WO2013103226A1 - Marqueur pour le diagnostic précoce de la rétinopathie diabétique et son utilisation - Google Patents

Marqueur pour le diagnostic précoce de la rétinopathie diabétique et son utilisation Download PDF

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WO2013103226A1
WO2013103226A1 PCT/KR2012/011806 KR2012011806W WO2013103226A1 WO 2013103226 A1 WO2013103226 A1 WO 2013103226A1 KR 2012011806 W KR2012011806 W KR 2012011806W WO 2013103226 A1 WO2013103226 A1 WO 2013103226A1
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diabetic retinopathy
protein
expression level
alpha
kit
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PCT/KR2012/011806
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English (en)
Korean (ko)
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김영수
유형곤
김경곤
김상진
유지영
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서울대학교 산학협력단
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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
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology
    • G01N2800/164—Retinal disorders, e.g. retinopathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • the present invention relates to a composition for diagnosing diabetic retinopathy and a kit for diagnosing diabetic retinopathy.
  • the present invention also relates to an analysis method for providing information necessary for diagnosing diabetic retinopathy.
  • Diabetic retinopathy is one of the three major ocular blindness disorders with macular degeneration and glaucoma. About 25% of the 5 million diabetic patients in Korea are known to have this disease, which occurs in about 40% of patients over 40 years of age with diabetes, and accounts for more than 60% of the blindness of elderly people over 65 years old. According to the National Health Insurance Corporation, the number of patients with diabetic retinopathy increased by 34% in four years from 153,000 in 2005 to 204,000 in 2009.
  • Diabetic retinopathy is caused by abnormal neovascularization in this area, not functioning properly as a blood vessel, and easily burst and cause bleeding.
  • Diabetic retinopathy is divided into non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR).
  • NPDR non-proliferative diabetic retinopathy
  • PDR proliferative diabetic retinopathy
  • NPDR non-proliferative diabetic retinopathy
  • PDR proliferative diabetic retinopathy
  • NPDR non-proliferative diabetic retinopathy
  • PDR proliferative diabetic retinopathy
  • Deepening in the back causes huge adverse effects on vision, so timely treatment is necessary.
  • abnormal neovascularization grows and gradually develops proliferative diabetic retinopathy, intraocular bleeding may occur and tractional retinal detachment may occur. Severe intraocular hemorrhage can lead to blindness.
  • Diabetic retinopathy is rarely diagnosed early due to little early symptoms, poor vision, poor focus, blurring, or glare. In spite of the adventitious treatment, many patients are suffering from severe disease and leading to blindness. However, more than 85% of patients can maintain visual acuity if they receive proper treatment at the early stage and thoroughly manage their blood sugar. Therefore, there is an increasing need for early detection and suppression of diabetic retina and early treatment for high risk groups. However, the etiology of diabetic retinopathy has not yet been accurately identified, and biomarkers for determining the extent of diabetic retinopathy are very limited. Therefore, by developing novel early diagnostic markers and antibodies with high specificity and sensitivity, the present inventors intended to invent a biomarker for early diagnosis of diabetic retinopathy in blood.
  • NPDR non-proliferative diabetic retinopathy
  • alpha-1-acid glycoprotein 2 (alpha-1-acid glycoprotein 2), alpha- which is a new diagnostic marker for diabetic retinopathy, which can effectively diagnose the early diagnosis and deepening of diabetic retinopathy.
  • Alpha-1-antitrypsin, Apolipoprotein A2, Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin Gelsolin is to provide a marker for diagnosing diabetic retinopathy at least one selected from.
  • Still another object of the present invention is to provide a composition for diagnosing diabetic retinopathy comprising an agent for measuring the level of mRNA or protein thereof of at least one gene selected from the above markers.
  • Another object of the present invention to provide a kit for diagnosing diabetic retinopathy comprising the composition.
  • Still another object of the present invention is to provide a method for providing information necessary for diagnosing diabetic retinopathy by using the composition or kit for diagnosing diabetic retinopathy.
  • Alpha-1-acid glycoprotein 2 (Alpha-1-acid glycoprotein 2), alpha-1-antitrypsin (Alpha-1-antitrypsin), apolipoprotein Providing one or more markers for diagnosing diabetic retinopathy selected from Apolipoprotein A2, Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin do.
  • diagnosis refers to identifying the presence or characteristic of a pathological condition.
