JP2018021882A - Method for inspecting periodontal disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for inspecting a periodontal disease and an inspection kit which can determine the onset of a periodontal disease and/or the stage of a periodontal disease easily, accurately, and rapidly.SOLUTION: The present invention relates to a method for inspecting a periodontal disease including a step of detecting or measuring ameloblastin in a sample collected from a mouth of a subject, and to a kit for inspecting a periodontal disease containing an antibody or a piece thereof which specifically binds to ameloblastin.SELECTED DRAWING: None

Description

  The present invention relates to a test method for periodontal disease by detecting or measuring ameloblastin in an oral sample, and a test kit for periodontal disease used in the method.

  Periodontal disease is a disease caused by the growth and infection of periodontal disease bacteria in plaque (plaque) on the gingival margin and subgingival margin, and gingivitis that causes bleeding and swelling limited to the gingiva due to inflammation. In addition to inflammation of the gingiva, it is divided into periodontitis in which the alveolar bone supporting the teeth is destroyed. So-called alveolar pyorrhea is called chronic periodontitis and does not only destroy periodontal tissue, but also varies depending on endotoxins produced by periodontal disease-causing bacteria and inflammatory substances released from white blood cells that resist bacteria. It is known to exacerbate various systemic diseases. For example, periodontal disease is associated with diseases such as diabetes, arteriosclerosis, myocardial infarction, cerebral infarction, premature birth, low birth weight premature birth, swallowing pneumonia, autoimmune diseases (allergy, rheumatism, etc.), and osteoporosis. Has been. Therefore, it is very important to detect periodontal disease as early as possible and to start treatment in order to prevent the progression of the disease and to perform appropriate treatment according to its symptoms and progression.

  As a diagnosis of periodontal disease, a probing test is known in which the depth of a periodontal groove (periodontal pocket) formed at the boundary between a tooth and a gingiva is measured with a probe called a periodontal probe. However, probing inspection requires skill and experience and may damage periodontal tissue. Moreover, since it is visual evaluation, objective judgment cannot be performed. Furthermore, since the probing test examines the extent to which the periodontal tissue has already been destroyed by periodontal disease, the probing pocket depth is shallow and early periodontal disease in which no lesion is observed cannot be found. Therefore, a simple method that can detect periodontal disease at an early stage using specimens such as saliva and gingival crevicular fluid, and know the degree of progression, reduces the burden on the test subject, and makes the test faster and more accurate. Desirable from the viewpoint of sex.

  So far, many methods have been proposed in which a specific protein contained in saliva or gingival crevicular fluid is used as a diagnostic marker for periodontal disease. For example, cysteine protease (patent document 1), lactoferrin and myeloperoxidase (patent document 2), α1-antitrypsin (patent document 3), histatin (patent document 4), 8-hydroxydeoxyguanosine (patent document 5), β- A method has been proposed in which defensin (Patent Document 6) or the like is detected or measured to determine whether or not periodontal disease is present or progress. These marker proteins are substances derived from periodontal pathogens such as Gindivarius, substances related to inflammation and bleeding, and bioprotective (antibacterial) substances against periodontal pathogens. However, there are problems such as lack of rapidity and accuracy, such as markers that appear after progressing to a certain time, and it takes time to obtain test results.

WO2004 / 106541 Publication Japanese Patent Laid-Open No. 10-2899 Japanese Unexamined Patent Publication No. 2000-28608 JP 2005-249567 A WO2002 / 079780 Publication JP 2009-145220 JP

  An object of the present invention is to provide a method and a test kit for periodontal disease, which can quickly, simply and accurately determine the presence and / or progression of periodontal disease.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that ameloblastin, which is one of the proteins constituting tooth enamel, is related to periodontal disease, and ameloblastin is used as an index. It was found that periodontal disease can be detected at an early stage. In addition, by detecting or measuring at least one inflammatory cytokine of IL-1β or IL-6 together with ameloblastin, it is possible to more accurately determine the state of periodontal disease including the degree of progression of periodontal disease. It was confirmed that it was possible to complete the present invention.

