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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • C—CHEMISTRY; METALLURGY
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders

Definitions

• the present invention relates to compositions of matter and antibodies for the detection of biomarkers. More particularly, the present invention provides for synthetic compositions of matter in addition to methods, processes, kits and in vitro diagnostic devices to assist with biomarker identification.
• S I 00 calcium-binding protein B (S lOOp) is a protein of the S-100 protein family. SI 00 proteins are localized in the cytoplasm and nucleus of a wide range of cells and are involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. Due to its prevalence in astrocytes, S lOOp has been heavily studied and identified as an important marker for diagnosing or predicting injuries and conditions of the central nervous system (CNS) including traumatic brain injury, neoplasia, Alzheimer' s disease, Down' s syndrome, epilepsy, and amyotrophic lateral sclerosis.
• CNS central nervous system
• S lOOp is expressed in several non-neuronal tissues, including adipose, skeletal muscle, cardiac, chondrocytes and epidermal cells. Therefore, these non-neuronal sources of S lOOp cannot be excluded as sources of elevation, thus precluding a clinical validation of an SlOOp diagnostic assay for any condition.
• SlOOp As a biomarker of any condition due to its lack of specificity. For this reason, SlOOp historically fails as a viable biomarker for many conditions which occur in a multi -trauma setting. For example, SlOOp is also present in human melanocytes, is a reliable marker for melanoma malignancy both in bioptic tissue and in serum. In addition, SlOOp has further been predictive of non-neuronal traumas, such as liver injury, femoral fracture, and myocardial infarction, in addition to soft tissue injuries, bone damage, and other diseases causing organ injury such as sepsis. Thus, the reliability of SlOOp as a measure of any particular condition depends upon the type of injury and the specificity of the assay.
• SlOOp tends to be a very sensitive biomarker, and is often still looked at as a potential candidate for diagnosing many different types of injuries and disorders.
• One such injury that SlOOp has been found to help diagnose is injuries to the CNS.
• elevated S 100B levels have been found to reflect presence of neuropathological conditions TBI or neurodegenerative diseases.
• Its potential clinical use is substantiated by standard modalities for prognosticating the extent of CNS damage: alterations in neuroimaging, cerebrospinal pressure, and other brain molecular markers (NSE and GFAP).
• SlOOp a prime candidate for CNS detection.
• SlOOp levels in patients reliably exclude major CNS pathology.
• levels have been reported to rise prior to detectable changes in intracerebral pressure, neuroimaging, and neurological examination findings.
• BBB blood-brain barrier
• SlOOp levels are elevated before seizures suggesting blood-brain barrier (BBB) leakage may be early event in seizure development.
• An important application for the use of SlOOp as a biomarker is selection of patients with minor head injury who need no further neuroradiological evaluation.
• Studies comparing CT scans and S lOOp levels demonstrate S lOOp values below 0.12 ng/mL are associated with low risk neuroradiological changes (e.g. intracranial hemorrhage or brain swelling) or significant clinical sequelae.
• S lOOp is non-specific to the CNS, despite many factors indicating that it is a prime candidate for detecting CNS pathologies.
• certain isoforms exist of S 100p which are specific to different organ types, that if identified, may differentially diagnose organ specific injuries, and thus providing a highly specific S lOOp assay.
• the present invention provides compositions of matter, antibodies and antigens related thereto, methods, kits and in vitro diagnostic processes specifically designed to detect protein markers that are differentially present in the samples of patients suffering from one of several injury and/or disorders alone or in a multi-trauma environment.
• inventive compositions of matter are those isolated nucleic acid sequence having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence of SEQ ID NO. 7 and SEQ ID NO. 8.
• compositions of matter, and antibodies raised against such compositions of matter assist with providing a sensitive, specific, quick, and non-invasive method to aid in diagnosis of a myriad of injuries and/or disorders that may be present in a subject, even in the event of a multi -trauma environment, by detecting and determining the amount of biomarkers that are indicative to the respective injury type.
• the measurement of these markers, alone or in combination with other markers for the injury type, in patient samples provides information that a diagnostician can correlate with a probable diagnosis of the extent of an injury.