  • diagnosis is to determine whether diabetic retinopathy develops.
  • NPDR non-proliferative diabetic retinopathy
  • the term "diagnostic marker” refers to a significant increase in gene expression or protein expression levels in individuals with non-proliferative diabetic retinopathy compared to normal controls (non-diabetic retinopathy) or proliferative diabetic retinopathy. Or organic biomolecules such as polypeptides or nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), which exhibit reduced behavior.
  • polypeptides or nucleic acids eg, mRNA, etc.
  • lipids lipids
  • glycolipids glycoproteins
  • sugars monosaccharides, disaccharides, oligosaccharides, etc.
  • Markers for diagnosing diabetic retinopathy for the purposes of the present invention are alpha-1-acid glycoprotein 2 (alpha-1-acid glycoprotein 2), alpha-1-antitrypsin (alpha-1-antitrypsin), apolipoprotein A2 (Apolipoprotein) A2), Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin.
  • Diabetic retinopathy is divided into non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR).
  • Non-proliferative diabetic retinopathy is characterized by the absence of blood vessel development, and proliferative diabetic retinopathy is different in its mechanism, such as the development of blood vessels, and non-proliferative diabetic retinopathy does not necessarily deepen into proliferative diabetic retinopathy.
  • a marker known as a diagnostic marker for proliferative diabetic retinopathy may not necessarily be used as a diagnostic marker for non-proliferative diabetic retinopathy.
  • alpha-1-acid glycoprotein 2 (Alpha-1-acid glycoprotein 2)
  • alpha-1-antitrypsin (Alpha-1-antitrypsin)
  • apolipoprotein A2 (Apolipoprotein A2)
  • Apolipoprotein A4 Beta-2-glycoprotein 1, and Gelsolin were identified as markers for diagnosing diabetic retinopathy.
  • biomarkers that overexpress or underexpress diabetic retinopathy by analyzing plasma samples from MH (macular hole), PDR (proliferative diabetic retinopathy), and NPDR (non-proliferative diabetic retinopathy) individuals Excavated. Specifically, samples of PDR and MH individuals were analyzed to identify PDR-specific candidate markers, and based on this, seven NPDR-specific markers were finally discovered.
  • the present invention provides alpha-1-acid glycoprotein 2, alpha-1-antitrypsin, apolipoprotein A2, A formulation for measuring mRNA or protein levels of one or more genes selected from Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin Provided is a composition for diagnosing diabetic retinopathy.
  • Gene information of the six proteins of the present invention are alpha-1-acid glycoprotein 2, GeneBank Accession No. AAA51549, Uniprot ID: P19652, alpha-1-antitrypsin (Alpha- 1-antitrypsin, GeneBank Accession No. AAA51547, Uniprot ID: P01009), Apolipoprotein A2 (Apolipoprotein A2, GeneBank Accession No. AAA51701, Uniprot ID: P02652), Apolipoprotein A4 (Apolipoprotein A4, GeneBank Accession No. AAA96731, Uniprot ID: P06727), Beta-2-glycoprotein 1 (GeneBank Accession No. AAA51766, Uniprot ID: P02749), and Gelsolin (Gelsolin, GeneBank Accession No. AAH26033, Uniprot ID: P06396) to be.
  • Alpha-1-acid glycoprotein 2 Alpha-1-antitrypsin, Apolipoprotein A2, Apolipoprotein A4 A4), Beta-2-glycoprotein 1, and Gelsolin have no known association with diabetes retinopathy.
  • Alpha-1-acid-glycoprotein 2 (Alpha-1-acid glycoprotein 2), alpha-1-antitrypsin (Alpha-1-antitrypsin), apolipoprotein A2 (Apolipoprotein A2), apolipoprotein A4 (Apolipoprotein A4) ), Beta-2-glycoprotein 1, and gelsolin are all specific gene expression levels in MO NPDR individuals compared to normal controls (nondiabetic retinopathy) or The expression level of the protein is reduced.
  • the diabetic retinopathy subject is preferably a non-proliferative diabetic retinopathy subject.
  • mRNA expression level measurement used in the present invention is to measure the amount of mRNA in the process of confirming the presence and expression of mRNA of the genes for diagnosing diabetic retinopathy in a biological sample to diagnose diabetic retinopathy.
