JP2020507622A - S100β and isoforms for neurological condition detection - Google Patents

Abstract

[Selection diagram] None

Description

  This application claims priority from US Provisional Application No. 62 / 455,787, filed February 7, 2017.

  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 assist in biomarker identification.

  S100 calcium binding protein B (S100β) is an S-100 protein family protein. S100 protein is present in the cytoplasmic matrix and nucleus of a wide variety of cells and is involved in the control of many cellular processes such as cell cycle progression and differentiation. Due to its abundance in astrocytes, S100β can be used to diagnose and predict central nervous system (CNS) damage and conditions, including traumatic brain injury, tumors, Alzheimer's disease, Down's syndrome, epilepsy, and amyotrophic lateral sclerosis Have been identified and studied as important markers for However, it has been found that S100β is also expressed in multiple non-neural tissues including adipocytes, skeletal muscle, heart, chondrocytes, and epidermal cells. Therefore, these S100β non-neural sources cannot be excluded as sources of elevation, thus preventing clinically validating S100β diagnostic assays in all conditions.

  S100β is involved in various types of cell activity and cell morphology control, albeit with some unclear cytological function within the cell (Goncalves et al., 2008, Kleindienst & Bullock 2006). However, S100β is known to play a role in toxicity / denaturation and nutrition / repair depending on its concentration when released outside the cell (Goncalves et al., 2008).

  The lack of specificity due to its abundance in many cell types makes it often difficult to use S100β as a biomarker in any situation. For this reason, S100β has not historically functioned as a viable biomarker for many conditions that occur in multiple trauma setting situations. For example, S100β is also present in human melanocytes and is a reliable marker of melanoma malignancy both in living cells and in serum. In addition to S100β, in addition to other diseases causing organ damage such as soft tissue damage, bone damage, and sepsis, non-neural trauma such as liver damage, femoral fracture, and myocardial infarction can be further predicted. Thus, the reliability of S100β as a measure of a particular condition depends on the type of injury and the specificity of the assay.

  Although abundant, S100β tends to be a very delicate biomarker, and is seen as a potential candidate for diagnosing a wide variety of injuries and diseases. One such damage for which S100β has been found to be diagnostic is damage to the CNS. 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 a reference format for the degree of prediction of CNS injury, ie, changes in neuroimaging, cerebrospinal fluid pressure, and other brain molecular markers (NSE and GFAP).

  There are several factors that make S100β the most important candidate for CNS detection. Initially, it has been found that normal S100β levels in patients reliably eliminate major CNS pathologies. In addition, levels have been reported to increase prior to detectable changes in intracerebral pressure, neuroimaging, and neurological findings. In addition, it has been found 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 use of S100β as a biomarker is in the selection of patients with minor head injuries that do not require further neuroradiologic evaluation. Studies comparing CT scans with S100β levels indicate that S100β values less than 0.12 ng / mL are associated with low-risk neuroradiological changes (eg, intracranial hemorrhage or brain swelling) or significant clinical sequelae. Is shown.

  One problem identified is the lack of specificity of S100β due to the widespread presence of S100β. Although originally thought to be specifically present in astroglia in the CNS, S100β is present in other extracerebral cells such as adipocytes, chondrocytes, and bone marrow cells (Donato 2001). Thus, it is a generally accepted theory that S100β is non-specific to the CNS, although many factors suggest that it is the first candidate to detect CNS pathology. As a result, an isoform of S100β that is specific for different organ types, if identified, differentially diagnoses organ-specific disorders, thus providing a very specific S100β assay. Therefore, even in a multiple trauma environment, a lesion-specific S100β assay that is sensitive to specific organ damage is needed but not addressed.

