will result in a detectable outcome such as a color change within the reagent that may be quantified, for example, using a spectrophotometer. This change in color is attributed to the surface plasmon resonance of the labelled-antibody conjugate, e.g. metal-antibody conjugate, which is induced by the binding of SIOOB.

[0049] The present methods advantageously provide a sensitive, specific, convenient method of detecting a target biomarker that may be used to diagnose or detect risk of various medical conditions, and in particular, TBI including concussion. The present method embodied in a test strip or a liquid reagent provides for quick and efficient on-site testing that is important for medical conditions that may occur outside of the clinical setting. Importantly, the method permits detection of a target biomarker at alevel that is indicative of a medical condition, such as concussion. For example, detection of SIOOB is achieved at a level of 5 ng / ml or less, preferably levels of 4 ng / ml, 3 ng / ml, 2.5 ng / ml, 2 ng / ml, 1 ng / ml or less, by the present methods. Further, the method permits detection of the target antigen on a target biomarker in saliva at a sensitivity level of at least about 80%, and preferably, at least about 85%, 90% or greater.

[0050] In another aspect, a kit is provided. In an embodiment, the kit comprises a lateral flow strip and one or more other components useful to conduct the method of identifying a biomarker. Such components may include, but are not limited to, a sample collection container, sample collection material such as swabs, cotton, gauze or the like, a pipette, diluent or assay buffer, or other related materials. The kit may also include instructions regarding the use of the lateral flow strip.

[0051] In another embodiment, a kit is provided comprising a liquid reagent and one or more other components useful to conduct the method of identifying the biomarker. Such components may include, but are not limited to, a sample collection container, sample collection material such as swabs, cotton, gauze or the like, a pipette, diluent or assay buffer, or other related materials.

[0052] Embodiments of the present invention are described by reference to the following specific example which is not to be construed as limiting.Example 1 - Conjugation of Antibody to Gold Nano-labels

[0053] Physical Conjugation - The application of gold nanoparticles (20-30 nm) for labeling SIOOB detection antibody was explored. To achieve conjugation of the gold nanoparticles to SIOOB antibody, SIOOB antibody (monoclonal (anti) SIOOB antibody from rabbit IGg cells purchased from Cell Signaling Technology) was combined with a gold nanoparticle solution (20 pl of SIOOB antibodies at 0.5 mg / ml in 200 pl of deionized water, and 40 pL of gold nanoparticles at 1.08 nM) and subjected to a conjugation process using electrostatic interaction (physical absorption).

[0054] An agglutination test was performed using varying concentrations of SIOOB protein to confirm formation of the conjugate, i.e. binding of the gold nanoparticles to SIOOB antibody.

[0055] Successful conjugation was achieved, and the conjugate was determined to effectively bind to the proteins A and G immobilized on strips (bacterial proteins from Streptococcal bacteria and S. aureus, respectively, which bind to different sites in the Fc region of IgG-type antibodies). Thesepositive results provided strong validation that the conjugation process was successful and that the gold nanoparticles served as an efficient and reliable label.

[0056] With the successful use of gold nanoparticles as the label, the specificity of the conjugate to SIOOB was confirmed. Conjugation check strips were prepared by immobilizing the unlabeled detection antibody (capture antibody) onto the test line and the SIOOB protein onto the control line of the strips. Results showed distinct binding, evidenced by a color change at the test and control lines, respectively. This color change indicated proper absorption of the antibody and protein onto the nitrocellulose membrane and successful immobilization. The limit of detection for the conjugate was determined to be 19 pg / ml.

[0057] Covalent conjugation - The detection antibody could also be covalently conjugated to gold nanoshells. BioReady Gold Nanoshells (150 nm) were purchased from nanoComposix and the carboxyl surface was activated using EDC / sulfo-NHS chemistry. For every ImL of the gold nanoshells at 20 OD, 160ug of sulfo-NHS and 80ug of EDC was added to form active NHS-ester end groups. These reagents were freshly dissolved in water before adding to the gold nanoshells. After incubation and multiple wash steps through centrifugation, the nanoshells were re-suspended to their original volume in a reaction buffer containing 0.5% polyethylene glycol (molecular weight 20,000g / mol) to promote the conjugation reaction. Monoclonal (anti) SIOOB antibodies (30ug) from rabbit IGg cells purchased from Cell Signaling Technology were added to the nanoshells and incubated for 1 hour. The antibodies were free from sodium azide and BSA preservatives which contain free amines that could disrupt conjugation chemistry. After intubation, a quencher (e.g, 50% w / v hydroxylamine) was added to deactivate any remaining active NHS-ester groups and multiple washing steps were performed by centrifugation.