  • Analytical methods for this purpose include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real-time reverse transcriptase (Real-time RT-PCR), RNase protection assay (RPA). assays, Northern blotting, DNA chips, etc., but are not limited to these.
  • the agent for measuring mRNA expression level of a gene is preferably a primer pair or probe, and since the nucleic acid information of the genes is known in GeneBank et al., Those skilled in the art will recognize primers or probes that specifically amplify specific regions of these genes based on the sequence. You can design
  • the term "measurement of protein expression level" used in the present invention is a process of confirming the presence and degree of expression of a protein expressed from a gene for diagnosing diabetic retinopathy in a biological sample in order to diagnose diabetic retinopathy.
  • Antibodies, interacting proteins, ligands, nanoparticles, or aptamers that specifically bind to protein or peptide fragments of the gene can be used to determine the amount of protein, but specific for the protein or peptide fragment. All detection means with an affinity can be included and preferably the protein expression level itself is measured without the use of antibodies, interacting proteins, ligands, nanoparticles or aptamers.
  • the protein expression level measurement or comparative analysis methods include protein chip analysis, immunoassay, ligand binding assay, Matrix Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF), and Surface Enhanced Laser Desorption / Ionization Time (SELDI-TOF).
  • diabetic retinopathy refers to a complication in which peripheral circulatory disorder occurs due to diabetes and a decrease in vision occurs due to a disorder in the microcirculation of the retina, and preferably may be non-proliferative diabetic retinopathy. .
  • the agent for measuring the mRNA level is alpha-1-acid glycoprotein 2 (Alpha-1-acid glycoprotein 2), alpha-1-antitrypsin (Alpha-1-antitrypsin), Apolipoprotein A2 (Apolipoprotein A2) Primer pairs, probes that specifically bind to one or more genes selected from Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin. Or antisense nucleotides.
  • primer pair includes primer pairs of all combinations of forward and reverse primers that recognize a target gene sequence, but is preferably a primer pair that provides assay results with specificity and sensitivity.
  • nucleic acid sequence of a primer is a sequence that is inconsistent with a non-target sequence present in a sample, it can give high specificity when the primer amplifies only a target gene sequence containing complementary primer binding sites and does not cause nonspecific amplification. .
  • the term “probe” refers to a substance that can specifically bind to a target substance to be detected in a sample, and means a substance that can specifically confirm the presence of the target substance in the sample through the binding. do.
  • the type of probe molecule is a material commonly used in the art, but is not limited.
  • the probe molecule may be a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a peptide, a polypeptide, a protein, an RNA, or a DNA. It is PNA.
  • the probes include those derived from or similar to organisms or produced in vitro as biomaterials, for example enzymes, proteins, antibodies, microorganisms, flora and fauna, neurons, DNA, and RNA.
  • DNA may include cDNA, genomic DNA, oligonucleotides, RNA includes genomic RNA, mRNA, oligonucleotides, and examples of proteins may include antibodies, antigens, enzymes, peptides, and the like.
  • antisense refers to a nucleotide in which an antisense oligomer hybridizes with a target sequence in RNA by Watson-Crick base pairing, allowing formation of mRNA and RNA: oligomeric heterodimers, typically within the target sequence. It refers to oligomers having a sequence of bases and a backbone between subunits. The oligomer may have exact sequence complementarity or approximate complementarity to the target sequence.
  • the agent for measuring the protein level is alpha-1-acid glycoprotein 2 (Alpha-1-acid glycoprotein 2), alpha-1-antitrypsin (Alpha-1-antitrypsin), Apolipoprotein A2 (Apolipoprotein A2) ), An antibody that specifically binds to one or more protein or peptide fragments selected from Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin. , Interaction proteins, ligands, nanoparticles, or aptamers.
  • the term “antibody” refers to a specific protein molecule directed against an antigenic site.
  • the antibody is the alpha-1-acid glycoprotein 2 (Alpha-1-acid glycoprotein 2), alpha-1-antitrypsin (Alpha-1-antitrypsin), apolipoprotein A2 (Apolipoprotein A2) Means an antibody that specifically binds to at least one protein selected from apolipoprotein A4, beta-2-glycoprotein 1, and gelsolin, Polyclonal antibodies, monoclonal antibodies and recombinant antibodies. Generating antibodies can be readily prepared using techniques well known in the art.
