How to measure the severity of periodontal disease

Abstract

To provide a method of measuring severity of periodontal disease.SOLUTION: A method of measuring severity of periodontal disease in a subject using a reflection value of the oxidized HDL level as an indicator is provided, the method comprising measuring the reflection value of the oxidized HDL level in a blood sample collected from the subject.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to a method for measuring the severity of periodontal disease. [Background technology]

[0002] Periodontal disease is an inflammatory disease caused by infection of periodontal tissues by periodontal pathogens, and is broadly divided into gingivitis and periodontitis depending on the stage of progression. Plaque caused by poor oral hygiene is thought to be one of the causes of periodontal disease, and gingivitis occurs when oral hygiene behavior is stopped, demonstrating the importance of oral hygiene behavior to remove plaque.

[0003] In addition, gingivitis is generally considered to be a reversible disease that can be restored to a healthy state by plaque control such as tooth brushing and cleaning, etc. On the other hand, periodontitis, which is an advanced stage of gingivitis, is considered to be an irreversible disease because it destroys the alveolar bone, and surgical treatment may be required for its treatment. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6310631 [Patent Document 2] Patent No. 6779776 Summary of the Invention [Problem to be solved by the invention]

[0005] The progression from gingivitis to periodontitis is thought to occur as an extension of persistent gingival inflammation caused by plaque adhesion, but because oral bacteria such as periodontal pathogens and biological factors (systemic diseases) are intricately involved, it is difficult to evaluate periodontal disease based solely on the presence or absence of plaque adhesion, and screening in places where dental professionals are not present is problematic.

[0006] In addition, for example, if a subject suffering from periodontal disease carefully brushes his / her teeth immediately before an oral examination, the plaque removal status will be good at the time of the examination, but the degree of periodontal disease is unlikely to improve as a result of the brushing. As can be seen from this, it is often difficult to evaluate periodontal disease based only on the presence or absence of plaque adhesion, and therefore there is a demand for the development of a simple method for diagnosing periodontal disease (Patent Document 1).

[0007] An object of the present disclosure is to provide a method for measuring the severity of periodontal disease. Another object of the present disclosure is to provide a method for measuring the severity of periodontal disease without a dental checkup. [Means for solving the problem]

[0008] The present inventors have found that when the value reflecting the amount of oxidized HDL in a blood sample is high, the possibility of severe periodontal disease increases compared to when the value is low, and have made further improvements.

[0009] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. A method for measuring the severity of periodontal disease in a subject, comprising measuring a value reflective of the amount of oxidized HDL in a blood sample collected from the subject, using the value reflective of the amount of oxidized HDL as an index. Section 2. Item 2. The method according to Item 1, further comprising measuring an antibody titer against periodontal pathogens in a biological sample collected from the subject, and measuring the severity of periodontal disease in the subject using the antibody titer against the periodontal pathogens as an index. Section 3. The measurement of the reflected value of the oxidized HDL amount (1) The method is carried out by a method including a step of mixing a blood sample with a solution containing a transition metal compound and an acidic buffer solution, Item 3. The method according to item 1 or 2. Section 4. The measurement of the reflected value of the oxidized HDL amount moreover, (2) The method is carried out by a method including a step of measuring the free radicals produced in the step (1), The method according to item 3. Section 5. Item 5. The method according to any one of Items 1 to 4, wherein the reflection value of the oxidized HDL amount is a change in absorbance per unit time. Section 6. Item 6. The method according to any one of Items 1 to 5, wherein the subject has plaque. Effect of the Invention

[0010] A method for determining the severity of periodontal disease is provided. Also provided is a method for determining the severity of periodontal disease without a dental examination. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a diagram showing tooth numbers and names. [Diagram 2] The results of periodontal disease classification in groups with and without plaque adhesion are shown. [Diagram 3] 1 shows the classification results of periodontal disease in the antibody titer group of 100 U / mL or more and the group of less than 100 U / mL. [Figure 4] The results of periodontal disease classification are shown for groups with and without plaque adhesion, and for groups with antibody titers of 100 U / mL or more and less than 100 U / mL. [Diagram 5] 1 shows the classification results of periodontal disease in a group whose oxidized HDL level was reflected as 31 mOD / min or more and a group whose oxidized HDL level was less than 31 mOD / min. [Figure 6] The results of periodontal disease classification are shown for groups with and without plaque adhesion, with values ​​reflecting oxidized HDL levels of 31 mOD / min or more and less than 31 mOD / min. [Figure 7] 1 shows the classification results of periodontal disease in groups with antibody titers of 100 U / mL or more and less than 100 U / mL, and in groups with values ​​reflecting oxidized HDL levels of 31 mOD / min or more and less than 31 mOD / min. [Figure 8]The graph shows the classification results of periodontal disease in groups with and without plaque adhesion, in groups with antibody titers of 100 U / mL or more or less than 100 U / mL, and in groups with values ​​reflecting oxidized HDL levels of 31 mOD / min or more or less than 31 mOD / min. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Each embodiment included in the present disclosure will be described in further detail below. The present disclosure encompasses a method for measuring the severity of periodontal disease in a subject. In this specification, the method may be referred to as the "measurement method of the present disclosure."

