JP2016031306A - Inspection method of periodontal disease and inspection diagnostic kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inspection method and an inspection diagnostic kit thereof capable of simply, quickly, and accurately determining the presence or absence or the progress state of periodontal disease.SOLUTION: An inspection method of periodontal disease includes: an inspection method of periodontal disease which detects or measures calprotectin contained in a specimen of the gingival crevicular fluid or the saliva by immunoassay; a technique of collecting the specimen of the gingival crevicular fluid or the saliva; and a technique of detecting or measuring calprotectin contained in the specimen of the gingival crevicular fluid or the saliva by immunoassay using immunochromatography. The inspection method of periodontal disease also includes: an inspection diagnostic kit of periodontal disease which detects or measures calprotectin.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for testing periodontal disease and a test diagnostic kit. More specifically, by detecting or measuring calprotectin contained in the gingival crevicular fluid (GCF) or saliva specimen by immunoassay, the presence or absence and progression of periodontal disease can be easily chairside. The present invention relates to an inspection method and an inspection diagnostic kit that can determine quickly and accurately.

Periodontal disease refers to all diseases that occur in the periodontal tissue consisting of gingiva, cementum, periodontal ligament and alveolar bone. It is an inflammatory disease mainly caused by oral bacteria in plaque. It is roughly divided into periodontitis. “Gingivitis” refers to a case where inflammation is confined to the gingiva, and “periodontitis” refers to a case where the inflamed site extends beyond the gingiva and the periodontal ligament and alveolar bone are damaged or destroyed.
In the case of gingivitis, even if the periodontal tissue is inflamed, the inflammation has not spread to the periodontal ligament or alveolar bone, and therefore it can be cured by removing the cause plaque and cleaning it. However, if the symptom progresses from gingivitis to periodontitis, especially in the case of severe periodontitis where the teeth become wobbled or the periodontal ligament and alveolar bone are damaged or destroyed, cleaning the oral cavity Plaque removal becomes difficult and it becomes difficult to return to the original health condition.

In recent years, periodontal disease has been positioned as a lifestyle-related disease and has spread to about 80% of adults in Japan, and is the leading cause of tooth loss in adults. In addition, recent research suggests a relationship with serious systemic diseases (diabetes, arteriosclerosis, heart disease, premature birth, low birth weight infants, cerebrovascular disease, etc.), and it has become clear that this is a risk factor. Yes.
In dental caries and periodontal disease, which are said to be the two major dentistry diseases, periodontal disease does not tend to decrease because it is an intractable disease, and countermeasures are important and urgent in preventive medicine. It is considered as an issue. For this reason, it is important to detect periodontal disease as early as possible and treat it immediately to prevent it from becoming severe.

At present, various clinical parameters are used to examine periodontal disease at general dental clinics, etc., and to determine the effects after periodontal treatment. Periodontal pocket examination, tooth mobility examination, and X-ray inspection. A standard diagnostic method is morphometric probing depth (PD). This is a method for evaluating the degree of destruction of periodontal tissue by inserting an instrument called a probe into the periodontal pocket and measuring its depth.

However, the examination with the probe knows the degree to which the periodontal tissue has already been destroyed due to periodontal disease, and the degree of disease activity according to the degree of inflammation at the time of examination cannot be known. Furthermore, it has been pointed out that measurement requires skill, damages to periodontal tissue, and judgment is subjective due to visual evaluation, and uncertainty and variation occur.

Periodontal disease is generally referred to as a chronic disease, but it is known that the destruction of periodontal tissue is not always at a constant rate and repeats an active period and a rest period. During the active period, the degree of disease activity is large, and attachment loss and alveolar bone resorption rapidly progress in a short period of time, but progress seems to stop in the rest period. However, a method for diagnosing these active periods and rest periods with a single test has not yet been established.

For this reason, in addition to the above-mentioned diagnostic methods, many methods related to bacteriological and biochemical tests have been proposed as objective indicators for predicting early diagnosis and progression of periodontal diseases, and non-invasive tests that do not use probes. Attempts have been made for clinical application.