  • the antibodies of the present invention also include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains.
  • a functional fragment of an antibody molecule refers to a fragment having at least antigen binding function, and includes Fab, F (ab '), F (ab') 2 and Fv.
  • the term "aptamer” refers to a biopolymer material that inhibits protein interaction through three-dimensional binding with a specific target protein in the form of DNA, RNA of a single, double helix, and various target molecules. It has the characteristic of binding to.
  • the aptamers may be small nucleic acids of 15-50 bases in length that are folded into defined secondary and tertiary structures, such as stem-loop structures.
  • the aptamers preferably bind to the target high or low expression protein with a kd of less than 10 ⁇ 6 , 10 ⁇ 8 , 10 ⁇ 10 , or 10 ⁇ 12 .
  • Aptamers can bind to high or low expression proteins with very high specificity, and aptamers can be composed of multiple ribonucleotide units, deoxyribonucleotide units, or a mixture of two types of nucleotide residues.
  • the aptamers of the present invention may further comprise one or more modified base, sugar or phosphate backbone units.
  • the present invention provides a kit for diagnosing diabetic retinopathy comprising the composition for diagnosing diabetic retinopathy.
  • the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit.
  • MRM multiple reaction monitoring
  • the diabetic retinopathy diagnostic kit may further include one or more other component compositions, solutions, or devices suitable for analytical methods.
  • the diagnostic kit may be a diagnostic kit comprising essential elements necessary to perform reverse transcriptase.
  • the reverse transcription polymerase kit contains each primer pair specific for the marker gene.
  • the primer is a nucleotide having a sequence specific to the nucleic acid sequence of each gene, and is about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length. It may also include primers specific for the nucleic acid sequence of the control gene.
  • reverse transcriptase kits include test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC- DEPC-water, sterile water, and the like.
  • reaction buffers pH and magnesium concentrations vary
  • dNTPs deoxynucleotides
  • enzymes such as Taq-polymerase and reverse transcriptase
  • DNAse DNAse
  • RNAse inhibitor DEPC- DEPC-water sterile water, and the like.
  • the DNA chip kit may include a substrate on which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and a reagent, a preparation, an enzyme, or the like for preparing a fluorescent probe.
  • the substrate may also comprise cDNA or oligonucleotide corresponding to the control gene or fragment thereof.
  • ELISA kits include antibodies, interacting proteins, ligands, nanoparticles or aptamers that specifically bind to the protein or peptide fragment.
  • Antibodies are antibodies that have high specificity and affinity for each marker protein and have little cross-reactivity to other proteins. They are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies.
  • the ELISA kit may also include antibodies specific for interacting proteins, ligands, nanoparticles, aptamers or control proteins that specifically bind to the protein or peptide fragment.
  • Other ELISA kits can bind reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (eg conjugated with the antibody) and substrates or antibodies thereof. Other materials and the like.
  • the present invention provides a method for providing information for diagnosing diabetic retinopathy using the diabetic retinopathy diagnostic composition or the diabetic retinopathy diagnostic kit.
  • the information providing method is alpha-1-acid glycoprotein 2, alpha-1-antitrypsin from a biological sample isolated from a suspected diabetic retinopathy. ), Expression of one or more genes selected from Apolipoprotein A2, Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin. Measuring the level or expression level of the protein; And it may be a method of providing information for diagnosing diabetic retinopathy comprising comparing the expression level of the gene or the expression level of the protein with a normal control sample.
  • biological sample includes tissues, cells, whole blood, serum, plasma, saliva, cerebrospinal fluid, or urine, which differ in gene expression levels or protein expression levels due to the development of diabetic retinopathy. This is not restrictive.
  • Alpha-1-acid-glycoprotein 2 (Alpha-1-acid glycoprotein 2), alpha-1-antitrypsin (Alpha-1-antitrypsin), apolipoprotein A2 (Apolipoprotein A2), apolipoprotein A4 (Apolipoprotein A4) ), Beta-2-glycoprotein 1, and gelsolin are all specific gene expression levels in MO NPDR individuals compared to normal controls (nondiabetic retinopathy) or Since the expression level of the protein is reduced, it is possible to provide information by diagnosing diabetic retinopathy when the level is decreased.
  • the diabetic retinopathy is non-proliferative diabetic retinopathy.