That is, the present invention includes the following inventions.
(1) A method for examining periodontal disease, comprising a step of detecting or measuring ameloblastin in a sample collected from the oral cavity of a subject.
(2) The periodontal disease test method according to (1), wherein the detection or measurement is performed by an immunological measurement method using an antibody or a fragment thereof that specifically binds to ameloblastin.
(3) The method for examining periodontal disease according to (1), further comprising the step of detecting or measuring at least one inflammatory cytokine of IL-1β or IL-6 in the sample.
(4) The detection or measurement is performed by an immunoassay using an antibody or a fragment thereof that specifically binds to at least one inflammatory cytokine of IL-1β or IL-6. The test method of periodontal disease as described.
(5) The method for examining periodontal disease according to any one of (1) to (4), wherein the sample is saliva.
(6) The method for examining periodontal disease according to any one of (1) to (4), wherein the sample is gingival crevicular fluid.
(7) The method for examining periodontal disease according to any one of (1) to (4), wherein the sample is serum derived from gingival blood.
(8) The periodontal disease examination method according to any one of (1) to (7), wherein the examination is for determining the onset and / or progression of periodontal disease.
(9) A test kit for periodontal disease comprising an antibody or a fragment thereof that specifically binds to ameloblastin.
(10) The test kit for periodontal disease according to (9), wherein the antibody or a fragment thereof that specifically binds to ameloblastin is held in an immunochromatographic test strip.
(11) The test kit for periodontal disease according to (9) or (10), further comprising an antibody or a fragment thereof that specifically binds to at least one inflammatory cytokine of IL-1β or IL-6.

  ADVANTAGE OF THE INVENTION According to this invention, the test | inspection method of periodontal disease and the kit for a test | inspection which can perform the determination of the presence or absence of a periodontal disease and / or a progress degree rapidly, simply, and correctly are provided. According to the present invention, it is possible to detect periodontal disease at an early stage by using an immunological measurement method, particularly a simple immunochromatography method, using saliva or gingival crevicular fluid that can be easily collected from a subject. . Therefore, the burden on the subject in the periodontal disease test is small, and in the stage of gingivitis and mild periodontitis, simple treatment such as tooth brushing and calculus removal can be performed, and the treatment cost of the patient can be reduced.

FIG. 1 shows the results of immunochromatographic detection of ameloblastin in saliva collected from healthy individuals, patients with early periodontitis, and patients with chronic periodontitis. FIG. 2 shows the quantification results by ELISA of inflammatory cytokines (IL-1β, IL-6) in saliva collected from healthy individuals, patients with early periodontitis, and patients with chronic periodontitis.

  The method for examining periodontal disease of the present invention includes a step of detecting or measuring ameloblastin in a sample collected from the oral cavity of a subject.

  Ameloblastin is a gene-specific protein present in dental enamel and constitutes enamel together with amelogenin, enamelin, tufterin and the like. Ameloblastin is formed by enamel blasts at the time of tooth development and is synthesized as a 62 kDa protein, but it is decomposed into 13-17 kDa fragments (enamel trabecular sheath) in the tissue surrounding the enamel crystals. Localize. Although the function of ameloblastin has not yet been elucidated, it has a calcium binding ability and is thought to play an important role in controlling the growth of enamel crystals and the promotion of calcification. The base sequence of human ameloblastin (Genbank number: Nucleotide NM — 016519; Protein NP — 057603.1) is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 2 in the sequence listing.

  In the present invention, the “subject” is not limited as long as it is a mammal capable of developing periodontal disease, but includes humans, monkeys, dogs, cats, cows, horses, pigs, rabbits, mice, rats, etc. preferable.

  In the inspection method of the present invention, it is preferable to use saliva, gingival crevicular fluid, or serum derived from gingival blood as the “sample collected from the oral cavity”.

  Saliva collection can be performed by a known method, for example, a method of discharging resting saliva accumulated in the mouth in a resting state into a container (vomiting saliva method), from a salivary gland opening using a dedicated saliva collecting tube Examples include a method of directly collecting pure saliva, a method of discharging stimulated saliva accumulated in the mouth by chewing paraffin gum, etc. into the container. .