[0013] The measurement method of the present disclosure involves measuring a value reflecting the amount of oxidized HDL in a blood sample taken from a subject.

[0014] The subject may be a human or a non-human mammal. The human may be, for example, a healthy individual, a human suffering from periodontal disease (a periodontal disease patient), or a human suspected of suffering from periodontal disease. The subject may be a subject with or without plaque adhesion. The non-human mammal is not particularly limited, and examples thereof include mammals kept as pets, livestock, laboratory animals, etc. Examples thereof include dogs, cats, monkeys, cows, horses, sheep, goats, pigs, rabbits, mice, rats, camels, llamas, etc.

[0015] As used herein, "plaque" refers to a mass of bacteria that adheres to the surface of teeth. Plaque is also sometimes called dental plaque.

[0016] The method for confirming the presence or absence of plaque adhesion is not particularly limited, and known methods can be adopted. For example, a method for judging the presence or absence of plaque adhesion by scraping the tooth surface with the tip of a periodontal probe or a dental probe, a method for judging the presence or absence of plaque adhesion by staining the tooth surface using a plaque staining solution, a method for judging the presence or absence of plaque adhesion by image diagnosis, a method for detecting plaque by a sensor, etc. are listed. In addition, since plaque adhesion occurs as a result of insufficient oral hygiene, it is already known that there is a relationship with the number of times teeth are brushed and the use of interdental cleaning tools, and that gingival bleeding occurs when plaque adhesion continues, and therefore, a questionnaire about oral hygiene behavior, a test for checking bleeding from the gums (occult blood), etc. can be used as a substitute for plaque adhesion.

[0017] The teeth to be checked for the presence or absence of plaque adhesion may be, for example, all teeth or target teeth. The target teeth may be, for example, a total of six teeth, including upper right 6, upper right 1, upper left 6, lower right 6, lower left 1, and lower left 6. The numbers correspond to the numbers shown in FIG. 1. Also, the teeth may be ones whose images can be captured by a smartphone or the like (mainly anterior teeth) or ones whose images can be captured by an intraoral camera.

[0018] If the above-mentioned method finds no plaque adhesion or it is below the detection limit, it can be determined that there is no plaque adhesion, and if not, it can be determined that there is plaque adhesion. As shown in the examples described later, when evaluating the presence or absence of plaque by Plaque Index (PlI), if the average value of the PlI measurement value is, for example, less than 0.5 (preferably less than 0.25, more preferably 0), it can be determined that there is no plaque adhesion, and if it is 0.5 or more (preferably 0.25 or more, more preferably greater than 0), it can be determined that there is plaque adhesion.

[0019] The blood sample is not particularly limited as long as it is a sample derived from blood collected from a subject, and may be, for example, whole blood or a blood separation product (e.g., serum, plasma, etc.). The method and conditions for storing the blood sample collected from the subject are not particularly limited, and may be carried out according to a conventional method.

[0020] The method for collecting a blood sample from a subject is not particularly limited and may be performed according to a conventional method. When serum or plasma is used as a blood sample, it is preferable to use a vacuum blood collection tube containing a serum or plasma separator from the viewpoints of ease of handling, prevention of infection, etc. The collected blood sample may be used as is, or may be freeze-dried or otherwise stored and then the freeze-dried product may be dissolved in a suitable solvent as described below and then used. The collected blood sample may be frozen and stored as is, or may be frozen and stored after being dissolved in a suitable solvent as described below and then thawed when ready for use.

[0021] The value reflecting the amount of oxidized HDL refers to a value reflecting the amount of oxidized HDL contained in a blood sample, and examples thereof include the weight (mass) and concentration of oxidized HDL. Alternatively, for example, a measured value reflecting the amount of oxidized HDL contained in a blood sample may be used, and examples of such values ​​include absorbance and fluorescence intensity. The measured value may be a value measured using a measurement auxiliary reagent. Examples of such measurement auxiliary reagents include fluorescent dyes, oxidized HDL-specific recognition molecules (e.g., antibodies or fragments thereof), and combinations of two or more of these.