  • Alpha-1-acid-glycoprotein 2 of the present invention alpha-1-antitrypsin, apolipoprotein A2, apolipoprotein A4 ( Apolipoprotein A4), Beta-2-glycoprotein 1, and Gelsolin are all diabetes mellitus because they specifically reduce the level of gene expression or the expression of the protein in MO NPDR individuals.
  • the expression level decreases as compared with the non-diabetic retinopathy (control), it can be judged as medium-term non-proliferative diabetic retinopathy (MO NPDR) during the advanced stage of diabetic retinopathy.
  • MO NPDR medium-term non-proliferative diabetic retinopathy
  • the expression level of the gene of the present invention can measure or compare the mRNA expression level.
  • the mRNA expression level measurement or comparison may use reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip, etc., but the present invention is limited thereto. It is not.
  • reverse transcriptase polymerase reaction competitive reverse transcriptase polymerase reaction
  • real time reverse transcriptase polymerase reaction RNase protection assay
  • Northern blotting or DNA chip etc.
  • the protein expression level of the present invention can be measured and compared using antibodies, interacting proteins, ligands, nanoparticles or aptamers that specifically bind to the protein or peptide fragment.
  • the antibody and the protein of interest in the biological sample form an antigen-antibody complex, and a method of detecting the antibody is used.
  • the term “antigen-antibody complex” means a combination of a protein antigen and an antibody that recognizes it to identify the presence or absence of the gene of interest in a biological sample.
  • the detection of the antigen-antibody complex can be detected using methods as known in the art, such as spectroscopic, photochemical, biochemical, immunochemical, electrical, absorbing, chemical and other methods.
  • the protein expression level measurement and comparison in the present invention is characterized by measuring and comparing the protein expression level itself without using an antibody.
  • the protein expression level measurement or comparative analysis methods include protein chip analysis, immunoassay, ligand binding assay, Matrix Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, Surface Enhanced Laser (SELDITOF) Desorption / Ionization Time of Flight Mass Spectrometry analysis, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass Liquid chromatography-Mass Spectrometry (LCMS), liquid chromatography-Mass Spectrometry / Mass Spectrometry (LC-MS / MS), western blot, and enzyme linked immunosorbentassay (ELISA), but are not limited thereto.
  • MALDI-TOF Matrix Desorption / Ionization Time of Flight Mass Spectrometry
  • SELDITOF Surface
  • the protein in the biological sample is passed through the LC analysis column with a concentration gradient of 5% to 85% for 30 minutes with a solution of 5% distilled water, 95% acetonitrile and 0.1% formic acid based on volume%.
  • concentration gradient was carried out because the resolution of a specific material may vary depending on the solution mixing ratio, and thus the range was selected for the optimal range for simultaneously separating various proteins.
  • MS mass spectrometry
  • MRM Multiple reaction monitoring
  • SRM selected reaction monitoring
  • SIM Selected Ion Monitoring
  • MRM selects a particular ion one more time from the broken ion to detect another continuously connected source of MS. It is a method of using the ions obtained from the collision after passing through it once more. More specifically, in SIM, there is a problem in that the selected ions may interfere with quantification when the selected ions are ions that are also detected in plasma.
  • the present invention provides a marker for early diagnosis of diabetic retinopathy, thereby measuring and comparing the expression level of the reduced expression gene or its protein in patients with non-proliferative diabetic retinopathy. Can significantly predict or identify early diagnosis and degree of disease
  • 1 is a flow chart schematically showing the experimental process of the present invention.
  • 2 to 4 are tables showing 142 transitions for 61 proteins presented as marker candidates in the Examples of the present invention.
  • FIG. 5 is a graph showing the duration of T2DM and the level of HbA1c for 60 plasma samples from ANOVA analysis results. Black represents the NoDR group, green represents the MI NPDR group, blue represents the MO NPDR group, and red represents the SV NPDR group.
  • FIG. 6 shows four protein markers of the present invention, Alpha-1-acid glycoprotein 2, Alpha-1-antitrypsin, and apolipoprotein A2.
  • A2 a box-plot showing the expression patterns of each of Apolipoprotein A4, Beta-2-glycoprotein 1, and Gelsolin will be.
  • FIG. 7 is a graph showing an interactive plot and an ROC curve of alpha-1-acid glycoprotein 2 according to an embodiment of the present invention.
