WO2002079780A1

WO2002079780A1 - Method of examining periodontal disease

Info

Publication number: WO2002079780A1
Application number: PCT/JP2002/003077
Authority: WO
Inventors: Naoyuki Sugano
Original assignee: Nihon University
Priority date: 2001-03-28
Filing date: 2002-03-28
Publication date: 2002-10-10
Also published as: JPWO2002079780A1

Abstract

A method of examining periodontal disease which involves the step of subjecting a sample collected from the oral cavity of a subject to an immunoassay with the use of an anti-8-hydroxydeoxyguanosine antibody and thus quantifying 8-hydroxydeoxyguanosine. Thus, periodontal disease can be conveniently examined.

Description

Technical analysis of periodontal disease

The present invention relates to the detection of periodontal disease. Tall.

Relationship between high health status and maintenance of oral malaise s Recently attracted attention. That is, it has been reported that a decrease in oral flare ability is associated with the development of various diseases. In addition, it has been reported that the decrease in 3¾1 ability is associated with senile dementia. Since periodontal disease is a major cause of tooth loss, prevention of periodontal disease is important to maintain oral malaise. Since periodontal disease progresses over a long period of time, it is desirable to find out periodontal disease on the shelf by mass examination and treat it.

¾¾, Periodontal disease is diagnosed by periodontal blowing. This is called a probe inserted into a periodontal pocket and measuring its depth to tell the damage of the periodontal fiber. Therefore, a highly specialized sickle is required for the diagnosis, and it is time-consuming, so it is difficult to perform a mass test. In addition, the probing method is used to know the inflammation (depth of the periodontal pocket) caused by periodontal disease, and it is not possible to know the pathological condition at the time of the examination and the inflamed flora.

It has also been proposed to diagnose periodontal disease by measuring substances secreted with inflammation. For example, the relationship between aspartate aminotransferase and periodontal disease has been reported. However, accurate measurement was not possible with this method due to the presence of inhibitors in the mouth. In addition, regarding other inflammation-related substances, the relationship with periodontal disease has been clarified, and it is difficult to more accurately measure inhibitors. Diagnosis by the periodontal probing method was still performed without being adopted for diagnosis. The inventor of the present invention has proposed that 8-hydroxydoxyguanosine (hereinafter, referred to as 8—OH dG) is related to periodontal disease, and it is possible to perform measurement without problems such as an inhibitory substance by immunoassay, and The present inventors have found that measured values are significantly related to the disease state of periodontal disease, and I made it true.

Therefore, an object of the present invention is to provide a method for periodontal disease. Disclosure of the invention

First, the present invention relates to the following:

(1) Periodontal disease analysis including the step of performing an immunoassay using an anti-8-hydroxydeoxyguanosine ^ body on a sample collected from the subject's mouth to quantify 8-hydroxydeoxyguanosine Leave.

(2) Knitting difficulty is 赚 The ^^ method of (1).

(3) Fujimi is on a fine night, and the saliva is subjected to centrifugal separation before the return to Immortality (1).

(4) The detection method according to (1), in which the immorality is performed by a competitive method.

(5) The detection method according to (2), wherein the imnoassay is performed by a competition method.

(6) The detection of (3), in which the disgusting imnoassy is conducted by a competitive law.

(7) Acknowledgment Imnoassy conducted by the sandwich method (1)

(8) Inspection of (2), in which the disgusting imnoassy is performed by the sandwich method.

(9) The detection of (3), in which the disgusting imnoassy is performed by the sandwich method.

In addition, the present invention relates to a labeled anti-8-hydroxydeoxyguanosine ^: body, which is used in a method for detecting periodontal disease based on a quantitative amount of 8-hydroxydeoxyguanosine collected from the oral cavity. Intellectual 8-hydroxydoxyguanosine ^: Immobilized or immobilized on a solid phase selected from the group consisting of body, labeled 8-hydroxydoxyguanosine and 8-hydroxydoxyguanosine For kits or assays containing at least one or more reagents that have not been converted.

Further, the present invention provides a method for binding 8-hydroxydeoxyguanosine in ^ collected from the oral cavity to transliteration using an immunospecific J¾S with anti-8-hydroxydoxyguanosine. A kit or detector for testing periodontal disease by quantifying 8-hydroxydeoxyguanosine, comprising a means for measuring or visualizing the activity of the label.

As used herein, “quantitative” refers to not only the age at which the exact amount is measured, but also the approximate amount. 彻 Including making a decision.

In addition, the test method of the present invention is particularly useful for examination of periodontal disease in the nursing period including humans. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing 8-OH dG marrow of healthy subjects and patients with periodontal disease.

