WO2020138965A1 - Method for diagnosis of periodontal disease, and composition and kit therefor - Google Patents

Method for diagnosis of periodontal disease, and composition and kit therefor Download PDF

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WO2020138965A1
WO2020138965A1 PCT/KR2019/018497 KR2019018497W WO2020138965A1 WO 2020138965 A1 WO2020138965 A1 WO 2020138965A1 KR 2019018497 W KR2019018497 W KR 2019018497W WO 2020138965 A1 WO2020138965 A1 WO 2020138965A1
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periodontal disease
mmp
diagnosis
prognosis
disease
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PCT/KR2019/018497
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French (fr)
Korean (ko)
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김은경
김바울
김진섭
박재형
유승범
손미진
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주식회사 수젠텍
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Publication of WO2020138965A1 publication Critical patent/WO2020138965A1/en

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    • 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
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • 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

Definitions

  • the present invention relates to a method for diagnosing periodontal disease, and more particularly, to a method for diagnosing periodontal disease by distinguishing active and inactive type matrix metalloproteinase 8 (MMP-8) from biological samples.
  • MMP-8 matrix metalloproteinase 8
  • Periodontal disease includes gingivitis occurring in the gums and periodontitis occurring in the alveolar bone, and is a disease in which the surrounding tissue supporting the tooth is destroyed, not a disease in which the tooth itself is damaged, and belongs to an inflammatory chronic disease.
  • the inflammation gradually progresses while the patient does not feel it, and the alveolar bone around the tooth is destroyed, and the disease is often recognized only when the tooth needs to be extracted. To prevent this, it is necessary to have a technique that can diagnose periodontal disease early.
  • the inventors of the present invention through the proteomic analysis of saliva or gingival crevicural fluid (GCF), which contains various inflammatory substances in response to the occurrence of periodontal disease, biomarker for periodontal disease And additionally, correlation between biomarkers for diagnosis of periodontal disease with high accuracy was analyzed.
  • GCF gingival crevicural fluid
  • An object of the present invention is to provide a method capable of diagnosing infection of bacteria causing periodontal disease with high accuracy.
  • Another object of the present invention is to provide a method of diagnosing periodontal disease with higher accuracy from the ratio of active and inactive forms of MMP-8 detected from a subject, and the ratio of active and inflammatory disease markers of MMP-8.
  • MMP-8 Microx Metalloproteinase-8 (Matrix Metalloproteinase-8) from a biological sample derived from a subject; An inflammatory disease marker, and a marker derived from a periodontal disease strain; detecting any one or more selected from the group consisting of; And when the level of the marker derived from the MMP-8, inflammatory disease marker or periodontal disease strain detected in the biological sample increases compared to a normal control sample, determining the subject from which the biological sample originated as periodontal disease; It provides a method for providing information necessary for the diagnosis or prognosis of periodontal disease.
  • the step of detecting the MMP-8 may be to distinguish and detect the active MMP-8 and the inactive MMP-8.
  • the step of determining the subject as periodontal disease is that the concentration or concentration ratio of the marker detected from the biological sample satisfies Equation 1 and Equation 2, and satisfies Equation 3 or Equation 4. In some cases, it may include judging as periodontal disease.
  • the biological sample may be any one or more selected from the group consisting of saliva, gingival fluid (GCF) and sublingual fluid (SLF).
  • GCF gingival fluid
  • SLF sublingual fluid
  • the biological sample may be any one or more selected from the group consisting of saliva, gingival fluid (GCF) and sublingual fluid (SLF).
  • GCF gingival fluid
  • SLF sublingual fluid
  • the inflammatory disease markers are C-reactive protein (CRP), secretary immunoglobulin A (sIgA), immunoglobulin (Immunoglobulin), interleukin-1 beta (IL-1beta), interleukin-6 (IL- 6), tumor necrosis factor-alpha (TNF- ⁇ ;Tumor necrosis factor-alpha), beta-glucuronidase ( ⁇ -glucuronidase), lysozyme (Lysozyme), lactoferrin (Lactoferrin) It may be abnormal.
  • the marker derived from the periodontal disease strain may be any one or more selected from the group consisting of Leukotoxin, Karilysin, Cytolysin, Gingipain.
  • the step of determining the periodontal disease may include that performed by a microarray, an antigen-antibody reaction or a mass spectrometry method.
  • the step of determining the periodontal disease may be to further use non-marker clinical information of the subject.
  • the non-marker clinical information may be any one or more selected from the group consisting of measuring the depth of the periodontal pocket of the subject, bleeding or X-ray examination.
  • the active MMP-8 has at least 95% or more homology with any one or more sequences selected from the group consisting of site 1, site 2, site 3 and site 4 listed in Table 1 below. It may be a peptide containing.
  • the inactive MMP-8 may include a peptide having at least 95% homology to any one or more sequences listed in Table 2 below.
  • the present invention is a diagnosis or prognosis of periodontal disease comprising an antibody that specifically binds to an active type MMP-8 (active matrix metalloproteinase-8) and an antibody that specifically binds to an inactive type MMP-8 (pro Matrix Metalloproteinase-8). It provides a composition for.
  • the present invention provides a composition for diagnosing or prognosing periodontal disease comprising a primer or probe specific for nucleic acids encoding active MMP-8 (active matrix metalloproteinase-8) and inactive MMP-8 (pro matrix metalloproteinase-8). to provide.
  • the present invention diagnoses periodontal disease comprising an antibody that specifically binds to the active type MMP-8 (active matrix metalloproteinase-8) and an antibody that specifically binds to the inactive type MMP-8 (pro matrix metalloproteinase-8) or
  • For the diagnosis or prognosis of periodontal disease comprising a prognostic composition and/or a primer or probe specific for nucleic acids encoding active MMP-8 (active matrix metalloproteinase-8) and inactive MMP-8 (pro matrix metalloproteinase-8)
  • a kit for diagnosing or prognosing periodontal disease comprising a composition.
  • the method of providing information necessary for diagnosing or prognosing periodontal disease according to the present invention can be easily determined whether or not periodontal disease occurs by confirming that the level of MMP-8, an inflammatory disease marker, or a marker derived from a periodontal disease strain is increased compared to a normal control sample. can do.
  • periodontal disease can be diagnosed with high accuracy by detecting MMP-8 as an active type and an inactive type, and the degree of disease progression can be grasped by additionally using information on an inflammatory disease marker. This can provide information that can be used for diagnosis, treatment and further monitoring of periodontal disease.
  • 1 is a result of predicting the epitope position of the entire amino acid sequence of the inactive and active MMP-8.
  • Figure 2 shows a sequence region and sequence that are likely to be epitope positions.
  • Figure 4 shows a gel image confirming this after forming the active MMP-8 using the inactive MMP-8.
  • Figure 5 shows the results of confirming the specific binding to each antigen by preparing antibodies that specifically bind to active MMP-8, inactive MMP-8 and sIgA, respectively, according to Experimental Example 2 of the present invention.
  • FIG. 6 is a graph showing a result of calculating a quantitative curve by measuring fluorescence signals according to concentrations of active MMP-8, inactive MMP-8, and sIgA according to Experimental Example 2 of the present invention.
  • FIG. 7 is a graph showing the results of comparing the concentrations of active MMP-8, inactive MMP-8, total MMP-8 and IL-1beta in the normal control group and periodontal disease patient group according to Experimental Example 3 of the present invention.
  • Figure 8 is the ratio of the active MMP-8 / total MMP-8, the ratio of the active MMP-8 / sIgA and the ratio of the total MMP-8 / sIgA of the normal control group and periodontal disease patient group according to Experimental Example 3 of the present invention This graph shows that there is a significant difference.
  • Method for providing information necessary for the diagnosis or prognosis of periodontal disease is selected from the group consisting of MMP-8 (Matrix Metalloproteinase-8), inflammatory disease markers and markers derived from periodontal disease strains from biological samples derived from a subject Detecting any one or more; And when the level of the marker derived from the MMP-8, inflammatory disease marker, or periodontal disease strain in the biological sample increases compared to a normal control sample, determining the subject from which the biological sample originated as periodontal disease.
  • MMP-8 Microx Metalloproteinase-8
  • Periodontal disease in the present invention refers to inflammatory reaction and/or degeneration of the gums and surrounding connective tissue and bone, and includes, but is not limited to, gingivitis, periodontitis, and peri-implantitis.
  • peri-implantitis means that an inflammatory reaction occurs around the implant, including loss of surrounding bone tissue and soft tissue infection after the implant procedure.
  • MMP-8 is a type of collagen degrading enzyme involved in the destruction of periodontal tissue and may be used as a biomarker for diagnosing periodontal disease according to the present invention together with markers derived from inflammatory disease markers and/or periodontal disease strains.
  • MMP-8 may be active type aMMP-8 (active MMP-8).
  • inflammatory disease markers include C-reactive protein (CRP), sIgA (secretary immunoglobulin A), immunoglobulin (Immunoglobulin), interleukin-1 beta (IL-1beta), interleukin-6 (IL-) 6), tumor necrosis factor-alpha (TNF- ⁇ ), beta-glucuronidase, lysozyme, lactoferrin, and destruction of host connective tissue Markers such as MMP, aspartate aminotransferase (AST), tissue (inhibitor of metalloprotease (TPM), Alkaline phosphatase (ALP) that can be used as a marker for bone tissue alteration, receptor activator of NF-kB ligand (RANKL), Osteocalcin, Osteonectin, Osteopontin Can be used without limitation.
  • CRP C-reactive protein
  • sIgA secretary immunoglobulin A
  • immunoglobulin immunoglobulin
  • Immunoglobulin immunoglobulin
  • CRP C-reactive protein
  • SIgA is the most abundant immunoglobulin among body secretions such as saliva, tears, colostrum, and gastrointestinal secretions, and its concentration increases by distinguishing between gingivitis and periodontitis.
  • a biomarker derived from a periodontal disease strain may include any one or more selected from the group consisting of Leukotoxin, Karilysin, Cytolysin and Gingipain possessed by the periodontal disease strain. It can, but is not limited to.
  • the detecting of MMP-8 may be characterized by distinguishing and detecting active MMP-8 and inactive MMP-8.
  • MMP a type of collagen degrading enzyme, is involved in the decomposition and reconstruction of the extracellular matrix, of which the active form of MMP-8 can be detected.
  • Proteins belonging to MMPs are classified into active and inactive forms depending on the presence or absence of a propeptide domain composed of 80 to 90 amino acids.
  • MMP-8 is active in inactive form. It is converted to and the change in the ratio of the inactive form to the active form occurs. These changes can be measured and used to diagnose periodontal disease.
  • the concentration or concentration ratio of the marker detected from the biological sample satisfies Equation 1 and Equation 2, and satisfies Equation 3 or Equation 4, the subject from which the biological sample is derived It can be judged as periodontal disease.
  • the biological sample of the subject when the biological sample of the subject satisfies the expressions 1 and 2, it can be regarded as a patient candidate for periodontal disease, and when the expression 1 or 2 does not satisfy, the periodontal disease is absent or is a mild normal group. I can judge.
  • the equation 3 or equation 4 when the equation 3 or equation 4 is satisfied from the sample of the candidate for the periodontal disease, it is possible to determine the periodontal disease with high accuracy through diagnosis through a multi-step algorithm by determining the patient with periodontal disease.