In bacteriological examinations, periodontal disease is an infection in which periodontal pathogenic bacteria are largely involved in its onset and progression, so identifying and diagnosing risk factors by identifying the bacterial flora and bacterial count, Necessary information such as determination of therapeutic effects may be obtained. For example, a method for specifically detecting ALP (alkaline phosphatase), an enzyme released by periodontopathic bacteria contained in saliva, and peptidase, a trypsin-like enzyme, has been devised and put into practical use. (See Patent Document 1 and Non-Patent Document 1). However, it has been pointed out as a problem that grasping of periodontal disease bacteria by these methods does not necessarily reflect the current state of periodontal tissue.

Many biochemical methods have been proposed for use in examinations and diagnoses as specific periodontal disease markers, such as exudates that leak into the gingival crevice (gingival crevicular fluid) and saliva. Yes. For example, a diagnostic kit that utilizes the fact that MMP (matrix metalloprotease), a type of protease that degrades collagen, which is the main component of gingival connective tissue, is related to the progress of periodontal disease (See Patent Document 2).

Furthermore, γ-GTP (γ-glutamyltransferase), an enzyme in gingival crevicular fluid, β-defensin, an antibacterial peptide, and calprotectin, a calcium-binding protein, are detected and measured for periodontal disease. A method for determining the presence or absence has also been proposed (see Patent Documents 3 to 4 and Non-Patent Document 2). However, the measurement by these methods uses an expensive device, the sample processing operation is complicated, it takes a long time to obtain the measurement result, and it is not suitable for the chair side. There were problems such as low reliability of the inspection, and it was not satisfactory as a diagnostic method.

JP-A-8-75729 Japanese Patent No. 3083325 Japanese Patent No. 35299283, JP 2009-145220 A

Hyoron Publishing Co., Ltd. “Saliva Test Handbook for Dentists and Dental Hygienists” 2008: 62-63, 68-69 Japanese Journal of Periodontology, Vol. 49 (2007), No. 1, P 13-19

Accordingly, an object of the present invention is to provide an inspection method and an inspection diagnostic kit that can easily and quickly determine the presence or absence and progression of periodontal disease.

As a result of intensive studies in view of the problems of the prior art, the present inventor has compared calprotectin, a calcium binding protein contained in gingival crevicular fluid (GCF) or saliva, with other periodontal disease markers. Focusing on the fact that the absolute amount is large and easy to detect, and that the concentration of calprotectin in gingival crevicular fluid or saliva is highly correlated with the degree of periodontal disease, By using calprotectin in saliva as a periodontal disease marker and detecting or measuring calprotectin in a specimen by immunological measurement, the presence or content of calprotectin can be judged visually or numerically with a measuring device The present invention was completed by finding that the presence / absence and progress of periodontal disease can be easily and quickly determined accurately on the chair side.

Therefore, the present invention is solved by the following means.
(1) A method for testing periodontal disease, wherein calprotectin contained in a gingival crevicular fluid or saliva specimen is detected or measured by an immunoassay.
(2) a method for collecting a specimen of gingival crevicular fluid or saliva and a technique for detecting or measuring calprotectin contained in a specimen of gingival crevicular fluid or saliva by an immunoassay, Diagnostic or diagnostic kit for periodontal disease characterized by detecting or measuring.
(3) The periodontal disease test method and test diagnostic kit according to (1) or (2) above, wherein the immunological assay is an immunochromatography method characterized by detecting calprotectin.

According to the periodontal disease test method and test diagnostic kit of the present invention, the presence or absence and progression of periodontal disease can be easily and quickly determined accurately without damaging the periodontal tissue. It can be suitably used for chair side, group examination, self-diagnosis and the like.

Plan view of immunochromatography device 1 is a longitudinal section of the immunochromatography apparatus shown in FIG.