  The present invention provides compositions, antibodies and related antigens, methods, kits, and protein markers that are differentially present in patient samples suffering from multiple disorders and / or diseases alone, or in multiple trauma settings. Provide an in vivo diagnostic process specifically designed for detection. The composition of the invention has SEQ ID NO. 7 and SEQ ID NO. 8 is an isolated nucleic acid sequence that is 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence. These compositions, and the antibodies produced against the compositions, can detect and identify the amount of biomarkers indicative of each trauma type, resulting in a myriad of potential in a subject or multiple trauma environment. Help provide a sensitive, specific, fast, non-invasive method to help diagnose trauma and disease. Measuring these markers in a patient sample, alone or together with other markers depending on the type of disorder, provides information that allows the diagnostician to correlate with a diagnosis of a range of possible disorders.

  The present invention further provides an in vitro diagnostic process and kit for detecting trauma and disease, even if the subject is suffering from multiple trauma prior to measurement of a particular trauma or biomarker.

  Other aspects of the invention are described below.

FIG. 1 shows a 2D gel for detecting the isoform of S100β in serum, wherein the nucleic acid sequences of SEQ ID NO: 7 and SEQ ID NO: 8, which represent the S100β isoform and S100β7-S100β, are 50%, 60%, 70%, and 80%, respectively. , 85%, 90%, 95%, 96%, 97%, 98% or 99% of the captured isolated nucleic acid sequences.

FIG. 2 shows that the nucleic acid sequence of SEQ ID NO: 7 and SEQ ID NO: 8 and 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% The method for generating an antibody against a peptide corresponding to the nucleic acid sequence of exon 2, exon 3, and exon 4 of the isolated S100β cDNA is shown.

FIG. 3 shows the exon sequences of the nucleic acid sequence forms 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).

FIG. 4 shows the positions of the various exons of the complete S100β mRNA. Exon refers to exon 1, exon 2, and exon 3 to the stop codon TGA or exon 4 to the stop codon TGA or TAG.

  The invention is useful in diagnosing and managing an injury or abnormal condition in a subject, where the injury or condition is alone or in combination with another set of injuries or disorders. An isolated nucleic acid alone that is 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of S100β7 or S100β8; Determining the trauma or condition of a patient through measuring biomarkers in a biological sample from a subject, such as, or in combination with each other or one or more additional biomarkers known in the art, Even in trauma patients, it is provided more specifically than previously achievable. The description is 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of S100β7 or S100β8. Directly directed to the first isoform of S100β selected from nucleic acids. Surprisingly, by combining the detection of one or more biomakers from S100β1 to S100β8, multiple traumas can be diagnosed simultaneously and distinguished from others.

  As used herein, the term "diagnosing" refers to identifying the presence or absence of other conditions, such as neurological or trauma or disease. Diagnosis is arbitrarily referred to as the result of an assay in which a particular ratio or proportion of a biomarker is detected or not detected.

  As used herein, a "ratio" is a positive ratio where the level of the target is greater than or the same as the target in a second sample, relative to a known or recognized baseline level. A negative ratio indicates that the level of the target is lower than the target of the second sample or lower compared to a known or recognized baseline level of the same target. A neutral ratio refers to no change in the target biomarker.

  Trauma, as used herein, is a mutation in the integrity or activity of a cell or molecule, activity, level, strength, condition, or other mutation that can be tracked for an event. Examples of trauma include features such as physical, mechanical, chemical, biological, functional, infectious, or other cellular or molecular repair. Illustrative examples of events include biological trauma such as stroke due to physical trauma such as impact or occlusion or leakage of blood vessels. An event refers to any transmission by an infectious agent. One skilled in the art will recognize many equivalent events that are encompassed by the terms trauma or event. Trauma refers to a physical event such as any impact. Trauma may further include diseases or genetic abnormalities such as Parkinson's disease, Alzheimer's disease, ALS, and the like.

  Where a range of values is recited, the range is explicitly included within the range, not just the final value of the range, but also includes the intermediate values of the range, which vary with the last significant digit in the range. By way of example, the range 1 to 4 is intended to include the listed 1 to 2, 1 to 3, 2 to 4, 3 to 4, 1 to 4.