  • FIG. 8 is a graph showing an interactive plot and ROC curve of alpha-1-antitrypsin according to an embodiment of the present invention.
  • FIG. 9 is a graph showing an interactive plot and ROC curve of apolipoprotein A2 according to an embodiment of the present invention.
  • FIG. 10 is a graph showing an interactive plot and ROC curve of apolipoprotein A4 according to an embodiment of the present invention.
  • FIG. 11 is a graph showing an interactive plot and ROC curve of beta-2-glycoprotein 1 according to an embodiment of the present invention.
  • FIG. 12 is a graph showing an interactive plot and ROC curve of gelsolin (Gelsolin) according to an embodiment of the present invention.
  • the present invention confirmed the efficacy of 61 protein marker candidates using MRM method in plasma samples of diabetic retinopathy patients.
  • the blood samples of non-proliferative diabetic retinopathy (NPDR) patients used in the present invention are 15 early non-proliferative diabetic retinopathy (mild NPDR, MI NPDR), 15 medium-term non-proliferative diabetic retinopathy (MO NPDR)
  • 15 non-proliferative diabetic retinopathy severe NPDR, SV NPDR
  • NoDR diabetic but not diabetic retinopathy
  • Six biomarkers that specifically reduce expression in NPDR was confirmed by MRM (Multiple Reaction Monitoring) experimental method using Triple Quadrupole LC-MS / MS to complete the present invention.
  • Bicinchoninic acid BCA, Cat. No: B9643
  • copper (II) sulfate aqueous solution Cat. No: C2284
  • Sequencing-grade modified trypsin Cat.No: V5111, porcine
  • IAA iodoacetamide
  • DTT dithiothreitol for protein denaturation
  • Urea urea, Cat.No: U6504
  • FFA formic acid
  • T62200 trifluoroacetic acid
  • HPLC-grade methanol HPLC-grade methanol (HPLC-grade methanol, MeOH, Cat.No: UN1230), acetonitrile (acetonitrile, ACN, Cat.No: UN1648), and HPLC-grade water (Duksan, Seoul, Korea) Purchased).
  • Sep-PAK Vac (1 cc, tC18 cartridges, Cat. No: WAT054960) used for peptide desalting was purchased from Waters (Milford, Mass.).
  • Peptide mixtures of beta-galactosidase (Cat. No: 4333606) from E. coli were purchased from Applied Biosystems (Foster City, CA).
  • Pico Tip emitter (stock no: FS360-20-10-N-20-C12, ID in tip 10 ⁇ m, ID 20 ⁇ m, OD 360 ⁇ m, length 20 cm) and IntegraFrit capillary (stock no: IF360-75-50- N-5, ID 75 ⁇ m, OD 360 ⁇ m, length 50 cm) was purchased from New Objective (Woburn, MA).
  • Sample vials (part no: LP1114-1265, 0.2-ml glass microinsert, silanized) were purchased from Interface Engineering (Seoul, Korea).
  • Protein LoBind tubes (Cat. No: 022431081, 1.5 ml) and low-retention tips (Rainin Instrument, Oakland, Calif.) Were used.
  • FIG. 1 A flow chart schematically illustrating the experimental process of the present invention is shown in FIG. 1.
  • PDR which is a protein having a difference in expression through vitreous proteome analysis in patients with proliferative diabetic retinopathy (PDR) and macular hole (MH, non-diabetic control), in order to select a protein marker candidate group Specific candidate markers were identified.
  • PDR proliferative diabetic retinopathy
  • MH macular hole
  • NPDR non-proliferative diabetic retinopathy
  • the AUC value is a calculated value derived from the ROC curve.
  • Plasma samples from 45 patients with non-proliferative diabetic retinopathy (NPDR) early in diabetic retinopathy (NPDR) and samples from control patients (diabetic but not diabetic retinopathy, NoDR) were obtained for LC MS / MS test samples.
  • Clinical characteristics of the 45 patients with non-proliferative diabetic retinopathy (NPDR) and control patients are shown in Table 2 below. The patients were divided into three groups: mild, moderate, and severe.
  • NoDR group without diabetic retinopathy
  • MI NPDR Mild NPDR
  • MO NPDR Moderate NPDR
  • SV NPDR Severe NPDR
  • HbA1c levels representing the amount of sugar molecules reflecting the blood glucose mean over the last two to three months, increased by 7.4% in all groups, and there was no difference between groups in the ANOVA analysis.