1 Immunochromatography device 2 Sample addition site (“sample pad”)
3 Labeling substance holding site (“conjugate pad”)
4 Chromatographic membrane carrier 4-1 Judgment site ("Judgment line")
4-2 Control site ("Control line")
4A Supplementary site 5 Absorption site ("Absorption pad")
6 Adhesive sheet ("lining adhesive sheet")

Hereinafter, the present invention will be described in detail. In the method for examining periodontal disease of the present invention, calprotectin contained in a gingival crevicular fluid or saliva specimen is detected or measured by an immunoassay to determine the presence or absence of periodontal disease or its progress. A diagnostic kit based on the test method is a method for collecting a specimen of gingival crevicular fluid or saliva, and a technique for detecting or measuring calprotectin contained in a specimen of gingival crevicular fluid or saliva. It is easy to determine quickly and accurately.

  Calprotectin according to the present invention is a heterodimer complex in which S100A8 and S100A9 (also known as MRP8 and MRP14) known as a series of calcium-binding proteins are paired one molecule at a time. It is a mediator of transmission. Calprotectin is known to be abundant in neutrophils, monocytes, and macrophages, and is also known to be involved in inflammatory effects by binding to vascular endothelial cells. Calprotectin is also used as a diagnostic marker for acute or homeostatic inflammation. Calprotectin is expressed in the plasma of appendicitis patients, in the stool of patients with inflammatory bowel disease, and in the joint bone fluid of patients with rheumatoid arthritis. Levels have been reported to rise.

  Calprotectin is also known to be expressed in normal mucosal epithelium and gingiva in the oral cavity. In the present invention, human gingival crevicular fluid or saliva is used as a specimen, and calprotectin in the specimen is used. By detecting or measuring the concentration of protectin, the presence or absence of periodontal disease or its progress is determined.

  When the gingival crevicular fluid is used as a specimen, the effusion from the gingival pocket in the oral cavity and the exudate from the tissue surrounding the dental implant are collected and used. As the collecting means, a known method may be used, and a sample collecting support having a property of absorbing liquid can be used. For example, a filter paper piece such as Perio paper or a twisted thread like paper point can be used. As a collecting method, a support is attached or inserted near the entrance of the gingival pocket, and the exudate is soaked and collected. The collection time is preferably 10 to 60 seconds, more preferably 10 to 30 seconds.

The amount of gingival crevicular fluid collected is not particularly limited as long as it is within the range in which the effects of the present invention can be obtained, but specifically 0.1 to 2.5 μL is preferable, and more preferably 0.5 to 2 0.0 μL. Calprotectin can be extracted from the support in which the gingival crevicular fluid has soaked using an extraction solvent, and subjected to measurement. Examples of the extraction solvent include phosphate buffers such as water, physiological saline, and PBS,
Good buffers such as HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) can be used. The extraction conditions are as follows. Place the support soaked with gingival crevicular fluid into a microtube or a centrifuge tube, and place 50 μL to 2.0 mL of extraction solvent in it, or leave it still, or stir using a high-speed stirrer. The calprotectin is extracted by shaking with a shaker, and this can be used as a sample for measurement.

  In the case of using saliva as a specimen, the collecting means may be performed by a known method, a method of discharging resting saliva accumulated in the mouth in a resting state into a container, and stimulating saliva accumulated in the mouth by chewing paraffin gum or the like. A method of discharging into a container, a commercially available saliva collection kit, or the like can be used. The amount of saliva to be collected is preferably 0.2 to 2.0 mL, and more preferably 0.5 to 1.0 mL.

  As the saliva to be used for the specimen, either the collected saliva can be used as it is, or any saliva supernatant obtained by centrifuging saliva to remove insoluble matters can be used. Although the saliva supernatant may be used as it is as a specimen, it is preferable to use the saliva supernatant diluted with a solvent because a more accurate measurement result can be obtained. Examples of the solvent for diluting saliva include distilled water, physiological saline, and phosphate buffer. The dilution rate of the saliva supernatant is preferably 2 to 6 times the supernatant obtained by centrifugation, and if the dilution rate is less than 2 times, the measurement result may be affected by saliva components, If the dilution factor exceeds 6 times, the calprotectin concentration may not reach the detection limit.

