WO2013009146A2 - Marker for diagnosing diabetic retinopathy - Google Patents

Abstract

The present invention relates to a composition and a kit comprising an agent for measuring a gene or a protein level that can be used to diagnose and determine the progression of a diabetic retinopathy patient, for diagnosing diabetic retinopathy, a method for providing information for diagnosing diabetic retinopathy, and to a method for diagnosing diabetic retinopathy using the composition and the kit.

Description

Markers for Diagnosing Diabetic Retinopathy and Uses thereof

The present invention is a composition, kit for diagnosing diabetic retinopathy, comprising a preparation for measuring the level of gene or protein that can be used for diagnosis and diagnosis of diabetic retinopathy patients, method for providing information for diagnosing diabetic retinopathy using the same and diabetes It relates to a method for diagnosing retinopathy.

Diabetes is a serious disease with increased blood sugar due to abnormalities in the production or use of insulin and accompanying various acute and chronic complications. In the United States, 9.6% (over 20 million people) of the population 20 years of age or older have diabetes, and an estimated 50 million pre-diabetes patients at high risk are estimated (2005 national diabetes facts in the United States. sheet). In 2002, $ 132 billion was spent on direct and indirect medical expenses related to diabetes.

In Korea, according to the National Health and Nutrition Survey conducted by the Centers for Disease Control and Prevention in 2005, 9.0% of men over 30 and 7.2% of women were diabetic. According to the 2007 Korean Diabetes Research Report, published by the Korean Diabetes Association, the prevalence of diabetes is about 8% and 10% of new patients occur each year. Diabetes-related medical expenses make up about 20% (about 3 trillion won) of health insurance expenses. Given the recent rapid increase in the number of diabetics currently estimated at 4-5 million, the number of diabetics can reach 10 million after 10-20 years.

Since diabetes has a long morbidity, it is accompanied by various complications of the whole system. Representative examples include cardiovascular disease, diabetes mellitus, diabetes neuropathy, and diabetic retinopathy. Among them, diabetic retinopathy (DR) occurs in over 60% of diabetic patients within 10 years of diabetic diagnosis and over 90% of diabetic patients in 20 years.

Diabetic retinopathy is a microangiopathy of diabetes characterized by changes in permeability of retinal vessels, vascular obstruction, ischemia, neovascularization, and thus fibrovascular proliferation.

Diabetic retinopathy is the largest cause of acquired blindness in adults, and in the United States, 12,000 to 24,000 people die from diabetes every year. In the United States, the prevalence of diabetic retinopathy is estimated to be about 40% of diabetic patients and 8% is reported to be a serious condition that can lead to blindness.

Diabetic retinopathy is divided into early non-proliferative diabetic retinopathy (NPDR) and late proliferative diabetic retinopathy (PDR) according to the degree of progression (FIG. 1).

 Non-proliferative diabetic retinopathy (NPDR) is characterized by retinal bleeding, microaneurysm, exudate, retinal edema, etc. due to obstruction of the retinal capillaries and changes in permeability. have. In addition, macular edema (DME, diabetic macular edema) can cause severe vision decline even in non-proliferative diabetic retinopathy.

Proliferative diabetic retinopathy (PDR) is a stage in which neovascularization proliferates due to an ischemia condition following extensive vessel occlusion of the retina. This proliferation progresses from the retina to the vitreous and fibrovascular proliferation causes complications such as vitreous hemorrhage, tractional retinal detachment, and neovascular glaucoma, in which the retina falls from its original attachment site. This is the stage of blindness.

In spite of laser treatment or surgical treatment, there are still many patients whose symptoms continue to lead to blindness. Therefore, there is an increasing need for early detection and suppression of diabetic retinopathy and early treatment for high risk groups. However, the pathogenesis of diabetic retinopathy has not yet been accurately identified, and biomarkers for determining the progression of retinopathy due to diabetes are very limited.

To date, diabetic retinopathy has been mainly focused on biochemical and molecular biological studies of individual proteins in the vitreous. In addition, the proteomics study of diabetic retinopathy is a profiling (discovery) step of the vitreous protein bodies that identify proteins in patient vitreous by 2-DE and mass spcectrometry. Very little verification and validation studies have been conducted to determine whether these vitreous proteins are expressed in the blood or can be used as clinical biomarkers.