• the present invention further provides in vitro diagnostic processes and kits for detecting injuries and disorders in a subject for a particular injury, even in the event that the subject has suffered multiple traumas prior to the measurement of the biomarkers.
• FIG. 1 illustrates a 2D gel detecting the isoforms of SIOOP in serum, showing the capture of the isolated nucleic acid sequence having 50%, 60%, 70%, 80%, 85%, 90%, 95%,
• FIG. 2 illustrates a method to generate antibodies to peptides corresponding to the isolated nucleic acid sequences of Exon 2, Exon 3 and Exon 4 of the S100p cDNA having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence of SEQ ID NO. 7 and SEQ ID NO. 8.
• FIG. 3 illustrates the Exon arrangements for the formation of nucleic acid sequence of SEQ ID NO. 7 and SEQ ID NO. 8.
• the isoforms so identified consist of Exon 1, Exon 2 and either that portion of Exon 3 (S100B7) or Exon 4 (S100B8) that completes the isoform sequence to the stop codon (TGA, TAG nucleic acid sequence).
• FIG. 4 illustrates the various Exon locations in the complete SIOOP mRNA.
• the Exons include Exon 1, Exon 2 and then either Exon 3 to stop codon TGA or Exon 4 to stop codon TGA or TAG. DETAILED DESCRIPTION OF THE INVENTION
• the present invention has utility in the diagnosis and management of an injury or abnormal condition in a subject, with the injury or condition being alone, or in combination with a series of other injuries or disorders.
• biomarkers in a biological sample from a subject, such as the isolated nucleic acid sequence having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence of S100p7 or S100p8, alone or in combination with each other or in combination with one or more additional biomarkers known in the art, a determination of subject's injury or condition is provided with greater specificity than previously attainable, even in the event of a multi-trauma patient.
• the present description is directed toward a first isoform of S100p, selected from the isolated nucleic acid sequence having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence of S 100p7 or 8100 ⁇ 8, as a biomarker.
• a biomarker selected from the isolated nucleic acid sequence having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence of S 100p7 or 8100 ⁇ 8, as a biomarker.
• diagnosis means recognizing the presence or absence of a neurological or other condition such as an injury or disease. Diagnosing is optionally referred to as the result of an assay wherein a particular ratio or level of a biomarker is detected or is absent.
• a ratio is either a positive ratio wherein the level of the target is greater than the target in a second sample or relative to a known or recognized baseline level of the same target.
• a negative ratio describes the level of the target as lower than the target in a second sample or relative to a known or recognized baseline level of the same target.
• a neutral ratio describes no observed change in target biomarker.
• an injury is an alteration in cellular or molecular integrity, activity, level, robustness, state, or other alteration that is traceable to an event.
• Injury illustratively includes a physical, mechanical, chemical, biological, functional, infectious, or other modulator of cellular or molecular characteristics.
• An event is illustratively, a physical trauma such as an impact (percussive) or a biological abnormality such as a stroke resulting from either blockade or leakage of a blood vessel.
• An event is optionally an infection by an infectious agent.
• An injury is optionally a physical event such as a percussive impact.
• Traum may further include a disease or genetic abnormality such as Parkinson's Disease, Alzheimer's disease, ALS, etc.
• range is intended to encompass not only the endpoint values of the range but also intermediate values of the range as explicitly being included within the range and varying by the last significant figure of the range.
• a recited range of from 1 to 4 is intended to include 1-2, 1-3, 2-4, 3-4, and 1-4
• the present invention provides compositions of matter, antibodies and antigens related thereto, methods, kits and in vitro diagnostic processes specifically used to detect protein markers that are differentially present in the samples of patients suffering from one of several injury and/or disorders alone or in a multi-trauma environment.
• inventive compositions of matter are those identified as the isolated nucleic acid sequence having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence of SEQ ID NO. 7 and SEQ ID NO. 8.
• compositions of matter, and antibodies raised against such compositions of matter assist with providing a sensitive, specific, quick, and non-invasive method to aid in diagnosis of a myriad of injury and/or disorders that may be present in a subject, even in the event of a multi-trauma environment, by detecting and determining the amount of biomarkers that are indicative to the respective injury type.