In the present invention, calprotectin contained in the gingival crevicular fluid or saliva specimen is determined by an immunological assay. As the immunological measurement method, a commonly used method can be used. For example, enzyme-labeled immunoassay (ELISA), radioimmunoassay (RIA), aggregation method, immunochromatography method, turbidimetric method, turbidity measurement method, Western blot method and the like can be mentioned. In the present invention, the immunochromatography method is most preferably used because accurate measurement results can be obtained easily and quickly on the chair side, and a special and expensive measurement device is not required.

The immunochromatography apparatus for detecting calprotectin used in the immunochromatography method is an immunological measurement method that applies capillary action, which is a property in which a test substance flows slowly on a membrane carrier while dissolving the reagent. Can be mentioned. The immunochromatography method is not particularly limited, but is an immunochromatography method which is a sandwich immunoassay method in which the antigen is sandwiched between a first antibody and a second antibody against the calprotectin antigen. The device is most preferably used.

Further, the immunochromatography apparatus comprises a chromatography membrane carrier provided with a capture site (determination line) formed by previously collecting and fixing a first antibody against a calprotectin antigen as a test sample in a line at a predetermined position. A mixture of a labeled substance modified with a second antibody against the calprotectin antigen and a predetermined amount of the test sample is developed on the chromatography membrane carrier toward the capture site, The immunocomplex of the calprotectin antigen and the labeling substance contained in is captured at the capture site and colored in a line shape, and the color protection state is present visually or using appropriate equipment. It is characterized by measuring it qualitatively and quantitatively.

The immunochromatography apparatus is not particularly limited as long as it is an apparatus that can be used for a normal immunochromatography method. For example, FIG. 1 schematically shows a plan view of an immunochromatography apparatus. FIG. 2 schematically shows a longitudinal section of the immunochromatography apparatus shown in FIG.

The specifications of the immunochromatography apparatus used in the present invention will be described in detail below.
The immunochromatography apparatus 1 includes a sample addition site 2 (“sample pad”) and a labeling substance holding site (“conjugate pad”) 3 from upstream to downstream in the development direction (indicated by arrow X in FIG. 1). The chromatographic membrane carrier 4 (“membrane”) and the absorption site 5 (“absorption pad”) are arranged in this order on the pressure-sensitive adhesive sheet 6 (“backing pressure-sensitive adhesive sheet”). Further, the chromatographic membrane carrier 4 has a supplementary site 4A and a determination site (“determination line”) 4-1, which is a region where an antibody or antigen that specifically binds to the analyte is immobilized. . The determination line is usually one, but two or more can be provided if desired. Furthermore, it can have a control site ("control line") 4-2, which is a region where a control antibody or antigen is immobilized.
The structure, specification, and mode of each part are as follows.

The sample addition site 2 (“sample pad”) is composed of a porous sheet having such a property that the sample is rapidly absorbed but the holding force is weak and the sample quickly moves to the reaction site. . As the porous sheet, for example, glass fiber, porous synthetic resin sheet or film such as porous polyethylene or polypropylene, cellulose paper such as filter paper and cotton cloth, or woven or non-woven fabric can be used. In the present invention, in order to further suppress non-specific reactions, the sample addition site 2 is preliminarily impregnated with and loaded with a reagent composition containing a protein such as albumin, a buffer solution, a surfactant, and the like. Can do.

The labeling substance holding site 3 (“conjugate pad”) can be prepared by preparing a suspension containing the labeling substance, applying the suspension to an appropriate absorbent pad, and then drying it. it can. Examples of the labeling substance holding part include glass fibers, cellulose, and a porous material such as polyethylene or polypropylene subjected to hydrophilic surface treatment.