GENERAL The present invention relates to compositions of matter, antibodies and antigens related thereto, methods, kits, and alone suffering from one of several injuries and / or disorders, or in multiple trauma environments. Provided are in vitro diagnostic processes that are particularly used to detect protein markers that are specifically present in patient samples. The composition of the present invention comprises 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% of the nucleic acid sequence of SEQ ID NO: 7 and the nucleic acid sequence of SEQ ID NO: 8. Or as an isolated nucleic acid sequence having 99% identity. These compositions, and the antibodies generated against such compositions, can be used to detect and determine the amount of biomarkers that indicate each type of injury, even in the case of multiple trauma environments, to subject. Help provide a sensitive, specific, rapid, non-invasive method to assist in diagnosing the myriad of injuries and / or disorders that may be present. Measuring these markers in a patient sample, alone or in combination with other markers depending on the type of injury, provides information that allows the diagnostician to correlate with a presumed diagnosis of the degree of injury.

Compositions To analyze a biological sample for the presence of a biomarker and one or more other biomarkers of a particular injury or condition, an antibody-based assay is preferred. Suitable Western blots are described in the Examples section below. For faster analysis (as is important in emergency medical situations), immunosorbent assays (such as ELISA and RIA) and immunoprecipitation assays can be used.

  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 antigens used in these detection methods and devices, they include antibodies produced by the subject's own tissue as an autoimmune response to the natural proteins of cells, tissues, or organs formed therein. It can be a protein or peptide produced for detection. Preferably, these antigens are peptides having the following sequences for each of the defined isoforms from which antibodies are produced, produced or produced.

SEQ ID NO: 1 is public (LOCUS NP_006263 92; CAG46920, aa (molecular weight about 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; general sequence referred to in embl accession CR542123.1) and commonly referred to, or linked to all S100β studies now recognized in the art. Due to the recent discovery of several isoforms heretofore unknown in the art, it is necessary to change the conventional name S100β to S100β1 according to custom within the scientific research community. The peptide, or antibodies raised against the peptide, are recognized in the art as not being specific for a particular injury or disorder. This nucleic acid sequence is known to have the following amino acid sequence as a result of the splicing of exon 1 and exon 2: MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEEIKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMVTTACHEFFEHE

SEQ ID NO: 2 is the first composition identified in U.S. Patent Application S / N: 14 / 293,758, formed as a result of exon 1, exon 2, exon 3 of a newly discovered human nucleic acid sequence, The protein resulting from the translation of the nucleic acid sequence is named S100β2. Protein S100β2 is known to have an estimated molecular weight of 11,295 daltons and is known to have the following amino acid sequence:
MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEEIKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMRRSCKKADSKGLQPSRS

SEQ ID NO: 3 is the second composition formed as a result of exon 1 (truncated) and exon 3 of the human nucleic acid sequence and identified in U.S. Patent Application S / N: 14 / 293,758, The protein resulting from the translation is named S100β3. Peptide S100β3, which is known to have a molecular weight of about 2,750 daltons, is known to have the following amino acid sequence:
MSELEKAMRRSCKKADSKGLQPSRS

SEQ ID NO: 4 is the third composition identified in US Patent Application No. 14 / 293,758, formed as a result of exon 1 (cleavage) and exon 2 of a human nucleic acid sequence, and As a result of the translation, the protein is named S100β4. Peptide S100β4, which is known to have a molecular weight of about 5,950 daltons, is known to have the following amino acid sequence:
MSELEKAMEIKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMVTTACHEFFEHE

  SEQ ID NO: 5 is the fourth of a composition of matter identified in US Patent Application No. 14 / 293,758, formed as a result of exon 1 (cleavage), exon 2 (cleavage # 1) and exon 3. You. The human nucleic acid sequence, and thus the protein as a result of translation of the nucleic acid sequence, is named S100β5. Peptide S100β5, which is known to have a molecular weight of about 6,500 daltons, is known to have the following amino acid sequence: MSELEKAMEIKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMRRSCKKADSKGLQPSRS

  SEQ ID NO: 6: fifth composition identified in US patent application S / N: 14 / 293,758 resulting from exon 1, exon 2 (truncated # 2) and exon 3 of human nucleic acid Yes, and thus the protein resulting from translation of the nucleic acid sequence is termed S100β6. Peptide S100β6, which is known to have a molecular weight of 6007,600 daltons, is known to have the following amino acid sequence: MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEEIKEQERRSCKKADSKGLQPSRS

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

  SEQ ID NO: 8 is a number of newly discovered fifth compositions formed as a result of exon 1, newly discovered exon 2 and exon 4a of the human nucleic acid sequence, and The resulting protein is named S100β8. Peptide S100β8, which has been found to have a molecular weight of 10,281.8 daltons or less, has been found to be at least 10% homologous to the following sequence or said sequence, or to have any portion of said sequence. MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLERWSLPMLPRLVSNFLCSSYPPALASQSAGITETVMQESRQQGLAA

  The above sequence is the S100β isoform found in the human polypeptide sequence, but similar isoforms are rat, mouse, rabbit, cow, Chinese hamster, rhesus monkey, zebrafish, goat, chicken, dog, domestic cat, pig, and human It can be used in other species having the same sequence alignment as S100β. These alternative sequences can be replaced with the inventive processes, methods, assays, and in vitro diagnostic devices described herein. Furthermore, while the discovery of these proteins is a breakthrough in the diagnosis of injury to multiple trauma patients, other compositions of interest are 50%, 60% , 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the antibody sequence raised against any of the peptide sequences represented by the nucleic acid sequence, or rat Antibodies raised against mice, antibodies raised against rabbits, cows, Chinese hamsters, rhesus monkeys, zebrafish, goats, chickens, dogs, domestic cats, pigs, and other similar species of similar sequence alignments It is.

  Antibodies are selectively labeled. One skilled in the art will recognize the numerous labels operable herein. Labels and labeling kits are commercially available. Labels illustratively include fluorescent labels, biotin, peroxidase, radionucleotides, or other labels known in the art. Alternatively, a detection species for another antibody or other compound known in the art is used as a form of detection of the biomarker to which the antibody binds. If the antibody is directly bound to a detectable label, such as an enzyme, fluorophore, radioisotope, etc., the presence of the label is optionally detected by examining the substrate for the detectable label. Alternatively, a detectably labeled secondary antibody that binds to the marker-specific antibody is added to the substrate.

  Due to the low antigenicity of the novel exon 2 and 3a and 4a peptide sequences, a better way to generate mouse responses for generating antibodies was needed. Consequently, in at least one embodiment, a modified novel MAP (multi-antigen peptide) system was used. In such an embodiment, FIG. 2 shows the basis for a new and explicit modification of the modified MAP method.

  The multiple antigen peptide (MAP) dendrimer system has four sections that increase the induction of a stronger immune response, reduce enzymatic degradation, and increase molecular recognition of the antigenic peptide for antibody production. A 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. A lipophilic moiety is attached to the peptide epitope to enhance functionality and immune stimulation. The MAP system is hydrophobic and requires formulation in an emulsion medium that is biocompatible for administration to animals commonly used to generate antibodies. The required emulsion medium is one that does not oxidize methionine, cysteine, and tryptophan within the peptide sequence, but can process their hydrophobic properties.

Biomarker MethodAn exemplary process for detecting the presence or absence of a biomarker alone or in combination in a biological sample includes obtaining a biological sample from a subject, such as a human, and contacting the biological sample with a detectable compound or agent. Detecting the marker to be analyzed, including, for example, antibodies or aptamers, and analyzing the binding of the compound or agent to the sample after washing. Samples that specifically bind to the compound or agent will express the marker being analyzed.

