S100β and isoforms for detection of neurological conditions

Abstract

A method for detecting traumatic brain injury or stroke in a subject in vitro is provided. [Solution] A method comprising measuring the amount of one or more biomarkers associated with traumatic brain injury or stroke in a body fluid sample obtained from a subject suspected of having suffered a traumatic brain injury or stroke, wherein the biomarker is S100β7 or S100β8, and comparing the amount of the measured biomarker with the amount of the biomarker in a healthy subject to determine the subject's injury or condition.

Description

[Technical field]

[0001] This application claims priority from U.S. Provisional Application No. 62 / 455,787, filed February 7, 2017.

[0002] The present invention relates to compositions and antibodies for the detection of biomarkers. In particular, the present invention provides methods, processes, kits, in vivo diagnostic devices, as well as synthetic compositions to aid in biomarker identification. [Background technology]

[0003] S100 calcium-binding protein B (S100β) is a member of the S-100 protein family. S100 proteins are present in the cytosol and nucleus of a wide variety of cells and are involved in the regulation of numerous cellular processes, such as cell cycle progression and differentiation. Due to its abundance in astrocytes, S100β has been identified and studied as an important marker for the diagnosis and prognosis of central nervous system (CNS) injuries and conditions, including traumatic brain injury, tumors, Alzheimer's disease, Down's syndrome, epilepsy, and amyotrophic lateral sclerosis. However, S100β has also been found to be expressed in multiple non-neuronal tissues, including adipocytes, skeletal muscle, heart, chondrocytes, and epidermal cells. Thus, these non-neuronal sources of S100β cannot be excluded as sources of elevation, thus preventing the clinical validation of diagnostic assays for S100β in all conditions.

[0004] Although its cytological function inside the cell remains somewhat unclear, S100β is involved in various types of cellular activities and in the regulation of cell morphology (Goncalves et al. 2008, Kleindienst & Bullock 2006). However, when released outside the cell, S100β is known to play toxic / degenerative and trophic / repair roles depending on the concentration (Goncalves et al. 2008).

[0005] Due to its abundance in many cell types, lack of specificity often makes it difficult to use S100β as a biomarker in all circumstances. For this reason, S100β has historically not served as a viable biomarker for many conditions occurring in the context of multiple traumatic environments. For example, S100β is also present in human melanocytes and is a reliable marker of melanoma malignancy both in vivo and in serum. S100β can also predict other diseases that cause soft tissue injury, bone injury, and organ injury such as sepsis, as well as non-neural trauma such as liver injury, femur fracture, and myocardial infarction. Thus, the reliability of S100β as a measure of a particular condition depends on the type of injury and the specificity of the assay.

[0006] Although abundant, S100β tends to be a very sensitive biomarker and is seen as a possible candidate for diagnosing many types of injuries and diseases. One such injury where S100β has been found to be diagnostically useful is CNS injury. For example, elevated S100β levels have been found to reflect the presence of neuropathological conditions TBI or neurodegenerative diseases. Its potential clinical use has been demonstrated by reference modalities for predicting the extent of CNS injury, namely neuroimaging, cerebrospinal fluid pressure, and changes in other brain molecular markers (NSE and GFAP).

[0007] Several factors make S100β the prime candidate for CNS detection. First, we know that normal S100β levels in patients reliably exclude major CNS pathology. Furthermore, it has been reported that levels are elevated prior to detectable changes in intracranial pressure, neuroimaging, and neurological examination findings. Furthermore, we know that S100β levels are elevated prior to epilepsy, suggesting that blood-brain barrier (BBB) ​​leakage may be an early event in the development of epilepsy. An important application of S100β as a biomarker is in the selection of patients with mild head injury who do not require further neuroradiological evaluation. Studies comparing CT scans with S100β levels have shown that S100β levels below 0.12 ng / mL are associated with low-risk neuroradiological changes (e.g., intracranial hemorrhage or brain swelling) or important clinical sequelae.