The labeling substance is not particularly limited, and examples thereof include a color labeling substance, an enzyme labeling substance, a radiation labeling substance, and the like, but a coloration labeling substance is most preferably used because of the rapid measurement. Examples of the color labeling substance include metal colloid and colored latex, and typical examples of the metal colloid include platinum colloid and gold colloid. The size of the metal colloid particles is not particularly limited, and usually a diameter of about 3 to 100 nm is preferable. Further, representative examples of the colored latex include synthetic latex or natural latex colored with pigments such as red and blue. The size of the colored latex is selected from a diameter of several nm to several hundred nm. These colored labeling substances can be produced by known methods, but commercially available products can also be used or processed. Further, a fluorescent label or the like can be used.

The chromatography membrane carrier 4 (“membrane”) is not particularly limited as long as it has an action of causing capillary action and can absorb and move a test substance or the like. For example, it is selected from the group consisting of nitrocellulose, cellulose acetate, nylon, polyethersulfone, polyvinyl alcohol, polyester, glass fiber, polyolefin, cellulose, and an artificial polymer composed of these mixed fibers. Most suitable for the invention.

  Absorption site 5 (“absorption pad”) is a site where the added sample is physically absorbed by chromatographic movement and absorbs and removes unreacted labeling substances from the chromatographic membrane carrier. Cellulose filter paper, A water-absorbing material such as nonwoven fabric or cloth is used.

  The calprotectin antibody used in the present invention is not particularly limited, and examples thereof include polyclonal antibodies and monoclonal antibodies commonly used in various immunoassays. For example, anti-human calprotectin monoclonal antibody, anti-human calprotectin polyclonal antibody, etc. are mentioned. Among them, anti-human calprotectin monoclonal antibody has the most excellent reaction specificity with human calprotectin antigen. preferable.

  The developing solution used in the present invention is not particularly limited as long as it is a developing solution that can be used in a normal immunochromatography method. The developing solution used in the present invention is a liquid constituting a mobile phase, and moves together with a test sample and a labeling substance on a chromatography membrane carrier that is a stationary phase. Further, the developing solution of the present invention can be used not only as a developing solution but also as a sample diluent, and the diluted solution used for dilution of the sample can be used as it is as a developing solution in the immunochromatography method.

As the developing solution, for example, one containing a nonionic surfactant is used, and a polyoxyethylene surfactant can be preferably used. Preferred examples of the polyoxyethylene surfactant include polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acid ester (trade name “Tween” series), polyoxyethylene pt-octylphenyl ether (trade name “Triton”). Series), polyoxyethylene pt-nonylphenyl ether (trade name "Triton N" series) and the like. The content of the above-described nonionic surfactant is not particularly limited, but is in the range of 0.01 to 10% by weight, preferably 0.05 to 5% by weight, based on the total weight of the composition. Moreover, the said nonionic surfactant can also be used individually or in mixture of 2 or more types.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to this Example at all.

(Reference Example 1) Preparation of calprotectin detection immunochromatography device (1) Preparation of anti-calprotectin antibody-modified gold colloid 50 mM KH2PO4 buffer in 10 mL of colloidal gold solution (WRGH1-50NM, Wine Red Chemical) with a diameter of 50 nm (Colour 7.0) 1 mL of a gold colloid solution to which 1 mL was added was added 1 mL of a 40 μg / mL anti-calprotectin monoclonal antibody (Company) solution and stirred. After standing for 15 minutes, 1% polyethylene glycol (PEG Mw. 20000, Wako Pure Chemical Industries, Ltd.) was added, and then 1.0 mL of 10% bovine serum albumin (BSA Fraction V, SIGMA) aqueous solution was added and stirred. After centrifuging this solution at 7500 rpm, 4 ° C. for 30 minutes, the supernatant was removed, leaving about 1 mL, and the gold colloid was redispersed with an ultrasonic cleaner. The colloidal gold labeled antibody was obtained by removing the supernatant. The colloidal gold labeled antibody was suspended in a 50 mM Tris-HCl buffer (pH 7.4) containing 10% sucrose, 1% BSA, 0.5% Triton-X100 to obtain a colloidal gold labeled antibody solution.