  The invention further provides for comparing the amount of one or more biomarkers to normal levels in an uninjured patient to determine the injury or condition in the subject. It is understood that by selecting additional biomarkers, the type of cells involved in the abnormal organ or physical condition, as well as the type of cell death, can be identified. The practice of the process of the present invention will allow the physician to determine the appropriate therapeutic agent and administer it for optimal benefit to a subject suffering from a particular condition in a multi-traumatic scenario, and to provide other treatments for other measured disorders. Provides a test to help select therapies that can be administered in combination with.

  In vitro techniques for the detection of markers include, by way of example, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, radioassays, Western blots, Southern blots, Northern blots, immunoprecipitations, 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. . In addition, in vivo techniques for detecting the marker include introducing a labeled agent that specifically binds the marker to a biological sample or subject.

  Various permutations of these basic immunoassays are also effective in the present invention. These methods involve a biomarker-specific antibody, as opposed to a sample being immobilized on a substrate, where the substrate binds to a detectable label under conditions that cause the antibody to bind to the labeled marker. Contact with biomarker. The substrate is then contacted with the sample under conditions that allow the analyzed marker to bind to the antibody. A decrease in the amount of detectable label on the substrate after washing indicates that the sample contains a marker.

Although antibodies are preferred for use in the present invention because of their broad characteristics, other suitable agents that specifically bind to a biomarker (eg, peptides, aptamers, or small organic molecules) can be used in the immunoassays described above. Is optionally used in place of an antibody. For example, aptamers that specifically bind to S100β7 and / or one or more other S100β protein isoforms or the same peptide can be used. Aptamers are nucleic acid-based molecules that bind to a specific ligand. Methods for making aptamers with specific binding specificities are described in detail 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.

kit
In yet another aspect, the invention provides a kit that supports the diagnosis of a particular injury, the severity of the injury, subcellular localization and / or a particular disorder, wherein the kit is used to detect a marker of the invention. Can be used. For example, the kit can be used to detect any one or more of the markers described herein, wherein the markers are present in a patient and a sample of a normal subject, respectively. The kits of the present invention have many uses. For example, the kit can be used to distinguish whether a subject has sepsis, bone damage, muscle or tissue damage, or brain damage. In another example, the kits can be used to identify compounds that modulate the expression of one or more markers in an ex vivo or in vivo animal model and determine the efficacy of the treatment.

  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 kits can be prepared from the materials described above, and previous discussion of materials (eg, antibodies, detection reagents, immobilized supports, etc.) is fully applicable to this section and will not be repeated. If desired, the kit may further include a pre-fractionation spin column. In one embodiment, the kit can further include instructions for appropriate operating parameters in the form of a label or a separate insert.

  In one embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the peptide of the invention. These antigens have 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleic acid sequence of SEQ ID NOs: 7-8 It can be any of the peptides represented by the nucleic acid molecule. The diagnostic kit includes a substantially isolated antibody that specifically immunoreacts with the peptide or polynucleotide antigen, and 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 present invention comprises 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of SEQ ID NOS: 7 to 8. Antibodies raised against any of the peptides represented by the nucleic acid molecules. The diagnostic kit includes a substantially isolated antibody that specifically immunoreacts with the peptide or polynucleotide antigen, and 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 present invention comprises 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of SEQ ID NOS: 7 to 8. Antibodies raised against any of the peptides represented by the nucleic acid molecules. In one embodiment, the antibody is a monoclonal or polyclonal antibody raised against a rat, rabbit or human breed of the isoform of S100β described herein. The detection means of the kit includes a labeled secondary monoclonal or polyclonal antibody. Alternatively or additionally, the detecting means comprises a labeled competitor antigen.