[0008] One problem identified is the lack of specificity of S100β due to its widespread presence. Originally thought to be specific to astroglia within the CNS, S100β is present in other extracerebral cells such as adipocytes, chondrocytes, and bone marrow cells (Donato 2001). Thus, the generally accepted theory is that S100β is non-specific to the CNS, even though many factors suggest it would be a prime candidate to detect CNS pathology. As a result, isoforms of S100β that are specific to different organ types, if identified, would provide a highly specific S100β assay, as they would differentially diagnose organ-specific disorders. Thus, there is a need, but an unmet need, for injury-specific S100β assays that are sensitive to specific organ injury, even in a polytrauma setting. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention provides compositions, antibodies and associated antigens, methods, kits, and in vivo diagnostic processes specifically designed to detect protein markers differentially present in patient samples suffering from multiple disorders and / or diseases alone or in the setting of multiple trauma. The compositions of the invention are isolated nucleic acid sequences that are 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences of SEQ ID NO.7 and SEQ ID NO.8. These compositions, and antibodies raised against the compositions, help provide a sensitive, specific, rapid, non-invasive method to aid in the diagnosis of a myriad of injuries and diseases that may be present in a subject or in the setting of multiple trauma, by detecting and identifying the amount of biomarkers indicative of each trauma type. Measuring these markers alone or in conjunction with other markers depending on the type of injury in patient samples provides information that allows diagnosticians to correlate the diagnosis of a range of possible disorders.

[0010] The present invention further provides in vitro diagnostic processes and kits for detecting injury and disease, even when a subject has suffered specific injury or multiple injuries prior to measurement of biomarkers.

[0011] Other aspects of the invention are described below. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows a 2D gel detecting S100β isoforms in serum, showing capture of isolated nucleic acid sequences that are 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences of SEQ ID NO:7 and SEQ ID NO:8, which represent the S100β isoforms, S100β7-S100β, respectively.

[0013] [Diagram 2]FIG. 2 shows a method for generating antibodies against peptides corresponding to the nucleic acid sequences of exon 2, exon 3, and exon 4 of isolated S100β cDNA having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the nucleic acid sequences of SEQ ID NO:7 and SEQ ID NO:8.

[0014] [Diagram 3] Figure 3 shows the exon sequences in the nucleic acid sequence form of SEQ ID NO: 7 and SEQ ID NO: 8. The identified isoforms include exon 1, exon 2 and a portion of exon 3 (S100B7) or exon 4 (S100B8) that complete the sequence of the isoform up to the stop codon (TGA, TAG nucleic acid sequence).

[0015] [Figure 4] Figure 4 shows the location of the various exons in the complete S100β mRNA, indicating exon 1, exon 2, and exon 3 until the stop codon TGA or exon 4 until the stop codon TGA or TAG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention is useful in the diagnosis and management of injury or abnormal conditions in a subject, whether the injury or condition is alone or in combination with a series of other injuries or disorders. Through measuring biomarkers in a biological sample from a subject, such as isolated nucleic acids that are 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of S100β7 or S100β8 alone, or in combination with each other or one or more additional biomarkers known in the art, determination of the injury or condition of the patient is provided with more specificity than previously achievable, even in multi-injured patients. The present description is directed to the first isoform of S100β selected from isolated nucleic acids that are 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of S100β7 or S100β8. Surprisingly, by combining detection of one or more biomarkers from S100β1 to S100β8, multiple lesions can be diagnosed simultaneously and differentiated from one another.

[0017] The term "diagnosing" as used herein means distinguishing the presence or absence of a neurological or other condition, such as trauma or disease. Diagnosis may optionally refer to an assay result in which a particular ratio or proportion of a biomarker is detected or not detected.

[0018] As used herein, "ratio" refers to a positive ratio of the level of the target to the target in the second sample or to the known or recognized baseline level of the same target. A negative ratio refers to a lower level of the target in the second sample or to the known or recognized baseline level of the same target. A neutral ratio refers to no change in the target biomarker.

[0019] Trauma, as used herein, is a change in the integrity, activity, level, strength, state, or other change that can be traced to an event. Examples of trauma are physical, mechanical, chemical, biological, functional, infectious, or other cellular or molecular repair characteristics. Examples of events are physical trauma, such as an impact, or biological abnormalities, such as a stroke caused by a blocked or leaking blood vessel. An event refers to any infection by an infectious agent. Those skilled in the art will recognize many equivalent events that are encompassed by the terms trauma or event. Trauma refers to any physical event, such as an impact. Trauma may further include diseases or genetic abnormalities, such as Parkinson's disease, Alzheimer's disease, ALS, etc.