Therefore, it is necessary to develop new clinical markers with high specificity and sensitivity, as well as antibodies capable of detecting them, and to easily predict the early diagnosis and progression of diabetic retinopathy.

The present inventors have made diligent efforts to develop markers useful for early diagnosis of diabetic retinopathy. As a result, we have discovered a diabetic retinopathy-specific protein and using the expression pattern of the specific protein that increases or decreases in diabetic retinopathy. It was confirmed that can be easily diagnosed to complete the present invention.

One object of the present invention is for diagnosing diabetic retinopathy in which at least one selected from alpha-1 acid glycoprotein (AGP) and pigment epithelium-derived factor (PEDF), which are new diabetic retinopathy markers for early diagnosis of diabetic retinopathy. To provide a marker.

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.

Another object of the present invention to provide a method for diagnosing diabetic retinopathy using the composition or kit for diagnosing diabetic retinopathy.

 The present invention provides a marker for diagnosing diabetic retinopathy, and by measuring and comparing the expression level of a gene or a protein whose expression is increased or decreased in a diabetic retinopathy, the early diagnosis and the degree of disease of the diabetic retinopathy is significant. Can be predicted or identified. In addition, the marker of the present invention enables a non-invasive diagnosis can be a simple and effective early diagnosis of diabetic retinopathy by blood, urine tests and the like.

1 is an eye photograph of a patient with non-proliferative diabetic retinopathy and proliferative diabetic retinopathy.

2 is a flow chart schematically showing the implementation process of the first to sixth embodiments.

3 is a graph illustrating an ROC curve and an interactive plot of AGP according to Example 6. FIG.

Figure 4 is a graph showing the ROC curve and interactive plot of PEDF according to Example 6.

5 is a graph showing an ROC curve and an interactive plot of APOC1 according to Example 6. FIG.

6 is a graph showing an ROC curve and an interactive plot of APOB100 according to Example 6. FIG.

FIG. 7 is a graph illustrating an ROC curve and an interactive plot of APOC3 according to Example 6. FIG.

8 is a graph illustrating an ROC curve and an interactive plot of the VTN according to the sixth embodiment.

9 is a graph illustrating an ROC curve and an interactive plot of PLG according to Example 6. FIG.

10 is a graph showing an ROC curve and an interactive plot of HRP according to Example 6. FIG.

FIG. 11 is a graph illustrating ROC curves and interactive plots of AFMs according to Example 6. FIG.

12 is a graph illustrating an ROC curve and an interactive plot of CP according to Example 6. FIG.

FIG. 13 is a graph illustrating an ROC curve and an interactive plot of CFB according to Example 6. FIG.

As one embodiment for achieving the above object, the present invention provides a marker for diagnosing one or more diabetic retinopathy selected from Alpha-1 acid glycoprotein (AGP) and Pigment epithelium-derived factor (PEDF).

As used herein, the term "diagnostic" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether diabetic retinopathy develops. Preferably it is to determine whether the early stage of diabetic retinopathy non-proliferative diabetic retinopathy.

As used herein, the term "diagnostic marker" refers to significant levels of gene expression or protein expression levels in individuals with non-proliferative diabetic retinopathy compared to normal controls (non-diabetic retinopathy) or proliferative diabetic retinopathy. Organic biomolecules such as polypeptides or nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), etc.

The marker for diagnosing diabetic retinopathy for the purposes of the present invention may be AGP (Alpha-1 acid glycoprotein) or PEDF (Pigment epithelium-derived factor). Preferably, the marker according to the present invention may be used in combination of AGP, PEDF alone, or AGP and PEDF.

More preferably, the diabetic retinopathy diagnostic marker is APOC1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), APOC3 (Apolipoprotein C3), VTN (Vitronectin), PLG (Plasminogen), HRP (Histidine-rich protein), AFM (Afamin) It may further include one or more selected from Ceruloplasmin (CP) and Complement factor B (CFB). Preferably, at least one selected from nine markers, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or at least nine may be used in combination. For example, APOC1; APOC1 and VTN; AFM, CP and CFB; APOC1, APOC3, PLG and HRP; And the like, but are not limited thereto.