• the measurement of these markers, alone or in combination of other markers for the injury type, in patient samples provides information that a diagnostician can correlate with a probable diagnosis of the extent of an injury.
• Antibody-based assays are preferred for analyzing a biological sample for the presence of a biomarker and one or more other biomarkers of a particular injury or condition. Suitable western blotting methods are described below in the examples section. For more rapid analysis (as may be important in emergency medical situations), immunosorbent assays (e.g., ELISA and RIA) and immunoprecipitation assays may be used.
• immunosorbent assays e.g., ELISA and RIA
• immunoprecipitation assays may be used.
• Reagents as described herein may be any antibody to a protein or peptide sequence or any antigen to detect an antibody formed as part of an autoimmune response in a subject.
• the antigens used for these detection methods and devices may be those proteins or peptides generated to detect an antibody that has been produced by a subject's own tissues as an autoimmune response to cells, tissues or native proteins of the organism in which it was formed.
• these antigens are peptides having the following sequence for each of the defined isoforms from which antibodies are, have been, or will be generated:
• SEQ ID NO. 1 is the publicly recognized (LOCUS NP_006263 92; CAG46920, aa (molecular wt. about 10,713 Daltons) linear, DEFINITION protein S 100p [Homo sapiens], ACCESSION NP_006263 VERSION NP_006263.1 GI: 5454034 or GI: 4957424 DBSOURCE REFSEQ: accession NM_006272.2; embl accession CR542123.1) generic sequence commonly referred to or linked to all SIOOP studies currently recognized in the art.
• SIOOP Because of the recent discoveries of the several isoforms, not previously known in the art, the conventional name SIOOP necessarily needs to be changed to SlOOpl as is the convention within the scientific research community.
• This peptide, or antibodies raised against this peptide, is recognized in the art as not being specific to any particular injury or disorder.
• This nucleic acid sequence is known to have the following amino acid sequence as noted is the result of splicing Exon 1 and Exon 2: MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELIN ELSHFLEEIKEQEVVDKVME TLDNDGDGECDFQEFMAFVAMVTTACHEFFEHE
• SEQ ID NO 2 is the first of the compositions of matter identified in U.S. Patent Application S/N: 14/293,758 formed as a result of the splicing Exon 1, Exon 2 (truncated #1) and the newly discovered Exon 3 of the human nucleic acid sequence, thus the protein as a result of the translation of the nucleic acid sequence is named S100p2.
• the protein S100p2 found to have a molecular weight of an estimated 11,295 Daltons, has been found to have the following amino acid sequence:
• SEQ ID NO 3 is the second of the compositions of matter 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, thus the protein as a result of the translation of the nucleic acid sequence is named S100p3.
• the peptide S100p3, found to have a molecular weight of about 2,750 Daltons has been found to have the following amino acid sequence:
• SEQ ID NO 4 is the third of the compositions of matter identified in U.S. Patent Application S/N: 14/293,758 formed as a result of Exon 1 (truncated) and Exon 2 of the human nucleic acid sequence, thus the protein as a result of the translation of the nucleic acid sequence is named S 100p4.
• the peptide S 100P4 found to have a molecular weight of about 5,950 Daltons has been found to have the following amino acid sequence:
• SEQ ID NO 5 is the fourth of the compositions of matter identified in U. S. Patent Application S/N: 14/293,758 formed as a result of Exon 1 (truncated), Exon 2 (truncated #1) and Exon 3 of the human nucleic acid sequence, thus the protein as a result of the translation of the nucleic acid sequence is named S 100p5.
• the peptide S100p5 found to have a molecular weight of about 6,500 Daltons has been found to have the following amino acid sequence:
• SEQ ID NO 6 is the fifth of the compositions of matter identified in U. S. Patent Application S/N: 14/293,758 formed as a result of Exon 1, Exon 2 (truncated #2) and Exon 3 of the human nucleic acid sequence, thus the protein as a result of the translation of the nucleic acid sequence is named S100p6.