(2) Preparation of colloidal gold labeled antibody retention site (conjugation pad) Colloidal labeled antibody solution prepared in (1) to glass fiber pad (Glass Fiber Conjugate Pad, Millipore) cut to 5 mm x 15 mm 40.0 μl was impregnated and dried overnight under reduced pressure to obtain a colloidal gold labeled antibody holding site.

(3) Preparation of antibody-immobilized membrane (chromatography membrane carrier)
An antibody was immobilized on a nitrocellulose membrane (HiFlow Plus HF180, Merck Millipore) cut to 25 mm × 200 mm by the following method to prepare an antibody-immobilized membrane. Using a coating machine, fix the anti-calprotectin monoclonal antibody solution for immobilization prepared at 1.5 μg / mL with the long side of the membrane down, and form a line with a width of about 1 mm at a position 7 mm from the bottom. It was applied to. Similarly, a control anti-mouse IgG antibody solution prepared so as to be 0.5 mg / mL was applied in a line at a position of 13 mm from the bottom, and dried at room temperature. Then, 500 mL of blocking solution (1.0 w% BSA, Merck Millipore) was immersed in the antibody-immobilized membrane in a liquid bath and left as it was for 15 minutes. The membrane was removed from the solution and dried overnight at room temperature to obtain an antibody-immobilized membrane.

(4) Preparation of test immunochromatography apparatus The antibody-immobilized membrane prepared in (3) was attached to the backing adhesive sheet. Attach the gold colloid-labeled antibody retention site prepared in (2) so that it overlaps about 1 mm below the antibody-immobilized membrane, and further cut it into a sample addition pad (20 mm x 150 mm) so that it overlaps about 2 mm. A glass fiber pad, Merck Millipore) was laminated and pasted. Further, an absorption pad (cellulose membrane cut to 80 mm × 150 mm, Merck Millipore) was overlaid on the upper side of the antibody-immobilized membrane so as to overlap by about 6 mm. These laminated members are cut with a cutter so that the long side is 4 mm wide and parallel to the short side to produce a 5 mm x 55 mm immunochromatographic strip, which is then placed in a plastic case for testing. An immunochromatographic apparatus was used.

<Example 1> Periodontal disease test by calprotectin detection in gingival crevicular fluid (GCF) (1) GCF collection and pretreatment 10 periodontal disease patients diagnosed with chronic periodontitis and 5 healthy subjects A total of 15 subjects were taken as subjects and collected from 1 to 3 sites per subject, and a total of 30 GCF specimens were collected. The collection method is as follows. Periodontal disease patients are visually inspected, and Periopaper (Oraflow) is applied to the gingival sulcus at sites where inflammation is observed and suspected to be affected, and in healthy individuals where inflammation is not observed and healthy. The GCF specimen was collected by inserting for 30 seconds, immersed in a microcentrifuge tube containing 1.0 mL of phosphate buffer, and allowed to stand for 5 minutes. Thereafter, the mixture was shaken for 5 minutes using a high-speed stirrer to perform an extraction operation, and 50 μL was taken out as a specimen.
(2) Calprotectin detection by immunochromatography method The 50 μLGCF sample obtained in (1) above was diluted 4-fold with 150 μL of a 0.10% nonionic surfactant Tween20 solution as a drop sample, Using a micropipette, 100 μL was dropped onto the sample addition pad site of the test immunochromatography device prepared in Reference Example 1 for chromatographic development. Leave at room temperature for 10 minutes and observe with naked eyes the amount captured in the judgment line. “−” Indicates that the red line is hardly visible, “+” indicates that the red line is slightly identifiable, and “+” indicates that it is clear. Is “++”, and “++” is the one that can be confirmed more strongly and clearly. Furthermore, the color intensity of the judgment line was quantified using an immunochromatographic reader (DiaScan, manufactured by Otsuka Electronics). Table 1 shows the test results.