[0022] The method for measuring the reflected value of the amount of oxidized HDL in a blood sample is not particularly limited, and can be performed according to a conventional method. For example, the method includes a method for measuring oxidized HDL (malondialdehyde-modified HDL) using an ELISA method, a method for measuring the nitration or chlorination of Apo-A1, a protein constituting HDL, by a mass spectrometer, the d-ROMs method (a method for measuring the concentration of hydroxyperoxide in a blood sample), and a method described in Japanese Patent No. 6779776 (Patent Document 2) that includes the step of (1) mixing a blood sample with a solution containing a transition metal compound and an acidic buffer solution. Among them, the method including the step of (1) mixing a blood sample with a solution containing a transition metal compound and an acidic buffer solution is preferable. In this specification, the step may be referred to as "step (1)". Furthermore, it is more preferable to measure by a method including the step of (2) measuring the free radicals produced by the step (1). In this specification, the step may be referred to as "step (2)".

[0023] The method for measuring the reflective value of the amount of oxidized HDL in a blood sample may include a step of separating HDL from the blood sample. In the present specification, this step may be referred to as "step (0)". The HDL separated in step (0) can be used as the blood sample in step (1). The method for separating HDL from a blood sample is not particularly limited, and can be performed according to a conventional method. For example, the dextran sulfate method using dextran sulfate and magnesium ions can be mentioned.

[0024] The transition metal compound is not particularly limited as long as it can be ionized in the mixed solution to become a metal ion and can react with hydroxyperoxide (R-OOH) to generate a free radical, and examples thereof include copper (II) compounds, iron (II) compounds (divalent iron compounds), and iron (III) compounds (trivalent iron compounds). Among these, iron compounds such as iron (II) compounds and iron (III) compounds are preferred. Examples of iron compounds include ammonium iron (II) sulfate hexahydrate and iron (III) chloride hexahydrate. By using a transition metal compound in step (1), advantageous effects such as 1) the presence of a sufficient amount of transition metal ions that react with hydroxyperoxide makes it possible to measure oxidized HDL with a small amount of blood sample and 2) the measurement of oxidized HDL can be achieved without being affected by the amount of iron contained in blood are achieved.

[0025] In addition, the reflected value of the amount of oxidized HDL is larger in a solution containing a divalent iron compound than in a solution containing a trivalent iron compound. In other words, a measurement method using a divalent iron compound can measure oxidized HDL with higher sensitivity than a trivalent iron compound. Therefore, among the above-mentioned iron compounds, a divalent iron compound is particularly preferred.

[0026] The concentration of the transition metal compound in the mixed solution is not particularly limited, and can be, for example, about 0.1 to 150 μM, preferably 1 to 100 μM.

[0027] The acidic buffer solution is not particularly limited as long as it has an acidic pH and a buffering effect, and any known buffer solution can be used, such as an acetate buffer solution.

[0028] The pH of the acidic buffer solution is not particularly limited as long as it is acidic, and is preferably about 2 to 6.9, more preferably about 3 to 6.5, and even more preferably 4.5 to 6. When a color development method is used as a method for measuring free radicals in step (2) described below, the pH of the acidic buffer solution is particularly preferably about 3 to 6.9.

[0029] The concentration of the acidic buffer solution is not particularly limited and is, for example, 0.005 to 0.5M, preferably 0.01 to 0.2M.

[0030] The reaction temperature in step (1) is not particularly limited and can be, for example, about 20 to 40°C.

[0031] As described above, in step (1), hydroperoxides (R-OOH) in oxidized HDL contained in a blood sample react with transition metal ions to generate peroxy radicals (R-OO · ) and alkoxy radicals (RO · By measuring the free radicals produced in step (1), oxidized HDL can be measured.

[0032] Therefore, it is preferable that the method for measuring a value reflecting the amount of oxidized HDL further comprises the step (2) of measuring the free radicals produced in the above step (1).

[0033] The method for measuring free radicals is not particularly limited, and any known method can be used, such as a color development method, a chemiluminescence method, or an electron spin resonance (ESR) method.

[0034] The color-developing method uses a substance that reacts with free radicals to produce color (a color-developing substance), and measures the absorbance of the colored substance using a spectrophotometer or the like.

[0035] The color-forming substance is not particularly limited as long as it is a compound that reacts with a free radical or an alkoxy radical to develop color, and any known compound can be used. For example, a compound represented by the following general formula (1) or a salt thereof can be used.

[0036] [ka]

[0037] In the above general formula (1), each R is the same or different and represents a hydrogen atom, a halogen atom, methyl, or ethyl. In particular, it is preferable that at least two of each R are methyl or ethyl, and it is more preferable that R substituting the same nitrogen atom are both methyl or ethyl.

[0038] As the compound represented by the general formula (1), N,N-dimethyl-p-phenylenediamine (DMPD) and N,N-diethyl-p-phenylenediamine (DPD) are preferred.