• the peptide S 100p6, found to have a molecular weight of -7,600 Daltons has been found to have the following amino acid sequence:
• SEQ ID NO 7 is the first of the several newly discovered compositions of matter formed as a result of Exon 1, a newly discovered Exon 2 and Exon 3b of the human nucleic acid sequence, thus the protein as a result of the translation of the nucleic acid sequence is named S 100p7.
• the peptide S 100p7 is found to have a molecular weight of -10,342.75 Daltons has been found to have the following sequence:
• SEQ ID NO 8 is the fifth of the several newly discovered compositions of matter formed as a result of Ex on 1, a newly discovered Ex on 2 and Ex on 4a of the human nucleic acid sequence, thus the protein as a result of the translation of the nucleic acid sequence is named S100p8.
• the peptide S100p8 found to have a molecular weight of -10,281.8 Daltons has been found to have the following sequence:
• sequences described above are those S100p isoforms found for the human polypeptide sequence, that similar isoforms may be available in rat, mouse, rabbit, bovine, Chinese hamster, Rhesus monkey, zebrafish, goat, chicken, dog, domestic cat, and pig and other species having a similar sequence alignment to the S100p for humans. These alternative sequences may be substituted in the inventive processes, methods, assays, and in vitro diagnostic devices described herein.
• compositions of matter of importance are those antibodies raised against any of the peptide sequences represented by the nucleic acid sequence having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence of SEQ ID NO's 7-8, or antibodies raised against the rat, mouse, rabbit, bovine, Chinese hamster, Rhesus monkey, zebrafish, goat, chicken, dog, domestic cat, and pig and other similar species formulations of the similar sequence alignment.
• An antibody is optionally labeled.
• Labels and labeling kits are commercially available. Labels illustratively include, fluorescent labels, biotin, peroxidase, radionucleotides, or other label known in the art.
• a detection species of another antibody or other compound known to the art is used as form detection of a biomarker bound by an antibody. If the antibody is directly conjugated with a detectable label, such as an enzyme, fluorophore, or radioisotope, the presence of the label is optionally detected by examining the substrate for the detectable label.
• a detectably labeled secondary antibody that binds the marker-specific antibody is added to the substrate.
• FIG. 2 presents the basis for the novel and distinct modifications of the modified MAP method.
• the Multiple Antigenic Peptide (MAP) dendrimer system has four sections to increase the induction of a stronger immune response, reduce enzymatic degradation and enhance molecular recognition to antigenic peptides for antibody generation.
• the MAP core which consists of a branched lysine core scaffold has a bare cell-penetrating peptide attached to one end and several copies of the peptide epitopes attached to the other.
• lipophilic moieties are attached to the peptide epitopes.
• the MAP system is hydrophobic and requires formulation in an emulsion medium that is biocompatible for administration into the animals normally used to generate the antibodies.
• the emulsion medium required is one that does not oxidize the methionines, cysteines and tryptophans in the peptide sequences yet is able to handle their hydrophobic characteristics.
• An exemplary process for detecting the presence or absence of a biomarker, alone or in combination, in a biological sample involves obtaining a biological sample from a subject, such as a human, contacting the biological sample with a compound or an agent capable of detecting of the marker being analyzed, illustratively including an antibody or aptamer, and analyzing binding of the compound or agent to the sample after washing. Those samples having specifically bound compound or agent express the marker being analyzed.
• the present invention further provides a step of comparing the quantity of one or more biomarkers to normal levels of uninjured patients to determine the injury or condition of the subject. It is appreciated that selection of additional biomarkers allows one to identify the types of cells implicated in an abnormal organ or physical condition as well as the nature of cell death.
• the practice of an inventive process provides a test which can help a physician determine suitable therapeutics to administer for optimal benefit of the subject suffering from a particular condition in a multi-trauma scenario and to select a therapeutic that may be administered in combination with other therapeutics for the other measured injuries.
• In vitro techniques for detection of a marker illustratively include enzyme linked immunosorbent assays (ELISAs), radioimmunoassay, radioassay, western blot, Southern blot, northern blot, immunoprecipitation, immunofluorescence, mass spectrometry, RT-PCR, PCR, liquid chromatography, high performance liquid chromatography, enzyme activity assay, cellular assay, positron emission tomography, mass spectroscopy, combinations thereof, or other technique known in the art.