  • the biological sample refers to saliva, gingival fluid (GCF), sublingual fluid (SLF), and other samples of biological origin.
  • the biological sample may be saliva, gingival fissure, or sublingual deceased solution obtainable from an animal, more preferably a mammal, most preferably a human.
  • the sample can be pretreated before being used for detection.
  • it may include cell lysis, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like.
  • a step of separating nucleic acids and proteins from the sample may be added to obtain more accurate results when used for detection.
  • the process of separating proteins from biological samples can be performed using known processes, and protein levels can be measured by various methods performed by those skilled in the art.
  • the step of determining periodontal disease through a marker detected from the biological sample may include being performed by a microarray, an antigen-antibody reaction, or mass spectrometry.
  • the amount of antigen-antibody response complex formation in normal subjects without periodontal disease and the amount of antigen-antibody complex formation in suspected periodontal disease patients can be compared, and information necessary for diagnosing periodontal disease through whether or not the above-mentioned concentrations of the proteins are detected Can provide.
  • the antigen-antibody complex means a combination of a periodontal disease biomarker protein and an antibody specific to it, and the amount of formation of the antigen-antibody complex can be quantitatively measured through the signal size of the detection label.
  • the marker protein of the present invention is a known protein
  • the antibody used in the present invention can be produced by a conventional method widely known in the field of immunology using the above-known protein as an antigen.
  • monoclonal antibodies can be prepared using known fusion methods, recombinant DNA and phage antibody library techniques.
  • Immunological analysis can be included without limitation as long as it can measure the binding of the antigen to the antibody.
  • These methods are known in the art, for example, immunocytochemistry and immunohistochemistry, radioimmunoassays, immunochromatograhy, enzyme linked immunoassay (ELISA), immunoblotting (immunoblotting), Farr assay, immunoprecipitation, and immunofluorescence.
  • non-marker clinical information of the subject may be additionally used.
  • the non-marker clinical information may be any one or more selected from the group consisting of measuring the depth of the subject's periodontal pocket, bleeding, and X-ray examination, but is not limited thereto.
  • the active MMP-8 comprises a peptide having at least 95% homology with any one or more sequences selected from the group consisting of site 1, site 2, site 3 and site 4 listed in Table 1 below. It may be.
  • the inactive MMP-8 may include a peptide having at least 95% homology to any one or more sequences listed in Table 2 below.
  • the present invention is a diagnosis or prognosis of periodontal disease comprising an antibody that specifically binds to an active type MMP-8 (active matrix metalloproteinase-8) and an antibody that specifically binds to an inactive type MMP-8 (pro Matrix Metalloproteinase-8). It provides a composition for.
  • the antibody may be a polyclonal antibody or a monoclonal antibody, but may preferably be a monoclonal antibody.
  • the monoclonal antibody can be quantitatively analyzed by performing a color development reaction using a secondary antibody bound with an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) and their substrate, or directly to the marker protein. Quantitative analysis can be performed using a combination of AP or HRP enzyme with a monoclonal antibody.
  • AP alkaline phosphatase
  • HRP horseradish peroxidase
  • FITC fluorescein isothiocyanat
  • PE phytoerythrin
  • the reaction between the marker protein and the antibody is immunochromatography, western blot, immunoprecipitation (IP), enzyme-linked immunoabsorbent assays (ELISA), immunostaining immunohistochemistry (IHC) and flow cytometry analysis (FACS) can be confirmed by any one or more protein identification experiments selected from the group consisting of, but is not limited thereto.
  • composition for diagnosing or prognosing periodontal disease may include primers or probes specific for nucleic acids encoding active MMP-8 and inactive MMP-8.
  • the present invention is an antibody specifically binding to the active type MMP-8 (active matrix metalloproteinase-8) and an antibody specifically binding to the inactive type MMP-8 (pro Matrix Metalloproteinase-8) or the active type MMP-8 And a primer or probe specific for nucleic acids encoding inactive MMP-8.
  • the kit according to the present invention can be used for microarray, gene amplification, dipstick rapid kit, ELISA analysis or immunoassay.
  • the kit may be a kit for diagnosing periodontal disease composed of a microarray.
  • the microarray is capable of detecting a protein specifically attached to the antibody by an antigen-antibody reaction by attaching the antibody on a slide glass surface treated with a specific reagent.
  • the kit comprising the antibody may further include one or more additional components necessary for analysis.
  • it may further include a buffer necessary for detection, a stabilizer, an inert protein, a sample collection tool, a negative and/or positive control, etc.
  • the antibody may include radionuclides and fluorescors. ), enzymes (enzyme) and the like.
  • the structure of the active MMP-8 is currently unknown (PDB ID: 2OY4), and the structure of the inactive MMP-8 is currently known as the inactive MMP-3 (PDB ID: 1SLM) and MMP-1 (PDB ID: 1SU3). ), and modeled using a modeler (Andrej Sali, USA, version 9.20) based on the structure of the active MMP-8 (FIG. 3 ).
  • the propeptide of the inactive MMP-8 was isolated and converted to the active MMP-8. At this time, under the judgment that the newly exposed portion is likely to exhibit the unique characteristics of the active type MMP-8 compared to the inactive type, surface exposure degree and polarity were calculated as properties that can affect the immune response. The values are shown in Table 3 below. This was calculated using ENVA (syntekabio, Korea), which can be used for environmental analysis of protein structures.
  • amino acid position including the active site of MMP-8 was selected by selecting a position where the difference in surface exposure and polarity between the active and inactive forms was large.
  • mice For the production of antibodies that specifically bind to active MMP-8, inactive MMP-8 and sIgA, these and adjuvants were mixed and injected into mice, and spleens were extracted from immunized mice.
  • the mouse cancer cells, SP2/0 cells and spleen cells were fused using PEG.
  • the fused cells were confirmed by ELISA, and the fused cells were selected and inoculated into the mouse peritoneal cavity. When ascites were formed in the abdominal cavity after inoculation, the samples were collected and purified using protein G gel.
  • FIG. 5(a) it was confirmed that the antibody that specifically binds to the active MMP-8 antigen does not react with the inactive MMP-8 antigen and has high reactivity with the active MMP-8.
  • FIG. 5(b) confirmed that the antibody that specifically binds to the inactive MMP-8 does not react with the active MMP-8 antigen, and has high reactivity with the inactive MMP-8.
  • Antibodies that specifically bind to sIgA were dispensed into the NC membrane. After mixing the sIgA-specific antibody-conjugated Europium bead and the sIgA antigen (Fitzgerald) in a development buffer, the antibody-dispersed NC membrane was immersed in the development buffer. After 10 minutes, the NC membrane was irradiated with a 365 nm UV lamp to confirm antigen-antibody binding. It can be seen through FIG. 5(c) that sIgA and the antibody formed a specific binding.
  • Active MMP-8 antigen and inactive MMP-8 antigen were diluted in saliva by concentrations according to Table 4 below, and then mixed 1:1 with a saliva storage solution to prepare an antigen solution.
  • the saliva storage solution was used by mixing 1.0 mM PMSF and 2.0 mM EDTA in PBS.
  • the antigen solution and the sample dilution were mixed at a ratio of 1:9 and then dispensed into the sample inlet of the MMP-8 measurement kit.
  • fluorescence signals of inactive MMP-8 and active MMP-8 were measured using a Time Resolved Fluorescence (TRF) measurement equipment. Quantitative curves were drawn using signals measured for each concentration. 6(a) and 6(b) show quantitative curves of the inactive type (pMMP-8) and the active type (aMMP-8), respectively.
  • saliva was collected from 14 subjects in the 20s and 80s who had no periodontal disease and 38 patients with periodontal disease among men and women in their 20s and 80s who visited the Seoul National University Dental Hospital Periodontology Department and One-Stop Consultation Center.
  • the normal control group is based on the case where there is no periodontal disease or the degree of gingivitis is very slight, and the depth of the periodontal pocket is less than 3 mm, and the group of periodontal disease patients is confirmed by bone loss progression on the radiograph and the depth of the periodontal pocket is 5 mm or more Classified as.
  • the clinical sample mixed 1:1 with the saliva storage solution was mixed with the sample diluent 1:9 and dispensed into the sample inlet of the MMP-8 measurement kit.
  • the MMP-8 measurement kit used a diagnostic kit based on Lateral Flow Assay for detecting MMP-8 by immunochromatography. After 10 minutes, fluorescence signals of active MMP-8 and inactive MMP-8 were measured using a Time Resolved Fluorescence (TRF) measurement equipment, and the measured signals were substituted into a quantitative curve to convert the concentration.
  • TRF Time Resolved Fluorescence
  • the fluorescence signal of sIgA was measured in the same manner as in the method of 3-1., except that the clinical sample was mixed with the sample dilution at 1:99, and the measured signal was substituted into a quantitative curve to convert the concentration.
  • the clinical sample mixed 1:1 with the saliva storage solution was mixed 1:1 with the sample diluent and dispensed at 100 ⁇ l in a 96-well plate, and the biotin-conjugate solution was dispensed at 50 ⁇ l and maintained at room temperature for 2 hours. Thereafter, after washing, 100 ⁇ l of streptavidin-HRP solution was dispensed and maintained at room temperature for 1 hour. After washing again, 100 ⁇ l of the TMB substrate reagent was dispensed, and the plate was wrapped with foil to maintain the temperature for 10 minutes at room temperature. After 100 ⁇ l of the reaction stop solution was dispensed, absorbance at 450 nm was measured.
  • Table 5 shows concentrations of inactive MMP-8, active MMP-8, sIgA, and IL-1beta.
  • inactive MMP-8, active MMP-8, sIgA and IL-1beta were all higher in the periodontal disease patient group than in the normal control group. Specifically, sIgA and IL-1beta showed a 1.45-fold and 3-fold concentration increase in the periodontal disease patient group compared to the normal control group. In particular, the inactive MMP-8 was 3.32 times, the active MMP-8 was 4.12 times, and the overall MMP Even at -8, it showed 3.72 times higher concentration difference compared to the normal control group.
  • the ratio of total MMP-8 to active MMP-8 and the ratio of active MMP-8 to sIgA which is one of the inflammatory disease markers, also determines periodontal disease through their significant difference. It can be confirmed that it can be an important factor.

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Abstract

The present invention relates to a method for diagnosis of a periodontal disease. More particularly, a periodontal disease is diagnosed when an inflammatory disease marker including MMP-8, or a marker derived from a periodontal disease-causing strain is detected at an increased concentration, compared to a sample from a normal control. The detection of MMP-8 is separately performed for active MMP-8 and inactive MMP-8, whereby a diagnosis method and a composition and kit for diagnosis or prognosis prediction, which are of high accuracy, can be provided.

Description

치주질환 진단 방법, 이를 위한 조성물 및 키트Periodontal disease diagnosis method, composition and kit therefor
본 발명은 치주질환 진단 방법에 관한 것으로, 보다 상세하게는 생물학적 시료로부터 활성, 비활성형의 MMP-8(matrix metalloproteinase 8)을 구별하여 치주질환을 진단하는 방법에 관한 것이다.The present invention relates to a method for diagnosing periodontal disease, and more particularly, to a method for diagnosing periodontal disease by distinguishing active and inactive type matrix metalloproteinase 8 (MMP-8) from biological samples.