Comparative Example 1 Periodontal Tissue Examination Using Visual Inspection and Probe Periodontal tissue examination was performed on 30 sites from which specimens were collected in Example 1, and the following clinical parameters were examined. Using a probe (periodontal pocket measuring device), the depth of periodontal pocket (Probing pocket depth, PPD) and bleeding reaction during probing (Bleeding on probing, BOP) were confirmed visually. Were recorded as positive “+”, and those without were recorded as negative “−”. In addition, the gingival inflammation index (Gingival index, GI), which is the degree of inflammation of the gingiva, was confirmed by visual inspection. No inflammation was observed in the gingiva, normal condition was "0", mild gingival inflammation was "1", moderate BOP was recorded as “2” for gingival inflammation, and spontaneous bleeding was recorded as “3” for severe inflammation. Table 1 shows the test results.

From the results of Example 1 and the results of Comparative Example 1 of the conventional method, comprehensively diagnose the site from which the sample was collected, the normal and healthy site is “normal”, and the affected site where inflammation is observed is: Classified as “gingivitis” or “periodontitis”. Furthermore, the inflammation state of the periodontal tissue was determined according to the criteria shown in Table 2.

From the result of the immunochromatography method of Example 1 in the present invention, all GCF samples collected from the site visually determined as the affected site of the periodontal disease affected group showed a positive value of “+” or more. However, the GCF samples visually judged as healthy sites in the healthy group were negative for “−” except for 2 of 11 samples. In the immunochromatography reader, the average value of GCF samples collected from periodontal patients was 166.9, but the average value of GCF samples from healthy subjects was significantly low at 35.8. Therefore, it was found that by using the method of the present invention, by detecting and measuring calprotectin in a GCF sample by immunochromatography, the site from which the sample was collected can be easily distinguished from an affected site and a healthy site.

Subsequently, the inflammation evaluation by the immunochromatography method of Example 1 in the present invention was compared with the inflammation evaluation by the presence or absence of bleeding reaction by BOP of the conventional method. In addition, as a reference for inflammation evaluation of the immunochromatography method of Example 1, an immunochromatography reader value was set as 50 as a cut-off value, 50 or less being “no inflammation” and 51 or more being “inflammation”. The results are shown in Table 3. As a result, the BOP of the conventional method of Comparative Example 1 showed positive bleeding reaction at 17 sites and was determined to be positive. However, according to the above criteria of Example 1, calprotectin was detected at 21 sites and determined as “inflamed”. It was. That is, the four sites that were negative in Comparative Example 1 may have been inflamed but could not be detected. Sample No. 24 and Sample No. 27 collected as normal sites of healthy individuals have an immunochromatography reader value of 50 or higher, are higher than those of other healthy sites, have a small PPD but have mild inflammation. There was a possibility that it was comprehensively diagnosed as the clinical parameter of Comparative Example 1 and classified as gingivitis. Therefore, it has been found that the use of the method of the present invention enables more precise detection of inflammation occurring in the periodontal tissue that could not be detected by the conventional method.

Further, when comparing both sites of specimen No. 8 and specimen No. 13, the PPD was 7 mm and the BOP was also positive, but the immunochromatography reader value of Example 1 was 89 for specimen No. 8 and specimen no. .13 is significantly different from 245, suggesting that the site of specimen No. 13 has a large degree of inflammation. In fact, in the visual GI, specimen No. 13 showed GI = 3 and more severe inflammation, which was consistent with clinical findings. Therefore, it was found that by using the method of the present invention, the inflammation of the periodontal tissue can be detected biochemically, and the difference in the degree of inflammation can also be determined.

That is, the periodontal tissue examination by the immunochromatography method of the present invention may be able to detect signs of inflammation that have been overlooked by the conventional examination method using a probe, and can clearly grasp the degree of inflammation. It has been shown that it can be a clinically effective testing tool.