[0020] When a range of values ​​is listed, the range includes not only the end values ​​of the range of values ​​but also intermediate values ​​that are expressly included within the range and are varied by the last significant digit of the range. By way of example, a range of 1 to 4 is intended to include 1 to 2, 1 to 3, 2 to 4, 3 to 4, 1 to 4 as recited.

[0021] General matters The present invention provides compositions of matter, antibodies and associated antigens, methods, kits, and in vitro diagnostic processes that are specifically used to detect protein markers specifically present in samples from patients suffering from one of several injuries and / or disorders alone or in multiple traumatic circumstances. The compositions of the present invention are identified as isolated nucleic acid sequences having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleic acid sequences of SEQ ID NO: 7 and SEQ ID NO: 8. These compositions, and antibodies generated against such compositions, help provide a sensitive, specific, rapid, non-invasive method to detect and determine the amount of biomarkers indicative of each injury type, even when multiple traumatic circumstances occur, to aid in the diagnosis of the myriad injuries and / or disorders that may be present in a subject. Measuring these markers in patient samples alone or in combination with other markers depending on the injury type provides information that a diagnostician can correlate with a probable diagnosis of the extent of the injury.

[0022] composition To analyze a biological sample for the presence of the biomarker and one or more other biomarkers of a particular injury or condition, antibody-based assays are preferred. A suitable Western blot method is described in the Examples section below. For more rapid analysis (such as may be important in emergency medical situations), immunosorbent assays (such as ELISA and RIA) and immunoprecipitation assays can be used.

[0023] The reagents described herein can be any antibody against a protein, peptide sequence or any antigen for detecting antibodies formed as part of an autoimmune response in a subject. In particular, in the case of the antigens used in these detection methods and devices, they can be proteins or peptides generated to detect antibodies produced by the subject's own tissues as an autoimmune response against native proteins of cells, tissues, or organs formed within. Preferably, these antigens are peptides with the following sequences for each of the defined isoforms to which the antibodies are generated, generated, or will be generated:

[0024] SEQ ID NO: 1 is a publicly recognized (LOCUS NP_006263 92; CAG46920, aa (molecular weight approximately 10,713 daltons) linear, DEFINITION protein S100β [Homo sapiens], ACCESSION NP_006263 VERSION NP_006263.1 GI:5454034 or GI:4957424 DBSOURCE REFSEQ: Accession NM_006272.2; embl Accession CR542123.1) common sequence that is commonly referred to or linked to all S100β studies currently recognized in the art. Due to the recent discovery of multiple isoforms previously unknown in the art, the traditional name S100β must necessarily be changed to S100β1, in accordance with convention within the scientific research community. It is recognized in the art that this peptide, or antibodies raised against this peptide, are not specific for any particular injury or disorder. This nucleic acid sequence is known to have the following amino acid sequence, as a result of splicing of exons 1 and 2: MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEEIKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMVTTACHEFFEHE

[0025] SEQ ID NO:2 is the original composition identified in US Patent Application S / N:14 / 293,758, which is formed as a result of exon 1, exon 2, and exon 3 of a newly discovered human nucleic acid sequence, and thus the protein resulting from the translation of the nucleic acid sequence is named S100β2. The protein S100β2 is found to have a predicted molecular weight of 11,295 daltons and is found to have the following amino acid sequence: MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEEIKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMRRSCKKADSKGLQPSRS

[0026] SEQ ID NO:3 is a second composition identified in U.S. Patent Application S / N:14 / 293,758, formed as a result of exon 1 (truncated) and exon 3 of the human nucleic acid sequence, and the protein resulting from translation of the nucleic acid sequence is designated S100β3. The peptide S100β3, found to have a molecular weight of approximately 2,750 daltons, has been found to have the following amino acid sequence: MSELEKAMRRSCKKADSKGLQPSRS

[0027] SEQ ID NO:4 is the third composition identified in US Patent Application Serial No. 14 / 293,758 that is formed as a result of exon 1 (truncated) and exon 2 of the human nucleic acid sequence, and thus as a result of translation of the nucleic acid sequence, a protein is designated S100β4. The peptide S100β4, which is found to have a molecular weight of approximately 5,950 daltons, has been found to have the following amino acid sequence: MSELEKAMEIKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMVTTACHEFFEHE

[0028] SEQ ID NO:5 is the fourth of the compositions of matter identified in US Patent Application Serial No. 14 / 293,758 and is formed as a result of exon 1 (truncated), exon 2 (truncated #1) and exon 3. The human nucleic acid sequence and therefore the protein resulting from translation of the nucleic acid sequence is designated S100β5. Peptide S100β5, known to have a molecular weight of approximately 6,500 daltons, has been found to have the following amino acid sequence: MSELEKAMEIKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMRRSCKKADSKGLQPSRS

[0029] SEQ ID NO: 6: The fifth composition identified in US Patent Application S / N: 14 / 293,758 formed as a result of exon 1, exon 2 (truncated #2) and exon 3 of human nucleic acid, and therefore the protein resulting from translation of the nucleic acid sequence is named S100β6. Peptide S100β6, known to have a molecular weight of ∼7,600 daltons, has been found to have the following amino acid sequence: MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEEIKEQERRSCKKADSKGLQPSRS

[0030] SEQ ID NO:7 is the first of several newly discovered compositions formed as a result of exon 1, the newly discovered exon 2, and exon 3b of the human nucleic acid sequence, and therefore the protein resulting from translation of the nucleic acid sequence is named S100β7. The molecular weight of peptide S100β7 has been determined to be approximately 10,342.75 daltons and has been found to have the following sequence or at least 10% homology to the above sequences, or a portion of any of the above sequences: MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLERWSLPMLPRLVSNFLCSSYPPALASQSAGITGNQRAGGCGQSHGNTGQ

[0031] SEQ ID NO:8 is some newly discovered fifth composition formed as a result of exon 1, newly discovered exon 2 and exon 4a of the human nucleic acid sequence, and therefore the protein resulting from translation of the nucleic acid sequence is named S100β8. Peptide S100β8, found to have a molecular weight of less than or equal to 10,281.8 daltons, has been found to have the following sequence or at least 10% homology thereto, or any portion of said sequence: MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLERWSLPMLPRLVSNFLCSSYPPALASQSAGITETVMQESRQQGLAA

[0032] The above sequences are S100β isoforms found in human polypeptide sequences, however similar isoforms are available in rat, mouse, rabbit, cow, Chinese hamster, rhesus monkey, zebrafish, goat, chicken, dog, domestic cat, pig, and other species with similar sequence alignments to human S100β. These alternative sequences may be substituted in the processes, methods, assays, and in vitro diagnostic devices of the invention described herein. Additionally, while the discovery of these proteins represents a breakthrough in the diagnosis of injuries in multiple trauma patients, other compositions of interest are antibodies raised against any of the peptide sequences represented by nucleic acid sequences that are 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences of SEQ ID NOs: 7-8, or antibodies raised against rats, mice, rabbits, cows, Chinese hamsters, rhesus monkeys, zebrafish, goats, chickens, dogs, domestic cats, pigs, and other similar species of similar sequence alignments.

[0033] The antibody is optionally labeled. Those skilled in the art will recognize numerous labels that can be used herein. Labels and labeling kits are commercially available. Exemplary labels include fluorescent labels, biotin, peroxidase, radioactive nucleotides, or other labels known in the art. Alternatively, a detection species of another antibody or other compound known in the art is used as a form of detection of the biomarker that the antibody binds. If the antibody is directly bound to a detectable label, such as an enzyme, fluorophore, or radioisotope, the presence of the label is optionally detected by examining the substrate of the detectable label. Alternatively, a detectably labeled secondary antibody that binds to the marker-specific antibody is added to the substrate.

[0034] Due to the low antigenicity of the novel exon 2 and 3a and 4a peptide sequences, a better method of generating mouse responses to generate antibodies was needed. As a result, in at least one embodiment, a novel modified MAP (multiple antigen peptide) system was used. In such an embodiment, Figure 2 shows the basis for the novel and distinct modifications of the modified MAP method.