치주질환은 잇몸에 발생하는 치은염과 치조골에 발생하는 치주염을 포함하며, 치아 자체가 손상되는 질환이 아닌 치아를 지지하는 주위 조직이 파괴되는 질환으로서 염증성 만성질환에 속한다. 이러한 치주질환은 환자가 느끼지 못하는 사이에 염증이 점차적으로 진행되어 치아 주변의 치조골이 파괴되어 치아를 뽑아야 하는 상태가 되어서야 발병을 인지하는 경우가 많다. 이를 막기 위해서는 치주질환을 조기에 진단할 수 있는 기술이 필요하다.Periodontal disease includes gingivitis occurring in the gums and periodontitis occurring in the alveolar bone, and is a disease in which the surrounding tissue supporting the tooth is destroyed, not a disease in which the tooth itself is damaged, and belongs to an inflammatory chronic disease. In the periodontal disease, the inflammation gradually progresses while the patient does not feel it, and the alveolar bone around the tooth is destroyed, and the disease is often recognized only when the tooth needs to be extracted. To prevent this, it is necessary to have a technique that can diagnose periodontal disease early.
현재까지 전 세계적으로 치주질환과 관련된 다양한 연구가 진행 중이지만 치주질환이 어느 정도 진행이 된 후에 이를 치료하는 것에 연구가 집중되어 있을 뿐, 치주질환의 진단과 관련하여서는 육안으로 확인하거나 X-ray촬영을 통한 정성적 진단에 그치고 있는 것이 현실이다. 따라서, 치주질환을 조기에 진단하여 질병이 악화되기 전 효과적으로 제어하기 위한 진단 기술 개발이 시급하다.To date, various studies related to periodontal disease have been conducted worldwide, but research is focused on treating periodontal disease after it has progressed to some extent, and the diagnosis of periodontal disease is visually confirmed or X-ray taken. It is a reality that it is only a qualitative diagnosis through. Therefore, it is urgent to develop a diagnostic technique for diagnosing periodontal disease early and effectively controlling it before the disease worsens.
한편, 치주질환을 진단하기 위한 전신성 바이오마커 및 국소(구강) 바이오마커에 대한 연구가 지속되어 오고 있지만, 최근까지도 전신성 바이오마커 또는 구강 바이오마커의 검출만으로는 높은 정확도나 민감도를 구현하고 있지 못하다.On the other hand, studies on systemic biomarkers and topical (oral) biomarkers for diagnosing periodontal disease have continued, but until recently, the detection of systemic biomarkers or oral biomarkers has not achieved high accuracy or sensitivity.
이에 본 발명자들은 상기 과제를 해결하기 위하여, 치주질환이 발병하면 이에 반응하여 여러 염증성 물질들을 함유하게 되는 타액 또는 치은열구액(gingival crevicural fluid; GCF)의 프로테옴 분석을 통하여, 치주질환에 대한 바이오마커를 찾고, 추가적으로 고정확도의 치주질환 진단을 위한 바이오마커 사이의 상관관계를 분석하였다.In order to solve the above problems, the inventors of the present invention, through the proteomic analysis of saliva or gingival crevicural fluid (GCF), which contains various inflammatory substances in response to the occurrence of periodontal disease, biomarker for periodontal disease And additionally, correlation between biomarkers for diagnosis of periodontal disease with high accuracy was analyzed.
본 발명의 목적은 치주질환을 일으키는 세균의 감염여부를 높은 정확도로 진단할 수 있는 방법을 제공하는 것이다.An object of the present invention is to provide a method capable of diagnosing infection of bacteria causing periodontal disease with high accuracy.
본 발명의 다른 목적은 대상체로부터 검출한 MMP-8의 활성형 및 비활성형의 비율, MMP-8의 활성형 및 염증성 질환 마커의 비율로부터 보다 높은 정확도로 치주질환을 진단하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method of diagnosing periodontal disease with higher accuracy from the ratio of active and inactive forms of MMP-8 detected from a subject, and the ratio of active and inflammatory disease markers of MMP-8.
대상체 유래의 생물학적 시료로부터 MMP-8(Matrix Metalloproteinase-8); 염증성 질환 마커, 및 치주질환 균주 유래의 마커;로 이루어지는 군에서 선택되는 어느 하나 또는 둘 이상을 검출하는 단계; 및 상기 생물학적 시료에서 검출된 상기 MMP-8, 염증성 질환 마커 또는 치주질환 균주 유래의 마커 수준이 정상 대조군 시료와 비교하여 증가한 경우, 상기 생물학적 시료가 유래한 대상체를 치주질환으로 판단하는 단계;를 포함하는 치주질환 진단 또는 예후에 필요한 정보를 제공하는 방법을 제공한다.MMP-8 (Matrix Metalloproteinase-8) from a biological sample derived from a subject; An inflammatory disease marker, and a marker derived from a periodontal disease strain; detecting any one or more selected from the group consisting of; And when the level of the marker derived from the MMP-8, inflammatory disease marker or periodontal disease strain detected in the biological sample increases compared to a normal control sample, determining the subject from which the biological sample originated as periodontal disease; It provides a method for providing information necessary for the diagnosis or prognosis of periodontal disease.
본 발명의 일 예에 있어서, 상기 MMP-8을 검출하는 단계는 활성형 MMP-8과 비활성형 MMP-8을 구별하여 검출하는 것일 수 있다.In one example of the present invention, the step of detecting the MMP-8 may be to distinguish and detect the active MMP-8 and the inactive MMP-8.
본 발명의 일 예에 있어서, 상기 대상체를 치주질환으로 판단하는 단계는 상기 생물학적 시료로부터 검출된 마커의 농도 또는 농도 비율이 하기 식 1 및 식 2를 만족하고, 하기 식 3 또는 식 4를 만족하는 경우 치주질환으로 판단하는 것을 포함하는 것일 수 있다.In one example of the present invention, the step of determining the subject as periodontal disease is that the concentration or concentration ratio of the marker detected from the biological sample satisfies Equation 1 and Equation 2, and satisfies Equation 3 or Equation 4. In some cases, it may include judging as periodontal disease.
[식 1][Equation 1]
Figure PCTKR2019018497-appb-I000001
Figure PCTKR2019018497-appb-I000001
[식 2][Equation 2]
Figure PCTKR2019018497-appb-I000002
Figure PCTKR2019018497-appb-I000002
[식 3][Equation 3]
Figure PCTKR2019018497-appb-I000003
Figure PCTKR2019018497-appb-I000003
[식 4][Equation 4]
Figure PCTKR2019018497-appb-I000004
Figure PCTKR2019018497-appb-I000004
본 발명의 일 예에 있어서, 상기 생물학적 시료는 타액, 치은열구액(Gingival Crevicular Fluid; GCF) 및 혀 밑 고인액(Sublingual Fluid;SLF)으로 이루어지는 군에서 선택되는 어느 하나 이상일 수 있다.In one example of the present invention, the biological sample may be any one or more selected from the group consisting of saliva, gingival fluid (GCF) and sublingual fluid (SLF).
본 발명의 일 예에 있어서, 상기 생물학적 시료는 타액, 치은열구액(Gingival Crevicular Fluid; GCF) 및 혀 밑 고인액(Sublingual Fluid; SLF)으로 이루어지는 군에서 선택되는 어느 하나 이상일 수 있다.In one example of the present invention, the biological sample may be any one or more selected from the group consisting of saliva, gingival fluid (GCF) and sublingual fluid (SLF).
본 발명의 일 예에 있어서, 상기 염증성 질환 마커는 CRP(C-reactive Protein), sIgA(secretary Immunoglobulin A), 면역글로불린(Immunoglobulin), 인터루킨-1베타(IL-1beta), 인터루킨-6(IL-6), 종양괴사인자-알파(TNF-α;Tumor necrosis factor-alpha), 베타-글루쿠로니다제(β-glucuronidase), 리소자임(Lysozyme), 락토페린(Lactoferrin) 으로 이루어진 군에서 선택되는 어느 하나 이상일 수 있다.In one embodiment of the present invention, the inflammatory disease markers are C-reactive protein (CRP), secretary immunoglobulin A (sIgA), immunoglobulin (Immunoglobulin), interleukin-1 beta (IL-1beta), interleukin-6 (IL- 6), tumor necrosis factor-alpha (TNF-α;Tumor necrosis factor-alpha), beta-glucuronidase (β-glucuronidase), lysozyme (Lysozyme), lactoferrin (Lactoferrin) It may be abnormal.
본 발명의 일 예에 있어서, 상기 치주질환 균주 유래의 마커는 류코톡신(Leukotoxin), Karilysin, 세포융해소(Cytolysin), 진지페인(Gingipain)으로 이루어진 군에서 선택되는 어느 하나 이상일 수 있다.In one example of the present invention, the marker derived from the periodontal disease strain may be any one or more selected from the group consisting of Leukotoxin, Karilysin, Cytolysin, Gingipain.
본 발명의 일 예에 있어서, 상기 치주질환으로 판단하는 단계는 마이크로어레이, 항원-항체 반응 또는 질량분석 방식으로 수행되는 것을 포함하는 것일 수 있다.In an example of the present invention, the step of determining the periodontal disease may include that performed by a microarray, an antigen-antibody reaction or a mass spectrometry method.
본 발명의 일 예에 있어서, 상기 치주질환으로 판단하는 단계는 상기 대상체의 비마커 임상정보를 추가로 사용하는 것일 수 있다.In one example of the present invention, the step of determining the periodontal disease may be to further use non-marker clinical information of the subject.
본 발명의 일 예에 있어서, 상기 비마커 임상정보는 상기 대상체의 치주낭의 깊이 측정, 출혈 여부 또는 X-ray 검사로 이루어지는 군에서 선택되는 어느 하나 이상일 수 있다.In one example of the present invention, the non-marker clinical information may be any one or more selected from the group consisting of measuring the depth of the periodontal pocket of the subject, bleeding or X-ray examination.
본 발명의 일 예에 있어서, 상기 활성형 MMP-8은 하기 표 1에 기재된 site 1, site 2, site 3 및 site 4로 이루어지는 군에서 선택되는 어느 하나 이상의 서열과 적어도 95% 이상의 상동성을 가지는 펩티드를 포함하는 것일 수 있다.In one example of the present invention, the active MMP-8 has at least 95% or more homology with any one or more sequences selected from the group consisting of site 1, site 2, site 3 and site 4 listed in Table 1 below. It may be a peptide containing.
Figure PCTKR2019018497-appb-T000001
Figure PCTKR2019018497-appb-T000001
본 발명의 일 예에 있어서, 비활성형 MMP-8은 하기 표 2에 기재된 어느 하나 이상의 서열과 적어도 95% 이상의 상동성을 가지는 펩티드를 포함하는 것일 수 있다.In one example of the present invention, the inactive MMP-8 may include a peptide having at least 95% homology to any one or more sequences listed in Table 2 below.
Figure PCTKR2019018497-appb-T000002
Figure PCTKR2019018497-appb-T000002
본 발명은 활성형 MMP-8(active Matrix Metalloproteinase-8)과 특이적으로 결합하는 항체 및 비활성형 MMP-8(pro Matrix Metalloproteinase-8)과 특이적으로 결합하는 항체를 포함하는 치주질환 진단 또는 예후용 조성물을 제공한다.The present invention is a diagnosis or prognosis of periodontal disease comprising an antibody that specifically binds to an active type MMP-8 (active matrix metalloproteinase-8) and an antibody that specifically binds to an inactive type MMP-8 (pro Matrix Metalloproteinase-8). It provides a composition for.