  • Table 4 group insulin Statins Hypertension Taking Unused Taking Unused Taking Unused NoDR 9 6 8 6 3 12 MI NPDR 10 5 7 3 One 10 MO NPDR 8 7 7 6 5 10 SV NPDR 7 8 5 10 4 11 Statistical analysis method Pearson chi-square test Fisher exact test Fisher exact test p -value 0.7155 0.3239 0.4966
  • the data indicate that the plasma samples taken from the four groups have a relatively homogeneous diabetes background, and the present inventors believe that the difference in the expression level of the target protein is primarily due to the difference in stages of diabetic retinopathy.
  • the hypothesis can be hypothesized.
  • Each plasma sample was analyzed using conventional methods (Park, J .; Kwon, H .; Kang, Y .; Kim, Y., Proteomic analysis of O-GlcNAc modifications derived from streptozotocin and glucosamine induced beta-cell apoptosis.J Biochem Mol Biol 2007 , 40, (6), 1058-68.).
  • blood was collected with a K2-EDTA-coated 10-ml tube (BD Sciences, NJ, P / N number: 367525) and centrifuged at 3000 g for 10 minutes at 4 ° C.
  • Each plasma sample was divided into 50 ⁇ l samples and stored at -80 ° C.
  • Plasma samples were quantified using Bradford, of which 200 ⁇ g of plasma was taken and denatured into 100 ⁇ l 6 M Urea. It was then reduced to 1.7 ⁇ l 200 mM DTT and then alkylated with 6.7 ⁇ l 200 mM iodoacetic acid. 500 ⁇ l of distilled water was added thereto to dilute urea to 1 M or less, and then trypsin was treated at a ratio of 50: 1 (protein: trypsin, w / w) to make the denatured protein into a peptide. After overnight storage in a shaker at 37 ° C., 50 ⁇ l of 0.1% trifluoroacetic acid (TFA) was added to terminate the reaction.
  • TFA trifluoroacetic acid
  • Denatured peptides were desalted using Sep-PAK tC18 cartridge (50 mg) and lyophilized with 2 mL 60% acetonitrile and 0.1% TFA solution. This was dissolved in 300 ⁇ l Solution A (95% distilled water, 5% acetonitrile, 0.1% formic acid), to which an internal standard beta-galactosidase peptide (residues 954-962, GDFQFNISR) was added. ) 50 fmol was spiked and analyzed by MRM.
  • Solution A 95% distilled water, 5% acetonitrile, 0.1% formic acid
  • MS / MS analysis was performed on the proteins selected in the excavation study. Based on this, a representative peptide for each protein was selected (Q1 transition), and the highest intensity ion (Q3) was selected among the fragmentation ions generated by electrically breaking the peptide. Two peptides per protein were selected and two fragmentation ions per peptide were selected to determine Q1 / Q3 as four transitions for one protein. Transitions were selected using the MRM Initiated Detection and Sequencing (MIDAS) workflow program (MRM Pliot, version 2.0, Applied biosystems, USA) for transitions that are difficult to select experimentally due to low peaks. Transitions that were not captured by the MIDAS workflow program were selected by selecting peptides with high observed numbers using the Peptide Atlas database.
  • MIDAS MRM Initiated Detection and Sequencing
  • LC used MDLC nanoflow Tempo LC of MDS.
  • C18 resin with a diameter of 3 ⁇ m and a pore size of 200 mm 3 was directly filled using a fused sillica capillary column having a length of 15 cm and an internal diameter of 100 ⁇ m.
  • Peptide samples were injected by direct injection method, 1.0 ⁇ l was injected directly into the analitical column without passing through the trap column. The flow rate was 400 nl / min.
  • Solution A (95% distilled water, 5% acetonitrile, 0.1% formic acid
  • Solution B 5% distilled water, 95% acetonitrile, 0.1% formic acid.