[0035] The multiple antigen peptide (MAP) dendrimer system has four sections that increase the induction of stronger immune responses, reduce enzymatic degradation, and enhance molecular recognition to antigen peptides for antibody generation. The MAP core, consisting of a branched lysine core scaffold, has a naked cell-penetrating peptide attached at one end and several copies of the peptide epitope attached at the other end. Lipophilic moieties are attached to the peptide epitopes to enhance functionality and immune stimulation. MAP systems are hydrophobic and require formulation in an emulsion vehicle that is biocompatible for administration to animals typically used to generate antibodies. The required emulsion vehicle is one that does not oxidize methionine, cysteine, and tryptophan within the peptide sequence but can handle their hydrophobic character.

[0036] Biomarker Methods An exemplary process for detecting the presence or absence of biomarkers, alone or in combination, in a biological sample includes obtaining a biological sample from a subject, such as a human, contacting the biological sample with a detectable compound or agent, detecting the marker to be analyzed, illustratively including an antibody or aptamer, and analyzing binding of the compound or agent to the sample after washing. Samples that specifically bind to the compound or agent will express the marker under analysis.

[0037] The invention further provides for the step of comparing the amount of one or more biomarkers to normal levels in non-injured patients to determine the injury or condition of the subject. It is understood that by selecting additional biomarkers, the type of cells involved in the abnormal organ or body condition, as well as the type of cell death, can be identified. Implementation of the process of the invention provides a test that helps a physician determine the appropriate therapeutic agent and select a treatment that can be administered for optimal benefit of a subject suffering from a particular condition in a polytrauma scenario, and that can be administered in combination with other treatments for other measured disorders.

[0038] In vitro techniques for detection of a marker include, by way of example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, radiometric assay, Western blot, Southern blot, Northern blot, immunoprecipitation, immunofluorescence, mass spectrometry, RT-PCR, PCR, liquid chromatography, high performance liquid chromatography, enzyme activity assays, cell assays, positron emission tomography, mass spectrometry, combinations thereof, or other techniques known in the art. Additionally, in vivo techniques for detecting a marker include introducing a labeling agent that specifically binds the marker to a biological sample or subject.

[0039] Various permutations of these basic immunoassay methods are also useful in the present invention. These methods include a biomarker-specific antibody, as opposed to a sample immobilized on a substrate, and the substrate is contacted with a biomarker conjugated with a detectable label under conditions that cause the antibody to bind to the labeled marker. The substrate is then contacted with the sample under conditions that allow the marker being analyzed to bind to the antibody. A decrease in the amount of detectable label on the substrate after washing indicates that the sample contains the marker.

[0040] Although antibodies are preferred for use in the present invention due to their broad characteristics, other suitable agents that specifically bind to biomarkers (e.g., peptides, aptamers, or small organic molecules) are optionally used in place of antibodies in the above immunoassays. For example, S100β7 and / or one or more other S100β protein isoforms or aptamers that specifically bind to the same peptides can be used. Aptamers are nucleic acid-based molecules that bind to specific ligands. Methods for generating aptamers with specific binding specificities are detailed in U.S. Patent Nos. 5,475,096, 5,670,637, 5,696,249, 5,270,163, 5,707,796, 5,595,877, 5,660,985, 5,567,588, 5,683,867, 5,637,459, and 6,011,020.

[0041] kit In yet another aspect, the present invention provides kits to aid in the diagnosis of a particular injury, injury severity, subcellular localization, and / or a particular disorder, which kits can be used to detect the markers of the present invention. For example, the kits can be used to detect any one or more markers described herein, which are present in samples from patients and normal subjects, respectively. The kits of the present invention have many uses. For example, the kits can be used to distinguish whether a subject has sepsis, bone injury, muscle or tissue injury, or brain injury. In another example, the kits can be used to identify compounds that modulate the expression of one or more markers in an in vitro or in vivo animal model to determine the efficacy of treatment.

[0042] In one embodiment, the kit comprises (a) an antibody that specifically binds to the marker (capture agent), and (b) a detection agent for detecting the amount of the marker. Such a kit can be prepared from the materials described above, and the previous discussion of materials (e.g., antibodies, detection reagents, immobilization supports, etc.) is fully applicable to this section and will not be repeated. Optionally, the kit may further comprise a pre-fractionation spin column. In one embodiment, the kit may further comprise instructions for appropriate operating parameters in the form of a label or separate insert.