Preferably, the combination of one or more selected from the nine markers and AGP or PEDF or AGP and PEDF may be used as a diagnostic marker for diabetic retinopathy.

Diabetic retinopathy is divided into early stage of non-proliferative diabetic retinopathy (NPDR) and late stage proliferative diabetic retinopathy (PDR). Non-proliferative diabetic retinopathy is characterized by not developing blood vessels, and proliferative diabetic retinopathy is different in its mechanism, such as the development of blood vessels. Since non-proliferative diabetic retinopathy does not necessarily progress to proliferative diabetic retinopathy, a marker known as a diagnostic marker for proliferative diabetic retinopathy cannot necessarily be used as a diagnostic marker for non-proliferative diabetic retinopathy.

The present inventors have found that AGP or PEDF can be used as a diagnostic marker for diabetic retinopathy through the following verification.

In one embodiment of the present invention, 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 these, 11 NPDR-specific markers (AGP, PEDF, APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP and CFB). ) Was finally unearthed.

In another embodiment of the present invention, the plasma samples of the normal control group (non-diabetic retinopathy) and NPDR (non-proliferative diabetic retinopathy) subjects were analyzed to identify the 11 types of NPDR (non-proliferative diabetic retinopathy) identified above. The validity of the enemy marker was established.

In one embodiment, the present invention provides a composition for diagnosing diabetic retinopathy comprising an agent for measuring mRNA or protein level of one or more genes selected from alpha-1 acid glycoprotein (AGP) and pigment epithelium-derived factor (PEDF). to provide.

Alpha-1 acid glycoprotein (AGP) is an acute plasma alpha-globulin glycoprotein that is regulated by two polymorphic genes and, according to the gene ontology classification, is a protein involved in immune response and fibrin lysis. Is AGP (GeneBank Accession No. AAB33887, Uniprot: P02763). However, the association of AGP with diabetic retinopathy is unknown at all. AGP is characterized by an increase in the gene expression level or the expression level of the protein in the diabetic retinopathy subject compared to the normal control (individual non-diabetic retinopathy). The diabetic retinopathy subject is preferably a non-proliferative diabetic retinopathy subject.

PEDF (Pigment epithelium-derived factor) is a multifunctional secretory protein with anti-angiogenic, anti-tumorgenic and neurotropic functions, according to the gene ontology classification according to retinoblastoma and preactivated B-lymphocytes B-lymphocytes) is a protein involved. The genetic information is PEDF (GeneBank Accession No. AAK92491, Uniprot: P36955). However, no association between PEDF and diabetic retinopathy is known at all. PEDF is characterized by an increase in the gene expression level or expression level of the protein in diabetic retinopathy individuals compared to normal controls (individuals not diabetic retinopathy). The diabetic retinopathy subject is preferably a non-proliferative diabetic retinopathy subject.

The composition is in addition to the agent for measuring the mRNA or protein level of one or more genes selected from AGP and PEDF, APOC1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), APOC3 (Apolipoprotein C3), VTN (Vitronectin), PLG ( Diabetes comprising agents for measuring mRNA or protein levels of one or more genes selected from the group consisting of Plasminogen, HRP (Histidine-rich protein), AFM (Afamin), Ceruloplasmin (CP) and Complement factor B (CFB) It may be a composition for diagnosing retinopathy.

The APOC1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), and APOC3 (Apolipoprotein C3) are all proteins involved in lipid metabolism according to the gene ontology classification, and the genetic information is APOC1 (GeneBank Accession No. AAD02506). , Uniprot: P02654), APOB100 (GeneBank Accession No. AAB04636, Uniprot: P04114), APOC3 (GeneBank Accession No. AAS68230, Uniprot: P02656). However, no association between APOC1, APOB100 and APOC3 with diabetic retinopathy is known at all.