[0039] Examples of the salt of the compound represented by the general formula (1) include sulfates, oxalates, and diacetates.

[0040] The compound represented by formula (1) or a salt thereof used in the reaction step may be used alone or in combination of two or more.

[0041] In addition, the compound represented by the general formula (1) can be used by dissolving it in a solvent as necessary. The solvent is not particularly limited, and examples thereof include pure water, a buffer solution, and dimethyl sulfoxide (DMSO). The concentration of the solution is not particularly limited, and can be, for example, about 0.1 to 50 mM, and preferably about 1 to 20 mM.

[0042] The method for measuring the absorbance of the colored substance is not particularly limited, and can be performed by a conventional method. For example, since a radical cation that exhibits a reddish purple color is generated by the reaction of a free radical with a color-forming substance, the radical cation can be measured by measuring the absorbance using a known device. The wavelength for measuring the absorbance can be, for example, 460 to 570 nm, preferably 500 to 560 nm.

[0043] In addition, when measuring absorbance, it is preferable to measure the time course of absorbance between the start time and the end time of measurement in order to measure quantitatively. For example, it is preferable to measure the rate of increase in absorbance (change in absorbance per unit time) with the time 3 minutes after the start of the reaction between the free radical and the color-developing substance as the starting point and the time 6 minutes after the start of the reaction as the end point.

[0044] The chemiluminescence method uses a substance (luminescent substance) that reacts with free radicals to emit light, and measures the light emitted when the excited substance returns to its ground state using a luminescence spectrometer or similar device.

[0045] The luminescent substance is not particularly limited as long as it is a compound that reacts with free radicals to emit light, and known compounds can be used, such as luminol, Dansyl-TEMPO, lucigenin, 2-methyl-6-p-methoxyphenylethynylimidazopyrazinone (MPEC), hydroxyphenyl fluorescein (HPF), aminophenyl fluorescein (APF), and Cypridina luciferin derivative (MCLA).

[0046] The method for measuring the light emitted by the luminescent substance is not particularly limited, and can be appropriately determined depending on the type of the luminescent substance used, etc.

[0047] The electron spin resonance method (ESR method) is a spectroscopic analysis that observes the transition between levels that occurs when an unpaired electron is placed in a magnetic field. The electron spin resonance method is not particularly limited and can be performed using a known method and device, and may be either a direct method in which free radicals are directly measured or an indirect method in which a spin trapping agent is reacted with a free radical. When an indirect method is adopted, the spin trapping agent is not particularly limited and a known spin trapping agent can be used. Examples of the spin trapping agent include nitrone-based spin trapping agents such as 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 2,5,5-triethyl-1-pyrroline-N-oxide (M3PO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (TMPO), and N-tert-α-phenylnitrone (PBN); and nitroso-based spin trapping agents such as 2-methyl-2-nitrosopropane (MNP) and nitrosodurene (ND).

[0048] The measurement method of the present disclosure may include measuring an antibody titer against periodontal pathogens in a biological sample collected from the subject.

[0049] The biological sample is not particularly limited, and examples thereof include samples that may contain antibodies against periodontal pathogens (e.g., IgG antibodies, IgA antibodies, IgM antibodies, etc.). From the viewpoints of ease of collection and ease of handling, etc., the biological sample is preferably blood, saliva, gingival crevicular fluid, feces, tears, nasal swabs, or amniotic fluid. Furthermore, organs such as the liver can also be used as samples that may contain antibodies against periodontal pathogens. Of these, blood is preferred. When blood is used as a biological sample, plasma or serum is more preferable, and serum is even more preferable. When plasma is used as a biological sample, plasma separated by filter paper or a plasma separator may be used. The method and conditions for storing the biological sample collected from the subject are not particularly limited, and can be performed according to a conventional method.

[0050] The method for collecting a biological sample from a subject is not particularly limited and may be performed according to a conventional method. When serum or plasma is used as a biological sample, it is preferable to use a vacuum blood collection tube containing a serum or plasma separator from the viewpoints of ease of handling, prevention of infection, etc. In addition, the collected biological sample may be used as is, or may be freeze-dried or otherwise stored and then the freeze-dried product may be dissolved in a suitable solvent as described below and then used. In addition, the collected biological sample may be frozen and stored as is, or may be frozen and stored after being dissolved in a suitable solvent as described below, and then thawed when ready for use.

[0051] Examples of solvents for dissolving biological samples include physiological saline, PBS, phosphate buffer, Tris buffer, borate buffer, Good Buffer, etc. These solvents may contain casein, skim milk, bovine serum albumin (BSA), gelatin, blood protein, or plant protein as an active ingredient, protein component stabilizers such as blood components of normal animals such as mice, rabbits, goats, and fetal cows, and normal human serum components, various preservatives, surfactants, and enzyme activators such as salts of metals such as zinc and magnesium.

