WO2007124439A2 - Procédés et kits de diagnostic d'un accident vasculaire cérébral - Google Patents

Procédés et kits de diagnostic d'un accident vasculaire cérébral Download PDF

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WO2007124439A2
WO2007124439A2 PCT/US2007/067117 US2007067117W WO2007124439A2 WO 2007124439 A2 WO2007124439 A2 WO 2007124439A2 US 2007067117 W US2007067117 W US 2007067117W WO 2007124439 A2 WO2007124439 A2 WO 2007124439A2
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stroke
sgot
fluid sample
mmp
concentration
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WO2007124439A3 (fr
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Mark Chandler
Mike Spain
James Mapes
Ralph Mcdade
Josh Kemp
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Myriad RBM Inc
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Rules Based Medicine Inc
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    • 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
    • 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
    • G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event

Definitions

  • the present invention relates to methods, kits and reagents for detection and/or diagnosis of stroke.
  • a method for rapid detection and/or accurate diagnosis of stroke is provided.
  • the method can be practiced with a determination of the concentrations of one or two biomarkers in a patient fluid sample. Elevated (or depressed, as the case might be) levels of the one or two biomarkers, which are statistically different from levels found in "normals" (that is, control subjects not suffering from stroke), support a positive diagnosis of stroke.
  • the method utilizes a panel of analytes or "biomarkers,” up to twelve or more substances found in a sample fluid (e.g., whole blood, serum, plasma, or urine), to help support a positive or negative diagnosis of stroke. Up to 99% accuracy in making a correct diagnosis may be provided by the method.
  • a method of diagnosing stroke in a human subject suspected of suffering from stroke comprises: (a) obtaining a fluid sample from a human subject suspected of suffering from stroke; (b) determining the concentration of Matrix Metallo Proteinase 3 (MMP-3) in said fluid sample; (c) deciding if the determined concentration of MMP-3 in said fluid sample is statistically different from that found in a control group of human subjects, whereby a statistically different elevated concentration of MMP-3 supports a positive diagnosis of stroke.
  • the human subject is complaining of sudden weakness of the face, arm or leg. Any one of a number of fluid samples can be tested.
  • the fluid sample is selected from whole blood, plasma, serum, or urine. It has been discovered that a measured concentration of about 1 ng/mL or above of MMP-3 in the fluid sample supports a positive diagnosis of stroke.
  • a method for diagnosing stroke in a human subject suspected of suffering from stroke comprises: (a) obtaining a fluid sample from a human subject suspected of suffering from stroke; (b) determining the concentration of Serum Glutamic Oxaloacetic Transaminase (SGOT) in said fluid sample; (c) deciding if the determined concentration of SGOT in said fluid sample is statistically different from that found in a control group of human subjects, whereby a statistically different depressed concentration of SGOT supports a positive diagnosis of stroke. It is expected that a measured concentration of about 10 ⁇ g/mL or below of SGOT in said fluid sample will support a positive diagnosis of stroke.
  • SGOT Serum Glutamic Oxaloacetic Transaminase
  • Still another aspect of the invention relates to a method of diagnosing stroke in a human subject suspected of suffering from stroke, comprising: (a) obtaining a fluid sample from a human subject suspected of suffering from stroke; (b) determining the concentrations of MMP-3 and SGOT in said fluid sample; and (c) deciding if the determined concentrations of MMP-3 and SGOT in said fluid sample are statistically different from that found in a control group of human subjects, whereby a statistically different elevated concentration of MMP-3 and a statistically different depressed concentration of SGOT together support a positive diagnosis of stroke.
  • the method of the invention further comprises determining the concentration in said fluid sample of at least one of IL-18, Factor VII, ICAM-I, Creatine Kinase-MB, MCP-I, Myoglobin, C Reactive Protein, TIMP-I, Ferritin, or Glutathione S-Transferase, or any combination thereof.
  • concentration in said fluid sample of at least one of IL-18, Factor VII, ICAM-I, Creatine Kinase-MB, MCP-I, Myoglobin, C Reactive Protein, TIMP-I, Ferritin, or Glutathione S-Transferase, or any combination thereof.
  • concentration in said fluid sample of at least one of IL-18, Factor VII, ICAM-I, Creatine Kinase-MB, MCP-I, Myoglobin, C Reactive Protein, TIMP-I, Ferritin, or Glutathione S-Transferase, or any combination thereof.