VTN (Vitronectin), PLG (Plasminogen), and HRP (Histidine-rich protein) are genes involved in immune response and fibrin lysis according to Gene ontology classification, and the genetic information is VBN (GeneBank Accession No. AAH05046). , Uniprot: P04004), PLG (GeneBank Accession No. AAA60113, Uniprot: P00747), HRP (GeneBank Accession No. AAI50592, Uniprot: P04196). However, no association between VTN, PLG and HRP with diabetic retinopathy is known at all.

AFM (Afamin) is a protein involved in transport according to the gene ontology classification, and the genetic information can be found in GeneBank Accession No. AAI09021, Uniprot: P43652. However, the association of AFM with diabetic retinopathy is unknown at all.

According to gene ontology classification, CP (ceruloplasmin) is a protein involved in transport, and its genetic information is CP (GeneBank Accession No. AAF02483, Uniprot: P00450). However, the association of AFM with diabetic retinopathy is unknown at all.

Complement factor B (CFB) is a protein involved in retinoblastoma and preactivated B-lymphocytes according to gene ontology classification.The genetic information is CFB (GeneBank Accession No. AAA16820, Uniprot). : P00751). However, no association between CFB and diabetic retinopathy is known.

The APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP and CFB are all reduced in gene expression level or expression level of the protein in diabetic retinopathy compared to normal control (individual not diabetic retinopathy). Has characteristics. The diabetic retinopathy subject is preferably a non-proliferative diabetic retinopathy subject.

As used herein, the term "mRNA expression level measurement" refers to measuring 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 in order 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 thereto. Since nucleic acid information of genes according to the present invention is known to GeneBank et al., Those skilled in the art can design primer pairs or probes that specifically amplify specific regions of these genes based on the sequences.

As used herein, the term "protein expression level measurement" refers to 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 to diagnose diabetic retinopathy. Protein expression level measurement is to determine the amount of protein using an antibody that specifically binds to the protein of the gene, preferably refers to measuring the protein expression level itself without using an antibody.

The protein expression level measurement or comparative analysis method is protein chip analysis, immunoassay, ligand binding assay, Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, Surface Enhanced Laser Desorption / SELDI-TOF Ionization Time of Flight Mass Spectrometry, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid phase chromatography-mass spectrometry liquid chromatography-Mass Spectrometry (LC-MS), liquid chromatography-Mass Spectrometry / Mass Spectrometry (LC-MS / MS), Western blotting, and enzyme linked immunosorbentassay (ELISA), but are not limited thereto.

As used herein, the term "diabetic retinopathy" refers to a diabetic complication in which peripheral circulatory disorder occurs due to diabetes, which causes disorders in the microcirculation of the retina, resulting in decreased vision. Preferably the diabetic retinopathy according to the invention is non-proliferative diabetic retinopathy.

Preferably, the agent measuring the mRNA level is specific for at least one gene selected from AGP and PEDF and additionally at least one gene selected from APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP and CFB. Primer pairs, probes or antisense nucleotides that bind to each other.

As used herein, the term “primer pair” includes primer pairs of all combinations of forward and reverse primers that recognize a target gene sequence, but preferably, a primer pair that provides an assay with specificity and sensitivity. to be. High specificity can be imparted when the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample so that only the target gene sequence containing the complementary primer binding site is amplified and does not cause nonspecific amplification. .

As used herein, the term "probe" refers to a substance that can specifically bind to a target substance to be detected in a sample, and through the binding, a substance that can specifically confirm the presence of the target substance in the sample. it means. The type of probe is a material commonly used in the art, but is not limited. Preferably, the probe 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. More specifically, the probes include those derived from or similar to organisms or produced ex vivo as biomaterials, for example enzymes, proteins, antibodies, microorganisms, flora and fauna, organ cells, 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.

As used herein, the term “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. An oligomer having a sequence of bases and an intersubunit backbone. The oligomer may have exact sequence complementarity or approximate complementarity to the target sequence.

Preferably, the agent measuring the protein level is specific for at least one protein selected from AGP and PEDF and additionally at least one protein selected from APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP and CFB. It may include an antibody that binds to.

As used herein, the term “antibody” refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody means an antibody that specifically binds to at least one protein selected from AGP, PEDF, APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP, and CFB. Antibodies of the invention include all 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. The functional fragment of an antibody molecule means the fragment which has at least antigen binding function, and includes Fab, F (ab '), F (ab') 2, Fv, etc.