[0052] As a method for measuring antibody titer against periodontal pathogens, for example, a method can be used in which an antigen-antibody reaction product (a complex containing an antibody and the polypeptide) is detected between an antibody (e.g., an IgG antibody, an IgA antibody, an IgM antibody, etc.) against periodontal pathogens present in a biological sample and the polypeptide, which is generated by contacting the biological sample with an antigenic polypeptide (which undergoes an antigen-antibody reaction with an antibody against periodontal pathogens).

[0053] In this specification, "having antigenicity" means undergoing an antigen-antibody reaction with an antibody against periodontal pathogens. Examples of the antibody include immunoglobulin antibodies such as IgG antibodies, IgM antibodies, and IgA antibodies, and the antigen-antibody reaction may be with any of the immunoglobulin antibodies. Among these, IgG antibodies are preferred.

[0054] Antigenicity can be evaluated by ELISA. More specifically, when the antigen-antibody reaction is detected by ELISA using the polypeptide to be evaluated as an antigen and the positive serum of a periodontal disease patient containing an antibody against periodontal pathogens as a primary antibody, the polypeptide is judged to have antigenicity.

[0055] The antigenic polypeptide is not particularly limited as long as it undergoes an antigen-antibody reaction with an antibody against periodontal pathogens, and any known polypeptide can be used. For example, it may be a polypeptide that includes the amino acid sequence shown in SEQ ID NO:1, or may be a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1.

[0056] The polypeptide can be produced by general chemical synthesis methods for proteins (e.g., liquid phase and solid phase methods) based on the amino acid sequence information. The above-mentioned polypeptide can also be produced by genetic engineering techniques using a polynucleotide encoding the polypeptide.

[0057] The polypeptide may further include a tag. Examples of the tag include peptide tags such as His tag and SBP (Streptavidin Binding Peptide) tag, and polypeptide tags such as GST (Glutathione-S-transferase) tag and MBP (Maltose Binding Protein) tag. Among them, polypeptide tags are preferred, and GST tags are more preferred.

[0058] The method for adding the tag is not particularly limited, and conventional methods and reaction conditions can be used.

[0059] The polypeptide may further have a labeling substance, such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-D-galactosidase, biotin, gold colloid, latex beads, etc.

[0060] The method for detecting the antigen-antibody reactant is not particularly limited, and conventional methods and reaction conditions can be used. For example, there is a method in which the antigen-antibody reactant is contacted with an anti-immunoglobulin (e.g., anti-IgG antibody, etc.) containing a labeling substance and the labeling substance is detected, or when the polypeptide contains a labeling substance, the labeling substance is detected. The method for detecting an antigen-antibody reaction product may include, as necessary, for example, a step of immobilizing an antigen on a solid phase, a washing step, a blocking step, a substrate reaction step, etc. Conventional methods and reaction conditions can be used for these steps.

[0061] As a method for detecting an antigen-antibody reaction product, an immunological method such as ELISA, FLEIA, CLEIA, dot blot, or immunochromatography is preferably used.

[0062] The antibody titer can be calculated, for example, as U / mL (concentration of antibodies against periodontal pathogens in a biological sample).

[0063] As shown in the examples below, when the reflected value of the amount of oxidized HDL is high, periodontitis is likely to become severe (progress). Therefore, according to the measurement method of the present disclosure, the reflected value of the amount of oxidized HDL in a blood sample collected from a subject is measured, and the reflected value of the amount of oxidized HDL can be used as an index to measure the severity of periodontal disease in the subject.

[0064] Periodontal disease is broadly divided into gingivitis and periodontitis (mild periodontitis and severe periodontitis). As used herein, "gingivitis" refers to a condition in which plaque accumulates on the gum line, causing inflammation of the gums, resulting in swelling and bleeding. In this specification, "periodontitis" refers to a more advanced state of gingivitis, in which plaque accumulates under the gum line, the alveolar bone is destroyed, the gap between the teeth and gums deepens, and a periodontal pocket is formed. In this specification, a periodontal pocket with a depth of 4 mm or more and less than 6 mm is defined as "mild periodontitis," and a periodontal pocket with a depth of 6 mm or more is defined as "severe periodontitis."

[0065] According to the measurement method of the present disclosure, for example, when the value reflecting the amount of oxidized HDL in a blood sample taken from a subject is equal to or higher than a standard value, it is possible to predict (determine) that the severity of periodontal disease is likely to be high, compared to when the value is below the standard value.