또한 본 발명은 활성형 MMP-8(active Matrix Metalloproteinase-8) 및 비활성형 MMP-8(pro Matrix Metalloproteinase-8)을 암호화하는 핵산에 특이적인 프라이머 또는 프로브를 포함하는 치주질환 진단 또는 예후용 조성물을 제공한다.In addition, the present invention provides a composition for diagnosing or prognosing periodontal disease comprising a primer or probe specific for nucleic acids encoding active MMP-8 (active matrix metalloproteinase-8) and inactive MMP-8 (pro matrix metalloproteinase-8). to provide.
본 발명은 상기 활성형 MMP-8(active Matrix Metalloproteinase-8)과 특이적으로 결합하는 항체 및 비활성형 MMP-8(pro Matrix Metalloproteinase-8)과 특이적으로 결합하는 항체를 포함하는 치주질환 진단 또는 예후용 조성물 및/또는 활성형 MMP-8(active Matrix Metalloproteinase-8) 및 비활성형 MMP-8(pro Matrix Metalloproteinase-8)을 암호화하는 핵산에 특이적인 프라이머 또는 프로브를 포함하는 치주질환 진단 또는 예후용 조성물을 포함하는 치주질환 진단 또는 예후용 키트를 제공한다.The present invention diagnoses periodontal disease comprising an antibody that specifically binds to the active type MMP-8 (active matrix metalloproteinase-8) and an antibody that specifically binds to the inactive type MMP-8 (pro matrix metalloproteinase-8) or For the diagnosis or prognosis of periodontal disease comprising a prognostic composition and/or a primer or probe specific for nucleic acids encoding active MMP-8 (active matrix metalloproteinase-8) and inactive MMP-8 (pro matrix metalloproteinase-8) Provided is a kit for diagnosing or prognosing periodontal disease comprising a composition.
본 발명에 따른 치주질환 진단 또는 예후에 필요한 정보를 제공하는 방법은 MMP-8, 염증성 질환 마커 또는 치주질환 균주 유래의 마커 수준을 정상 대조군 시료와 비교하여 증가됨을 확인함으로써 손쉽게 치주질환 발병 여부를 판단할 수 있다. The method of providing information necessary for diagnosing or prognosing periodontal disease according to the present invention can be easily determined whether or not periodontal disease occurs by confirming that the level of MMP-8, an inflammatory disease marker, or a marker derived from a periodontal disease strain is increased compared to a normal control sample. can do.
또한, 본 발명은 MMP-8을 활성형과 비활성형으로 구분하여 검출함으로써 치주질환을 높은 정확도로 진단할 수 있고, 염증성 질환 마커의 정보를 추가로 활용함으로써 질병의 진행 정도를 파악할 수 있다. 이는 치주질환을 진단, 치료하고 나아가 모니터링에 활용할 수 있는 정보를 제공할 수 있다.In addition, according to the present invention, periodontal disease can be diagnosed with high accuracy by detecting MMP-8 as an active type and an inactive type, and the degree of disease progression can be grasped by additionally using information on an inflammatory disease marker. This can provide information that can be used for diagnosis, treatment and further monitoring of periodontal disease.
도 1은 비활성형 및 활성형 MMP-8 전체 아미노산 서열의 에피토프 위치를 예측한 결과이다.1 is a result of predicting the epitope position of the entire amino acid sequence of the inactive and active MMP-8.
도 2는 에피토프 위치로 가능성이 높은 서열부위 및 서열을 나타낸 것이다.Figure 2 shows a sequence region and sequence that are likely to be epitope positions.
도 3은 비활성형 MMP-8 구조를 모델링한 이미지를 나타낸 것이다.3 shows an image modeling an inactive MMP-8 structure.
도 4는 비활성형 MMP-8을 이용하여 활성형 MMP-8를 형성한 후, 이를 확인한 겔 이미지를 나타낸 것이다.Figure 4 shows a gel image confirming this after forming the active MMP-8 using the inactive MMP-8.
도 5는 본 발명의 실험예 2에 따라 활성형 MMP-8, 비활성형 MMP-8 및 sIgA에 각각 특이적으로 결합하는 항체를 제작하여 각 항원과의 특이적 결합을 확인한 결과를 나타낸 것이다.Figure 5 shows the results of confirming the specific binding to each antigen by preparing antibodies that specifically bind to active MMP-8, inactive MMP-8 and sIgA, respectively, according to Experimental Example 2 of the present invention.
도 6은 본 발명의 실험예 2에 따라 활성형 MMP-8, 비활성형 MMP-8 및 sIgA의 농도별 형광 신호를 측정함으로써 정량 곡선을 산출한 결과 그래프를 나타낸 것이다.6 is a graph showing a result of calculating a quantitative curve by measuring fluorescence signals according to concentrations of active MMP-8, inactive MMP-8, and sIgA according to Experimental Example 2 of the present invention.
도 7은 본 발명의 실험예 3에 따른 정상 대조군과 치주질환 환자군의 활성형 MMP-8, 비활성형 MMP-8, 전체 MMP-8 및 IL-1beta의 농도를 비교한 결과 그래프를 나타낸 것이다.7 is a graph showing the results of comparing the concentrations of active MMP-8, inactive MMP-8, total MMP-8 and IL-1beta in the normal control group and periodontal disease patient group according to Experimental Example 3 of the present invention.
도 8은 본 발명의 실험예 3에 따라 정상 대조군과 치주질환 환자군의 활성형 MMP-8/전체 MMP-8의 비율, 활성형 MMP-8/sIgA의 비율 및 전체 MMP-8/sIgA의 비율에서 유의적 차이가 있음을 보여주는 그래프이다.Figure 8 is the ratio of the active MMP-8 / total MMP-8, the ratio of the active MMP-8 / sIgA and the ratio of the total MMP-8 / sIgA of the normal control group and periodontal disease patient group according to Experimental Example 3 of the present invention This graph shows that there is a significant difference.
이하에서 본 발명에 대하여 구체적으로 설명한다. 본 명세서에서 사용되는 용어는 따로 정의하지 않는 경우 해당 분야에서 통상의 지식을 가진 자가 일반적으로 이해하는 내용으로 해석되어야 할 것이다. 본 명세서의 도면 및 실시예는 통상의 지식을 가진 자가 본 발명을 쉽게 이해하고 실시하기 위한 것으로 도면 및 실시예에서 발명의 요지를 흐릴 수 있는 내용은 생략될 수 있으며, 본 발명이 도면 및 실시예로 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail. Unless otherwise defined, terms used in this specification should be interpreted as contents generally understood by a person having ordinary skill in the relevant field. The drawings and examples in this specification are for those of ordinary skill in the art to easily understand and implement the present invention. In the drawings and examples, contents that may obscure the subject matter of the invention may be omitted, and the present invention may be used in the drawings and embodiments. It is not limited to.
본 발명에 따른 치주질환 진단 또는 예후에 필요한 정보를 제공하는 방법은, 대상체 유래의 생물학적 시료로부터 MMP-8(Matrix Metalloproteinase-8), 염증성 질환 마커 및 치주질환 균주 유래의 마커로 이루어지는 군에서 선택되는 어느 하나 또는 둘 이상을 검출하는 단계; 및 상기 생물학적 시료에서 상기 MMP-8, 염증성 질환 마커 또는 치주질환 균주 유래의 마커 수준이 정상 대조군 시료와 비교하여 증가한 경우, 상기 생물학적 시료가 유래한 대상체를 치주질환으로 판단하는 단계;를 포함한다.Method for providing information necessary for the diagnosis or prognosis of periodontal disease according to the present invention is selected from the group consisting of MMP-8 (Matrix Metalloproteinase-8), inflammatory disease markers and markers derived from periodontal disease strains from biological samples derived from a subject Detecting any one or more; And when the level of the marker derived from the MMP-8, inflammatory disease marker, or periodontal disease strain in the biological sample increases compared to a normal control sample, determining the subject from which the biological sample originated as periodontal disease.
본 발명에서 치주질환이란 잇몸 및 주변 결합조직 및 뼈의 염증반응 및/또는 퇴행을 의미하는 것으로 치은염(gingivitis), 치주염(periodontitis), 임플란트 주위염(peri-implantitis)을 포함하는 것이지만 이에 제한되는 것은 아니다. 여기서 임플란트 주위염이란 임플란트 시술 후 주위 뼈조직 소실과 연조직 감염을 포함한 임플란트 주위에 염증 반응이 생기는 것을 의미한다.Periodontal disease in the present invention refers to inflammatory reaction and/or degeneration of the gums and surrounding connective tissue and bone, and includes, but is not limited to, gingivitis, periodontitis, and peri-implantitis. . Here, peri-implantitis means that an inflammatory reaction occurs around the implant, including loss of surrounding bone tissue and soft tissue infection after the implant procedure.
MMP-8은 치주조직 파괴에 관여하는 콜라겐 분해효소의 일종으로서 염증성 질환 마커 및/또는 치주질환 균주 유래의 마커와 함께 본 발명에 따른 치주질환 진단을 위한 바이오마커로 이용될 수 있다. 바람직하게는 MMP-8은 활성형인 aMMP-8(active MMP-8)일 수 있다.MMP-8 is a type of collagen degrading enzyme involved in the destruction of periodontal tissue and may be used as a biomarker for diagnosing periodontal disease according to the present invention together with markers derived from inflammatory disease markers and/or periodontal disease strains. Preferably, MMP-8 may be active type aMMP-8 (active MMP-8).
구체적으로 염증성 질환 마커로는 염증 지표가 될 수 있는 CRP(C-reactive Protein), sIgA(secretary Immunoglobulin A), 면역글로불린(Immunoglobulin), 인터루킨-1베타(IL-1beta), 인터루킨-6(IL-6), 종양괴사인자-알파(Tumor necrosis factor-alpha: TNF-α), 베타-글루쿠로니다제(β-glucuronidase), 리소자임(Lysozyme), 락토페린(Lactoferrin), 숙주 결합 조직의 파괴를 나타내는 마커인 MMP, AST(aspartate aminotransferase), TIMP(tissue　inhibitor　of　metalloprotease), 골조직 개조의 마커로 이용될 수 있는 ALP(Alkaline phosphatase), RANKL(receptor activator of NF-kB ligand), Osteocalcin, Osteonectin, Osteopontin가 제한없이 사용될 수 있다. 바람직하게는 CRP, sIgA, IL-1beta 또는 이들의 조합일 수 있다.Specifically, inflammatory disease markers include C-reactive protein (CRP), sIgA (secretary immunoglobulin A), immunoglobulin (Immunoglobulin), interleukin-1 beta (IL-1beta), interleukin-6 (IL-) 6), tumor necrosis factor-alpha (TNF-α), beta-glucuronidase, lysozyme, lactoferrin, and destruction of host connective tissue Markers such as MMP, aspartate aminotransferase (AST), tissue (inhibitor　of　metalloprotease (TPM), Alkaline phosphatase (ALP) that can be used as a marker for bone tissue alteration, receptor activator of NF-kB ligand (RANKL), Osteocalcin, Osteonectin, Osteopontin Can be used without limitation. Preferably CRP, sIgA, IL-1beta or a combination thereof.