  • certain threshold levels of analytes in the sample fluids may be important in the detection or diagnosis of stroke, including IL-18 (about 300 pg/mL or above), Factor VII (about 320 ng/mL or above), ICAM-I (about 170 ng/mL or above), Creatine Kinase-MB (about 5 ng/mL or above), MCP-I (about 275 pg/mL or above), Myoglobin (about 30 ng/mL or above), C Reactive Protein (about 11 ⁇ g/mL or above), TIMP-I (about 120 ng/mL or above), Ferritin (about 300 ng/mL or above), and Glutathione S-Transferase (about 2 ng/mL or above.
  • IL-18 about 300 pg/mL or above
  • Factor VII about 320 ng/mL or above
  • ICAM-I about 170 ng/mL or above
  • Creatine Kinase-MB about 5 ng/m
  • biomarkers may also be useful in arriving at a positive or negative diagnosis of stroke.
  • biomarkers include, in addition to those already disclosed, Prostate Specific Antigen (free), IL-3, Tissue Factor, alpha-Fetoprotein, Prostatic Acid Phosphatase, Stem Cell Factor, MIP-I -beta, Carcinoembryonic Antigen, IL- 13, TNF-alpha, IgE, Fatty Acid Binding Protein, ENA-78, IL-I -beta, Brain-Derived Nerotrophic Factor, Apolipoprotein Al, Serum Amyloid P, Growth Hormone, Beta-2 microglobulin, Lipoprotein (a), MMP-9, Thyroid Stimulating hormone, alpha-2 Macroglobulin, Complement 3, IL-7, Leptin, and IL-6.
  • Various techniques for assessing the importance of certain biomarkers in arriving at a diagnosis is also described herein.
  • One such technique is a projection of compiled results on a proximity map, whereby the proximity of a subject's determined concentrations to a cluster of other subjects' determined concentrations, who were previously diagnosed as having suffered from stroke, contributes to a positive diagnosis of stroke.
  • Other techniques include the application of one or more statistical methods (e.g., linear regression analysis, classification tree analysis, heuristic nave Bayes analysis and the like).
  • kits comprising reagents for determining the concentration in a fluid sample of a panel of analytes including MMP-3, SGOT and one or more of IL-18, Factor VII, ICAM-I, Creatine Kinase-MB, MCP-I, Myoglobin, C Reactive Protein, TIMP-I, Ferritin, or Glutathione S-Transferase.
  • the reagents may include antibodies against the members of a given panel of analytes.
  • the reagent may be immobilized on a substrate, which substrate may comprise a two-dimensional array, a microtiter plate, or multiple bead sets.
  • the methods may further comprise comparing the levels of the one, two, or more biomarkers in a patient's blood with levels of the same biomarkers in one or more control samples by applying a statistical method such as: linear regression analysis, classification tree analysis and heuristic nave Bayes analysis.
  • the statistical method may be, and typically is performed by a computer process, such as by commercially available statistical analysis software.
  • the statistical method is a classification tree analysis, for example CART (Classification and Regression Tree). Results for a particular patient or subject, whose sample fluid is tested against a panel of biomarkers according to the method, can be projected onto a proximity map. The proximity of a particular patient's biomarker concentration results to one of at least two populations (those previously diagnosed as having suffered a stroke and normals) supports a either a positive or negative diagnosis of stroke.
  • An article of manufacture which comprises binding reagents specific for at least one of MMP-3 and SGOT, preferably both biomarkers. More preferably, a kit is provided which comprises binding reagents specific for MMP-3, SGOT, IL- 18, Factor VII, ICAM-I, Creatine Kinase-MB, MCP-I, Myoglobin, C Reactive Protein, TIMP-I, Ferritin, or Glutathione S-Transferase. In a preferred embodiment, each binding reagent is immobilized on a substrate.
  • monoclonal antibodies against MMP-3, SGOT and the other biomarkers described herein are immobilized independently to one or more discrete locations on one or more surfaces of one or more substrates.
  • the substrates may be beads comprising an identifiable biomarker, wherein each binding reagent is attached to a bead comprising a different identifiable biomarker than beads to which a different binding reagent is attached.
  • the identifiable biomarker may comprise a fluorescent compound, a quantum dot, or the like.
  • a method for determining the occurrence of a stroke in a patient comprising determining levels of at least one of MMP-3 and SGOT.
  • a method of predicting onset of stroke comprising determining the change in concentration at two or more points in time of two or more markers in a patient's blood, wherein an observed increase in the concentration of MMP-3, a decrease in the concentration of SGOT or both, in the patient's blood between the two time points, is predictive of the onset of stoke.