[0066] An example of a standard value to be compared with the value reflecting the amount of oxidized HDL in a blood sample collected from a subject can be 31 mOD / min, which is the rate of increase in absorbance (change in absorbance per unit time), with the value reflecting the amount of oxidized HDL in the blood sample taken from the subject's blood sample starting at 3 minutes after the start of the reaction between the free radicals and the color-forming substance and ending at 6 minutes after the start of the reaction.

[0067] In addition, the reference value to be compared with the value reflecting the amount of oxidized HDL in the blood sample collected from the subject can be, for example, a value reflecting the amount of oxidized HDL in a blood sample collected from a subject (healthy individual) without periodontal disease. The value may be standardized based on data obtained in the past.

[0068] Furthermore, the reference value to be compared with the value reflecting the amount of oxidized HDL in a blood sample collected from a subject may be, for example, the average value, median value, mode value, or the like of the values ​​reflecting the amount of oxidized HDL in blood samples in a group consisting of multiple subjects. Among these, the median value is preferable.

[0069] In addition, according to the measurement method of the present disclosure, the change in the severity of periodontal disease over time can also be measured in the same subject.For example, when the reflected value of the amount of oxidized HDL in the blood sample taken from the subject increases over time, it can be predicted (determined) that periodontal disease may worsen.For example, when the reflected value of the amount of oxidized HDL in the blood sample taken from the subject decreases over time, it can be predicted (determined) that periodontal disease may improve.

[0070] In addition, as shown in the examples below, when the reflected value of the amount of oxidized HDL and the antibody titer are high, it is considered that periodontitis may be particularly likely to become severe (progress). Therefore, according to the measurement method of the present disclosure, by measuring the antibody titer against periodontal pathogens in a biological sample collected from a subject, the severity of periodontal disease of the subject can be measured using the antibody titer against the periodontal pathogens as an index.

[0071] According to the measurement method of the present disclosure, for example, when a value reflecting the amount of oxidized HDL in a blood sample collected from a subject is equal to or above a standard value, and the antibody titer against periodontal pathogens in a biological sample collected from the subject is equal to or above a standard value, it is possible to predict (determine) that the severity of periodontal disease is particularly likely to be high, compared with when these values ​​are below the standard values.

[0072] The standard value for comparison with the antibody titer against periodontal pathogens in a biological sample collected from a subject can be, for example, an antibody titer of 100 U / mL.

[0073] In addition, the standard value to be compared with the antibody titer against periodontal pathogens in a biological sample collected from a subject can be, for example, the antibody titer against periodontal pathogens in a biological sample collected from a subject (healthy individual) without periodontal disease. The value may be standardized based on data obtained in the past.

[0074] In addition, the standard value to be compared with the antibody titer against periodontal pathogens in a biological sample collected from a subject can be, for example, the average, median, or mode of the antibody titer against periodontal pathogens in biological samples from a group consisting of multiple subjects.

[0075] In addition, the measurement method of the present disclosure can also measure the change in the severity of periodontal disease over time in the same subject.For example, if the value reflecting the amount of oxidized HDL in the blood sample collected from the subject and the antibody titer against periodontal pathogens in the biological sample increase over time, it can be predicted (determined) that periodontal disease may have worsened.For example, if the value reflecting the amount of oxidized HDL in the blood sample collected from the subject and the antibody titer against periodontal pathogens in the biological sample decrease over time, it can be predicted (determined) that periodontal disease may have improved.

[0076] Although not particularly limited, the measurement method of the present disclosure preferably uses as an index a combination of a reflection value of the amount of oxidized HDL in a blood sample collected from a subject and an antibody titer against periodontal pathogens in a biological sample, and preferably uses as the reflection value the rate of increase in absorbance (change in absorbance per unit time) with the starting point being 3 minutes after the start of the reaction between the free radicals and the color-forming substance and the ending point being 6 minutes after the start of the reaction.

[0077] According to the present disclosure, the severity of periodontal disease can be measured based on the results of blood tests, etc., during health checkups, etc., even when a dental professional is not present or a dental checkup has not been conducted, by using a value reflecting the amount of oxidized HDL in a blood sample. Furthermore, according to the present disclosure, the severity of periodontal disease can be measured based on the results of blood tests, for example, during health checkups, by using the value reflecting the amount of oxidized HDL in a blood sample and the antibody titer against periodontal pathogens in a biological sample, even when a dental professional is not present or a dental checkup has not been conducted.

[0078] The present disclosure also encompasses a method for assisting in determining the severity of periodontal disease in a subject. In the present specification, the method may be referred to as the "assisted method of the present disclosure."

[0079] The assisting method of the present disclosure includes measuring a value reflecting the amount of oxidized HDL in a blood sample collected from a subject. The assisting method of the present disclosure may further include measuring an antibody titer against periodontal pathogens in a biological sample collected from the subject.