CRP(C-reactive protein)는 치주질환이 발생하는 경우 간에서 생성, 분비되어 급성 단계 혈액에서 나타나며, 치료가 진행되면 전신염증정도가 감소되어 농도가 줄어든다. SIgA는 타액, 눈물, 초유 및 위장 분비액 등 신체 분비물 중 가장 풍부하게 포함된 면역글로불린으로서 치은염과 치주염을 구분하여 그 농도가 증가한다.When periodontal disease occurs, CRP (C-reactive protein) is produced and secreted in the liver and appears in acute stage blood. As treatment progresses, the degree of systemic inflammation decreases and the concentration decreases. SIgA is the most abundant immunoglobulin among body secretions such as saliva, tears, colostrum, and gastrointestinal secretions, and its concentration increases by distinguishing between gingivitis and periodontitis.
또한, 구체적으로 치주질환 균주 유래의 바이오마커로는 치주질환 균주가 가지는 류코톡신(Leukotoxin), Karilysin, 세포융해소(Cytolysin)및 진지페인(Gingipain)으로 이루어지는 군에서 선택되는 어느 하나 이상을 포함할 수있으나, 이에 제한되지 않는다.In addition, specifically, a biomarker derived from a periodontal disease strain may include any one or more selected from the group consisting of Leukotoxin, Karilysin, Cytolysin and Gingipain possessed by the periodontal disease strain. It can, but is not limited to.
상기 MMP-8을 검출하는 단계는 활성형 MMP-8과 비활성형 MMP-8을 구별하여 검출하는 것을 특징으로 할 수 있다. 치주질환이 있는 경우 콜라겐 분해효소의 일종인 MMP가 세포외 기질의 분해 및 재건축 작용에 관여하게 되는데, 이 중 MMP-8의 활성형을 검출할 수 있다.The detecting of MMP-8 may be characterized by distinguishing and detecting active MMP-8 and inactive MMP-8. In the case of periodontal disease, MMP, a type of collagen degrading enzyme, is involved in the decomposition and reconstruction of the extracellular matrix, of which the active form of MMP-8 can be detected.
MMPs에 속하는 단백질은 80 내지 90개의 아미노산으로 구성된 프로펩티드 도메인의 존재 유무에 따라 활성형(active form)과 비활성형(pro form)으로 구분되는데 치주질환이 발병하면 MMP-8은 비활성형에서 활성형태로 전환되게 되어 비활성형과 활성형의 비율에 변화가 생기게 된다. 이러한 변화를 측정하여 치주질환 진단에 이용할 수 있다.Proteins belonging to MMPs are classified into active and inactive forms depending on the presence or absence of a propeptide domain composed of 80 to 90 amino acids. When periodontal disease develops, MMP-8 is active in inactive form. It is converted to and the change in the ratio of the inactive form to the active form occurs. These changes can be measured and used to diagnose periodontal disease.
본 발명의 일 예에 있어서, 상기 생물학적 시료로부터 검출된 마커의 농도 또는 농도 비율이 하기 식 1 및 식 2를 만족하고, 하기 식 3 또는 식 4를 만족하는 경우, 상기 생물학적 시료가 유래한 대상체를 치주질환으로 판단할 수 있다.In an example of the present invention, when the concentration or concentration ratio of the marker detected from the biological sample satisfies Equation 1 and Equation 2, and satisfies Equation 3 or Equation 4, the subject from which the biological sample is derived It can be judged as periodontal disease.
[식 1][Equation 1]
Figure PCTKR2019018497-appb-I000005
Figure PCTKR2019018497-appb-I000005
[식 2][Equation 2]
Figure PCTKR2019018497-appb-I000006
Figure PCTKR2019018497-appb-I000006
[식 3][Equation 3]
Figure PCTKR2019018497-appb-I000007
Figure PCTKR2019018497-appb-I000007
[식 4][Equation 4]
Figure PCTKR2019018497-appb-I000008
Figure PCTKR2019018497-appb-I000008
구체적으로 예를 들면, 대상체의 생물학적 시료가 상기 식 1 및 식 2를 만족하는 경우 치주질환의 환자 후보로 볼 수 있고, 상기 식 1 또는 식 2를 만족하지 않는 경우 치주질환이 없거나 경미한 정상군으로 판단할 수 있다. 또한, 상기 치주질환의 환자 후보의 시료로부터 상기 식 3 또는 식 4를 만족하는 경우에는 치주질환을 가지는 환자로 판단함으로써, 다단계 알고리즘을 통한 진단으로 높은 정확도의 치주질환 판단이 가능하다.Specifically, for example, when the biological sample of the subject satisfies the expressions 1 and 2, it can be regarded as a patient candidate for periodontal disease, and when the expression 1 or 2 does not satisfy, the periodontal disease is absent or is a mild normal group. I can judge. In addition, when the equation 3 or equation 4 is satisfied from the sample of the candidate for the periodontal disease, it is possible to determine the periodontal disease with high accuracy through diagnosis through a multi-step algorithm by determining the patient with periodontal disease.
상기 생물학적 시료는 타액, 치은열구액(Gingival Crevicular Fluid; GCF), 혀 밑 고인액(Sublingual Fluid; SLF) 및 생물학적 기원의 기타 시료를 말한다. 바람직하게는 상기 생물학적 시료는 동물, 보다 바람직하게는 포유동물, 가장 바람직하게는 인간으로부터 수득될 수 있는 타액, 치은열구액 또는 혀 밑 고인액일 수 있다.The biological sample refers to saliva, gingival fluid (GCF), sublingual fluid (SLF), and other samples of biological origin. Preferably, the biological sample may be saliva, gingival fissure, or sublingual deceased solution obtainable from an animal, more preferably a mammal, most preferably a human.
상기 시료는 검출에 사용되기 전에 전처리할 수 있다. 예를 들어, 세포 용해, 여과, 증류, 추출, 농축, 방해 성분의 불활성화, 시약의 첨가 등을 포함할 수 있다. 또한 상기 전처리 외에, 상기 시료로부터 핵산 및 단백질을 분리하는 단계를 추가하여 검출에 사용하는 경우 보다 정확도 높은 결과를 얻을 수 있다.The sample can be pretreated before being used for detection. For example, it may include cell lysis, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like. In addition, in addition to the pre-treatment, a step of separating nucleic acids and proteins from the sample may be added to obtain more accurate results when used for detection.
생물학적 시료에서 단백질을 분리하는 과정은 공지의 공정을 이용하여 수행할 수 있으며 단백질 수준은 이 기술 분야에서 통상의 기술자가 실시하는 다양한 방법으로 측정될 수 있다.The process of separating proteins from biological samples can be performed using known processes, and protein levels can be measured by various methods performed by those skilled in the art.
상기 생물학적 시료로부터 검출된 마커를 통하여 치주질환으로 판단하는 단계는 마이크로어레이, 항원-항체 반응 또는 질량분석 방식으로 수행되는 것을 포함할 수 있다. 치주질환을 가지지 않은 정상 대상체의 항원-항체 반응 복합체 형성량과 치주질환 의심환자에서의 항원-항체 복합체의 형성량을 비교할 수 있고, 일정 농도 이상의 상기 단백질들의 검출 여부를 통하여 치주질환 진단에 필요한 정보를 제공할 수 있다.The step of determining periodontal disease through a marker detected from the biological sample may include being performed by a microarray, an antigen-antibody reaction, or mass spectrometry. The amount of antigen-antibody response complex formation in normal subjects without periodontal disease and the amount of antigen-antibody complex formation in suspected periodontal disease patients can be compared, and information necessary for diagnosing periodontal disease through whether or not the above-mentioned concentrations of the proteins are detected Can provide.
본 발명에서 항원-항체 복합체란 치주질환 바이오마커 단백질과 이에 특이적인 항체의 결합물을 의미하고, 항원-항체 복합체의 형성량은 검출 라벨의 시그널의 크기를 통하여 정량적으로 측정이 가능하다.In the present invention, the antigen-antibody complex means a combination of a periodontal disease biomarker protein and an antibody specific to it, and the amount of formation of the antigen-antibody complex can be quantitatively measured through the signal size of the detection label.
본 발명의 마커 단백질은 공지된 단백질이므로 본 발명에 사용되는 항체는 상기 공지된 단백질을 항원으로 하여 면역학 분야에서 널리 알려져 있는 통상의 방법으로 제조할 수 있다. 예를 들어, 단일클론항체는 공지된 융합방법, 재조합 DNA 및 파지 항체 라이브러리 기술을 이용하여 제조할 수 있다.Since the marker protein of the present invention is a known protein, the antibody used in the present invention can be produced by a conventional method widely known in the field of immunology using the above-known protein as an antigen. For example, monoclonal antibodies can be prepared using known fusion methods, recombinant DNA and phage antibody library techniques.
면역학적 분석은 항원과 항체의 결합을 측정할 수 있는 방법이라면 제한되지 않고 포함될 수 있다. 이러한 방법들은 당 분야에서 공지되어 있으며, 예를 들어 면역세포화학 및 면역조직화학, 방사선 면역 분석법(radioimmunoassays), 면역크로마토그래피법(immunochromatograhy), 효소결합면역법(ELISA:Enzyme Linked Immunoabsorbent assay), 면역블롯(immunoblotting), 파아르 분석법(Farr assay), 면역침강, 면역형광법 등이 있다.Immunological analysis can be included without limitation as long as it can measure the binding of the antigen to the antibody. These methods are known in the art, for example, immunocytochemistry and immunohistochemistry, radioimmunoassays, immunochromatograhy, enzyme linked immunoassay (ELISA), immunoblotting (immunoblotting), Farr assay, immunoprecipitation, and immunofluorescence.
상기 치주질환으로 판단하는 단계는 상기 대상체의 비마커 임상정보를 추가로 사용할 수 있다. 비마커 임상정보로는 대상체의 치주낭의 깊이 측정, 출혈 여부 및 X-ray 검사로 이루어지는 군에서 선택되는 어느 하나 이상일 수 있으나, 이로 제한되는 것은 아니다.In the determining of the periodontal disease, non-marker clinical information of the subject may be additionally used. The non-marker clinical information may be any one or more selected from the group consisting of measuring the depth of the subject's periodontal pocket, bleeding, and X-ray examination, but is not limited thereto.
본 발명에 따를 때, 활성형 MMP-8은 하기 표 1에 기재된 site 1, site 2, site 3 및 site 4로 이루어지는 군에서 선택되는 어느 하나 이상의 서열과 적어도 95% 이상의 상동성을 가지는 펩티드를 포함하는 것일 수 있다.According to the present invention, the active MMP-8 comprises a peptide having at least 95% homology with any one or more sequences selected from the group consisting of site 1, site 2, site 3 and site 4 listed in Table 1 below. It may be.
[표 1][Table 1]
Figure PCTKR2019018497-appb-I000009
Figure PCTKR2019018497-appb-I000009
본 발명에 따를 때, 비활성형 MMP-8은 하기 표 2에 기재된 어느 하나 이상의 서열과 적어도 95% 이상의 상동성을 가지는 펩티드를 포함하는 것일 수 있다.According to the present invention, the inactive MMP-8 may include a peptide having at least 95% homology to any one or more sequences listed in Table 2 below.