FIG. 2 is a daraf showing 8-OHdG concentrations before and after treatment of periodontal disease patients. BEST MODE FOR CARRYING OUT THE INVENTION

(8-hydroxydeoxyxiguanosine antibody)

The anti-8-hydroxydeoxyguanosine ^ used in the present invention (hereinafter referred to as an anti-8-OH dG antibody) is a monoclonal antibody or a polyclonal antibody. This is an antibody.

The polyclonal antibody is purified from, for example, a sample obtained by associating 8-OHdG with a protein-polypeptide as an immunogen and then temporarily administering it to a warm-blooded animal and collecting the ascites, blood, etc. It was manufactured.

Purification of 8-OH dG antibody from serum is difficult to separate and refine immunoglobulins, such as salting, alcohol, isoelectric focusing, electrophoresis, iontophoresis, ultracentrifugation, It is performed by gel filtration, specific elaboration in which only antibodies are collected using an active adsorbent such as anti-glycan compound or protein A or protein G, and the bonds are dissociated to obtain the antibodies. More specifically, it can be separated by chromatography using recombinant Protein G lacking the albumin binding region as a ligand. The base support is preferably highly cross-linked Sepharose, with a purification time of, for example, 5 to 20 minutes, and a maximum flow rate of, for example, 1 to: L0m1Z minutes.

Monoclonal antibodies are commercially available (Niken Food Co., Ltd., Nippon Daidani Control Laboratory, anti-8-OHdG monoclonal antibody). It can also be obtained, for example, from Koehler and Milstein in the same manner as in [Nature, Vol. 256 (1975), p. 495]. Collect spleens or lymph nodes from animals that have been infected with 8-OHdG and produce antibodies contained in them. By fusing the vesicles to the bone cells, it is possible to obtain a monoclonal antibody-producing hybridoma. Monoclonal / antibodies are preferred for their ability to be obtained using rats and mice. For example, it is preferable to ¾λ at the age at which mice are immunized, subcutaneously, intravenously or intravenously. For example, it is common to use a spleen cell that has been immunized about 2 to 6 times at a 2-week immunization interval, and excised about 1 to 5 days, preferably about 2 to 4 days after the final immunization. Dose per mouse for example about ΰ Λ μ g or more, preferably about 10 μ _g ~300 _g. Also, before removing the spleen, perform partial removal to reduce the rise in antibody titer in the blood, and remove the limb of the individual with the increased antibody titer.

The fusion operation can be performed by the method described above; for example, the above-mentioned Koehler and Milstein removal or a method analogous thereto. Examples of bone transducing spores include NS-1, P3U1, and SP2 / 0. The preferred ratio between the number of antibody-producing vesicles and the number of bone fiber vesicles is from about 1: 1 to about 20: 1.

Examples of the fusion promoter include polyethylene glycol (PEG) Ndai virus and the like, but PEG is preferably used. The polymerization degree of PEG is usually about 1,000 to 6,000, and the concentration is used at about 10% to 80%. The fusion time is about 0.5-30 minutes, and the separation is, for example, 20-40 ° C, preferably 30-70. As an example of preferable conditions, efficient fusion can be achieved by treating PEG 6,000 with about 35 to 55% at about 4 to: L 0 min. The fused cells can be selectively grown using hypoxanthine-aminopterin-thymidine medium [HAT medium; Nature, 256, 495 (1975)] or the like.

The culture supernatant of the proliferated cells can be screened for the production of the desired antibody, but the antibody titer can be screened as follows. In other words, in this method, the presence or absence of antibody production that is exempted from the tl stage can be examined by a method such as the radioimmunoassay (RIA) method or the enzymatic immunoassay (EIA) method. Various modifications are also possible for the above method. One method using EIA is described as an example of a preferable measurement method. For example, a heron anti-mouse immunoglobulin is coupled to a carrier such as cellulose beads according to a conventional method, and the culture serum to be measured is added thereto, and mouse serum is added thereto. At 40 ° C). After that, wash the substance well, add the immunogen labeled with «, and let it stand for a certain period of time (about 4 to 40 ° C). After thoroughly washing the reaction product, add the enzyme substrate, and react for a certain period of time at a constant temperature (approximately 4 to 40 ° C). Alternatively, it can be measured by fluorescence intensity or the like. It is desirable to clone the anti- # ゥ cells that have been shown to grow in the selective medium and have anti- # ¾ activity by the marginal method. The same screening is performed on the supernatant of the cloned cells to increase the number of cells having a high antibody titer, thereby obtaining a monoclonal antibody-producing hybridoma clone.