In still another aspect, the present invention provides a kit for diagnosing diabetic retinopathy comprising the composition for diagnosing diabetic retinopathy. Preferably, 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.

In addition, preferably, the diabetic retinopathy diagnostic kit may further include one or more other component compositions, solutions, or devices suitable for analytical methods.

Preferably, 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. Other reverse transcriptase kits include test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), enzymes such as deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC -May include DEPC-water, sterile water, and the like.

Preferably, the diagnostic kit may be a diagnostic kit comprising an essential element necessary to perform a DNA chip. 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.

Also preferably, the diagnostic kit may be a diagnostic kit comprising essential elements necessary for performing an ELISA. ELISA kits include antibodies specific for the protein. 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 can also include antibodies specific for the control protein. 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.

As another aspect, 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.

Preferably, the information providing method is an expression level or protein expression level of one or more genes selected from alpha-1 acid glycoprotein (AGP) and pigment epithelium-derived factor (PEDF) from biological samples isolated from patients with suspected diabetic retinopathy. Measuring; 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.

As used herein, the term "biological sample" means a tissue, cell, whole blood, serum, plasma, saliva, cerebrospinal fluid or urine, or the like, in which the gene expression level or protein expression level is different due to the development of diabetic retinopathy. However, it is not limited thereto.

The AGP and PEDF has a characteristic that the expression level of the gene or protein expression level is increased compared to the normal control, and when the level is increased can be diagnosed with diabetic retinopathy and provide information. Preferably the diabetic retinopathy is non-proliferative diabetic retinopathy.

Preferably, the measuring and comparing step is APOC1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), APOC3 (Apolipoprotein C3), VTN (Vitronectin), PLG (Plasminogen), HRP (Histidine-rich protein), AFM ( Afamin), Ceruloplasmin (CP) and Complement factor B (CFB) may further comprise the step of measuring and comparing the expression level of the protein or the expression level of the protein selected from the group consisting of.

The APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP, and CFB are characterized by a decrease in the expression level of the gene and the expression level of the protein compared to the normal control, diabetic retinopathy Can diagnose and provide information. At this time, the diabetic retinopathy is non-proliferative diabetic retinopathy.

In addition, preferably, comparing the expression level of the gene of AGP or PEDF or the expression level of the protein with the expression level of the gene of the individual sample of proliferative diabetic retinopathy or the expression level of the protein, determining that the increase is non-proliferative diabetic retinopathy It may further include.

Since the AGP and PEDF of the present invention increase the gene or protein expression level in non-proliferative diabetic retinopathy, when it is increased compared to the gene or protein expression level of proliferative diabetic retinopathy (PDR) individuals, It can be determined as dietary diabetic retinopathy (NPDR).

In addition, at least one selected from the group consisting of APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP, and CFB as a complex marker decreases the gene or protein expression level in non-proliferative diabetic retinopathy, thereby proliferating diabetic retina. A decrease in comparison with the level of gene or protein expression in an individual (PDR) can be determined as non-proliferative diabetic retinopathy (NPDR).

Preferably, 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 include, but is not limited to, reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, northern blotting or DNA chip. Through the above measurement methods, it is possible to confirm the mRNA expression level of the normal control group and the mRNA expression level of the diabetic retinopathy patient, and to compare or determine the level of these expressions to diagnose or predict the onset of diabetic retinopathy.

Preferably the protein expression level of the present invention can be measured and compared using an antibody that specifically binds to the protein. 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.

As used herein, 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.

More preferably, 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.

For the purpose of the present invention, the protein expression level measurement or comparative analysis methods include protein chip analysis, immunoassay, ligand binding assay, Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid phase Liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), western blot, and enzyme linked immunosorbentassay (ELISA). .

In specific embodiments of the invention, the LC-MRM method was used to measure and compare the protein expression levels of AGP, PEDF, APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP or CFB itself.

Specifically, the protein in the biological sample was 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%. Since the resolution of a specific material may vary depending on the solution mixing ratio, a concentration gradient was performed, and the above range was selected for an optimal range for separating various proteins at the same time.