 Optionally, the kit further comprises a standard or control information, wherein the test sample is compared to a control information standard to provide a diagnostic amount in which the test amount of the marker detected in the sample is consistent with the diagnosis of the particular injury, the severity of the injury. The degree of subcellular localization, the particular disorder, and / or the effect of treatment on the patient can be determined.

In one embodiment, the kit comprises (a) 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of SEQ ID NO: 7 or SEQ ID NO: 8. And (b) contacting the sample with an adsorbent and detecting the marker or biological sample, thereby comprising instructions for detecting the marker. In some embodiments, the kit can include an eluent (alternatively or in combination with the instructions) or instructions for making the eluent, wherein the combination of the sorbent and the eluent comprises gas phase ion spectroscopy. Allows the detection of markers using the method. Such kits can be prepared from the materials described above, and previous discussions of these materials (eg, probe substrates, sorbents, cleaning solutions, etc.) are fully applicable to this section and will not be repeated.

  In one embodiment, the kit comprises a first substrate having an adsorbent thereon (eg, particles functionalized with the adsorbent), and the first substrate disposed and removably insertable into a gas phase ion spectrometer. And a second substrate on which a simple probe can be formed. Alternatively, the kit may include a single substrate in the form of a removably insertable probe with an adsorbent on a substrate. The kit may further include a pre-fractionation spin column (eg, Cibacron Blue Agarose column, anti-HSA agarose column, size exclusion column, Q-anion exchange spin column, single-stranded DNA column, lectin column, etc.).

  If desired, the kit can further include instructions for appropriate operating parameters in the form of a label or a separate insert. For example, a kit may include standard instructions that inform the consumer how to wash the probe after the sample has contacted the probe. In one embodiment, the kit may include instructions for pre-fractionating the sample to reduce the complexity of proteins in the sample. In one embodiment, the kit may include instructions for automating the fractionation or other method.

Biological Fluids The compositions, processes, methods, and in vitro diagnostic devices of the present invention provide the ability to detect and monitor the levels of proteins or autoantibodies released into the body following injury, allowing clinicians to (1) Determine the severity of the disorder in the patient with the disorder, (2) monitor the patient for the development of signs of the disorder or secondary damage that may trigger these cellular changes, (3) blood, plasma, Testing for these proteins in body fluids, such as serum, CSF, urine, saliva, and sweat, enhances diagnostic capacity by continuously monitoring the progress of damage and the effects of treatment.

  The sample is preferably a biological sample. Preferred examples of biological samples include cells, tissues, cerebrospinal fluid (CSF), artificial CSF, whole blood, serum, plasma, cytoplasm, urine, feces, gastric juice, digestive juice, saliva, nasal or other airway fluid, vagina 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 first flow into the extracellular fluid or space, and ultimately into other fluids in the body, including circulating blood. Through normal bodily functions (such as removing impurities from the kidney), proteins are transferred to other bodily fluids such as urine, sweat, and saliva. Accordingly, other suitable biological samples include, but are not limited to, these cells or body fluids secreted from these cells. It should also be understood that collecting body fluids such as cerebrospinal fluid, blood, plasma, serum, saliva, urine, etc. from a subject is generally less invasive and less traumatic than collecting solid tissue biopsy samples . Therefore, samples that are biological fluids are preferred for use in the present invention.

  Biological samples of CSF, blood, urine, and saliva are collected using conventional collection techniques. For example, a CSF lumbar puncture (LP) 20 gauge introducer needle is inserted and a certain amount of CSF is withdrawn so as not to limit sample collection to the procedures contained herein. For blood, samples are collected by venipuncture of Vacutainer tubes, centrifuged if necessary, and separated into serum and plasma. For urine and saliva, it is desirable to collect the sample while avoiding contamination of the sample with contaminants. All biological samples can be stored in aliquots at -80 ° C for later assays. Surgical techniques for obtaining solid tissue samples are well known in the art. Any suitable biological sample can be obtained from the subject to detect the marker. It should be understood that the methods used herein can be replicated identically for any biological fluid to detect a marker in a sample.