  • a method of diagnosing stroke in a human subject suspected of suffering from stroke comprising: (a) obtaining a fluid sample from a human subject suspected of suffering from stroke; (b) determining the concentration of MMP-3 or SGOT or both in said fluid sample; (c) deciding if the determined concentration of MMP-3 or SGOT or both, respectively, in said fluid sample is statistically different from that found in a control group of human subjects, whereby a statistically different elevated concentration of MMP-3 or SGOT or both, respectively, supports a positive diagnosis of stroke.
  • FIG. 1 is an example of a projection of a proximity map of patients whose fluid samples are tested against a panel of biomarkers listed on the right-hand margin.
  • the results of this proximity map analysis indicate that consideration of all the biomarkers listed provides a degree of accuracy of a correct diagnosis of stroke of about 98%, with subjects having suffered a stroke positioned on the left-hand side of the figure (red or light gray dots) and subjects who have not suffered a stroke positioned on the right-hand side of the figure (blue or dark gray spots).
  • FIG. 2 provides an example of a proximity map of patients whose fluid samples have been tested against a panel of biomarkers listed on the right-hand margin.
  • FIG. 3 is another example of a proximity map of patients whose fluid samples have been tested against a panel of biomarkers listed on the right-hand margin (except that the results for the biomarkers Tissue Factor and vWF are excluded from this analysis).
  • FIG. 4 is yet another example of a proximity map of patients whose fluid samples are tested against a panel of biomarkers listed on the right-hand margin.
  • the results of this proximity map analysis would indicate that consideration of all the biomarkers listed provides a degree of accuracy of a correct diagnosis of stroke of about 94%, with subjects having suffered a stroke positioned on the left-hand side of the figure (red or light gray dots) and subjects who have not suffered a stroke positioned on the right-hand side of the figure (blue or dark gray spots).
  • FIG. 5 is still another example of a proximity map of patients whose fluid samples were tested against a panel of biomarkers listed on the right-hand margin.
  • Stroke as defined herein, broadly refers as the sudden deficit in brain function usually related to impaired cerebral blood flow. Strokes may be further defined by the nature of the deficiency (e.g., thrombosis, embolism, hemorrhage, ischemia). However, solely in the interest of clarity in drafting, the term "stroke” is meant to encompass any deficiency in brain function related to impaired cerebral blood flow, and not limited to a particular source for said impairment.
  • the parameters for establishing the significance of one or more biomarkers for the diagnosis of stroke are determined statistically by comparing normal or control blood (preferably, e.g., serum or plasma) levels of these biomarkers with blood levels in patients clinically and properly diagnosed as having suffered from or is having a stroke.
  • normal or control blood preferably, e.g., serum or plasma
  • the statistical data presented below in Table 1 identify certain mean values and accompanying standard deviations of the above-described biomarkers, which may be found in the blood of stroke patients in comparison to normals.
  • MMP-3 about 1 ng/niL or above
  • SGOT about 10 ⁇ g/mL or below
  • IL- 18 about 300 pg/mL or above
  • Factor VII about 320 ng/mL or above
  • ICAM-I about 170 ng/mL or above
  • Creatine Kinase-MB about 5 ng/mL or above
  • MCP-I about 275 pg/mL or above
  • Myoglobin about 30 ng/mL or above
  • C Reactive Protein about 11 ⁇ g/mL or above
  • TIMP-I about 120 ng/mL or above
  • Ferritin about 300 ng/mL or above
  • Glutathione S-Transferase about 2 ng/mL or above.
  • Certain statistical methods can be used to identify discriminating biomarkers and panels thereof. These statistical methods may include, but are not limited to: 1) linear regression; 2) classification tree methods; and 3) statistical machine learning to optimize the unbiased performance of algorithms for making predictions. Each of these statistical methods is well-known to those of ordinary skill in the field of biostatistics and can be performed as a process in a computer. A large number of software products are available commercially to implement statistical methods, such as, without limitation, S-PLUSTM, commercially available from Insightful Corporation of Seattle, Washington; however, the instant invention is not limited to the use of any one specific software.
  • biomarkers useful in the determination and/or diagnosis of stroke and by use of statistical methods to identify which biomarkers and groups of biomarkers are particularly useful in identifying stroke-at-risk patients, a person of ordinary skill in the art, based on the disclosure herein, can compose panels of biomarkers having superior selectivity and sensitivity.