  In the test method of the present invention, when saliva is used as a sample, the collected saliva can be used as it is, but the saliva supernatant obtained by centrifuging saliva to remove insoluble matters, or a surfactant (for example, Tween 20) You may use the processing liquid by a positive surfactant. Centrifugation is performed at 5000-12000 rpm, preferably 800-10000 rpm, for about 10-30 minutes. When the saliva supernatant is used, it may be used as it is, but it is preferable to dilute the saliva supernatant about 2 to 5 times with a solvent from the viewpoint of obtaining a more accurate measurement result. Examples of the solvent for diluting saliva include distilled water, physiological saline, and phosphate buffer. The amount of saliva collected is preferably about 0.1 to 1.0 ml.

  The collection of gingival crevicular fluid can also be performed by a known method. For example, methods for collecting gingival crevicular fluid include a method of inserting a periopaper into the gingival crevice, a method using a micropipette, a method of inserting a thread or paper point twisted into the gingival crevice, etc. The method of inserting into the gingival crevice and confirming the amount of gingival crevicular exudate collected with Periotron is more preferable because the operation time is short and the patient is not burdened. When collecting gingival crevicular fluid with filter paper such as perio paper, the time for insertion into the gingival crevice is preferably about 15 to 60 seconds, and the amount of collected gingival crevicular fluid is preferably about 0.5 to 5.0 μl.

  In the inspection method of the present invention, it is possible to directly use the collected gingival crevicular fluid, but use a sample extracted from a filter paper such as perio paper in which the gingival crevicular fluid has soaked, using an extraction solvent. It is preferable. Examples of the extraction solvent include water, physiological saline, and phosphate buffer solutions such as PBS. Extraction can be performed by immersing a filter paper such as perio paper in which gingival crevicular exudate has soaked in an extraction solvent in a container such as a microtube.

  Moreover, when using the serum isolate | separated from the blood extract | collected from the gingiva as a sample, the blood from a gingiva can be extract | collected with a dental injection needle, and serum can also be obtained by normal methods, such as a centrifugation method. The amount of serum to be collected is preferably about 0.5 to 1.0 μl.

  In the test method of the present invention, an antibody (anti-ameloblastin antibody) that can specifically bind to ameloblastin is used for detection or measurement of ameloblastin in a sample collected from the oral cavity of such a subject. Performed by immunoassay.

  The antibody used for the above-described immunological measurement of ameloblastin can be obtained by a method well known to those skilled in the art. The antibody may be either a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is preferred.

  The antibody can be prepared according to a known method using ameloblastin of the animal species to be measured or a partial polypeptide thereof as an antigen. Ameloblastin or a partial polypeptide thereof used as an antigen is, for example, a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 or a partial sequence thereof is incorporated into an expression vector and introduced into an appropriate host cell to transform the transformant. The recombinant protein can be obtained by culturing the transformant and expressing the recombinant protein, and purifying the expressed recombinant protein from the culture or culture supernatant. Alternatively, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or a partial sequence thereof can be chemically synthesized and used as an immunogen. When a polypeptide consisting of a partial sequence of ameloblastin is used as an antigen, a polypeptide comprising an amino acid sequence of at least 30 consecutive amino acid sequences (SEQ ID NO: 2) of ameloblastin, preferably 50 or more amino acids. preferable.

  In order to obtain a monoclonal antibody, antibody-producing cells (spleen cells, lymph node cells, etc.) are removed from a mammal sensitized with the antigen and fused with myeloma cells. The thus obtained hybridoma can be cloned, and the antibody can be recovered from the culture to obtain a monoclonal antibody. A polyclonal antibody can be prepared by collecting blood from a mammal (eg, rabbit, rat, mouse, etc.) sensitized with an antigen and separating the serum from the blood by a known method.

  The antibody can also be used as a fragment as long as it can specifically bind to ameloblastin. Examples of active fragments of antibodies include Fab fragments, F (ab ′) 2 fragments, single chain antibodies (scFv), and the like. These active fragments can be prepared by applying known methods such as papain, pepsin and trypsin treatment.