  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 should be understood that variations and modifications can be made without departing from the spirit and scope of the invention. Although the examples are generally directed to the analysis of mammalian tissues, particularly mouse tissues, those of skill in the art will appreciate that similar techniques and other techniques known in the art will readily adapt the examples to other mammals, such as humans. Recognize that it translates into. The reagents set forth 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 are available . Variations within the concept of the invention will be apparent to those skilled in the art.

Example 1

  Three clusters were tested for increased mRNA size (mRNA was converted to cDNA to prevent enzymatic degradation of the RNA sequence). Cluster 1 focused on testing and sequencing all mRNAs, which were approximately 1,000 to 2,000 bases in length, and gave and received hundreds of bases (see histogram). Clusters 2 and 3 were 2,000-3,000 bases and 3,000-6,000 bases, respectively.

Cluster 1's report contained approximately 25,000 to 30,000 potentially unique mRNA sequences, and thus 9,137 page sequences representing proteins, such as: The following is the mRNA sequence of S100β7 (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
CCGCCCAGGACCCGCAGCAGAGACGACGCCTGCAGCAAGGAGACCAGGAAGGGGTGAGAC
AAGGAAGAGGATGTCTGAGCTGGAGAAGGCCATGGTGGCCCTCATCGACGTTTTCCACCA
ATATTCTGGAAGGGAGGGAGACAAGCACAAGCTGAAGAAATCCGAACTCAAGGAGCTCAT
CAACAATGAGCTTTCCCATTTCTTAGAGAGATGGAGTCTCCCTATGTTGCCCAGGCTGGT
CTCAAACTTCCTGTGCTCAAGTTATCCTCCTGCCTTAGCTTCCCAAAGTGCTGGGATTAC
AGGAAATCAAAGAGCAGGAGGTTGTGGACAAAGTCATGGAAACACTGGACAATGATGGAG
ACGGCGAATGTGACTTCCAGGAATTCATGGCCTTTGTTGCCATGGTTACTACTGCCTGCC
ACGAGTTCTTTGAACATGAGTGAGATTAGAAAGCAGCCAAACCTTTCCTGTAACAGAGAC
GGTCATGCAAGAAAGCAGACAGCAAGGGCTTGCAGCCTAGTAGGAGCTGAGCTTTCCAGC
CGTGTTGTAGCTAATTAGGAAGCTTGATTTGCTTTGTGATTGAAAAATTGAAAACCTCTT
TCCAAAGGCTGTTTTAACGGCCTGCATCATTCTTTCTGCTATATTAGGCCTGTGTGTAAG
CTGACTGGCCCCAGGGACTCTTGTTAACAGTAACTTAGGAGTCAGGTCTCAGTGATAAAG
CGTGCACCGTGCAGCCCGCCATGGCCGTGTAGACCCTAACCCGGAGGGAACCCTGACTAC
AGAAATTACCCCGGGGCACCCTTAAAACTTCCACTACCTTTAAAAAACAAAGCCTTATCC
AGCATTATTT

Example 2
ELISA assays show that two have no known history of neuronal damage and six have one or more clinical symptoms of injury or disease resulting from neuronal damage (three with traumatic brain injury, one with stroke 1). Human, Alzheimer's disease 1), and serum from 8 humans. Microplates were coated with antibodies raised against the sequence of SEQ ID NO: 7. Wells were washed. 100 μl of subject serum was added to each well. The microplate was incubated at 37 ° C. for 1 hour and then washed again to remove unbound antibody. Thereafter, the protein-antibody complex was measured. Comparing the levels of protein-antibody complexes formed by the ELISA method, six subjects exhibiting one or more clinical symptoms of a disease or injury resulting from nerve cell damage show that the protein formed by the ELISA method has The antibody matrix concentration was found to be significantly higher than in two intact patients.