  • biomarkers examples include: MMP-3, SGOT, IL-18, Factor VII, ICAM-I, Creatine Kinase-MB, MCP-I, Myoglobin, C Reactive Protein, von Willebrand Factor, TIMP-I, Ferritin, Glutathione S-Transferase, Prostate Specific Antigen (free), IL-3, Tissue Factor, alpha- Fetoprotein, Prostatic Acid Phosphatase, Stem Cell Factor, MIP-I -beta, Carcinoembryonic Antigen, IL- 13, TNF-alpha, IgE, Fatty Acid Binding Protein, ENA-78, IL-I -beta, Brain-Derived Nerotrophic Factor, Apolipoprotein Al, Serum Amyloid P, Growth Hormone, Beta-2 microglobulin, Lipoprotein (a), MMP-9, Thyroid Stimulating hormone, alpha-2 Macroglobulin, and others.
  • Examples of specific panels comprising selected biomarkers from the above-mentioned list include, but are not limited to: (i) CRP, CKMB, Factor VII, Ferritin, GST, ICAM-I, IL-18, IL-IB, IL-3, MCP-I, MMP-3, Myoglobin, SGOT, TIMP-I and vWF; (ii) CRP, CKMB, Factor VII, Ferritin, GST, ICAM-I, IL-18, MCP-I, MMP-3, Myoglobin, SGOT, TIMP-I, Tissue Factor and vWF; (iii) CRP, CKMB, Factor VII, Ferritin, GST, ICAM-I, IL-18, MCP-I, MMP-3, Myoglobin, SGOT and TIMP-I; (iv) SGOT, CKMB, MMP-3, GST, Factor VII, IL-18, IL-3, MCP-I, ICAM-I and IL-
  • the invention is based on an evaluation of at least MMP-3 levels, alone or in combination with levels of immunological SGOT and/or other biomarkers, in serum for diagnosis of stroke in all stages of their occurrence.
  • the invention is also based on the evaluation of at least immunological SGOT levels, optionally in combination with levels of at least MMP-3. Patients with stroke are at considerable risk for death and serious complications, and outcomes can be improved with appropriate diagnosis and therapy. Thus, rapid and accurate diagnosis of patients complaining of the symptoms of stroke is critical for patient care.
  • MMP-3 can be used as an early biomarker of inflammatory cardiovascular conditions, including stroke.
  • SGOT levels are appear depressed in stroke patients.
  • SGOT can also be used as an early biomarker of stroke.
  • the present method includes measuring the level of MMP-3 and/or SGOT in a biological sample (e.g. , whole blood, plasma, serum or urine and the like) from a patient; comparing the respective levels with that of control subjects; and diagnosing the state of disease based on the level of MMP-3 or SGOT relative to that of control subjects. In this way, a patient can be diagnosed with stroke if the level of MMP-3 is increased relative to that of control subjects or if SGOT is decreased relative to controls.
  • a typical control value for MMP-3 is in the range of about 0.1-0.8 ng/niL.
  • a concentration of about 1 ng/niL or above in a patient sample can support a positive diagnosis.
  • the general range for elevated values of MMP-3 is about 1.5-20 ng/niL.
  • a typical control value for SGOT is in the range of about 17-25 ⁇ g/mL.
  • An immunological concentration of about 10 ⁇ g/mL or below in a patient sample can support a positive diagnosis.
  • SGOT is often measured enzymatically. However, here, the sum of protein is presented, which may include enzymatically inactive plus enzymatically active SGOT.
  • the general range for depressed values of immunological SGOT concentration is about 1-15 ⁇ g/mL.
  • MMP-3 and SGOT can be captured with anti-MMP-3 and anti-SGOT polyclonal antibodies, respectively, or with corresponding monoclonal antibodies.
  • the diagnostic method may also include measuring the levels of one or more additional analytes selected from the group consisting of: IL- 18, Factor VII, ICAM-I, Creatine Kinase-MB, MCP-I, Myoglobin, C Reactive Protein, von Willebrand Factor, TIMP-I, Ferritin, Glutathione S-Transferase, Prostate Specific Antigen (free), IL-3, Tissue Factor, alpha-Fetoprotein, Prostatic Acid Phosphatase, Stem Cell Factor, MIP- 1-beta, Carcinoembryonic Antigen, IL- 13, TNF-alpha, IgE, Fatty Acid Binding Protein, ENA-78, IL-I -beta, Brain-Derived Nerotrophic Factor, Apolipoprotein Al, Serum Amyloid P, Growth Hormone, Beta-2 microglobulin, Lipoprotein (a), MMP-9, Thyroid Stimulating hormone, alpha-2 Macro globulin
  • MMP-3 levels above about 1 ng/mL has been identified in unstable angina patients and myocardial infarction patients.
  • SGOT levels below 10 ⁇ g/mL were also identified in unstable angina patients and myocardial infarction patients. Both biomarkers are associated with unfavorable outcomes when elevated.