In mass spectrometry, quantification was performed by MRM (Multiple reaction monitoring) in MS / MS mode. Whereas Selected Ion Monitoring (SIM) is a method that uses ions generated by collisions once in the source section of a mass spectrometer, MRM selects one ion from one broken ion one more time to source another MS in series. It is a method of using ions obtained from the collision after passing through it once more. In SIM, there is a problem that the selected quantitative ion may interfere with the quantification when the selected quantitative ion is an ion that is also detected in plasma. On the other hand, in case of using MRM, even if ions with the same mass are broken once more, the molecular structure is different and tends to be differentiated. Can be obtained. Thus, by using the MRM mode in mass spectrometry, it was possible to simultaneously analyze the desired materials with better analytical sensitivity.

 Through the above analysis methods, it is possible to compare the protein expression level in the normal control group and the protein expression level in the diabetic retinopathy individual, and to determine whether there is a significant increase or decrease in the expression level of the diabetic retinopathy marker gene to the protein. Diagnosis can be made.

Accordingly, as another aspect, the present invention is to measure the expression level or expression level of one or more genes selected from the group consisting of Alpha-1 acid glycoprotein (AGP) and Pigment epithelium-derived factor (PEDF) from the biological sample ; And it provides a method for diagnosing diabetic retinopathy comprising comparing the expression level of the gene or the expression level of the protein with a normal control sample.

The measuring and comparing step are APOC1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), APOC3 (Apolipoprotein C3), VTN (Vitronectin), PLG (Plasminogen), HRP (Histidine-rich protein), AFM (Afamin), The method may further include measuring and comparing the expression level of one or more genes selected from the group consisting of Ceruloplasmin (CP) and Complement factor B (CFB) or expression levels of proteins.

In another embodiment, the present invention provides Alpha-1 acid glycoprotein (AGP), Pigment epithelium-derived factor (PEDF), APOC1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), APOC3 (Apolipoprotein C3), VTN (Vitronectin), PLG (Plasminogen), Hisidine-rich protein (HRP), AFM (Afamin), Ceruloplasmin (CP), and Complement factor B (CFB) are provided for use as a marker for diagnosing diabetic retinopathy.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the present invention, and the present invention is not limited by these examples.

Example 1 Diagnosis of Diabetic Retinopathy Overexpression or Low Expression Protein

 First, MRM analysis of vitreous proteins in patients with proliferative diabetic retinopathy (PDR) and macular hole (MH) revealed MDR-specific candidate markers, which differ in expression.

Expression of proteins in vitreous and corresponding plasma samples from secondary PDR, macular hole (MH), and non-proliferative diabetic retinopathy (NPDR) patients based on the degree of PDR protein expression obtained in the above process. Aspects were analyzed by MRM. As a result, it was possible to find proteins exhibiting differences in expression compared to macular hole (MH) and diabetic retinopathy patients (PDR) (Table 1). Of these, two and nine overexpressions and nine underexpressions were able to finally select proteins specifically expressed in non-proliferative diabetic retinopathy (NPDR).

Table 1 SEQ ID NO: Protein name Expression Gene Accession number Uniprot One Alpha-1 acid glycoprotein (AGP) Increased expression AAB33887 P02763 2 Pigment epithelium-derived factor (PEDF) Increased expression AAK92491 P36955 3 APOC1 (Apolipoprotein C1) Reduced expression AAD02506 P02654 4 APOB100 (Apolipoprotein B100) Reduced expression AAB04636 P04114 5 Apolipoprotein C3 (APOC3) Reduced expression AAS68230 P02656 6 Vitronectin (VTN) Reduced expression AAH05046 P04004 7 PLG (Plasminogen) Reduced expression AAA60113 P00747 8 Histidine-rich protein (HRP) Reduced expression AAI50592 P04196 9 AFM (Afamin) Reduced expression AAI09021 P43652 10 Ceruloplasmin (CP) Reduced expression AAF02483 P00450 11 Complement factor B (CFB) Reduced expression AAA16820 P00751

Example 2: Patient Selection and Plasma Collection

Plasma samples from 45 patients with non-proliferative diabetic retinopathy (NPDR) early in diabetic retinopathy (NPDR) (samples for diabetic but without 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 three stages were mild, moderate and severe according to the progression of non-proliferative diabetic retinopathy.