• ELISAs enzyme linked immunosorbent assays
• radioimmunoassay radioassay
• radioassay western blot
• Southern blot Southern blot
• northern blot immunoprecipitation
• immunofluorescence mass spectrometry
• mass RT-PCR PCR
• liquid chromatography high performance liquid chromatography
• enzyme activity assay enzyme activity assay
• cellular assay positron emission tomography
• mass spectroscopy combinations thereof,
• any other suitable agent e.g., a peptide, an aptamer, or a small organic molecule
• a biomarker e.g., an aptamer that specifically binds 8100 ⁇ 7 and/or one or more of the other SIOO protein isoforms or peptides of the same might be used.
• Aptamers are nucleic acid-based molecules that bind specific ligands. Methods for making aptamers with a particular binding specificity are known as detailed in U.S. Patent Nos.
• kits for aiding a diagnosis of a particular injury, degree of severity of injury, subcellular localization and/or a particular disorder wherein the kits can be used to detect the markers of the present invention.
• the kits can be used to detect any one or more of the markers described herein, which markers are differentially present in samples of a patient and normal subjects.
• the kits of the invention have many applications.
• the kits can be used to differentiate if a subject has sepsis, bone injury, muscle or tissue injury, or brain injury.
• the kits can be used to identify compounds that modulate expression of one or more of the markers in in vitro or in vivo animal models to determine the effects of treatment.
• kits comprises (a) an antibody that specifically binds to a marker (a capture agent); and (b) a detection agent for detecting the amount of the marker.
• a capture agent for detecting the amount of the marker.
• the kit may further comprise pre- fractionation spin columns.
• the kit may further comprise instructions for suitable operation parameters in the form of a label or a separate insert.
• the invention includes a diagnostic kit for use in screening serum containing antigens of the peptide of the invention.
• antigens may be one of any of the peptides represented by nucleic acid molecules having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence of SEQ ID NO. 7-8.
• the diagnostic kit includes a substantially isolated antibody specifically immunoreactive with peptide or polynucleotide antigens and means for detecting the binding of the polynucleotide or peptide antigen to the antibody.
• the antibody is attached to a solid support.
• Antibodies used in the inventive kit are those raised against any one of the peptides represented by nucleic acid molecules having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% SEQ ID NO's 7-8.
• the antibody is a monoclonal or polyclonal antibody raised against the rat, rabbit or human forms of the isoforms of S100p described herein.
• the detecting means of the kit includes a second, labeled monoclonal or polyclonal antibody. Alternatively, or in addition thereto, the detecting means includes a labeled, competing antigen.
• the kit may further comprise a standard or control information so that the test sample can be compared with the control information standard to determine if the test amount of a marker detected in a sample is a diagnostic amount consistent with a diagnosis of a particular injury, degree of severity of the injury, subcellular localization, a particular disorder and/or effect of treatment on the patient.
• a kit comprises: (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% SEQ ID NO. 7 or SEQ ID NO. 8 and (b) instructions to detect the marker or markers by contacting a sample with the adsorbent and detecting the marker or markers with a biological sample.
• the kit may comprise an eluant (as an alternative or in combination with instructions) or instructions for making an eluant, wherein the combination of the adsorbent and the eluant allows detection of the markers using gas phase ion spectrometry.
• Such kits can be prepared from the materials described above, and the previous discussion of these materials (e.g., probe substrates, adsorbents, washing solutions, etc.) is fully applicable to this section and will not be repeated.
• the kit comprises a first substrate comprising an adsorbent thereon (e.g., a particle functionalized with an adsorbent) and a second substrate onto which the first substrate can be positioned to form a probe which is removably insertable into a gas phase ion spectrometer.
• the kit may comprise a single substrate which is in the form of a removably insertable probe with adsorbents on the substrate.
• the kit may further comprise a pre-fractionation spin column (e.g., Cibacron blue agarose column, anti-HSA agarose column, size exclusion column, Q-anion exchange spin column, single stranded DNA column, lectin column, etc.).