  • MMP-3 is a valuable unstable plaque biomarker even when troponins and C-reactive protein are not elevated, potentially identifying high-risk patients who otherwise might remain undiagnosed.
  • MMP-3 may be directly involved in the pathophysiology of stroke as well.
  • the role SGOT plays in the pathophysiology of stroke is also not known; however, the same maker can be used to diagnose the disease.
  • the analytes used in the method of the invention can be detected, for example, by a binding assay.
  • a sandwich immunoassay can be performed by capturing MMP-3 and SGOT from a biological sample with antibodies having specific binding affinity for each protein, which then can be detected with a labeled antibody having specific binding affinity for each analyte.
  • standard immunohistochemical techniques can be used to detect MMP-3 and SGOT using such antibodies. Antibodies having affinity for MMP-3 and SGOT are available.
  • binding reagent refers to any compound, composition or molecule capable of specifically or substantially specifically (that is with limited cross-reactivity) binding another compound or molecule, which, in the case of immune-recognition is an epitope.
  • the binding reagents typically are antibodies, preferably monoclonal antibodies, or derivatives or analogs thereof, but also include, without limitation: Fv fragments; single chain Fv (scFv) fragments; Fab' fragments; F(ab')2 fragments; humanized antibodies and antibody fragments; camelized antibodies and antibody fragments; and multivalent versions of the foregoing.
  • Multivalent binding reagents also may be used, as appropriate, including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((scFv)2 fragments), diabodies, tribodies or tetrabodies, which typically are covalently linked or otherwise stabilized (i.e., leucine zipper or helix stabilized) scFv fragments.
  • Boding reagents also include aptamers, as are described in the art.
  • Antigen-specific binding reagents including antibodies and their derivatives and analogs and aptamers
  • Polyclonal antibodies can be generated by immunization of an animal.
  • Monoclonal antibodies can be prepared according to standard (hybridoma) methodology.
  • Antibody derivatives and analogs, including humanized antibodies can be prepared recombinantly by isolating a DNA fragment from DNA encoding a monoclonal antibody and subcloning the appropriate V regions into an appropriate expression vector according to standard methods.
  • Phage display and aptamer technology is described in the literature and permit in vitro clonal amplification of antigen-specific binding reagents with very affinity low cross-reactivity. Phage display reagents and systems are available commercially, and include Aptamer technology described for example and without limitation in U.S. Pat. Nos. 5,270,163, 5,475096, 5,840867 and 6,544,776.
  • sandwich assay refers to an immunoassay where the antigen is sandwiched between two binding reagents, which are typically antibodies.
  • the first binding reagent/antibody being attached to a surface and the second binding reagent/antibody comprising a detectable group.
  • detectable groups include, for example and without limitation: fluorochromes, enzymes, epitopes for binding a second binding reagent (for example, when the second binding reagent/antibody is a mouse antibody, which is detected by a fluorescently-labeled anti-mouse antibody), for example an antigen or a member of a binding pair, such as biotin.
  • the surface may be a planar surface, such as in the case of a typical grid-type array (for example, but without limitation, 96-well plates and planar microarrays), as described herein, or a non-planar surface, as with coated bead array technologies, where each "species" of bead is labeled with, for example, a fluorochrome (such as the Luminex technology described herein and in U.S. Pat. Nos. 6,599,331, 6,592,822 and 6,268,222), or quantum dot technology (for example, as described in U.S. Pat. No. 6,306,610).
  • a fluorochrome such as the Luminex technology described herein and in U.S. Pat. Nos. 6,599,331, 6,592,822 and 6,268,222
  • quantum dot technology for example, as described in U.S. Pat. No. 6,306,610.
  • the Luminex xMAP system may be utilized.
  • the xMAP system incorporates polystyrene microspheres that are dyed internally with two spectrally distinct fluorochromes. Using precise ratios of these fluorochromes, an array is created consisting of 100 different microsphere sets with specific spectral addresses. Each microsphere set can possess a different reactant on its surface. Because microsphere sets can be distinguished by their spectral addresses, they can be combined, allowing up to 100 different analytes to be measured simultaneously in a single reaction vessel. A third fluorochrome coupled to a reporter molecule quantifies the biomolecular interaction that has occurred at the microsphere surface.
  • Microspheres are interrogated individually in a rapidly flowing fluid stream as they pass by two separate lasers in the Luminex analyzer.
  • High-speed digital signal processing classifies the microsphere based on its spectral address and quantifies the reaction on the surface in a few seconds per sample.