[0058] Similar to the gold nanoparticles, the gold nanoshells effectively labelled the conjugate and could be detected on conjugation check strips.Example 2 - Lateral Flow Strip

[0059] A device in accordance with an embodiment of the invention is shown in Figure 2 and was prepared as follows.Test Strip Preparation Protocol

[0060] The test strip was assembled by applying a 1.5-2.5 cm absorption pad made of cellulose fiber to the downstream side of nitrocellulose membrane, ensuring some overlap of approximately 1-3millimeters (mm) on the nitrocellulose membrane which is approximately 0.5 cm wide and 5 cm long. A 0.5-1 cm strip of material the same as the absorption pad was then attached to the upstream side of the nitrocellulose membrane with 1-2 mm overlapping the nitrocellulose membrane. This optional sensitivity pad reduces the flow rate and improves binding between the conjugate particles and SIOOB. A 0.5-1 cm strip of conjugate pad (glass fiber pad of thickness 370pm) was then attached to the sensitivity pad (upper side) and was overlapping by 1-2 mm. Note that detection antibody conjugate and preservative were previously applied to the conjugate pad before assembly as set out below. Then, a 0.5-1 cm strip of sample pad (made of the same material as the absorption pad) was attached to the conjugate pad (upper side) with a 1-3 mm overlap. Attachment was via adhesive on the lower side of the pads. The strip was trimmed to result in a 0.5 cm width along its length. The test strips were assembled into cassettes with the following dimensions; length of 7cm, width of 2.5cm and height of 0.5cm. The well of the cassette has a maximum diameter of 1.5cm and hole diameter of 0.3cm.

[0061] The conjugate pad material was made from glass fiber pad of thickness 370pm obtained from Whatman Life Sciences. A mixture of 8uL of detection antibody conjugates, prepared as described in Example 1 using covalent conjugation, at 20 OD and 2pL of 10% sucrose were combined and deposited by pipetting onto the conjugate pad. The conjugate pad was then dried for 1 hour at 37°C prior to assembly in the strip.

[0062] To prepare the test line, monoclonal (anti) SIOOB capture antibody from mouse cells purchased from Sigma were dispensed 1.0 cm from the start of the nitrocellulose membrane using a 0.9mm gauge printing tip. Prior to their application to test strip, the antibodies which had been stored in 40% glycerol preservative, were subjected to wash filtration with an Amicon lOkDa ultrafilter and centrifugation. The wash filtration was performed once for 10 min to remove the majority of glycerol present and the antibody was collected in concentrated form. The washed antibodies were diluted to a concentration of 0.5 mg / ml in a buffer of lOmM PBS and 1% sucrose. The control line was immobilized with SIOOB (Human SIOOB His-tag Recombinant Protein) purchased from Thermo Fisher Scientific. SIOOB was used at a concentration of 50ug / ml in a lOmM PBS buffer. SIOOB was printed 1.5 cm from the start of the nitrocellulose membrane using a 0.45mm gauge printer tip. The nitrocellulose membrane was then oven dried at about 37°C for 45 minutes.Analysis of Strip

[0063] Saliva spiked with human SIOOB protein (0.1-1 ml of His-tag Recombinant Protein purchased from Invitrogen or Sigma-Aldrich) was applied to the sample pad of the strip. Within 5-15minutes both the test line and control line showed positive blue color. 0.1-1 ml of saliva with no SIOOB (control) was applied to the sample pad of another strip and showed positive color at the control line but no color at the test line. Thus, the strips were effective to accurately identify SIOOB in saliva sample.Example 3 - Full-Strip Limit of Detection Experiment

[0064] Full test strips were prepared using the Test Strip Preparation Protocol set out in Example2. Dilutions of SIOOB (Invitrogen Human SIOOB His-tag Recombinant Protein) in 5 mM PBS were combined with artificial saliva to prepare samples each with a resulting volume of 120 uL. The resulting concentration of SIOOB in respective samples was 100, 50, 25, 15, 10, 5, 2, 1, 0.5 and 0 ng / ml. The samples were separately dispensed onto the sample pad of a test strip. After 15 minutes, the strips were checked visually for binding of SIOOB protein to conjugate. The visual limit-of-detection was determined to be 5 ng / ml. This was based on examination of the strips which revealed that the intensity of the test line at SIOOB concentrations from 5 - 100 ng / ml was visually differentiated from the intensity of the test line in the absence of SIOOB (e.g. at an SIOOB concentration of 0 ng / ml).

[0065] To further validate the utility of the strips, Image analysis software called ImageJ was utilized to confirm the limit of detection by analyzing the signal intensity at the test line. Images were captured using a smartphone camera at 15 minutes from the addition of the samples to the strip. The images were uploaded to ImageJ and were converted to 32-bit greyscale format and inverted. A border was created to capture the pixels of the test line and the mean intensity of all the pixels within those borders was calculated. The mean intensity of the same pixel dimensions was taken at another spot on the nitrocellulose membrane that did not consist of the test or control line. To account for differences in the lighting of each photo, the relative mean intensity of the test line was determined by subtracting the mean intensity of the nitrocellulose membrane from the mean intensity of the test line. Statistical analysis of the test line relative mean intensity at each concentration of SIOOB yielded 0.3 ng / ml as the limit of detection as illustrated in Fig. 4.