[0043] In one embodiment, the invention includes a diagnostic kit for use in screening sera containing antigens of the peptides of the invention. These antigens can be any of the peptides represented by nucleic acid molecules having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleic acid sequences of SEQ ID NOs: 7-8. The diagnostic kit includes a substantially isolated antibody that specifically immunoreacts with the peptide or polynucleotide antigen and a means for detecting binding of the polynucleotide or peptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. The antibody used in the kit of the invention is an antibody raised against any of the peptides represented by nucleic acid molecules having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 7-8. The diagnostic kit includes a substantially isolated antibody that specifically immunoreacts with the peptide or polynucleotide antigen and a means for detecting binding of the polynucleotide or peptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. The antibodies used in the kits of the invention are antibodies raised against any of the peptides represented by nucleic acid molecules having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of SEQ ID NO: 7-8. In one embodiment, the antibodies are monoclonal or polyclonal antibodies raised against rat, rabbit or human varieties of the S100β isoforms described herein. The detection means of the kit includes a labeled secondary monoclonal or polyclonal antibody. Alternatively, or in addition, the detection means includes a labeled competing antigen.

[0044] Optionally, the kit can further include a standard or control information so that the test sample can be compared to the control information standard to determine whether the test amount of the marker detected in the sample is a diagnostic amount consistent with a diagnosis of a particular injury, severity of injury, subcellular localization, particular disorder, and / or effect of treatment for the patient.

[0045] In one embodiment, the kit includes (a) at least one agent for detecting a biomarker of a nucleic acid molecule having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of SEQ ID NO: 7 or SEQ ID NO: 8, and (b) instructions for detecting the marker by contacting the sample with an adsorbent and detecting the marker or biological sample. In some embodiments, the kit can include an eluent (alternatively or in combination with the instructions) or instructions for creating an eluent, where the combination of the adsorbent and the eluent allows for detection of the marker using gas phase ion spectroscopy. Such a kit can be prepared from the materials described above, and the previous discussion of these materials (e.g., probe substrate, adsorbent, wash solution, etc.) is fully applicable to this section and will not be repeated.

[0046] In one embodiment, the kit includes a first substrate having an adsorbent thereon (e.g., particles functionalized with an adsorbent) and a second substrate on which the first substrate can be positioned to form a removably insertable probe into a gas phase ion spectrometer. Alternatively, the kit may include a single substrate in the form of a removably insertable probe with an adsorbent on the substrate. The kit may further include a pre-fractionation spin column (e.g., a cibacron blue agarose column, an anti-HSA agarose column, a size exclusion column, a Q-anion exchange spin column, a single stranded DNA column, a lectin column, etc.).

[0047] If desired, the kit may further include instructions for appropriate operating parameters in the form of a label or separate insert. For example, the kit may include standard instructions informing the consumer how to wash the probe after the sample has contacted it. In one embodiment, the kit may include instructions for pre-fractionating the sample to reduce the protein complexity in the sample. In one embodiment, the kit may include instructions for automating the fractionation or other process.

[0048] Biological fluids The compositions, processes, methods, and in vitro diagnostic devices of the present invention provide the ability to detect and monitor levels of proteins or autoantibodies released in the body following injury, enhancing diagnostic capabilities to enable clinicians to (1) determine the severity of injury in patients with multiple disorders, (2) monitor patients for developing signs of injury or secondary injury that may induce these cellular changes, and (3) continually monitor the progression of injury and the effectiveness of treatment by testing for these proteins in bodily fluids such as blood, plasma, serum, CSF, urine, saliva, sweat, etc.

[0049] The sample is preferably a biological sample. Preferred examples of biological samples are cells, tissues, cerebrospinal fluid (CSF), artificial CSF, whole blood, serum, plasma, cytoplasmic fluid, urine, feces, gastric fluid, digestive fluid, saliva, nasal or other airway fluid, vaginal fluid, semen, buffered saline, saline, water, or other biological fluids recognized in the art. After injury, the cell membrane of any muscle, tissue, or organ is compromised, and its constituent proteins are first released into the extracellular fluid or space, and eventually into other fluids in the body, including circulating blood. Normal bodily functions (such as kidney removal of impurities) cause the proteins to migrate into other bodily fluids, such as urine, sweat, saliva, etc. Thus, other suitable biological samples include, but are not limited to, these cells or bodily fluids secreted by these cells. It is also understood that obtaining bodily fluids, such as cerebrospinal fluid, blood, plasma, serum, saliva, urine, etc., from a subject is generally less invasive and less traumatic than obtaining a solid tissue biopsy sample. Thus, samples that are biological fluids are preferred for use in the present invention.