  • the peptide was eluted through a concentration gradient of
  • Mass spectrometry was monitored in MRM mode for transitions to selected proteins using 4000 QTrap instrument, Applied Biosystems' hybrid triple quadrupole / linear ion trap. Ion voltage was used at 2000 Volt and the resolution at Quadruple 1 (Q1) and Quadruple 3 (Q3) was set in units. The dwell time for the transition was set to 20 milliseconds so that the total cycle time was 2.5 seconds. Neubulizing gas was used in 5 units, and heater temperature was set at 150 °C for analysis. To demonstrate batch-to-batch variation, 50 fmole beta-galactosidase peptide (beta-galactosidase peptide, Transition 542.3 / 636.3) spiked into each sample was also monitored at the same time. The MS run time was run for 60 minutes in time synchronization with the LC and the MS and LC were run using Analyst 2.1.2.
  • beta-galatosidase peptide (Transition 542.3 / 636.3) was used to quantify MRM at 8 concentration points of blank, 0.5, 1.0, 5.0, 10.0, 25.0, 50.0, 100.0 fmol. A standard curve was determined.
  • the individual MRM results were generated by extracting the ion extraction chromatography (XIC) of the MRM transitions using MultiQuant (AppliedBiosystems, ver1.0), calculating the peak area of each transition, and plotting it over time. It was. The area of each XIC peak is normalized to the XIC peak area of the beta-galatosidase peptide (Transition 542.3 / 636.3), an internal standard. Was performed.
  • ROC Receiveiver Operating Characteristic Curve and Interactive plot were prepared using MedCalc (MedCalc Software, Belgium, vesion 11.3.3) and ANOVA (Analysis of variance) statistical analysis was performed.
  • Sigma Plot Systat Software Inc, USA, version 10.1
  • Multivariable Analysis was performed to correlate many target proteins in classifying disease groups. The algorithm used was PLS-DA (Partial Least Square Discriminant Analysis) and SIMCA-P + (Umetrics, USA, demo version) software.
  • SV NPDR causes bleeding on the surface of the retina, resulting in symptoms of decreased vision in the patient, resulting in diagnosis by ophthalmic examination. Therefore, early diagnosis of SV NPDR using markers is meaningless, and patients with SV NPDR were excluded from expression pattern analysis for diagnostic marker discovery.
  • Sensitivity and specificity of each interactve plot of FIGS. 7 to 12 are expressed as “Sens” and “Spec”, and are expressed as% values.
  • the blue circle represents the standardized concentration of each protein in NoDR against MO NPDR.
  • the AUC value of each latent marker is shown in each ROC curve.
  • the marker of the present invention enables a non-invasive diagnosis, if a diagnostic method using a simple and effective biomarker of diabetic retinopathy, such as blood, urine tests, etc. is developed, will be very useful for early detection in home and general clinic For example, medical costs can be reduced from a national perspective.

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Abstract

La présente invention concerne une composition destinée au diagnostic de la rétinopathie diabétique et un kit de diagnostic de la rétinopathie diabétique. La présente invention concerne en outre une méthode d'analyse destinée à fournir des informations nécessaires pour diagnostiquer la rétinopathie diabétique. Comme mentionné ci-dessus, en fournissant un marqueur pour le diagnostic précoce de la rétinopathie diabétique, la présente invention permet de mesurer et de comparer les gènes avec une expression réduite ou un taux d'expression d'une protéine de ceux-ci chez un patient souffrant de rétinopathie diabétique, ce qui permet un diagnostic précoce de la rétinopathie diabétique et la prédiction significative ou l'identification de la progression de la maladie. Le marqueur selon la présente invention permet un diagnostic non invasif et serait donc très utile dans la détection précoce à la maison et dans des polycliniques lorsqu'une méthode de diagnostic simple et valide, qui utilise un biomarqueur de la rétinopathie diabétique par le biais d'une analyse de sang ou d'urine, est développée. Le marqueur peut être avantageux pour réduire les coûts médicaux au niveau national.
PCT/KR2012/011806 2012-01-03 2012-12-28 Marqueur pour le diagnostic précoce de la rétinopathie diabétique et son utilisation WO2013103226A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
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CN108137660A (zh) * 2014-10-07 2018-06-08 印度科学工业研究所 用于纯化和定量凝溶胶蛋白及其变体的适体
CN108137660B (zh) * 2014-10-07 2021-06-01 印度科学工业研究所 用于纯化和定量凝溶胶蛋白及其变体的适体
CN115927602A (zh) * 2022-12-30 2023-04-07 深圳市慢性病防治中心(深圳市皮肤病防治研究所、深圳市肺部疾病防治研究所) 一种甲基化CpG位点的应用以及试剂盒

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