In addition, when labeling an antibody, the labeling substance is not particularly limited as long as it is a labeling substance that enables immunological measurement of ameloblastin, for example, an enzyme (peroxidase, β-galactosidase, alkaline phosphatase, etc.) , Fluorescent substances (FITC, RITC, etc.), chemiluminescent substances (acridinium, ruthenium, lophine, etc.), radioisotopes ( ³ H, ¹⁴ C, ³⁵ S, ³² P, ¹²⁵ I, ¹³¹ I, etc.), biotin, digoxigenin, Examples include polypeptides containing tag sequences, colloidal gold particles, and the like. The labeling substance may be directly bound to the antibody, or may be indirectly labeled using an antibody that recognizes the labeling substance or an avidin-biotin system.

  The immunological measurement method is not particularly limited, and is a conventionally known method such as an enzyme-linked immunosorbent assay (Enzyme-Linked ImmunoSorbent Assay: ELISA method), latex agglutination method, immunochromatography method, Western blot method, radioimmunity Measurement method (RIA method), fluorescence immunoassay method (FIA method), luminescence immunoassay method, spin immunoassay method, turbidimetric method for measuring turbidity associated with antigen-antibody complex formation, and antibody solid phase membrane electrode An enzyme sensor electrode method, an immunoelectrophoresis method, or the like that detects a potential change due to binding to an antigen is used. ELISA methods include competitive methods, sandwich methods, and direct adsorption methods. Of these, immunochromatography, ElISA, and Western blot are preferred, but immunochromatography is preferred because accurate measurement results can be obtained easily and quickly on the chair side, and no special measuring device is required. Is more preferable.

  When the immunochromatography method is used as the immunological measurement method of the present invention, the procedure is as follows. First, a sample in which a capture site (judgment line) in which an anti-ameloblastin antibody (first antibody) is bound in a line at a predetermined position and an anti-ameloblastin antibody (second antibody) labeled with colored microparticles are bound A test piece for immunochromatography provided with a dropping part is prepared. Next, a sample collected from the subject's oral cavity (hereinafter sometimes simply referred to as a sample) is added to the sample dropping part and left for a certain period of time, so that it is directed to the capture site by the action of the capillary action of the carrier. To diffuse and move sequentially. Thereafter, an immune complex of ameloblastin contained in the sample and an anti-ameloblastin antibody labeled with colored microparticles is captured at the capture site, colored in a line, and the coloration state is visually determined. To do.

  The colored fine particles used in the immunochromatography are not particularly limited as long as they can be detected with the naked eye. For example, colloidal particles made of metals such as gold, silver, copper, sudan blue, sudan red, oil orange, quinizarin green. A colored latex obtained by coloring a latex with a pigment or a dye such as can be used. The test piece used for the said immunochromatography can be produced using a commercially available material by a well-known method. For example, a porous sheet having such a property that the sample is rapidly absorbed and quickly moves to the reaction part is preferable. Examples of the porous sheet include cellulose filter paper, glass fiber filter paper, polyurethane, polyacetate, acetic acid. Examples thereof include cellulose, nylon, and cotton cloth.