[표 2][Table 2]
Figure PCTKR2019018497-appb-I000010
Figure PCTKR2019018497-appb-I000010
본 발명은 활성형 MMP-8(active Matrix Metalloproteinase-8)과 특이적으로 결합하는 항체 및 비활성형 MMP-8(pro Matrix Metalloproteinase-8)과 특이적으로 결합하는 항체를 포함하는 치주질환 진단 또는 예후용 조성물을 제공한다.The present invention is a diagnosis or prognosis of periodontal disease comprising an antibody that specifically binds to an active type MMP-8 (active matrix metalloproteinase-8) and an antibody that specifically binds to an inactive type MMP-8 (pro Matrix Metalloproteinase-8). It provides a composition for.
상기 항체는 다클론성 항체(polyclonal antibody) 또는 단일클론항체(monoclonal antibody)일 수 있으나, 바람직하게는 단일클론항체일 수 있다.The antibody may be a polyclonal antibody or a monoclonal antibody, but may preferably be a monoclonal antibody.
상기 단일클론항체는 일반적으로 AP(alkaline phosphatase) 또는 HRP(horseradish peroxidase)등의 효소가 결합된 2차 항체 및 이들의 기질을 사용하여 발색반응 시킴으로써 정량분석할 수 있고, 또는 직접 상기 마커 단백질에 대한 단일클론항체에 AP 또는 HRP효소 등이 결합된 것을 사용하여 정량분석할 수 있다. 또한 FITC(fluorescein isothiocyanat), PE(phycoerythrin) 등을 포함하는 다양한 형광 표지물질이 결합된 항체를 사용할 수 있다.The monoclonal antibody can be quantitatively analyzed by performing a color development reaction using a secondary antibody bound with an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) and their substrate, or directly to the marker protein. Quantitative analysis can be performed using a combination of AP or HRP enzyme with a monoclonal antibody. In addition, it is possible to use antibodies with various fluorescent labeling substances, including FITC (fluorescein isothiocyanat), PE (phycoerythrin), and the like.
상기 마커 단백질과 항체의 반응은 면역크로마토그래피법(immunochromatography), 웨스턴 블랏(western blot), 면역침강법(Immunoprecipitation; IP), 효소결합면역흡착분석법(enzyme-linked immunoabsorbent assays; ELISA), 면역염색법(immunohistochemistry;IHC) 및 유세포분석법(flow cytometry analysis; FACS)으로 이루어지는 군에서 선택되는 어느 하나 이상의 단백질 확인 실험을 통해 확인할 수 있으나, 이에 제한되는 것은 아니다.The reaction between the marker protein and the antibody is immunochromatography, western blot, immunoprecipitation (IP), enzyme-linked immunoabsorbent assays (ELISA), immunostaining immunohistochemistry (IHC) and flow cytometry analysis (FACS) can be confirmed by any one or more protein identification experiments selected from the group consisting of, but is not limited thereto.
또한, 본 발명에 따른 치주질환 진단 또는 예후용 조성물은 활성형 MMP-8 및 비활성형 MMP-8을 암호화하는 핵산에 특이적인 프라이머 또는 프로브를 포함할 수 있다.In addition, the composition for diagnosing or prognosing periodontal disease according to the present invention may include primers or probes specific for nucleic acids encoding active MMP-8 and inactive MMP-8.
본 발명은 상기 활성형 MMP-8(active Matrix Metalloproteinase-8)과 특이적으로 결합하는 항체 및 비활성형 MMP-8(pro Matrix Metalloproteinase-8)과 특이적으로 결합하는 항체 또는 상기 활성형 MMP-8 및 비활성형 MMP-8을 암호화하는 핵산에 특이적인 프라이머 또는 프로브를 포함하는 치주질환 진단 또는 예후용 키트를 제공한다.The present invention is an antibody specifically binding to the active type MMP-8 (active matrix metalloproteinase-8) and an antibody specifically binding to the inactive type MMP-8 (pro Matrix Metalloproteinase-8) or the active type MMP-8 And a primer or probe specific for nucleic acids encoding inactive MMP-8.
본 발명에 따른 키트는 마이크로어레이용, 유전자 증폭용, 딥스틱 래피드 키트, ELISA 분석용 또는 면역분석용으로 사용될 수 있다.The kit according to the present invention can be used for microarray, gene amplification, dipstick rapid kit, ELISA analysis or immunoassay.
바람직하게 상기 키트는 마이크로어레이로 구성된 치주질환 진단용 키트일 수 있다. 상기 마이크로어레이는 특정 시약으로 처리된 슬라이드 글라스 표면 위에 항체를 부착하여 항원-항체 반응에 의해 상기 항체에 특이적으로 부착하는 단백질을 검출할 수 있도록 하는 것이다.Preferably, the kit may be a kit for diagnosing periodontal disease composed of a microarray. The microarray is capable of detecting a protein specifically attached to the antibody by an antigen-antibody reaction by attaching the antibody on a slide glass surface treated with a specific reagent.
상기 항체를 포함하는 키트는 추가로 분석에 필요한 하나 이상의 부가 성분을 포함할 수 있다. 예를 들어, 검출에 필요한 버퍼, 안정화제, 불활성 단백질, 시료 채취용 도구, 음성 및/또는 양성 대조군 등을 추가로 포함할 수 있다.또한, 상기 항체는 방사종(radionuclides), 형광원(fluorescors), 효소(enzyme) 등에 의해 표지화될 수 있다.The kit comprising the antibody may further include one or more additional components necessary for analysis. For example, it may further include a buffer necessary for detection, a stabilizer, an inert protein, a sample collection tool, a negative and/or positive control, etc. In addition, the antibody may include radionuclides and fluorescors. ), enzymes (enzyme) and the like.
이하, 본 발명을 실험예 및 실시예에 의하여 더욱 상세하게 설명한다. 단, 하기 실험예 및 실시예들은 본 발명을 더욱 쉽게 이해할 수 있도록 예시하는 것으로 본 발명의 내용이 실험예 및 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail by experimental examples and examples. However, the following experimental examples and examples are intended to illustrate the present invention more easily, and the contents of the present invention are not limited by the experimental examples and examples.
<실험예 1><Experimental Example 1>
1-1. 활성형 MMP-8과 비활성형 MMP-8의 펩티드 서열 분리1-1. Separation of peptide sequence between active MMP-8 and inactive MMP-8
활성형 MMP-8의 구조는 현재 밝혀져 있고(PDB ID: 2OY4), 비활성형 MMP-8의 구조는 현재 밝혀져 있는 비활성형의 MMP-3(PDB ID: 1SLM) 및 MMP-1(PDB ID: 1SU3)의 구조, 활성형 MMP-8의 구조를 기반으로 모델러(Andrej Sali, USA, version 9.20)를 이용하여 모델링하였다(도 3).The structure of the active MMP-8 is currently unknown (PDB ID: 2OY4), and the structure of the inactive MMP-8 is currently known as the inactive MMP-3 (PDB ID: 1SLM) and MMP-1 (PDB ID: 1SU3). ), and modeled using a modeler (Andrej Sali, USA, version 9.20) based on the structure of the active MMP-8 (FIG. 3 ).
비활성형 MMP-8의 프로펩티드를 분리시켜 활성형 MMP-8로 전환시켰다. 이때, 새롭게 노출되는 부분은 비활성형과 대비하여 활성형 MMP-8의 고유한 특성을 나타낼 가능성이 높다는 판단하에, 면역 반응에 영향을 줄 수 있는 특성으로서 표면노출도 및 극성을 계산하였다. 하기 표 3에 그 값을 나타내었다. 이는 단백질 구조의 환경 분석에 이용할 수 있는 ENVA(syntekabio, 한국)를 이용하여 계산하였다.The propeptide of the inactive MMP-8 was isolated and converted to the active MMP-8. At this time, under the judgment that the newly exposed portion is likely to exhibit the unique characteristics of the active type MMP-8 compared to the inactive type, surface exposure degree and polarity were calculated as properties that can affect the immune response. The values are shown in Table 3 below. This was calculated using ENVA (syntekabio, Korea), which can be used for environmental analysis of protein structures.
Figure PCTKR2019018497-appb-T000003
Figure PCTKR2019018497-appb-T000003
활성형과 비활성형 사이에서 표면노출도와 극성의 차가 크게 나타나는 위치를 선별하여 MMP-8의 활성부위를 포함하는 아미노산 위치임을 확인하였다.It was confirmed that the amino acid position including the active site of MMP-8 was selected by selecting a position where the difference in surface exposure and polarity between the active and inactive forms was large.
이를 토대로 아미노산 서열 기반의 단백질 에피토프 위치들을 예측할 수 있는 프로그램 7종(Bepipred, Bepipred 2, Chou & Fasman, Emini, Karplus & Schulz, Kolaskar & Tongaonkar, Parker)을 이용하여 도 1에 예상되는 에피토프의 위치를 나타내었다. 이러한 7종의 프로그램들이 공통적으로 에피토프라 예측하는 위치를 포함하면서, 표 3에서의 활성형과 비활성형 MMP-8의 표면노출도 및 극성의 차이가 높은 서열들을 최종 선별하였다. Based on this, 7 types of programs capable of predicting protein epitope positions based on amino acid sequence (Bepipred, Bepipred 2, Chou & Fasman, Emini, Karplus & Schulz, Kolaskar & Tongaonkar, Parker) were used to predict the epitope positions predicted in FIG. 1. Shown. Sequences having high differences in surface exposure and polarity of active and inactive MMP-8 in Table 3 were finally selected, while these seven programs included positions predicting epitopes in common.
펩타이드를 합성 후 항체형성을 유도할 시, 단일클론항체를 선별하기 위해서는 펩타이드 내 시스테인 잔기에 BSA 또는 KLH의 접합이 필요하기 때문에, 도 1 위치들을 일부 포함하면서 시스테인 잔기가 추가된 펩타이드 서열을 합성하였다.When inducing antibody formation after synthesizing the peptide, in order to select a monoclonal antibody, since the conjugation of BSA or KLH to the cysteine residue in the peptide is required, a peptide sequence in which cysteine residue is added while partially including the positions of FIG. 1 was synthesized. .
<실험예 2><Experimental Example 2>
2-1. 항체 제작2-1. Antibody production
활성형 MMP-8, 비활성형 MMP-8 및 sIgA에 특이적으로 결합하는 항체 제작을 위하여 이들과 면역보강제를 혼합하여 마우스에 주입하고, 면역이 완료된 마우스에서 비장을 적출하였다. 마우스의 암세포인 SP2/0 세포 및 비장세포를 PEG를 이용하여 융합하였다. 융합된 세포는 ELISA를 통하여 확인하였고, 융합된 세포를 선별하여 마우스 복강에 접종하였다. 접종 후 복강에 복수가 생성되면 이를 채취하여 protein G gel을 이용하여 항체를 정제하였다.For the production of antibodies that specifically bind to active MMP-8, inactive MMP-8 and sIgA, these and adjuvants were mixed and injected into mice, and spleens were extracted from immunized mice. The mouse cancer cells, SP2/0 cells and spleen cells, were fused using PEG. The fused cells were confirmed by ELISA, and the fused cells were selected and inoculated into the mouse peritoneal cavity. When ascites were formed in the abdominal cavity after inoculation, the samples were collected and purified using protein G gel.