• the kit can further comprise instructions for suitable operational parameters in the form of a label or a separate insert.
• the kit may have standard instructions informing a consumer how to wash the probe after a sample is contacted on the probe.
• the kit may have instructions for pre-fractionating a sample to reduce complexity of proteins in the sample.
• the kit may have instructions for automating the fractionation or other processes.
• inventive compositions of matter, processes, methods and in vitro diagnostic devices provide the ability to detect and monitor levels of those proteins or autoantibodies which are released into the body after injury to provide enhanced diagnostic capability by allowing clinicians (1) to determine the level of injury severity in patients with various injuries, (2) to monitor patients for development of disorders or signs of secondary injuries that may elicit these cellular changes and (3) to continually monitor the progress of the injury and the effects of therapy by examination of these proteins in biological fluids, such as blood, plasma, serum, CSF, urine, saliva or sweat.
• a sample is preferably a biological sample.
• biological samples are illustratively cells, tissues, cerebral spinal fluid (CSF), artificial CSF, whole blood, serum, plasma, cytosolic fluid, urine, feces, stomach fluids, digestive fluids, saliva, nasal or other airway fluid, vaginal fluids, semen, buffered saline, saline, water, or other biological fluid recognized in the art.
• the biological samples comprise CSF, blood, serum, plasma, sweat, saliva and urine. It should be appreciated that after injury, the cell membrane in any muscle, tissue or organ becomes compromised, leading to the efflux of its constituent proteins first into the extracellular fluid or space and eventually into other fluids in the body, including the circulating blood.
• samples which are biological fluids, are preferred for use in the invention.
• Biological samples of CSF, blood, urine and saliva are collected using normal collection techniques. For example, and not to limit the sample collection to the procedures contained herein, CSF Lumbar Puncture (LP) a 20-gauge introducer needle is inserted and an amount of CSF is withdrawn. For blood, the samples may be collected by venipuncture in Vacutainer tubes, and if preferred spun down and separated into serum and plasma. For urine and saliva, samples are collected avoiding the introduction of contaminants into the specimen is preferred. All biological samples may 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 samples can be obtained from a subject to detect markers. It should be appreciated that the methods employed herein may be identically reproduced for any biological fluid to detect a marker or markers in a sample.
• Cluster 1 focused on testing for and sequencing all mRNAs that were from about 1,000 to 2,000 bases in length, give or take several hundred bases (see histograms).
• Clusters 2 and 3 were from 2,000 to 3,000 bases and 3,000 to 6,000 bases, respectively.
• the report for cluster 1 contained 9, 137 pages of sequences like the one below representing about 25,000 to 30,000 potentially unique mRNA sequences and thus proteins.
• the one below is the mRNA sequence for S100p7 (the 13,714 th sequence tested and sequenced in cluster 1).
• ELISA assays were performed with sera from eight humans, two with no known history of neural ceil damage, and six presenting with one or more clinical symptoms of an injur ⁇ ' or disease resulting f om neural cell damage (three with traumatic brain injur ⁇ ', one with stroke, one with Alzheimer's Disease).
• Microplates were coated with antibodies raised against the sequence of SEQ ID NO 7. Wells were washed. One hundred ⁇ serum from the test subjects was added to each well. The microplates were incubated at 37° C. for one hour and then washed again to remove any unbounded antibodies. The protein-antibody complex was then subsequently measured.
• Example 2 The test of Example 2 was repeated with antibodies raised against the sequence of SEQ ID NO 8. ELISA. Similarly, in comparing the levels of the protein-antibody complex fomied by the ELISA method, it was found that the six subjects presenting with one or more clinical symptoms of a disease or injury resulting from neural cell damage had significantly higher concentrations of the protein-antibody matrix fomied in the ELISA method than the two uninjured patients.

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  • 2018-02-07 EP EP18751838.6A patent/EP3580355A4/fr active Pending
  • 2018-02-07 JP JP2019563344A patent/JP2020507622A/ja active Pending
• 2022
  • 2022-10-24 JP JP2022169855A patent/JP7597770B2/ja active Active

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