  When the ELISA method is used as the immunoassay method of the present invention, an anti-ameloblastin antibody is immobilized on a carrier, a sample is added and incubated, a labeled secondary antibody is added, and the bound secondary antibody is added. This is done by measuring. As the carrier for immobilizing the anti-ameloblastin antibody, for example, polyvinyl chloride, polystyrene, polyethylene, polypropylene, polyester, fluororesin, glass, metal and the like can be used. Examples of the shape of the carrier include plates, beads, cells, test tubes and the like. Preferably, a commercially available solid phase carrier such as a microtiter plate for enzyme immunoassay (96-well microtiter plate) can be used. The method for immobilizing the anti-ameloblastin antibody may be performed according to a known method. For example, an anti-ameloblastin antibody solution dissolved in an appropriate buffer is brought into contact with a carrier and left at 4 to 37 ° C. for 1 to 24 hours to immobilize the anti-ameloblastin antibody. At this time, in order to prevent non-specific adsorption, a buffer solution such as phosphate buffered saline (PBS) containing 1-10% albumin, gelatin, milk powder or fetal bovine serum, or Tris buffer (TBS) is used. It is preferable to use as a blocking solution to block unadsorbed portions. After immobilization, the carrier is washed with an appropriate buffer. Next, the sample is diluted about 50 to 5000 times before incubation. By adding the diluted sample to the carrier prepared as described above and incubating it, the ameloblastin present in the sample is reacted with the anti-ameloblastin antibody on the carrier. Incubation is performed at 4 to 37 ° C. for 1 to 24 hours, preferably about 16 hours. After the reaction, the reaction solution is removed and the carrier is washed. Next, a secondary antibody against ameloblastin is added. The secondary antibody against ameloblastin is an antibody that can bind to ameloblastin to be measured, and those labeled with a labeling substance such as an enzyme, a radioisotope, a fluorescent substance, or a luminescent substance can be used. Among these, enzyme-labeled secondary antibodies are preferable. For example, goat anti-human immunoglobulin, rabbit anti-human immunoglobulin, or sheep anti-human immunoglobulin labeled with an enzyme such as peroxidase, alkaline phosphatase, or β-galactosidase is used. Can do. The secondary antibody is diluted about 1000 to 10,000 times before incubation. The reaction is carried out at 4 to 37 ° C. for about 1 to 2 hours. After completion of the reaction, the reaction solution is removed and the carrier is washed.

  The measurement of the secondary antibody can be performed according to a known method according to the type of the labeling substance of the secondary antibody used. When an enzyme-labeled secondary antibody is used, a color development reaction is performed by adding a known color former in accordance with the selected enzyme. For example, when peroxidase is used, alkaline phosphatase is used with a substrate such as o-phenylenediamine, ABTS [2,2′-azino-bis- (3′-ethylbenzodiazolinesulfonic acid)] and hydrogen peroxide. In the case of using a substrate such as p-nitrophenyl phosphate or 4-methylumbeferyl phosphate, in the case of using β-galactosidase, o-nitrophenyl-β-D-galactoside, 4-methylumbelliferyl-β- A substrate such as D-galactoside may be used as the color former. The color development produced depending on the amount of ameloblastin in the sample can be quantified by measuring the absorbance of the reaction solution using a spectrophotometer, a fluorometer, or the like.

  When Western blotting is used as an immunoassay method of the present invention, a sample is added onto a polyacrylamide gel containing sodium dodecyl sulfate, subjected to electrophoresis with a certain voltage, and separated on the gel by electrophoresis. Is electrically transferred to a blotting membrane such as a PVDF (polyvinylidene difluoride) membrane. After blocking the membrane with skim milk, etc., react the anti-ameloblastin antibody with the membrane, then bind the labeled secondary antibody labeled with an enzyme, fluorescent substance, etc., and add a detection reagent according to the labeling substance. To detect.

  In addition, when detecting or measuring ameloblastin by the said immunoassay, any method can also use a commercially available kit.

  In the present invention, periodontal disease is examined or diagnosed by detecting or measuring ameloblastin (confirmation of the presence or absence of ameloblastin) using the immunological assay described above. In the present invention, “examination” or “diagnosis” typically means determination of whether or not a subject suffers from periodontal disease, but is not limited thereto, and the degree of progression of the disease. Determination of the therapeutic effect on the disease, determination of whether there is a risk of recurrence of the disease after treatment, and the like. In addition, “examination” or “diagnosis” can be replaced by the words “assistance for examination” or “assistance for diagnosis” that does not include diagnosis by a doctor. Specifically, for “examination” or “diagnosis” It means getting the data.