<Example 2> Periodontal disease screening test by detection of calprotectin in saliva (1) Periodontal disease patients who were diagnosed with chronic periodontitis by collecting saliva and performing pretreatment periodontal tissue examination, and healthy A total of 23 saliva samples were collected from each subject. The collection method is as follows. Each subject was allowed to chew paraffin gum for 3 minutes, and then saliva was collected with a commercially available saliva collection tube (Salstat). After the saliva collection tube containing saliva was centrifuged (3000 rpm, 2 minutes), 200 μL of the supernatant was taken out and used as a specimen.
(2) Calprotectin detection by immunochromatography method The 50 μL saliva specimen obtained in (1) above was diluted 4-fold with 150 μL of 0.10% nonionic surfactant Tween20 solution as a drop sample, 100 microliters was dripped at the sample addition pad site | part of the test immunochromatography apparatus produced in the said reference example 1 using the micropipette, and chromatographic development was carried out. Leave at room temperature for 10 minutes and observe with naked eyes the amount captured in the judgment line. “−” Indicates that the red line is hardly visible, “+” indicates that the red line is slightly identifiable, and “+” indicates that it is clear. Is “++”, and “++” is the one that can be confirmed more strongly and clearly. Furthermore, the color intensity of the judgment line was quantified using an immunochromatographic reader (DiaScan, manufactured by Otsuka Electronics). Table 5 shows the test results.

<Comparative Example 2> For periodontal disease test by CPI (Community Periodontal Index, regional periodontal disease index) in Example 2, for a total of 20 patients including 12 periodontal disease patients and 8 healthy subjects, A CPI test for periodontal disease was performed using a WHO probe developed for regional periodontal disease index measurement, and recording was performed according to the CPI code shown in Table 4. The inspection method is as follows. The oral cavity was divided into six blocks, upper and lower jaw and anterior molar, and the highest CPI code was selected for each block. Regarding the detailed examination site, the anterior tooth block is the upper right central incisor and the lower left central incisor, and the molar block is the first molar and the second molar. Table 5 shows the test results.

From the results of the immunochromatography method of Example 2 in the present invention, all saliva samples collected from 12 subjects of periodontal disease patients showed a positive value of “+” or more. However, saliva samples collected from healthy subjects were negative for “−” except for 2 of 8 samples. In the immunochromatography reader, the average value of saliva samples collected from patients with periodontal disease was 129.3, but the average value of GCF samples from healthy subjects was significantly low at 27.9. Therefore, it was found that by using the method of the present invention, the periodontal disease patient group and the healthy subject group can be generally discriminated by detecting and measuring calprotectin in the saliva specimen by immunochromatography.

Subsequently, the examination by the immunochromatography method of Example 2 in the present invention and the periodontal disease examination by CPI of Comparative Example 2 were compared. The “IC level” shown in Table 5 means that the immunochromatography reader value of Example 2 is 50 or less, level 0, 51 to 100 is level 1, 101 to 150 is level 2, 151 to 200 is level 3, and 201 or more. Level 4 was set at 5 levels and recorded. As a result of obtaining Spearman's rank correlation coefficient for the IC level and the values of CPI codes of Comparative Example 2 from 0 to 4, r = 0.950 and a significant correlation was obtained between the two determination methods.

That is, the periodontal disease test using saliva as a specimen by the immunochromatography method of the present invention shown in Example 2 showed a high correlation as compared with the periodontal disease test by CPI performed in the conventional mass screening and the like. Therefore, it can be an alternative method for periodontal disease testing by CPI. Furthermore, the test method of the present invention enables sample collection in a simple and non-invasive manner compared to the conventional method using a probe, and there is a possibility of periodontal disease particularly in a health examination such as a mass examination. It is effective as a screening test method for selecting a certain examinee and receiving a precise periodontal tissue examination by a dentist.

Claims (3)

  A method for examining periodontal disease, wherein calprotectin contained in a gingival crevicular fluid or saliva specimen is detected or measured by an immunoassay.   Including a method of collecting a specimen of gingival crevicular fluid or saliva and a method of detecting or measuring calprotectin contained in a specimen of gingival crevicular fluid or saliva by an immunoassay, and detecting or protecting calprotectin A diagnostic kit for periodontal disease characterized by measuring. The periodontal disease test method and test diagnostic kit according to claim 1 or 2, wherein the immunological measurement method is an immunochromatography method that detects calprotectin.