TABLE 2 group Gender (female / male) age DM elapsed period (years) HbAlc (%) Plasma concentration (µg / µl) CV of plasma concentration (%) NoDR 7/8 63.8 ± 9.5 7.9 ± 5.4 7.5 ± 1.6 63.8 9.1 MI NPDR 7/8 61.4 ± 6.7 12.3 ± 6.0 7.4 ± 1.3 61.4 9.4 MO NPDR 8/7 60.6 ± 9.9 15.9 ± 7.7 7.4 ± 1.1 60.6 7.9 SV NPDR 9/6 61.8 ± 9.1 11.8 ± 6.4 7.8 ± 0.8 61.8 9.7

(NoDR: Diabetic but no diabetic retinopathy, MI NPDR: Mild NPDR, MO NPDR: Moderate NPDR, SV NPDR: Severe NPDR)

Example 3: Pretreatment of Plasma Samples

Plasma samples were quantified using Bradford, of which 200 μg of plasma was taken, denatured into urea, reduced and alkylated with DTT and iodoacetic acid. Here, trypsin was treated at a ratio of 50: 1 (protein: trypsin, w / w) to make denatured proteins into peptides, and the peptides were desalted using C18 ZipTip and lyophilized. This was dissolved in solution A (95% distilled water, 5% acetonitrile, 0.1% formic acid) and spiked with 100 fmol of beta-galactosidase peptide, an internal standard, and analyzed by MRM. .

Example 4: Selection of Transition

In order to select the transitions of the proteins, 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 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 (MRMPliot, version 2.0, Appliedbiosystems, USA) for some transitions that were difficult to select due to low peaks. For transitions not captured by the MIDAS workflow program, the peptides were selected by selecting peptides with high observed numbers using the Peptide Atlas database.

Example 5: LC and MRM

LC used MDLC nanoflow TempoLC of MDS. To isolate the peptide, C18 resin with a diameter of 3 µm and a pore size of 200 mm was directly filled using a fused sillica capillary column of 15 cm in length and 100 µm in diameter. Peptide samples were injected by direct injection method, 1.0 μl was injected directly into the analitical column without passing through the trap column, and the flow rate was used as 400nl / min. Equilibrate the column with Solution A (95% distilled water, 5% acetonitrile, 0.1% formic acid) for 10 minutes and then from Solution 5 (5% distilled water, 95% acetonitrile, 0.1% formic acid) for 5 minutes to 85%, Peptides were eluted through a concentration gradient of 85% for 5 minutes.

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 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 would be 2.5 seconds. Neubulizing gas was used in 5 units and heater temperature was set at 150 ℃. In order to demonstrate the batch-to-batch variation, the 50 fmole beta-galactosidase peptide (beta-galactosidase peptide, Transition 542.3 / 636.3) spiked in each sample was also monitored at the same time. MS run time was run for 60 minutes in time synchronization with LC. MS and LC were run using Analyst 2.1.2.

Example 6 Data Analysis

For relative quantification, 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, and 100.0 fmol. Standard curve was determined. The individual MRM results were generated by extracting ion ion chromatography (XIC) of the MRM transitions using MultiQuant (AppliedBiosystems, ver1.0), and the peak area of each transition was calculated and plotted again over time. . Normalize the area of each XIC peak to the XIC peak area of the beta-galatosidase peptide (Transition 542.3 / 636.3), an internal standard, and use this value to perform relative quantitative analysis for each protein. Was performed. For statistical analysis, ROC (Receiver 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) was used for some plotting and t-test analysis.

As a result of the analysis, 11 proteins having significant differences among the proteins with different expressions were identified, and ROC curves and interactive plots of the proteins are shown in FIGS. 3 to 13.