  For example, the determination of the presence and / or progression of periodontal disease caused by ameloblastin compares the amount of ameloblastin in a sample taken from the oral cavity of a subject with the amount of ameloblastin in a control sample Can be done. The control sample refers to a sample to be used as a reference for comparison, and may be either a sample collected from the oral cavity of a healthy person (negative control) or a sample collected from the oral cavity of a periodontal disease patient (positive control). Measurement of the amount of ameloblastin in the control sample can be performed in the same procedure as the measurement of the amount of ameloblastin in the subject sample. The amount of ameloblastin in the control sample may be measured each time the amount of ameloblastin in the subject sample is measured, or may be measured in advance. If the amount of ameloblastin in the subject sample is significantly higher than the amount of ameloblastin in the negative control, the subject is determined to have periodontal disease and is equivalent to the amount of ameloblastin in the negative control. If so, the subject is determined not to have periodontal disease. Also, if the amount of ameloblastin in the subject sample is significantly less than the amount of ameloblastin in the positive control, it is determined that the subject does not suffer from periodontal disease or the symptoms have been improved by treatment. it can. The amount of ameloblastin in a subject sample may be measured by comparison with the amount of ameloblastin in a control sample, but the absolute amount of ameloblastin is not necessarily measured. It is not necessary, and it is sufficient if the relative relationship with the amount of ameloblastin in the control sample becomes clear.

  In addition to the step of detecting or measuring ameloblastin in a sample collected from the oral cavity of a subject, the method for examining periodontal disease of the present invention includes at least one of IL-1β or IL-6 in the sample. The step of detecting or measuring the inflammatory cytokine may be further performed. The detection or measurement of these inflammatory cytokines is also preferably performed by an immunological method, and the above-mentioned ameloblastin can be obtained using a commercially available kit equipped with anti-IL-1β antibody and anti-IL-6 antibody. What is necessary is just to follow the measurement method.

  The periodontal pocket is about 0.5 to 2 mm for healthy teeth, about 2 to 4 mm for gingivitis, about 3 to 4 mm for mild, and 4 for moderate. ~ 7mm, 7mm or more if severe. That is, if the periodontal pocket is 3 mm or less, health or health can be maintained, and if it is 4 mm or more, periodontal disease is progressing. Gingivitis, or the period from gingivitis to early periodontitis (referred to herein as the “initial stage”), there may be some bleeding or swelling in the gingiva, but there are almost no subjective symptoms Even if there is, it often proceeds by neglecting it and leaving it alone. As will be described later in Examples, according to the method for examining periodontal disease of the present invention, it is possible to determine the presence or absence of this periodontal disease by detecting or measuring ameloblastin. In the initial stage, inflammation of the gingiva only, and there is no abnormality in the alveolar bone, so it can be improved by brushing teeth and removing the calculus. And enables minimally invasive treatment.

  In the method for testing periodontal disease of the present invention, the accuracy of the test can be further ensured by performing detection or measurement of at least one inflammatory cytokine of IL-1β or IL-6. Specifically, even if the detection of ameloblastin is positive, if detection of IL-1β and / or IL-6 is negative, periodontal disease can be determined as an early stage, and if both are positive It can be determined that the patient has chronic periodontal disease. In the method for examining periodontal disease of the present invention, as long as the effect of assisting the determination of the onset and / or progression of periodontal disease due to ameloblastin can be achieved, the periodontal disease bacteria and their production substances ( (Toxins, enzymes, etc.) may be detected or measured together.

  The method for examining periodontal disease of the present invention can be applied to diagnosis of inflammation around the implant body (peri-implantitis) as well as natural teeth. Generally, peri-implantitis differs from periodontitis of natural teeth and does not form deep pockets. Since the diagnosis of peri-implantitis is mainly performed by the operator's visual observation, the quantification of inflammation according to the present invention is a new diagnostic index for peri-implantitis.

  The present invention also provides a test kit for periodontal disease for carrying out the above test method. The kit for testing periodontal disease of the present invention only needs to contain at least an antibody that specifically binds to ameloblastin, and specifically binds to at least one inflammatory cytokine of IL-1β or IL-6. Antibodies may be included. In addition, the antibody that specifically binds to ameloblastin included in the test kit for periodontal disease of the present invention is preferably in an aspect that is retained on a test piece for immunochromatography. The test kit for periodontal disease of the present invention further comprises an immobilization carrier, a labeled secondary antibody, a detection reagent, a reaction buffer, a washing buffer, a reaction stop solution, a coloring reagent, a standard Substances (positive control, negative control), reagents for dilution, instructions for use, etc. may be included.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