[0050] Biological samples of CSF, blood, urine, and saliva are collected using conventional collection techniques. For example, and not to limit sample collection to the procedures included herein, a CSF lumbar puncture (LP) involves inserting a 20-gauge introducer needle and withdrawing a volume of CSF. For blood, samples are collected by venipuncture in Vacutainer tubes and, if necessary, centrifuged to separate serum and plasma. For urine and saliva, it is desirable to collect samples while avoiding the introduction of contaminants into the sample. All biological samples can be stored in aliquots at -80°C for later assay. Surgical techniques for obtaining solid tissue samples are well known in the art. Any suitable biological sample can be obtained from a subject to detect markers. It is understood that the methods used herein can be identically reproduced for any biological fluid to detect markers in the sample. EXAMPLES

[0051] Various aspects of the present invention are illustrated by the following non-limiting examples. These examples are for illustrative purposes and do not limit the practice of the present invention. It is understood that variations and modifications can be made without departing from the spirit and scope of the present invention. Although the examples are generally directed to the analysis of mammalian tissues, particularly mouse tissues, those skilled in the art will recognize that similar techniques and other techniques known in the art will readily translate the examples to other mammals, such as humans. The reagents presented herein are generally cross-reactive between mammalian species, or alternative reagents with similar properties are commercially available, and those skilled in the art will readily understand where such reagents can be obtained. Variations within the concept of the present invention will be apparent to those skilled in the art.

[0052] Example 1

[0053] Three clusters were tested for increasing mRNA size (mRNA was converted to cDNA to prevent enzymatic degradation of the RNA sequence): Cluster 1 focused on testing and sequencing total mRNA that was approximately 1,000 to 2,000 bases in length, give or take a few hundred bases (see histogram); Clusters 2 and 3 were 2,000-3,000 bases and 3,000-6,000 bases, respectively.

[0054] The Cluster 1 report contained 9,137 pages of sequences representing approximately 25,000 to 30,000 potentially unique mRNA sequences and therefore proteins, such as the following: Below is the mRNA sequence for S100β7 (the 13,714th sequence tested and sequenced in Cluster 1): >c13714 / f2p4 / 850 isoform=c13714, full length coverage=2, non-full length coverage=4, isoform length=850 CCGCCCAGGACCCGCAGCAGGACGACGCCTGCAGCAAGGAGACCAGGAAGGGGTGAGAC AAGGAAGAGGATGTCTGAGCTGGAGAAGGCCATGGTGCCCTCATCGACGTTTTCCACCA ATATTCTGGAAGGGAGGGAGACAAGCACAAGCTGAAGAAATCCGAACTCAAGGAGCTCAT CAACAATGAGCTTTCCCATTTCTTAGAGAGATGGAGTCTCCCTATGTTGCCCAGGCTGGT CTCAAACTTCCTGTGCTCAAGTTATCCTCCTGCCTTAGCTTCCCAAAGTGCTGGGATTAC AGGAAATCAAAGAGCAGGAGGTTGTGGACAAAGTCATGGAAACACTGGACAATGATGGAG ACGGCGAATGTGACTTCCAGGAATTCATGGCCTTTGTTGCCATGGTTACTACTGCCTGCC ACGAGTTCTTTGAACATGAGTGAGATTAGAAAGCAGCCAAACCTTTCCTGTAACAGAGAC GGTCATGCAAGAAAGCAGACAGCAAGGGCTTGCAGCCTAGTAGGAGCTGAGCTTTCCAGC CGGTGTTGTAGCTAATTAGGAAGCTTGATTTGCTTTGTGATTGAAAAATTGAAAACCTCTT TCCAAAGGCTGTTTTAACGGCCTGCATCATTCTTTCTGCTATATTAGGCCTGTGTGTAAG CTGACTGGCCCCAGGGACTCTTGTTACAGTAACTTAGGAGTCAGGTCTCAGTGATAAAG CGTGCACCGTGCAGCCCCGCCATGGCCGTGTAGACCCTAACCCGGAGGGAACCCTGACTAC AGAAATTACCCCGGGGCACCCTTAAAACTTCCACTACCTTTAAAAAACAAAGCCTTATCC AGCATTATTTT