  • the bead-type immunoassays are preferable for a number of reasons. As compared to ELISAs, costs and throughput are far superior. As compared to typical planar antibody microarray technology the beads are superior for quantitation purposes because the bead technology does not require pre-processing or titering of the plasma or serum sample, with its inherent difficulties in reproducibility, cost and technician time. For this reason, although other immunoassays, such as, without limitation, ELISA, RIA and antibody microarray technologies, are capable of use in the context of the present invention, but they are not preferred.
  • immunoassays refer to immune assays, typically, but not exclusively sandwich assays, capable of detecting and quantifying a desired blood biomarker, namely at least one of MMP-3, SGOT, IL-18, Factor VII, ICAM-I, Creatine Kinase-MB, MCP-I, Myoglobin, C Reactive Protein, TIMP-I, Ferritin and Glutathione S-Transferase, or any combination of the foregoing.
  • a desired blood biomarker namely at least one of MMP-3, SGOT, IL-18, Factor VII, ICAM-I, Creatine Kinase-MB, MCP-I, Myoglobin, C Reactive Protein, TIMP-I, Ferritin and Glutathione S-Transferase, or any combination of the foregoing.
  • MMP-3 about 1 ng/niL or above
  • SGOT about 10 ⁇ g/mL or below
  • IL- 18 about 300 pg/mL or above
  • Factor VII about 320 ng/mL or above
  • ICAM-I about 170 ng/mL or above
  • Creatine Kinase-MB about 5 ng/mL or above
  • MCP-I about 275 pg/mL or above
  • Myoglobin about 30 ng/mL or above
  • C Reactive Protein about 11 ⁇ g/mL or above
  • TIMP-I about 120 ng/mL or above
  • Ferritin about 300 ng/mL or above
  • Glutathione S-Transferase about 2 ng/mL or above
  • blood includes any blood fraction, for example plasma, that can be analyzed according to the methods described herein.
  • Serum is a standard blood fraction that can be tested, and is tested in the Examples below.
  • blood levels of a particular biomarker it is meant that any appropriate blood fraction can be tested to determine blood levels and that data can be reported as a value present in that fraction.
  • the blood levels of a biomarker can be presented as 50 pg/mL serum.
  • methods for diagnosing stroke by determining levels of specific identified blood biomarkers are provided. Also provided are methods of detecting preclinical stroke comprising determining the presence and/or velocity of specific identified biomarkers in a patient's blood. By velocity it is meant the changes in the concentration of the biomarker in a patient's blood over time. Longitudinal data has value in determining the velocity of specific biomarkers in a patient's blood for predicting the onset of clinical stroke.
  • Biomarkers with velocity indicative of preclinical stroke include: MMP-3, IL-18, Factor VII, ICAM-I, Creatine Kinase-MB, MCP-I, Myoglobin, C Reactive Protein, von Willebrand Factor, TIMP-I, Ferritin, Glutathione S-Transferase, Prostate Specific Antigen (free), IL-3, Tissue Factor, alpha-Fetoprotein, Prostatic Acid Phosphatase, Stem Cell Factor, MIP- 1-beta, Carcinoembryonic Antigen, IL- 13, TNF-alpha, IgE, Fatty Acid Binding Protein, ENA-78, IL- 1-beta, Brain-Derived Nerotrophic Factor, Apolipoprotein Al, Serum Amyloid P, Growth Hormone, Beta-2 microglobulin, Lipoprotein (a), MMP-9, Thyroid Stimulating hormone, alpha-2 Macroglobulin, Complement 3, IL-7, Leptin and
  • Patient Population In one study, a patient population is chosen based on a strong predisposition to stroke indicated by such factors as age, diet, exercise, medical history and familial background; or suffering from stroke as indicated by CT scans or MRI. A normal or control patient population can be chosen from a wellness clinic. These control patients have no indication of suffering from cardiovascular disease. Consent and blood specimens from all participants should be obtained under IRB Protocol.
  • All blood samples can be logged on the study computer to track information such as storage date, freeze/thaw cycles and distribution.
  • Luminex assay Development of Luminex assay.
  • the reagents for multiplex system may be developed using commercially available antibody or produced by well known immunological methods.
  • Capture antibodies are generally monoclonal and detection antibodies polyclonal.
  • Capture Abs are covalently coupled to carboxylated polystyrene microspheres (Luminex Corporation (Austin, Tex.)). Covalent coupling of the capture antibodies to the microspheres is preferably performed by following the procedures recommended by Luminex. In short, the microspheres' stock solutions are dispersed in a sonif ⁇ cation bath for 2 min. An aliquot of 2.5 x 10 6 microspheres is resuspended in microtiter tubes containing 0.1 M sodium phosphate buffer, pH 6.1 (phosphate buffer), to a final volume of 80 ⁇ L.