Example 3
The test of Example 2 was repeated with antibodies raised against the sequence of SEQ ID NO: 8. ELISA. Similarly, comparing the levels of protein-antibody complexes formed by ELISA, six subjects exhibiting one or more clinical symptoms of a disease or injury resulting from neuronal damage were formed by ELISA. The protein antibody matrix concentration was found to be significantly higher than the two uninjured patients.

Other Embodiments While the foregoing detailed description has presented at least one example embodiment, there are numerous variants. In addition, one or more exemplary embodiments are examples only and do not limit the scope, applicability, or configuration of the described embodiments in any way. It should also be understood that the exemplary embodiments are examples only, and do not limit the scope, applicability, or configuration of the described embodiments in any way. Rather, the foregoing detailed description will provide those skilled in the art with a convenient road map for implementing the exemplary embodiment. Various changes may be made in the function and arrangement of elements without departing from the scope described in the appended claims and their legal equivalents.

The patent documents and publications mentioned in the specification are indicative of the level of those skilled in the art to which the invention pertains. These documents and publications are incorporated herein by reference as if each individual document or publication was specifically and individually incorporated herein by reference.

The foregoing description is illustrative of particular embodiments of the present invention, but is not meant to be limiting on its implementation. The following claims, including all equivalents, 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 (14)

  A composition comprising an amino acid sequence that is 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 7.   Antibodies raised against peptides that are 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 7 A composition comprising:   A composition comprising an amino acid sequence that is 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 7.   Antibodies raised against peptides that are 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 8 A composition comprising: The nucleic acid sequence of S100β7, S100β8 and SEQ ID NO: 7-8 and 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96 of the sample obtained the first time from the subject or the cells of the subject Determining the amount of one or more biomarkers selected from the group consisting of biomarkers that are%, 97%, 98% or 99% identical, and their degradation products;
Comparing the normal level of the biomarker and the amount of the biomarker to determine the damage or condition of the subject,
A method of determining the damage or condition of a subject.   6. The method of claim 5, wherein said sample is cerebrospinal fluid (CSF), blood, plasma, serum, saliva or urine.   6. The method of claim 5, further comprising measuring a second amount of the biomarker a second time to obtain a kinetic profile of the biomarker. (A) a substrate holding a sample isolated from a subject,
(B) An isolated nucleic acid that is 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of S100β7 or S100β8. And one or more agents that specifically interact with one or more biomarkers selected from the group consisting of and their degradation products and their antibodies thereto, and (c) diagnosing a neurological condition in the subject Printed instructions on the reaction of an agent sample or a portion of the sample to perform
A kit for using the method according to claim 5, comprising:   One or more agents is 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NOs: 7-8. The kit according to claim 8, which is an antibody produced against a certain peptide.   An isolated antibody that specifically binds to the amino acid sequence of SEQ ID NOs: 7-8. Produced for a peptide that is 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NOs: 7-8. Contacting said sample with said antibody,
Detecting a protein that binds to the antibody,
A method for detecting the presence of a protein in a sample due to nerve cell damage. Obtaining a sample from the subject's body fluid or cells;
Produced for a peptide that is 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NOs: 7-8. Contacting said sample with said antibody,
Detecting the sample protein bound to the antibody, wherein the presence of the protein bound to the antibody is indicative of a disease or injury that causes nerve cell damage,
In vitro diagnosis of diseases and injuries that cause nerve cell damage. An isolated nucleic acid sequence that is 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NOs: 7-8. Contacting with the sample;
Detecting an autoantibody that binds to the protein,
A method for detecting the presence of autoantibodies derived from nerve cell damage in a sample. Obtaining a body fluid or tissue from the subject;
Contacting the sample with a protein having the amino acid sequence of SEQ ID NOs: 7-8,
Detecting the sample autoantibody, wherein the presence of the autoantibody binding to the protein is indicative of a disease or injury that causes neuronal damage.
In vitro diagnosis of diseases and injuries that cause nerve cell damage.