As a result, FIGS. 3 and 4 show that ROG curves and interactive plots of AGP and PEDF, respectively, are increased in expression compared to NoDR samples, and thus can be used as diagnostic markers specific to non-proliferative diabetic retinopathy. 5 to 13 illustrate APOC1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), APOC3 (Apolipoprotein C3), VTN (Vitronectin), PLG (Plasminogen), HRP (Histidine-rich protein), AFM (Afamin), ROC curves and interactive plots of CP (Ceruloplasmin) and Complement factor B (CFB) are reduced in expression compared to NoDR samples, respectively, and can be used as diagnostic markers specific for additional nonproliferative diabetic retinopathy with AGP and PEDF It was.

Claims (16)

A composition for diagnosing diabetic retinopathy comprising an agent for measuring mRNA or protein level of one or more genes selected from alpha-1 acid glycoprotein (AGP) and pigment epithelium-derived factor (PEDF). According to claim 1, APOC1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), APOC3 (Apolipoprotein C3), VTN (Vitronectin), PLG (Plasminogen), HRP (Histidine-rich protein), AFM (Afamin), CP (Ceruloplasmin) ) And CFB (Complement factor B) composition for diagnosing diabetic retinopathy comprising an agent for further measuring the mRNA or protein level of one or more genes selected from the group consisting of. The composition of claim 1 or 2, wherein the diabetic retinopathy is non-proliferative diabetic retinopathy. The composition for diagnosing diabetic retinopathy of claim 1, wherein the agent for measuring mRNA level of the gene is a primer pair, a probe, or an antisense nucleotide that specifically binds to the gene. The composition for diagnosing diabetic retinopathy of claim 1, wherein the agent for measuring protein level comprises an antibody that specifically binds to the protein. Kit for diagnosing diabetic retinopathy comprising a composition according to any one of claims 1 to 5. The kit of claim 6, wherein the kit is a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme linked immnosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM). Diabetic retinopathy diagnostic kit, characterized in that the kit. Measuring the expression level of at least one gene selected from alpha-1 acid glycoprotein (AGP) and pigment epithelium-derived factor (PEDF) or expression level of the protein from the biological sample; And Method for 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. The method of claim 8, wherein the measuring and comparing are carried out: APOC1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), APOC3 (Apolipoprotein C3), VTN (Vitronectin), PLG (Plasminogen), HRP (Histidine-rich protein) Diagnosing diabetic retinopathy, further comprising measuring and comparing the expression level of one or more genes selected from the group consisting of AFM (Afamin), Ceruloplasmin (CP), and Complement factor B (CFB) or protein levels. How to Provide Information The method of claim 8, wherein the expression level of the gene or the expression level of the protein is compared with the expression level of the gene of the individual sample of proliferative diabetic retinopathy or the expression level of the protein. Method for providing information for diagnosing diabetic retinopathy further comprising. 10. The method of claim 8 or 9, wherein the expression level of the gene is the mRNA expression level of the gene. 12. The method of claim 11, wherein the measurement of mRNA levels comprises diagnosing diabetic retinopathy by reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip. How to Provide Information The method of claim 8, wherein the protein expression level is measured using an antibody that specifically binds to the protein. The method of claim 8, wherein the protein expression level is measured or compared by protein chip analysis, immunoassay, ligand binding assay, Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, or Surface Enhanced (SELDI-TOF). Laser 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 Performed using one selected from the group consisting of liquid chromatography-Mass Spectrometry (LC-MS), liquid chromatography-Mass Spectrometry / Mass Spectrometry (LC-MS / MS), western blot and enzyme linked immunosorbentassay (ELISA). Information providing method for diagnosing diabetic retinopathy, characterized in that. Measuring the expression level of at least one gene selected from alpha-1 acid glycoprotein (AGP) and pigment epithelium-derived factor (PEDF) or expression level of the protein from the biological sample; And Diagnosis of diabetic retinopathy comprising comparing the expression level of the gene or the expression level of the protein with a normal control sample. The method of claim 15, wherein the measuring and comparing are carried out: APOC1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), APOC3 (Apolipoprotein C3), VTN (Vitronectin), PLG (Plasminogen), HRP (Histidine-rich protein) Diagnosing diabetic retinopathy, further comprising measuring and comparing the expression level of one or more genes selected from the group consisting of AFM (Afamin), Ceruloplasmin (CP), and Complement factor B (CFB) or protein levels. How to Provide Information

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