  • Microspheres are then incubated with 250 ⁇ L of PBS- 0.05% Tween 20 for 4 h. After aspiration, the beads are blocked with 1 mL of PBS- 1% BSA-0.1% sodium azide. The microspheres are counted with a hemacytometer and stored at a final concentration of 10 6 microspheres per mL in the dark at 4 0 C. Coupling efficiency of monoclonal antibodies can be tested by staining about 2,000 microspheres with PE-conjugated goat anti-mouse IgG. Detection Abs are biotinylated and the extent of biotin incorporation, which may be determined using HABA assay, was 20 moles of biotin per mole of protein. The assays may be further optimized for concentration of detection Ab and for incubation times. [0055] Examples of some commercial sources of matched antibody cytokine pairs include R&D Systems, Minneapolis, MN and Fitzgerald Industries International, Inc., Concord, MA.
  • Sensitivity of the newly developed assay, determined in a Luminex assay using serially diluted purified CA- 125, is typically 20 IU.
  • Intra-assay variability, expressed as a coefficient of variation, is preferably calculated based on the average for patient samples and measured twice at least two different time points. The intra- assay variability within the replicates presented as an average coefficient of variation is generally about 8.5%.
  • Table 2 illustrates the diagnostic accuracy, which may be obtained by testing for each individual analyte and determining their usefulness as a diagnostic tool.
  • the best individual biomarkers would appear to be MMP-3 and SGOT.
  • the best combination of biomarkers would appear to be the twelve analytes that are shown in FIG. 4.
  • Proximity Map Analysis The proximity map data analysis can be conducted with a software program that groups samples by their similarities in analyte concentration patterns. A unique chemical signature is generated using the concentration of the analytes measured in each sample. The relationship of each sample signature is visualized in the GalaxyTM projection. The GalaxyTM is a proximity map, such that the closer two objects are in the visualization, the closer their chemical signatures are, and thus the more similar they are to one another.
  • the axes are dimensionless (a result of being derived from a principal component analysis), and thus the visualization is not a typical X-Y scatter plot in which moving along one axis means increasing or decreasing a single value.
  • the two axes of the GalaxyTM are defined by the first two principal components, a common method to reduce complex data.
  • the placement of objects (record points) is done using a set of heuristics that have been designed to maximize the preservation of spatial relationships that existed in the high-dimensional space of the original data while minimizing the overlap that can occur when doing simple projections.
  • An examination of the FIGs shows that the red circles (light gray, the stroke patients) are separated from the blue circles (dark gray, controls) to various degrees with all of the plots attaining fairly good separation.
  • FIG. 3 provides what is possibly the best separation. If an unknown sample is tested for the analytes listed in FIG.
  • Rates of classification accuracy can then be obtained using 10-fold cross-validation and generation of a Receiver Operating Characteristic (ROC) curve.
  • the sensitivity and specificity of the method depend on the cut-point (i.e., predicted probability from the classification tree) used to classify each subject as either a case or control.
  • cut-point i.e., predicted probability from the classification tree
  • using the standard cut-point of 0.5 i.e., everyone with a predicted probability above 0.5 is classified as a cancer case
  • Fixing the specificity at 91% still leads to a very high sensitivity, at 95.5% (again with 93% correctly classified).
  • a specificity of 95.3% corresponds to a sensitivity of 84.1% (and 90.0% correctly classified).
  • the total area under the receiver operating characteristic (ROC) curve is near one (which would represent perfect classification), at 0.966.
  • Assays may be performed in filter-bottom 96-well microplates. Purified antigens of interest are coupled to Luminex beads as described for antibodies. Antigen-coupled beads are pre-incubated with blocking buffer containing 4% BSA for 1 h at room temperature on microtiter shaker. Beads are then washed three times with washing buffer (PBS, 1% BSA, 0.05% Tween 20) using a vacuum manifold followed by incubation with 50 ⁇ L blood serum diluted 1 :250 for 30 min at 4 0 C. This dilution is selected as an optimal for recovery of anti-IL-18 IgG based on previous serum titration.