[0080] Regarding the method for measuring the value reflecting the amount of oxidized HDL in a blood sample, and the method for measuring the antibody titer against periodontal pathogens in a biological sample, the description of the "measurement method of the present disclosure" mentioned above can be cited.

[0081] According to the assisting method of the present disclosure, it is possible to assist in determining the severity of periodontal disease.

[0082] In addition, in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes all arbitrary combinations of the constituent features described in this specification.

[0083] In addition, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter included in the present disclosure. In other words, the present disclosure includes all subject matter consisting of all combinations of each combinable characteristic described in this specification. EXAMPLES

[0084] The contents of the present disclosure will be specifically explained using the following test examples. However, the present disclosure is in no way limited to these. In the following, unless otherwise specified, the experiments were performed under atmospheric pressure and room temperature conditions.

[0085] The following items were confirmed for 585 subjects (396 men, 189 women, average age 40.2 years) according to the methods described below.

[0086] Test Example 1 Measurement of PlI (Plaque Index) PlI was measured for six representative teeth per person (Figure 1). The representative teeth were the upper right 6, upper right 1, upper left 6, lower right 6, lower left 1, and lower left 6. However, if tooth 6 was missing, tooth 7 was used for measurement. PlI is an index showing the degree of plaque adhesion. PlI was measured for six representative teeth according to the following criteria. <Standards> 0: No adhesion observed 1: Plaque adhesion on less than 1 / 3 of the tooth surface 2: Plaque adhesion on 1 / 3 to less than 2 / 3 of the tooth surface 3: Plaque adhesion on more than 2 / 3 of the tooth surface Regarding the PlI of the six representative teeth, if the maximum PlI value was 0, it was judged as "no plaque," and if the maximum PlI value was 1 or more, it was judged as "plaque present."

[0087] Test Example 2: Blood antibody titer Measurement was performed by the CLEIA method (more specifically, the method described in Japanese Patent No. 6716241) using antigen protein 35N (SEQ ID NO: 1) having a GST tag on the N-terminus as an antigen.

[0088] ALP-labeled antigen and biotin-labeled antigen were prepared, and the target serum sample was mixed with a solution containing the ALP-labeled antigen and a solution containing the biotin-labeled antigen to prepare a complex of the antibody against periodontal pathogens contained in the biological sample, the ALP-labeled antigen, and the biotin-labeled antigen. The complex was bound to the porous carrier by contacting it with a porous carrier carrying anti-biotin antibodies, and the intensity of the chemiluminescence obtained by adding the ALP substrate was measured (40°C, 1 minute). A calibration curve was created using a standard positive serum with a clear antibody concentration against periodontal pathogens, and the antibody titer (U / mL) was calculated. The cutoff value for blood antibody titer was set at 100 U / mL.

[0089] Test Example 3 Calculation of the value reflecting the amount of oxidized HDL (oxHDL) in blood samples (1) Preparation of blood samples Blood was collected from the subject's vein using a vacuum blood collection tube, left at room temperature for 1 hour, and then centrifuged under general serum separation conditions to obtain the supernatant (serum) as a blood sample, which was then stored at -80°C.

[0090] (2) Measurement of oxidized HDL (oxHDL) A mixture of 0.1M acetate buffer (pH 4.8) and 100μM ammonium iron(II) sulfate hexahydrate was added to each well of the microplate and kept at 37℃. Next, N,N-diethyl-p-phenylenediamine sulfate solution (solvent: DMSO) was added to each well, and then the serum obtained in (1) above was added. The absorbance at a wavelength of 505nm was measured using a microplate reader (Bio-Rad) set at 37℃. The absorbance was measured by setting the instrument to Kinetics, measuring 48 times (8 minutes in total) every 10 seconds, and calculating the absorbance change per unit time (unit: mOD / min) from each value 3 minutes to 6 minutes after the start of the measurement. The cutoff value was set to 31mOD / min, which is the median value of the measured values ​​of all subjects.

[0091] Test Example 4 Classification of Periodontal Disease Community Periodontal Index (CPI) was measured by dental professionals. <Standards> Code 0: Healthy Code 1: Bleeding Code 2: Tartar buildup Code 3: Shallow periodontal pockets (PD=4-5mm) Code 4: Deep periodontal pockets (PD≧6mm) The code of each representative tooth was measured, and the maximum code was taken as the representative value for each subject. Codes 1 and 2 were combined to represent gingivitis, and each subject was classified into the following four groups.

[0092] [Table 1]

[0093] Reference example 1 The subjects were classified into groups with and without plaque adhesion, and the classification results of periodontal disease in each group are shown in Figure 2.