The hybridoma thus cloned is grown in a liquid medium. Specifically, for example, a liquid medium such as RPMI-1640 [Moore, G.E., et al., Journal of American Medical 'Association (J. Am. Med. Assoc.) 199, 549 (1967)] The monoclonal antibody can be obtained from the culture medium by culturing for about 2 to 10 days, preferably for about 3 to 5 days in a medium or the like supplemented with about 0.1 to 40% bovine serum. Antibodies can be obtained by exposing the cells to the contact space of Bf! L animals, proliferating the cells, and collecting fllTK. For this purpose, for example, about 1 × 10 ⁴ to 1 × 10 ⁷ mice, preferably about 5 × 10 ⁵ to 2 × 10 ⁶ mice, such as BALB / c, which were previously inoculated with ^^ of the mice, mineral oil, etc. The hybridoma is kept in the whole body, and the liquid is collected after about 7 to 20 days, preferably about 10 to 14 days. The antibody thus produced and accumulated can be easily converted into a monoclonal immunoglobulin, for example, by fractionation, DEAE-cellulose column chromatography, etc.

(Return)

The tongue collected from the mouth of the person used in the present invention is, for example, mouse, rinsing liquid in the mouth, gingival crevicular oozing, etc., but preferably saliva. The saliva is cleaned by rinsing the mouth after a meal, for about 10 minutes or more, preferably for about 30 minutes or more, more preferably for about 60 minutes or more, for example, about 60 to 180 minutes. It is desirable to collect after fractionation.

It is also possible to measure the overall symptom of periodontal disease using the nighttime as a dip, and then conduct a detailed examination of each tooth by collecting the gingival crevicular fluid.

The saliva is preferably collected by chewing paraffin gum or the like for about 10 seconds to about 10 minutes and then discharging the saliva into the container.

Removal of interdental crevicular fluid is performed by inserting an absorbent material such as hawk paper into the periodontal pocket.

Mouth] 3 ^ of the empty rinsing liquid is preferably about 10 seconds to about 10 It is collected by chewing for a minute and then discharging into the container with water in the mouth with water. ^^ Imnoassesse may be performed immediately after taking it, but it is also possible to take a tomato and perform imnoattesse several hours to several days later. In other words, in a group test, the collection up to the return can be performed, and the subsequent analysis can be performed at a location such as a clinical laboratory center. The age at which the immunotherapy is performed after several hours to several days, preferably 140. Store in a closed container at a temperature of from C to 20 ° C, more preferably from 120 to 4 ° C.

It is preferable that the dough be analyzed after pretreatment such as centrifugation, surface activity '14 ^ 1, etc. at night.

The centrifugation is carried out at 100 to 1200 rpm, preferably at 450 to 1200 rpm, in particular at 100 to 1200 rpm for 1 to 30 minutes, The reaction is preferably performed for 10 to 30 minutes, particularly for 20 to 30 minutes, and the supernatant is used as ^ I ·.

The surfactant active agent is preferably a cationic active agent active agent, such as Tween 20.

(Imno Atssey)

The imminor assays are, for example, radio imminor arts, enzymimimorsees, and fluorescent imminor arts. The transliteration used in the Immunity assay includes a thigh isotope, chromium, a fluorescent substance, a luminescent substance, and the like, and it is preferable to use an enzyme, or a so-called close label of a colloidal gold or a dye zo /.

Enzymimnoassy can be the Sandwich method, any of the conflicts. The ELISA method is particularly preferred. As the satu, one that is stable and has a large specific activity is preferable, and peroxidase, alkaline phosphatase, -D-galactosidase, darcosoxidase and the like can be used, but peroxidase is preferred. Examples of peroxidases include those that can be used from various sources. Horseradish peroxidase (HRP) extracted from horseradish is preferred.

In addition, in the case of the present invention, if the quantitative value is equal to or more than a certain value, there is a possibility that the patient has periodontal disease, and if the quantitative value is constant, there is no risk of periodontal disease. May be included. (Several areas of judgment) · The detection method of the present invention is particularly suitable for selecting a patient with a possibility of periodontal disease in a healthy test such as a mass test, and further having a doctor diagnose the disease. It is ^ の to use it as the scripting method of. At this age, when the concentration of 8 _OH dG is, for example, 3 ng / ml or more, preferably 4 ng / m1 or more, particularly 5 ng / m1 or more, there is a possibility of periodontal disease. In addition, since the examination according to the present invention can know the state of the disease at the time, the examination method for the female doctor to know the effect of the treatment during the periodontal disease treatment It can also be performed as.

(Kit, detector)

The present invention also relates to a kit or detection for performing a superior intellectual method using an anti-8-OH dG antibody.

For example, the present invention relates to a method for detecting or treating periodontal disease by quantifying 8-hydroxydeoxyguanosine in the oral cavity, which comprises a marker or a pharmacologic 8-hydroxydoxyguanosine / ^ body, and _ And / or a kit or test containing labeled or unlabeled 8-hydroxydoxyguanosine.