(Example 1) Preparation of immunochromatographic test piece holding anti-ameloblastin antibody (1) Preparation of colloidal gold-labeled anti-ameloblastin antibody Commercially available colloidal gold solution (EMGC40, BBI Solutions, particle size: 40 nm) Add 9 ml of 50 mM KH ₂ PO ₄ buffer (pH 7.5) to 9 ml, and then add 1 ml of 51.1 μg / ml anti-ameloblastin (AM-1) monoclonal antibody (Gennet) solution And stirred. After standing for 30 minutes, 1% polyethylene glycol (PEG Mw. 20000, Nacalai Tesque) was added, and 1.1 ml of 10% bovine serum albumin (BSA Fraction V, Nacalai Tesque) was added and stirred. After centrifuging this solution at 11000 rpm and 4 ° C. for 15 minutes, the supernatant was removed leaving about 0.5 ml, and the gold colloid was redispersed with an ultrasonic cleaner. To this dispersion, add 20 mM Tris-HCl buffer (pH 8.2) containing 0.05% PEG, 150 mM NaCl, 1% BSA, and 0.1% NaN ₃ and perform the same stirring, centrifugation, and supernatant removal operations as above. Was repeated 3 times for washing. After washing, the colloidal gold obtained by removing the supernatant is suspended in 20 mM Tris-HCl buffer (pH 8.0) containing 0.05% PEG, 1% BSA, 5% sucrose, and colloidal gold-labeled anti-Ameloblasts A tin antibody solution was obtained.

(2) Production of gold colloid-labeled anti-ameloblastin antibody retention site
30 μl of gold colloid-labeled anti-ameloblastin antibody solution prepared in (1) is impregnated into a glass fiber pad (GLASSFIBER DIAGNOSTIC PAD, Merck Millipore) cut into 5 mm × 10 mm, dried under reduced pressure overnight, and gold colloid A labeled antibody holding site was prepared.

(3) Preparation of antibody-immobilized membrane
Anti-ameloblastin (AM-2) monoclonal for immobilization prepared at 533 μg / ml with the long side of the nitrocellulose membrane (HiFlow Plus HF135, Merck Millipore) cut to 26 mm x 250 mm The antibody (manufactured by Genenet) solution was applied in a line at 1.46 μl / cm at a position of 8.5 mm from the top using an applicator (Nippon Engineering). Similarly, a control anti-mouse IgG antibody (Santa Cruz) solution prepared to 133 μg / ml was applied in a line at 1.43 μl / cm at a position 13 mm from the top of the membrane. After coating, the membrane was dried at 50 ° C. for 30 minutes to obtain an antibody-immobilized membrane.

(4) Preparation of immunochromatographic test piece The antibody-immobilized membrane prepared in (3) was attached to a backing adhesive sheet (pre-cut backing sheet, NIPPON ENGINEERING CO., LTD.). Attach the gold colloid-labeled antibody retention site prepared in (2) to the lower side of the antibody-immobilized membrane so that it overlaps by approximately 4 mm, and further cut the sample addition pad (17 mm x 250 mm) so that it overlaps approximately 3 mm. CELLULOSE FIBER SAMPLE PADS, Merck Millipore). Furthermore, an absorption pad (CELLULOSE FIBER SAMPLE PADS, Merck Millipore) cut to 17 mm × 250 mm was overlaid and pasted on the upper side of the antibody-immobilized membrane so as to overlap about 3 mm. The long side of the sheet in which a plurality of members were stacked and bonded in this way was cut with a cutter parallel to the short side at intervals of 5 mm to obtain a test piece for immunochromatography of 5 mm × 60 mm.

(Example 2)
1. Test Method (1) Collection of Saliva Sample Saliva samples were collected from the following three groups of subjects.
(i) Patients with early periodontitis: 3 patients with probing pocket depth (use attachment level if retraction is less than 3mm), alveolar bone resorption is less than 1/3 of root length, and no root bifurcation lesions
(ii) Patients with chronic periodontitis: Probing pocket depth of 4 mm or more, alveolar bone resorption rate of more than 1/3 of the root length, root bifurcation lesions, 3 patients with shaking
(iii) Healthy people: None of the above symptoms is observed. 3 subjects Each group of subjects is instructed to refrain from swallowing and to exhale saliva secreted into the oral cavity into the cup. Saliva was collected (vomiting saliva method) and stored at -80 ° C.