2-2. 제작된 항체의 검증2-2. Validation of the produced antibodies
(1) 활성형 MMP-8 및 비활성형 MMP-8에 특이적으로 결합하는 항체 검증(1) Validation of antibodies specifically binding to active MMP-8 and inactive MMP-8
NC 막(Nitrocellulose membrane)에 활성형 및 비활성형 MMP-8와 특이적으로 결합하는 항체를 분주(dotting)하였다. Anti MMP-8 항체(Sino biological)가 접합된 Europium bead 및 활성형 MMP-8 항원을 전개 버퍼(running buffer)에 혼합한 후, 항체가 분주된 NC 막을 전개 버퍼에 담갔다. 동일한 방법으로 Anti MMP-8 항체(Sino biological)가 접합된 Europium bead 및 비활성형 MMP-8 항원을 전개 버퍼에 혼합한 후, 항체가 분주된 NC 막을 전개 버퍼에 담갔다. 10분 경과 후 NC 막을 365 nm UV 램프로 비추어 항원-항체 결합을 확인하였다.Antibodies that specifically bind to active and inactive MMP-8 on the NC membrane (Nitrocellulose membrane) were dosed. After mixing the Anti MMP-8 antibody (Sino biological) conjugated Europium bead and the active MMP-8 antigen in a running buffer, the NC membrane dispensed with the antibody was immersed in the development buffer. In the same manner, the anti-MMP-8 antibody (Sino biological)-conjugated Europium bead and the inactive MMP-8 antigen were mixed in the development buffer, and then the antibody-dispersed NC membrane was immersed in the development buffer. After 10 minutes, the NC membrane was irradiated with a 365 nm UV lamp to confirm antigen-antibody binding.
도 5(a)의 결과를 통하여 활성형 MMP-8 항원에 특이적으로 결합하는 항체가 비활성형 MMP-8 항원과는 반응하지 않으면서, 활성형 MMP-8과는 반응성이 높은 것을 확인할 수 있었고, 도 5(b)를 통하여 비활성형 MMP-8에 특이적으로 결합하는 항체가 활성형 MMP-8 항원과는 반응하지 않으면서, 비활성형 MMP-8과는 반응성이 높은 것을 확인하였다.Through the results of FIG. 5(a), it was confirmed that the antibody that specifically binds to the active MMP-8 antigen does not react with the inactive MMP-8 antigen and has high reactivity with the active MMP-8. , FIG. 5(b) confirmed that the antibody that specifically binds to the inactive MMP-8 does not react with the active MMP-8 antigen, and has high reactivity with the inactive MMP-8.
(2) sIgA에 특이적으로 결합하는 항체 검증(2) Validation of antibodies specifically binding to sIgA
NC 막에 sIgA와 특이적으로 결합하는 항체를 분주하였다. sIgA와 특이적으로 결합하는 항체가 접합된 Europium bead와 sIgA 항원(Fitzgerald)을 전개 버퍼에 혼합한 후, 항체가 분주된 NC 막을 전개 버퍼에 담갔다. 10분 경과 후 NC 막을 365 nm UV 램프로 비추어 항원-항체 결합을 확인하였다. 도 5(c)를 통해 sIgA와 항체가 특이적 결합을 형성한 것을 확인할 수 있다.Antibodies that specifically bind to sIgA were dispensed into the NC membrane. After mixing the sIgA-specific antibody-conjugated Europium bead and the sIgA antigen (Fitzgerald) in a development buffer, the antibody-dispersed NC membrane was immersed in the development buffer. After 10 minutes, the NC membrane was irradiated with a 365 nm UV lamp to confirm antigen-antibody binding. It can be seen through FIG. 5(c) that sIgA and the antibody formed a specific binding.
2-3. 정량 곡선 산출2-3. Quantitative curve calculation
(1) 활성형 MMP-8 항원과 비활성형 MMP-8 항원을 타액에 하기 표 4에 따른 농도별로 희석한 후 타액 보관용액과 1:1로 혼합하여 항원용액을 제조하였다. 타액 보관용액은 PBS에 1.0 mM PMSF 및 2.0 mM EDTA를 혼합하여 사용하였다. 상기 항원용액과 검체 희석액을 1:9 비율로 혼합 후 MMP-8 측정 키트의 검체 주입구에 분주하였다. 검체를 주입하고 10분 경과 후, TRF(Time Resolved Fluorescence) 측정 장비를 이용하여 비활성형 MMP-8과 활성형 MMP-8의 형광 신호를 측정하였다. 농도별로 측정된 신호를 이용하여 정량 곡선을 그렸다. 도 6의 (a) 및 (b)는 각각 비활성형(pMMP-8)과 활성형(aMMP-8)의 정량 곡선을 나타낸 것이다.(1) Active MMP-8 antigen and inactive MMP-8 antigen were diluted in saliva by concentrations according to Table 4 below, and then mixed 1:1 with a saliva storage solution to prepare an antigen solution. The saliva storage solution was used by mixing 1.0 mM PMSF and 2.0 mM EDTA in PBS. The antigen solution and the sample dilution were mixed at a ratio of 1:9 and then dispensed into the sample inlet of the MMP-8 measurement kit. After 10 minutes of injecting the sample, fluorescence signals of inactive MMP-8 and active MMP-8 were measured using a Time Resolved Fluorescence (TRF) measurement equipment. Quantitative curves were drawn using signals measured for each concentration. 6(a) and 6(b) show quantitative curves of the inactive type (pMMP-8) and the active type (aMMP-8), respectively.
(2) sIgA 항원 용액과 검체 희석액을 1:99의 비율로 혼합한 것을 제외하면, 상기 (1)과 동일한 방법으로 sIgA의 형광 신호를 측정하고 농도별로 측정된 신호를 이용하여 정량 곡선을 그렸다. 도 6의 (c)는 sIgA의 정량 곡선을 나타낸 것이다.(2) Except for mixing the sIgA antigen solution and the sample dilution at a ratio of 1:99, the fluorescence signal of sIgA was measured in the same manner as in (1) above, and a quantitative curve was drawn using the signal measured for each concentration. Figure 6 (c) shows a quantitative curve of sIgA.
Figure PCTKR2019018497-appb-T000004
Figure PCTKR2019018497-appb-T000004
<실험예 3> 임상검체 테스트<Experimental Example 3> Clinical sample test
임상 검체 수집은 서울대학교 치과병원 치주과, 원스톱 협진센터를 내원한 20대 내지 80대 성인 남녀 중 치주질환이 없는 대상자 14명 및 치주질환자 38명의 타액을 채집하였다. 정상 대조군은 치주질환이 없거나 치은염이 아주 경미한 정도 및, 치주낭의 깊이가 3 mm 미만이며, 치주질환 환자군은 방사선 사진상으로 골소실 진행이 확인되고, 치주낭의 깊이가 5 mm이상으로 확인되는 경우를 기준으로 분류하였다.To collect clinical specimens, saliva was collected from 14 subjects in the 20s and 80s who had no periodontal disease and 38 patients with periodontal disease among men and women in their 20s and 80s who visited the Seoul National University Dental Hospital Periodontology Department and One-Stop Consultation Center. The normal control group is based on the case where there is no periodontal disease or the degree of gingivitis is very slight, and the depth of the periodontal pocket is less than 3 mm, and the group of periodontal disease patients is confirmed by bone loss progression on the radiograph and the depth of the periodontal pocket is 5 mm or more Classified as.
3-1. 활성형 및 비활성형 MMP-83-1. Active and inactive MMP-8
타액 보관용액과 1:1로 혼합되어 있는 임상 검체를 검체 희석액과 1:9로 혼합한 후, MMP-8 측정 키트의 검체 주입구에 분주하였다. 상기 MMP-8 측정 키트는 MMP-8을 면역 크로마토그래피법으로 검출하기 위한 측면유동법(Lateral Flow Assay) 기반의 진단 키트를 사용하였다. 10분 경과 후, TRF(Time Resolved Fluorescence) 측정 장비를 이용하여 활성형 MMP-8과 비활성형 MMP-8의 형광신호를 측정하여, 측정된 신호를 정량 곡선에 대입하여 농도를 환산하였다.The clinical sample mixed 1:1 with the saliva storage solution was mixed with the sample diluent 1:9 and dispensed into the sample inlet of the MMP-8 measurement kit. The MMP-8 measurement kit used a diagnostic kit based on Lateral Flow Assay for detecting MMP-8 by immunochromatography. After 10 minutes, fluorescence signals of active MMP-8 and inactive MMP-8 were measured using a Time Resolved Fluorescence (TRF) measurement equipment, and the measured signals were substituted into a quantitative curve to convert the concentration.
3-2. sIgA3-2. sIgA
임상 검체를 검체 희석액과 1:99로 혼합한 것을 제외하면 상기 3-1.의 방법과 동일한 방법으로 sIgA의 형광신호를 측정하고, 측정된 신호를 정량 곡선에 대입하여 농도를 환산하였다.The fluorescence signal of sIgA was measured in the same manner as in the method of 3-1., except that the clinical sample was mixed with the sample dilution at 1:99, and the measured signal was substituted into a quantitative curve to convert the concentration.
3-3. IL-1beta3-3. IL-1beta
타액 보관용액과 1:1로 혼합되어 있는 임상검체를 검체 희석액과 1:1로 혼합하여 96well plate에 100 ㎕씩 분주하고, Biotin-conjugate 용액을 50 ㎕씩 분주하여 상온에서 2시간 항온유지하였다. 이후, 세척한 다음 streptavidin-HRP 용액 100 ㎕씩 분주하여 상온에서 1시간 항온유지하였다. 다시 세척 후, TMB 기질 시약 100 ㎕씩 분주하였고, 플레이트를 호일로 감싸 상온에서 10분간 항온유지하였다. 반응 종결액(stop solution)을 100 ㎕ 분주 후, 450 nm에서의 흡광도를 측정하였다.The clinical sample mixed 1:1 with the saliva storage solution was mixed 1:1 with the sample diluent and dispensed at 100 µl in a 96-well plate, and the biotin-conjugate solution was dispensed at 50 µl and maintained at room temperature for 2 hours. Thereafter, after washing, 100 µl of streptavidin-HRP solution was dispensed and maintained at room temperature for 1 hour. After washing again, 100 µl of the TMB substrate reagent was dispensed, and the plate was wrapped with foil to maintain the temperature for 10 minutes at room temperature. After 100 μl of the reaction stop solution was dispensed, absorbance at 450 nm was measured.
하기 표 5에 비활성형 MMP-8, 활성형 MMP-8, sIgA 및 IL-1beta의 농도를 나타내었다.Table 5 below shows concentrations of inactive MMP-8, active MMP-8, sIgA, and IL-1beta.