A means for binding the difficult 8-OHdG collected from the oral cavity to a labeling agent by utilizing an immunospecific reaction with an anti-8-OHdG8 antibody, and a method for measuring the activity of the labeling agent It also relates to a kit or a detection device for the examination of periodontal disease by quantification of 8-OH dG, including means for visualization or visualization. Such kits can be used in health examinations, clinical examinations, medical treatments, etc., but can also be used by individuals to manage their own periodontal disease.

The means of conjugation can be selected from labeled or unlabeled anti-8-OHdG antibody, labeled or unlabeled, or not 8-OHdG, by leakage of Imnoase. Is done. The above-mentioned anti-8-OHdG anti-f and the above-mentioned 8-OHdG may be immobilized on a solid phase or non-immobilized according to the view of Immonoassembly.

The force for measuring or visualizing the activity of the labeled IJ is, for example, in the case of the labeling agent S enzyme, its substrate and terminator.

The solid phase can be, for example, polystyrene beads, plates, tubes, and the like, as well as nylon, nitrocellulose, cellulose acetate, glass, or other porous polymers. The solid phase, for example a porous solid polymer, may be in the form of a dipstick.

The kit may contain the above means, for example, anti-8-OHdG antibody, 8-OHdG, substrate, etc., as well as Nada, mi target quality and the like.

In the detection, for example, a solid phase composed of a porous material on which an anti-8-OHdG antibody is immobilized and a labeled anti-8-OHdG antibody is supported in a movable state when wet, It may be an inspection consisting of a casing in which a part of the phase is stored so as to be removed. Difficult case

Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not intended to limit the present invention.

Fiber example 1

64 patients with periodontal disease (from Kasumi to Kazu) and 10 healthy subjects were designated as ¾a. The paraffin gum was chewed for 5 minutes, and the saliva was collected. After subjecting saliva to centrifugation at 1000 Orpm for 10 minutes, the supernatant is then added to a commercially available ELISA kit for 8-OHdG (trade name: 8—OHdG Check, NOF Corporation). Pear) was used to measure the concentration of 8-OHdG. The results are shown in the graph of FIG.

As is evident from the graph, the average value of the periodontal disease group was 4.82 ng / m1, which varied up to 50 ng Zm1 depending on the disease state, but the average value of healthy ¾ was 2.3 ng / m2. 1 and at most 9. SngZml. In the periodontal disease group, the concentration of 8-OHdG was almost proportional to the progression of periodontal disease.

Difficult example 2:

In 14 patients with periodontal disease, the concentration of 8-OHdG was measured at the same time as in Example 1 after »treatment and after treatment. The results are shown in the graph of FIG. From the figure, it is clear that the treatment resulted in a decrease in the concentration S of 8-OHdG as well as an improvement in the medical condition. Availability

According to the present invention, periodontal disease can be tested in a simple manner. As a result, periodontal disease can be examined by mass screening and the like, periodontal disease can be prevented and treated, and ultimately a high health condition can be improved.

Claims

The scope of the claims

(1) A periodontal disease analysis including a step of performing immunoassay using an anti-8-hydroxydeoxyguanosine compound on a funnel collected from a subject's mouth to quantify 8-hydroxydeoxyguanosine Leave.

(2) The decoration according to claim 1, wherein the disgusting tomato is saliva.

(3) The quarantine according to claim 1, wherein the disgusting tongue is saliva, and the saliva is subjected to centrifugal separation before performing the touching.

(4) The inspection according to claim 1, wherein the disgust imnoassy is conducted by a competition law.

(5) The wing test according to claim 2, wherein the imunoassay is performed by a competition law.

(6) The male sexual analyst according to claim 3, wherein the editing is performed by a competition law.

(7) The inspection according to claim 1, wherein the tfffB ^ Munoassay is performed by a sandwich method.

(8) The test method according to claim 2, wherein the test is performed by the sandwich method.

(9) The inspection according to claim 3, wherein the inspection is performed by an abominable immortal sandwich method.

(10) Labeled anti-8-hydroxydeoxyguanosine ^:,, which is converted into a periodontal test for periodontal disease by the determination of 8-hydroxydeoxyguanosine in ^ f funnel collected from the oral cavity Anti-8-hydroxydeoxyguanosine ^: immobilized or immobilized on a solid phase selected from the group consisting of: body, labeled 8-hydroxydeoxyguanosine and 8-hydroxydeoxyguanosine Kit or test kit containing at least one or more reagents

(11) 8-Hydroxydoxyguanosine in ^! · Collected from the oral cavity is converted to a labeling agent using the anti-specific 8- & A kit or a detection device for testing periodontal disease by quantifying 8-hydroxydeoxyguanosine, comprising: means for aging; and means for measuring or visualizing the activity of the labeling agent.