[0055] Example 2 The ELISA assay was performed on sera from eight humans, two with unknown history of neuronal injury and six with one or more clinical symptoms of injury or disease resulting from neuronal injury (three with traumatic brain injury, one with stroke, and one with Alzheimer's disease). Microplates were coated with antibodies raised against the sequence of SEQ ID NO: 7. The wells were washed. 100 μl of subject serum was added to each well. The microplates were incubated at 37°C for 1 hour and then washed again to remove unbound antibody. Protein-antibody complexes were then measured. Comparing the levels of protein-antibody complexes formed by the ELISA method, it was found that the six subjects with one or more clinical symptoms of disease or injury resulting from neuronal injury had significantly higher concentrations of protein-antibody matrix formed by the ELISA method than the two uninjured patients.

[0056] Example 3 The test of Example 2 was repeated with antibodies raised against the sequence of SEQ ID NO: 8. ELISA. Similarly, when the levels of protein-antibody complexes formed were compared by ELISA, it was found that six subjects exhibiting one or more clinical symptoms of disease or injury resulting from neuronal cell injury had significantly higher protein-antibody matrix concentrations formed by ELISA than the two uninjured patients.

[0057] Other embodiments Although at least one exemplary embodiment has been presented in the foregoing detailed description, numerous variations exist. It should also be understood that the exemplary embodiment or embodiments are merely examples and are in no way intended to limit the scope, applicability, or configuration of the described embodiments. It should also be understood that the exemplary embodiments are merely examples and are in no way intended to limit the scope, applicability, or configuration of the described embodiments. Rather, the foregoing detailed description provides those skilled in the art with a convenient path for implementing the exemplary embodiments. Various changes may be made in the function and arrangement of elements without departing from the scope set forth in the appended claims and their legal equivalents.

[0058] The patent documents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are hereby incorporated by reference to the same extent as if each individual document or publication was specifically and individually incorporated by reference herein.

[0059] The foregoing description is illustrative of particular embodiments of the present invention, but is not meant to be a limitation on its practice. The following claims, including all equivalents thereof, are intended to define the scope of the invention. Sequence Listing SEQ ID NO:1: MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEEIKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMVTTACHEFFEHE SEQ ID NO:2: MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEEIKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMRRSCKKADSKGLQPSRS SEQ ID NO:3: MSELEKAMRRSCKKADSKGLQPSRS SEQ ID NO:4: MSELEKAMEIKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMVTTACHEFFEHE SEQ ID NO:5: MSELEKAMEIKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMRRSCKKADSKGLQPSRS SEQ ID NO:6: MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEEIKEQERRSCKKADSKGLQPSRS SEQ ID NO: 7: MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLERWSLPMLPRLVSNFLCSSYPPALASQSAGITGNQRAGGCGQSHGNTGQ SEQ ID NO:8: MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLERWSLPMLPRLVSNFLCSSYPPALASQSAGITETVMQESRQQGLAA

Claims

1. 1. A method for aiding in the ex vivo detection of traumatic brain injury or stroke in a subject, comprising: measuring the amount of one or more biomarkers associated with traumatic brain injury or stroke in a bodily fluid sample obtained from a subject suspected of having suffered a traumatic brain injury or stroke, the biomarker being S100β7 or S100β8; comparing the amount of the measured biomarker to the amount of the biomarker in a healthy individual to determine the injury or condition of the subject; The method, wherein the body fluid sample is cerebrospinal fluid (CSF), blood, plasma, serum, saliva or urine.

2. 10. The method of claim 1, further comprising measuring a second amount of the biomarker a second time to obtain a kinetic profile of the biomarker.

3. 1. A method for aiding in the ex vivo detection of traumatic brain injury or stroke in a subject, comprising: contacting a body fluid sample obtained from a subject suspected of having suffered a traumatic brain injury or stroke with S100β7 protein or S100β8 protein; The method includes analyzing the sample to determine whether the S100β7 protein or the S100β8 protein is bound to one or more antibodies, wherein the bodily fluid sample is cerebrospinal fluid (CSF), blood, plasma, serum, saliva, or urine.

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