  • washing buffer PBS, 1% BSA, 0.05% Tween 20

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Abstract

L'invention concerne des procédés de détection et de diagnostic d'un accident vasculaire cérébral. Ces procédés se fondent sur la découverte que des taux anormaux d'analytes choisis dans un fluide échantillon, typiquement dans des échantillons de sang, de patients à risque corroborent un diagnostic d'accident vasculaire cérébral. Ainsi, l'invention concerne au moins deux nouveaux biomarqueurs de l'accident vasculaire cérébral, la MMP-3 et la SGOT. En tout, les concentrations de douze analytes donnent un tableau sensible et sélectif de la condition du patient, c'est-à-dire indiquent si le patient subit un accident vasculaire cérébral. L'invention concerne également d'autres biomarqueurs importants de l'accident vasculaire cérébral, comprenant non limitativement l'IL-18, le facteur VII, l'ICAM-I, la créatine kinase MB, la MCP-1, la myoglobine, la protéine C réactive, le facteur de von Willebrand, la TIMP-1, la ferritine, la glutathion S-transférase, l'antigène prostatique spécifique (libre), l'IL-3, le facteur tissulaire, l'alpha-fœtoprotéine, la phosphatase acide prostatique, le facteur des cellules souches, la MIP-1 bêta, l'antigène carcino-embryonnaire, l'IL-13, le TNF alpha, l'IgE, la protéine de liaison aux acides gras, l'ENA-78, l'IL-1 bêta, le facteur neurotrophique dérivé du cerveau, l'apolipoprotéine A1, l'amyloïde P sérique, l'hormone de croissance, la microglobuline bêta 2, la lipoprotéine (a), la MMP-9, l'hormone thyréotrope, la macroglobuline alpha 2, le complément 3, l'IL-7, la leptine et l'IL-6. L'invention concerne également des kits contenant des réactifs permettant d'analyser des échantillons de fluides.
PCT/US2007/067117 2006-04-21 2007-04-20 Procédés et kits de diagnostic d'un accident vasculaire cérébral Ceased WO2007124439A2 (fr)

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Cited By (19)

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WO2009049189A3 (fr) * 2007-10-10 2009-06-18 Bg Medicine Inc Procédés pour détecter des événements cardiovasculaires et cérébrovasculaires préjudiciables majeurs
CN101566636B (zh) * 2008-04-24 2012-10-24 北京科美生物技术有限公司 一种定量检测血液中甲胎蛋白的磁性免疫层析试纸条的制备方法
US9958442B2 (en) 2009-02-11 2018-05-01 Duke University Sensors incorporating antibodies and methods of making and using the same
US9274126B2 (en) 2009-11-13 2016-03-01 Bg Medicine, Inc. Risk factors and prediction of myocardial infarction
US10215760B2 (en) 2009-11-25 2019-02-26 Hologic, Inc. Detection of intraamniotic and/or infection
CN102667486B (zh) * 2009-11-25 2016-03-09 霍洛吉克股份有限公司 羊膜内感染的检测
CN102667486A (zh) * 2009-11-25 2012-09-12 霍洛吉克股份有限公司 羊膜内感染的检测
US9164092B2 (en) 2009-11-25 2015-10-20 Hologic, Inc. Detection of intraamniotic infection
WO2011121362A2 (fr) 2010-04-01 2011-10-06 Cambridge Enterprise Limited Marqueurs biologiques
WO2011121362A3 (fr) * 2010-04-01 2011-11-24 Cambridge Enterprise Limited Marqueurs biologiques
US20170184611A1 (en) * 2011-12-02 2017-06-29 Randox Laboratories Ltd. Biomarker-based methods and biochips for aiding the diagnosis of stroke
US10914745B2 (en) * 2011-12-02 2021-02-09 Randox Laboratories Ltd. Biomarker-based methods for aiding the diagnosis of stroke
WO2014108396A1 (fr) * 2013-01-08 2014-07-17 Sphingotec Gmbh Taux à jeun d'hormone de croissance comme marqueur prédictif d'un risque cardiovasculaire
RU2677895C2 (ru) * 2013-01-08 2019-01-22 Сфинготек Гмбх Уровни гормона роста натощак в качестве прогностического маркера сердечно-сосудистого риска
US10407716B2 (en) 2014-03-13 2019-09-10 Duke University Electronic platform for sensing and control of electrochemical reactions
CN104849450A (zh) * 2015-05-31 2015-08-19 中国烟草总公司郑州烟草研究院 一种卷烟烟气体外免疫毒性的Luminex悬浮芯片测试方法
JP2020508444A (ja) * 2017-02-20 2020-03-19 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 無症候性脳虚血に関する血清学的アッセイ
JP7211626B2 (ja) 2017-02-20 2023-01-24 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 無症候性脳虚血に関する血清学的アッセイ
CN111122871A (zh) * 2018-10-31 2020-05-08 博阳生物科技（上海）有限公司 一种三碘甲状腺原氨酸的均相检测试剂盒、方法及应用

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