WO2002079780A1 - Procede permettant de controler la parodontopathie - Google Patents

Procede permettant de controler la parodontopathie Download PDF

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Publication number
WO2002079780A1
WO2002079780A1 PCT/JP2002/003077 JP0203077W WO02079780A1 WO 2002079780 A1 WO2002079780 A1 WO 2002079780A1 JP 0203077 W JP0203077 W JP 0203077W WO 02079780 A1 WO02079780 A1 WO 02079780A1
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WO
WIPO (PCT)
Prior art keywords
hydroxydeoxyguanosine
periodontal disease
test
saliva
collected
Prior art date
Application number
PCT/JP2002/003077
Other languages
English (en)
Japanese (ja)
Inventor
Naoyuki Sugano
Original Assignee
Nihon University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nihon University filed Critical Nihon University
Priority to JP2002577559A priority Critical patent/JPWO2002079780A1/ja
Publication of WO2002079780A1 publication Critical patent/WO2002079780A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56955—Bacteria involved in periodontal diseases

Definitions

  • the present invention relates to the detection of periodontal disease. Tall.
  • 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.
  • 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.
  • 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.
  • 8—OH dG 8-hydroxydoxyguanosine
  • an object of the present invention is to provide a method for periodontal disease. Disclosure of the invention
  • the present invention relates to the following:
  • 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.
  • Knitting difficulty is ⁇ The ⁇ method of (1).
  • 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
  • kits or assays containing at least one or more reagents that have not been converted.
  • the present invention provides a method for binding 8-hydroxydeoxyguanosine in ⁇ collected from the oral cavity to transliteration using an immunospecific J3 ⁇ 4S with anti-8-hydroxydoxyguanosine.
  • Quantitative refers to not only the age at which the exact amount is measured, but also the approximate amount. ⁇ Including making a decision.
  • test method of the present invention is particularly useful for examination of periodontal disease in the nursing period including humans.
  • 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.
  • 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.
  • 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.
  • mice are immunized, subcutaneously, intravenously or intravenously.
  • 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 ⁇ ⁇ ⁇ g or more, preferably about 10 ⁇ g ⁇ 300 g.
  • 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.
  • 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.
  • the fusion promoter examples 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.
  • 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.
  • 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.
  • RIA radioimmunoassay
  • EIA enzymatic immunoassay
  • Various modifications are also possible for the above method.
  • One method using EIA is described as an example of a preferable measurement method.
  • 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.
  • the hybridoma thus cloned is grown in a liquid medium.
  • 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.
  • mice 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.
  • 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.
  • 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.
  • ⁇ 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.
  • 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.
  • 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.
  • peroxidases include those that can be used from various sources. Horseradish peroxidase (HRP) extracted from horseradish is preferred.
  • 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 ⁇ ⁇ to use it as the scripting method of.
  • 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.
  • 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.
  • the present invention also relates to a kit or detection for performing a superior intellectual method using an anti-8-OH dG antibody.
  • 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.
  • 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 Imnados. 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.
  • 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
  • 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 3 ⁇ 4 was 2.3 ng / m2. 1 and at most 9. SngZml.
  • the concentration of 8-OHdG was almost proportional to the progression of periodontal disease.
  • periodontal disease can be tested in a simple manner.
  • 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.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
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  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé permettant de contrôler la parodontopathie, comprenant l'étape consistant à soumettre un échantillon prélevé de la cavité buccale d'un sujet à un essai immunologique mettant en oeuvre un anticorps anti-8-hydroxydéoxyguanosine pour quantifier ainsi la 8-hydroxydéoxyguanosine. Ainsi, la parontopathie peut être contrôlée de façon pratique.
PCT/JP2002/003077 2001-03-28 2002-03-28 Procede permettant de controler la parodontopathie WO2002079780A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2002577559A JPWO2002079780A1 (ja) 2001-03-28 2002-03-28 歯周病の検査方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2001-093700 2001-03-28
JP2001093700 2001-03-28

Publications (1)

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WO2002079780A1 true WO2002079780A1 (fr) 2002-10-10

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1640721A1 (fr) * 2003-06-30 2006-03-29 Applied Cell Biotechnologies, Inc. Methode permettant de detecter l'apparition d'une parodontite
US7330259B2 (en) 2004-05-27 2008-02-12 Nova Measuring Instruments Ltd. Optical measurements of patterned articles
WO2013108561A1 (fr) * 2012-01-16 2013-07-25 ライオン株式会社 Peptide marqueur pour la détermination du risque de développer un syndrome métabolique, et son utilisation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
SCHMIDT ET AL.: "AGEs induce oxidant stress in the gingiva", JOURNAL OF PERIODONTAL RESEARCH, vol. 31, no. 7, 1996, pages 508 - 515, XP002951563 *
TOYOKUNI ET AL.: "Quantitative immunohistochemical determination of 8-hydroxy-2'-deoxyguanosine by a monoclonal antibody", LABORATORY INVESTIGATION, vol. 76, no. 3, 1997, pages 365 - 374, XP002951569 *
YIN ET AL.: "Determination of 8-hydroxydeoxyguanosine by an immunoaffinity chromatography-monoclonal antibody-based ELISA", FREE RADICAL BIOLOGY & MEDICINE, vol. 18, no. 6, 1995, pages 1023 - 1032, XP002951570 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1640721A1 (fr) * 2003-06-30 2006-03-29 Applied Cell Biotechnologies, Inc. Methode permettant de detecter l'apparition d'une parodontite
EP1640721A4 (fr) * 2003-06-30 2009-06-17 Applied Cell Biotechnologies I Methode permettant de detecter l'apparition d'une parodontite
US7330259B2 (en) 2004-05-27 2008-02-12 Nova Measuring Instruments Ltd. Optical measurements of patterned articles
WO2013108561A1 (fr) * 2012-01-16 2013-07-25 ライオン株式会社 Peptide marqueur pour la détermination du risque de développer un syndrome métabolique, et son utilisation
US10168339B2 (en) 2012-01-16 2019-01-01 Lion Corporation Marker peptide for determining risk of developing metabolic syndrome, and use thereof

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