Figure PCTKR2019018497-appb-T000005
Figure PCTKR2019018497-appb-T000005
상기 표 5에서 확인할 수 있는 바와 같이, 비활성형 MMP-8, 활성형 MMP-8, sIgA 및 IL-1beta 모두 정상 대조군에 비하여 치주질환 환자군에서 농도가 높게 나타났다. 구체적으로 sIgA 및 IL-1beta는 정상 대조군에 비하여 치주질환 환자군에서 1.45배, 3배의 농도 증가를 나타내었고, 특히, 비활성형 MMP-8은 3.32배, 활성형 MMP-8은 4.12배, 전체 MMP-8에 있어서도 3.72배로 정상 대조군과 비교하여 높은 농도 차이를 보여주었다. 이러한 결과는 상기 마커들의 비약적인 농도 증가를 통하여 치주질환 여부의 진단이 가능하고, 여러 마커들의 농도 증가를 통하여 정확도 높은 진단이 가능함을 나타낸다.As can be seen from Table 5, inactive MMP-8, active MMP-8, sIgA and IL-1beta were all higher in the periodontal disease patient group than in the normal control group. Specifically, sIgA and IL-1beta showed a 1.45-fold and 3-fold concentration increase in the periodontal disease patient group compared to the normal control group. In particular, the inactive MMP-8 was 3.32 times, the active MMP-8 was 4.12 times, and the overall MMP Even at -8, it showed 3.72 times higher concentration difference compared to the normal control group. These results indicate that it is possible to diagnose periodontal disease through an increase in the concentration of the markers, and that it is possible to make an accurate diagnosis through an increase in the concentration of several markers.
또한, 도 8에 나타난 결과와 같이, 전체 MMP-8과 활성형 MMP-8의 비율 및 활성형 MMP-8과 염증성 질환 마커의 하나인 sIgA의 비율 역시, 이들의 유의적인 차이를 통하여 치주질환 판단에 중요한 인자가 될 수 있음을 확인할 수 있다.In addition, as shown in FIG. 8, the ratio of total MMP-8 to active MMP-8 and the ratio of active MMP-8 to sIgA, which is one of the inflammatory disease markers, also determines periodontal disease through their significant difference. It can be confirmed that it can be an important factor.
이상 첨부된 도면을 참조하여 본 발명의 실험예 및 실시예를 설명하였지만, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 실시태양일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아니다. 따라서 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해되어야 한다.The experimental examples and examples of the present invention have been described above with reference to the accompanying drawings, but for those skilled in the art to which the present invention pertains, these specific technologies are only preferred embodiments, thereby making the present invention The scope is not limited. Therefore, one of ordinary skill in the art will understand that the invention may be implemented in other specific forms without changing the technical spirit or essential features of the present invention. It should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (14)

  1. 대상체 유래의 생물학적 시료로부터 MMP-8(Matrix Metalloproteinase-8), 염증성 질환 마커, 및 치주질환 균주 유래의 마커로 이루어지는 군에서 선택되는 어느 하나 또는 둘 이상을 검출하는 단계; 및Detecting any one or more selected from the group consisting of MMP-8 (Matrix Metalloproteinase-8), an inflammatory disease marker, and a marker derived from a periodontal disease strain from a biological sample derived from a subject; And
    상기 생물학적 시료에서 검출된 상기 MMP-8, 염증성 질환 마커 또는 치주질환 균주 유래의 마커 수준이 정상 대조군 시료와 비교하여 증가한 경우, 상기 생물학적 시료가 유래한 대상체를 치주질환으로 판단하는 단계;를 포함하는 치주질환 진단 또는 예후에 필요한 정보를 제공하는 방법.Comprising the step of determining the subject from which the biological sample originated as periodontal disease when the level of the marker derived from the MMP-8, inflammatory disease marker or periodontal disease strain detected in the biological sample increases compared to a normal control sample. A method of providing information necessary for the diagnosis or prognosis of periodontal disease.
  2. 제 1항에 있어서,According to claim 1,
    상기 MMP-8을 검출하는 단계는 활성형 MMP-8과 비활성형 MMP-8을 구별하여 검출하는 것을 특징으로 하는 치주질환 진단 또는 예후에 필요한 정보를 제공하는 방법.The step of detecting the MMP-8 is a method of providing information necessary for the diagnosis or prognosis of periodontal disease, characterized in that it detects by distinguishing the active MMP-8 from the inactive MMP-8.
  3. 제 1항에 있어서,According to claim 1,
    상기 대상체를 치주질환으로 판단하는 단계는 상기 생물학적 시료로부터 검출된 마커의 농도 또는 농도 비율이 하기 식 1 및 식 2를 만족하고, 하기 식 3 또는 식 4를 만족하는 경우 치주질환으로 판단하는 것을 포함하는, 치주질환 진단 또는 예후에 필요한 정보를 제공하는 방법.The step of determining the subject as periodontal disease includes determining the periodontal disease when the concentration or concentration ratio of the marker detected from the biological sample satisfies Equation 1 and Equation 2 and satisfies Equation 3 or Equation 4 below. How to provide information necessary for the diagnosis or prognosis of periodontal disease.
    [식 1][Equation 1]
    Figure PCTKR2019018497-appb-I000011
    Figure PCTKR2019018497-appb-I000011
    [식 2][Equation 2]
    Figure PCTKR2019018497-appb-I000012
    Figure PCTKR2019018497-appb-I000012
    [식 3][Equation 3]
    Figure PCTKR2019018497-appb-I000013
    Figure PCTKR2019018497-appb-I000013
    [식 4][Equation 4]
    Figure PCTKR2019018497-appb-I000014
    Figure PCTKR2019018497-appb-I000014
  4. 제 1항에 있어서,According to claim 1,
    상기 생물학적 시료는 타액, 치은열구액(Gingival Crevicular Fluid; GCF) 및 혀 밑 고인액(Sublingual Fluid; SLF)으로 이루어지는 군에서 선택되는 어느 하나 이상인 치주질환 진단 또는 예후에 필요한 정보를 제공하는 방법.The biological sample is salivary, gingival fissure fluid (Gingival Crevicular Fluid; GCF) and sublingual sublingual fluid (Sublingual Fluid; SLF) at least one selected from the group consisting of periodontal disease diagnosis or prognosis.
  5. 제 1항에 있어서,According to claim 1,
    상기 염증성 질환 마커는 CRP(C-reactive Protein), sIgA(secretary Immunoglobulin A), 면역글로불린(Immunoglobulin), 인터루킨-1베타(IL-1beta), 인터루킨-6(IL-6), 종양괴사인자-알파(TNF-α;Tumor necrosis factor-alpha), 베타-글루쿠로니다제(β-glucuronidase), 리소자임(Lysozyme), 락토페린(Lactoferrin) 으로 이루어진 군에서 선택되는 어느 하나 이상인 치주질환 진단 또는 예후에 필요한 정보를 제공하는 방법.The inflammatory disease markers are C-reactive protein (CRP), secretary immunoglobulin A (sIgA), immunoglobulin, interleukin-1 beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α;Tumor necrosis factor-alpha), beta-glucuronidase, lysozyme, lactoferrin, or any one or more selected from the group consisting of periodontal disease diagnosis or prognosis How to provide information.
  6. 제 1항에 있어서,According to claim 1,
    상기 치주질환 균주 유래의 마커는 류코톡신(Leukotoxin), Karilysin, 세포융해소(Cytolysin), 진지페인(Gingipain)으로 이루어진 군에서 선택되는 어느 하나 이상인 치주질환 진단 또는 예후에 필요한 정보를 제공하는 방법.The marker derived from the periodontal disease strain is Leukotoxin (Leukotoxin), Karilysin, Cytolysin, Gingipain (Gingipain) at least one selected from the group consisting of periodontal disease diagnosis or prognosis.
  7. 제 1항에 있어서,According to claim 1,
    상기 치주질환으로 판단하는 단계는 마이크로어레이, 항원-항체 반응 또는 질량분석 방식으로 수행되는 것을 포함하는 치주질환 진단 또는 예후에 필요한 정보를 제공하는 방법.The step of determining the periodontal disease is a method for providing information necessary for diagnosing or prognosing periodontal disease, which is performed by microarray, antigen-antibody reaction, or mass spectrometry.
  8. 제 1항에 있어서,According to claim 1,
    상기 치주질환으로 판단하는 단계는 상기 대상체의 비마커 임상정보를 추가로 사용하는 것인 치주질환 진단 또는 예후에 필요한 정보를 제공하는 방법.The step of determining the periodontal disease is a method of providing information necessary for the diagnosis or prognosis of periodontal disease, wherein the non-marker clinical information of the subject is additionally used.
  9. 제 8항에 있어서,The method of claim 8,
    상기 비마커 임상정보는 상기 대상체의 치주낭의 깊이 측정, 출혈 여부 또는 X-ray 검사로 이루어지는 군에서 선택되는 어느 하나 이상인 치주질환 진단 또는 예후에 필요한 정보를 제공하는 방법.The non-marker clinical information is a method for providing information necessary for the diagnosis or prognosis of periodontal disease, which is at least one selected from the group consisting of measuring the depth of the periodontal pocket of the subject, whether it is bleeding or X-ray examination.
  10. 제 2항에 있어서,According to claim 2,
    상기 활성형 MMP-8은 하기 표 6에 기재된 site 1, site 2, site 3 및 site 4로 이루어지는 군에서 선택되는 어느 하나 이상의 서열과 적어도 95% 이상의 상동성을 가지는 펩티드를 포함하는 것인, 치주질환 진단 또는 예후에 필요한 정보를 제공하는 방법.The active MMP-8 is a periodontal group comprising a peptide having at least 95% or more homology with any one or more sequences selected from the group consisting of site 1, site 2, site 3 and site 4 listed in Table 6 below. How to provide the information needed to diagnose or prognosis a disease.
    [표 6][Table 6]
    Figure PCTKR2019018497-appb-I000015
    Figure PCTKR2019018497-appb-I000015
  11. 제 2항에 있어서,According to claim 2,
    상기 비활성형 MMP-8은 하기 표 7에 기재된 어느 하나 이상의 서열과 적어도 95% 이상의 상동성을 가지는 펩티드를 포함하는 것인, 치주질환 진단 또는 예후에 필요한 정보를 제공하는 방법.The inactive MMP-8 is a method of providing information necessary for the diagnosis or prognosis of periodontal disease, comprising a peptide having at least 95% or more homology with any one or more sequences listed in Table 7.
    [표 7][Table 7]
    Figure PCTKR2019018497-appb-I000016
    Figure PCTKR2019018497-appb-I000016
  12. 활성형 MMP-8(active Matrix Metalloproteinase-8)과 특이적으로 결합하는 항체 및 비활성형 MMP-8(pro Matrix Metalloproteinase-8)과 특이적으로 결합하는 항체를 포함하는 치주질환 진단 또는 예후용 조성물.A composition for diagnosing or prognosing periodontal disease comprising an antibody that specifically binds to an active type MMP-8 (active matrix metalloproteinase-8) and an antibody that specifically binds to an inactive type MMP-8 (pro Matrix Metalloproteinase-8).
  13. 활성형 MMP-8(active Matrix Metalloproteinase-8) 및 비활성형 MMP-8(pro Matrix Metalloproteinase-8)을 암호화하는 핵산에 특이적인 프라이머 또는 프로브를 포함하는 치주질환 진단 또는 예후용 조성물.A composition for the diagnosis or prognosis of periodontal disease comprising a primer or probe specific for nucleic acids encoding active matrix metalloproteinase-8 (MMP-8) and pro matrix metalloproteinase-8 (active matrix metalloproteinase).
  14. 제 12항 또는 제 13항의 조성물을 포함하는 치주질환 진단 또는 예후용 키트.A kit for diagnosing or prognosing periodontal disease comprising the composition of claim 12 or 13.
PCT/KR2019/018497 2018-12-26 2019-12-26 Method for diagnosis of periodontal disease, and composition and kit therefor WO2020138965A1 (en)

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