(2) Detection of ameloblastin by immunochromatography The test sample for immunochromatography in which the collected saliva sample was diluted 5 times with PBS containing 0.5% Tween20 and the anti-ameloblastin antibody prepared in Example 1 was retained. 120 μl was applied. A band pattern 30 minutes after the application was measured to confirm the presence or absence of ameloblastin.

(3) Quantification of Inflammatory Cytokines by Enzyme-Linked ImmunoSorbent Assay (ELISA) The concentrations of IL-1β, IL-6, and TNF-α contained in the collected saliva samples were quantified using a commercially available ELISA kit. The concentration of IL-1β was measured using a human IL-1β ELISA kit (manufactured by BioLegend) as follows.

  First, 50 μl of saliva sample was added to each well of the kit immobilized with anti-IL-1β antibody, incubated at room temperature for 2 hours, and washed 4 times with washing buffer after the reaction was completed. Next, 100 μl of labeled anti-IL-1β antibody was added to each well, incubated at room temperature for 30 minutes, and washed 5 times with a washing buffer after the reaction was completed. Thereafter, 100 μl of the substrate was added, incubated at room temperature for 20 minutes, 100 μl of the reaction stop solution was added, and the absorbance at 450 nm was measured using a microplate reader. Based on the calibration curve prepared using the dilution series of the calibrator, the IL-1β concentration was calculated from the absorbance of the sample.

  In addition, human IL-6 ELISA kit (manufactured by BioLegend) was used for IL-6 concentration measurement, and human TNF-α ELISA kit (manufactured by BioLegend) was used for TNF-α concentration measurement. The procedure was the same as that for measuring 1β.

2. Results FIG. 1 shows the results of detection of ameloblastin by immunochromatography. Ameloblastin was detected in all test groups. In comparison between samples, a tendency to enhance the band expression pattern was observed in patients with early periodontitis compared with healthy individuals or patients with chronic periodontitis. Moreover, the measurement result of the inflammatory cytokine density | concentration by ELISA method is shown in FIG. Increased concentrations of IL-1β and IL-6 were observed in saliva samples from patients with chronic periodontitis. TNF-α was not detectable in all experimental groups.

  The present invention can be used in the field of manufacturing a test agent for periodontal disease.

Claims (11)

  A method for examining periodontal disease, comprising a step of detecting or measuring ameloblastin in a sample collected from the oral cavity of a subject.   The method for examining periodontal disease according to claim 1, wherein the detection or measurement is performed by an immunoassay using an antibody or a fragment thereof that specifically binds to ameloblastin.   The method for examining periodontal disease according to claim 1, further comprising a step of detecting or measuring at least one inflammatory cytokine of IL-1β or IL-6 in the sample.   The tooth according to claim 3, wherein the detection or measurement is performed by an immunoassay using an antibody or a fragment thereof that specifically binds to at least one inflammatory cytokine of IL-1β or IL-6. Inspection method of peri-disease.   The method for examining periodontal disease according to any one of claims 1 to 4, wherein the sample is saliva.   The method for examining periodontal disease according to any one of claims 1 to 4, wherein the sample is gingival crevicular fluid.   The method for examining periodontal disease according to any one of claims 1 to 4, wherein the sample is serum derived from gingival blood.   The periodontal disease inspection method according to any one of claims 1 to 7, wherein the inspection is to determine the presence and / or progress of the onset of periodontal disease.   A test kit for periodontal disease comprising an antibody or a fragment thereof that specifically binds to ameloblastin.   The test kit for periodontal disease according to claim 9, wherein the antibody or a fragment thereof that specifically binds to ameloblastin is held in an immunochromatographic test strip.   The test kit for periodontal disease according to claim 9 or 10, further comprising an antibody or a fragment thereof that specifically binds to at least one inflammatory cytokine of IL-1β or IL-6.

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