Claims

CLAIMS1. A method of detecting a protein biomarker in a biological sample comprising the steps of:(a) combining the biological sample with a conjugate to form a mixture, wherein the conjugate comprises a metal particulate detectable label linked to a detection antibody that binds to an antigen on the biomarker, and wherein the conjugate binds with the biomarker to form a detectable complex;(b) contacting the mixture with a capture antibody that binds to the biomarker; and(c) detecting the presence of the biomarker in the sample when the detectable complex binds to the capture antibody and the label in the detectable complex is detected.2 The method of claim 1, wherein the biomarker is a biomarker of brain injury.3 The method of claim 1, wherein the biomarker is selected from the group of consisting of the calcium-binding protein SIOOB, ubiquitin carboxy-terminal hydrolase LI (UCH-L1), glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), tau proteins, a-II spectrin N-terminal fragment (SNTF), and neuron specific enolase (NSE).4 The method of claim 1, wherein the biomarker is SIOOB.5 The method of any one of claims 1-4, wherein the metal particulate detectable label comprises gold, silver, platinum, iron, copper, selenium, chromium, vanadium, titanium, manganese or an alloy thereof.6 The method of any one of claims 1-5, wherein the detectable label comprises nanoparticles of about 1 to 100 nm in size.7 The method of any one of claims l-,5 wherein the detectable label comprises nanoshells of 50- 150 nm in size.8 The method of any one of claims 1-7, wherein the biological sample is saliva.9 The method of claim 1, wherein the biomarker is SIOOB, the biological sample is saliva and the metal particulate detectable label is gold nanoparticles.10 The method of any one of claims 1-9, which enables visual detection of the protein biomarker at a level of 5 ng / ml or less.11 A test strip for the detection of a protein biomarker comprising: a sample pad; a conjugate pad comprising a detectable label / detection antibody conjugate; a detection zone comprising a test line withimmobilized capture antibody and a control line with immobilized protein that binds to the conjugate; and an adsorbent pad, wherein the detection antibody of the conjugate binds to the protein biomarker, and the capture antibody binds to the protein biomarker to immobilize the conjugate at the test line.

12. The test strip of claim 11, wherein the biomarker is a biomarker of brain injury.

13. The test strip of claim 11 or claim 12, wherein the biomarker is selected from the group of consisting of the calcium-binding protein SIOOB, ubiquitin carboxy-terminal hydrolase LI (UCH-L1), glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), tau proteins, a-II spectrin N- terminal fragment (SNTF), and neuron specific enolase (NSE).

14. The test strip of claim 11, wherein the biomarker is SIOOB.

15. The test strip of any one of claims 11-14, wherein the detectable label is a metal particulate label comprising gold, silver, platinum, iron, copper, selenium, chromium, vanadium, titanium, manganese or an alloy thereof.

16. The test strip of any one of claims 11-15, wherein the detectable label comprises nanoparticles of about 1 to 100 nm in size.

17. The test strip of any one of claims 11-16, wherein the detectable label comprises gold nanoparticles.

18. The test strip of any one of claims 11-15, wherein the detectable label comprises nanoshells of 50-150 nm in size.

19. The test strip of any one of claims 11-18, additionally comprising a sensitivity pad between the conjugate pad and the detection zone to increase contact time between the sample and the metal particulate detectable label / detection antibody conjugate.

20. The test strip of any one of claims 11-19, wherein the detection and capture antibodies are monoclonal antibodies which are from different sources.

21. A kit comprising the test strip as defined in any one of claims 11 -20, with one or more components useful to conduct the method of detecting a biomarker selected from a sample collection container, sample collection material such as swabs, cotton, gauze or the like, a pipette, diluent or assay buffer.

22. A method of detecting SIOOB biomarker in a biological sample comprising the steps of:(a) combining the biological sample with a conjugate to form a mixture, wherein the conjugate comprises a detectable label linked to a detection antibody that binds to an antigen on the biomarker, and wherein the conjugate binds with the biomarker to form a detectable complex;(b) contacting the mixture with a capture antibody that binds to the biomarker; and(c) detecting the presence of the biomarker in the sample when the detectable complex binds to the capture antibody and the label in the detectable complex is detected.

23. The method of claim 22, wherein the biological sample is saliva.

24. The method of claim 22 or 23, wherein the detection and capture antibodies are monoclonal antibodies which are different.

25. The method of any one of claims 22-24, wherein the detection and capture antibodies are from different sources.

26. The method of any one of claims 22-25, wherein the detectable label is a metal particulate label.

27. The method of any one of claims 22-26, wherein the detectable label is covalently linked to the detection antibody.

28. The method of claims 26 or 27, wherein the detectable label comprises nanoparticles of about 1 to 100 nm in size.

29. The method of claims 26 or 27, wherein the detectable label comprises nanoshells of 50-150 nm in size.

30. The method of any one of claims 26-29, wherein the detectable label comprises gold.

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