[0094] It was confirmed that the rate of gingivitis and PD=4-5mm (mild periodontitis) was higher in the group with plaque adhesion compared to the group without it. In particular, a large difference was confirmed in the rate of gingivitis. This suggests that when plaque adhesion is present, bleeding due to inflammation is more likely to occur, which in turn leads to the development of gingivitis and periodontitis (mild). In other words, it is suggested that the presence or absence of plaque adhesion may be an important indicator in the early stages of the onset of periodontal disease.

[0095] Reference example 2 The subjects were classified into two groups: those with a blood antibody titer of 100 U / mL or more, and those with a blood antibody titer of less than 100 U / mL. The classification results for periodontal disease in each group are shown in Figure 3.

[0096] It was confirmed that the proportion of patients with PD=4-5mm (mild periodontitis) and PD≧6mm (severe periodontitis) was higher in the group with an antibody titer of 100U / mL or more than in the group with an antibody titer of less than 100U / mL. In particular, a large difference was confirmed in the proportion of patients with PD≧6mm. This suggests that periodontitis may be more likely to become severe (progress) when antibody titers are high.

[0097] Reference example 3 The subjects were classified into groups with or without plaque, and further classified into groups with blood antibody titers of 100 U / mL or more and groups with titers below 100 U / mL. The classification results for periodontal disease in each group are shown in Figure 4.

[0098] In both the groups with and without plaque adhesion, it was confirmed that the proportion of patients with PD≧6mm (severe periodontitis), PD=4-5mm (mild periodontitis), and PD≧6mm (severe periodontitis) was higher in the group with an antibody titer of 100U / mL or higher compared to the group with an antibody titer of less than 100U / mL.

[0099] Example 1 The subjects were classified into a group whose oxidized HDL level was reflected as 31 mOD / min or more and a group whose level was less than 31 mOD / min. FIG. 5 shows the classification results of periodontal disease in each group.

[0100] It was confirmed that the proportion of PD≧6mm (severe periodontitis) was higher in the group with a value reflecting oxidized HDL of 31mOD / min or more than in the group with a value reflecting oxidized HDL of less than 31mOD / min. This suggests that when the value reflecting oxidized HDL is high, periodontitis may be more likely to become severe (progress).

[0101] Example 2 The subjects were classified into those with or without plaque adhesion, and further classified into those with values ​​reflecting oxidized HDL levels of 31 mOD / min or more and those with values ​​less than 31 mOD / min. The classification results for periodontal disease in each group are shown in Figure 6.

[0102] In both the groups with and without plaque, it was confirmed that the proportion of patients with PD≧6mm (severe periodontitis), PD=4-5mm (mild periodontitis), and PD≧6mm (severe periodontitis) was higher in the group with a value reflecting oxidized HDL of 31mOD / min or more compared to the group with a value less than 31mOD / min. This suggests that the severity of periodontitis can be measured by combining the value reflecting oxidized HDL and the presence or absence of plaque.

[0103] Example 3 The subjects were classified into two groups: those with blood antibody titers of 100 U / mL or more and those with titers below 100 U / mL, and further classified into those with titers reflecting oxidized HDL levels of 31 mOD / min or more and those with titers below 31 mOD / min. The classification results for periodontal disease in each group are shown in Figure 7.

[0104] It was confirmed that the proportion of PD≧6mm (severe periodontitis) was particularly high in the group with a value reflecting oxidized HDL of 31mOD / min or more and a blood antibody titer of 100U / mL or more compared to the other groups. This suggests that when the value reflecting oxidized HDL and the antibody titer are high, periodontitis may be particularly likely to become severe (progress). This suggests that the severity of periodontitis can be measured more accurately by combining the value reflecting oxidized HDL and the antibody titer.

[0105] Example 4 The subjects were classified into those with or without plaque, those with blood antibody titers of 100 U / mL or more, and those with titers less than 100 U / mL, and those with titers reflecting oxidized HDL levels of 31 mOD / min or more, and those with titers less than 31 mOD / min. The classification results for periodontal disease in each group are shown in Figure 8.

Claims

1. A method for measuring the severity of periodontal disease in a subject, comprising measuring a value reflective of the amount of oxidized HDL in a blood sample collected from the subject, and using the value reflective of the amount of oxidized HDL as an index.

2. The method according to claim 1, further comprising measuring an antibody titer against periodontal pathogens in a biological sample taken from the subject, and measuring the severity of the subject's periodontal disease using the antibody titer against the periodontal pathogens as an indicator.

3. The measurement of the reflection value of the amount of oxidized HDL (1) The method is carried out by a method including a step of mixing a blood sample with a solution containing a transition metal compound and an acidic buffer solution, The method according to claim 1 or 2.

4. The measurement of the reflection value of the amount of oxidized HDL moreover, (2) The method is carried out by a method including a step of measuring the free radicals produced in the step (1). The method according to claim 3.

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