WO1992021952A1 - Instrument servant a echantillonner une quantite minuscule d'un specimen - Google Patents

Instrument servant a echantillonner une quantite minuscule d'un specimen Download PDF

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Publication number
WO1992021952A1
WO1992021952A1 PCT/JP1992/000694 JP9200694W WO9221952A1 WO 1992021952 A1 WO1992021952 A1 WO 1992021952A1 JP 9200694 W JP9200694 W JP 9200694W WO 9221952 A1 WO9221952 A1 WO 9221952A1
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WO
WIPO (PCT)
Prior art keywords
test substance
water
biological test
sample
labeled
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PCT/JP1992/000694
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English (en)
Japanese (ja)
Inventor
Kenji Yasuda
Shusaburo Hokukoku
Takao Ogawa
Saeko Ito
Original Assignee
Meito Sangyo Kabushiki Kaisha
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Publication of WO1992021952A1 publication Critical patent/WO1992021952A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B01L2300/0838—Capillaries
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces

Definitions

  • the present invention relates to a tool for collecting a small amount of a sample that can be used for detecting a biological test substance, for example, a periodontopathogenic bacterium in the oral cavity or a specific antibody against the periodontopathogenic bacterium in the oral cavity, and a sample using the same.
  • a biological test substance for example, a periodontopathogenic bacterium in the oral cavity or a specific antibody against the periodontopathogenic bacterium in the oral cavity, and a sample using the same.
  • the present invention relates to a method for measuring a biological test substance and a kit for measurement.
  • Periodontal disease is divided into gingivitis and periodontitis. Further, periodontitis includes adult periodontitis and localized juvenile periodontitis. These periodontal diseases can lead to gingival inflammation, bleeding, drainage, periodontal pocket formation, periodontal ligament destruction, alveolar bone resorption, tooth sway, and tooth loss.
  • P gingivalis Prevotella intermedia (P. interme dia), Actinobacillus actinomycetemcomitans (A. actinomycet emcomitans), Treponema denticola (T. denticola), Bacteroi des forsythus (B.
  • Porphyromonas gingivalis ⁇ Prevotella intermedia corresponds to the conventional Bacteroides gingivalis ⁇ Bacteroides inter maxims, and according to a recent new classification (according to TJM van Steenbergen at the International Conference on Periodontal Disease in 1990). So called.
  • periodontopathogenic bacteria living in samples from periodontal pockets such as gingival crevicular fluid and subgingival plaque (plaque) of periodontal disease patients are cultured under anaerobic conditions on a blood agar medium. It has been reported that periodontal pathogenic bacteria can be detected by examining the detailed biochemical properties of various colonies (Loes che, f.J., Syed, SA, Schmidt, E. and Morrison, EC: J. Perio dont. 56, 447—4556, 1985).
  • Periodontopathic bacteria inhabiting periodontal bockets can be examined under a microscope after Gram staining or by dark field microscopy (Listgarten, HA et al .: J. Clin. Periodontol. 5, 1 15— 1332, 1978), and detection of periodontal pathogenic bacteria by combining antibodies against periodontopathogenic bacteria and fluorescent dyes has also been performed (Zambon, JJ, Bochacki, V. and Genco, KJ: Oral Microbiol. Immunol. 1, 39-44, 1986).
  • periodontal pathogenic bacteria living in periodontal pockets have been detected using antibodies to the respective periodontal pathogenic bacteria using an enzyme-linked immunosorbent assay (ELISA) (Zambon, JJ, Bochacki). , V. and Genco, RJ: Oral Microbiol. Immunol. 1, 39-44, 1986).
  • ELISA enzyme-linked immunosorbent assay
  • the present inventor has developed a tool which can efficiently collect a biological test substance in a sample as an index of diagnosis of any disease including periodontal disease, even if the amount is very small.
  • the present inventors have conducted intensive research on a measurement method and a measurement kit capable of directly, quickly and easily detecting a biological test substance in a sample using the same, and have completed the present invention. Disclosure of the invention
  • the present invention relates to a biological test substance in a specimen, characterized by having a biological test substance collection portion made of a water-absorbing material coated with a substantially water-insoluble and water-permeable porous film.
  • a collection tool is provided.
  • the present invention also relates to a stick-shaped or strip-shaped water-absorbing material, which may be in contact with at least a sample adjacent to a biological test substance collecting portion having a length of 1 to 5 orchids of the water-absorbing material. It is intended to provide a device for collecting a biological test substance, characterized in that the surface of a certain portion of the sample is waterproofed.
  • the present invention further comprises contacting a biological test substance collecting portion of the above-mentioned collection tool with a sample, and then contacting the sample with a reagent comprising a labeled substance capable of specifically binding to the biological test substance.
  • Another object of the present invention is to provide a method for measuring a biological analyte in a sample, which comprises qualitatively or quantitatively detecting a labeled reagent bound to the collection portion.
  • the present invention further provides a biological sample in a specimen, comprising a combination of the above-mentioned collection tool and a reagent comprising a labeled substance capable of specifically binding to the biological test substance to be detected in the sample.
  • a biological sample in a specimen comprising a combination of the above-mentioned collection tool and a reagent comprising a labeled substance capable of specifically binding to the biological test substance to be detected in the sample.
  • Provide kit for measurement of test substance c
  • FIG. 1 is a schematic cross-sectional view of an example of the sampling tool of the present invention.
  • the collection tool of the present invention can take various forms depending on the type of the target sample, etc., but is generally a stick (a thin stick that may be tapered). It is desirable to be in the form of strips (narrow sheets or films).
  • the collection tool may be made of any of natural, semi-synthetic, and synthetic types as long as the whole or at least the biological test substance collection portion is made of a water-absorbing material and the material is highly absorbent.
  • superabsorbent starch polymers for example, superabsorbent starch polymers, superabsorbent cellulose polymers, dextran polymers, superabsorbent synthetic polymers (for example, polyacrylates, polyvinyl alcohols, polyacrylamides, Polyoxyethylene-based polymers) and the like, and among them, highly water-absorbing cellulose-based polymers and highly water-absorbing synthetic polymers are preferable.
  • superabsorbent starch polymers for example, superabsorbent cellulose polymers, dextran polymers, superabsorbent synthetic polymers (for example, polyacrylates, polyvinyl alcohols, polyacrylamides, Polyoxyethylene-based polymers) and the like, and among them, highly water-absorbing cellulose-based polymers and highly water-absorbing synthetic polymers are preferable.
  • a paper stick for example, paper koyori
  • paper point in the dental field
  • a filter paper cut into a narrow strip is also used as a material. Can be used conveniently.
  • At least the surface of at least the tip portion of the biological material to be sampled of the absorbent material as described above, for example, the stick-shaped or strip-shaped absorbent material is substantially water-insoluble. It is coated with a permeable porous film.
  • a substantially water-insoluble and water-permeable porous film as shown in ⁇ , the water in the sample passes through the pores of the film and is absorbed by the water-absorbing material inside.
  • adsorption and immobilization can be performed while being concentrated on an L-type film coating, and that there is a remarkable action and effect that determination upon detection with a labeled reagent becomes extremely easy.
  • the pore size of the porous film to be coated depends on the moisture content in the sample. It is desirable to set the size so that it does not substantially pass through the biological test substance to be detected, for example, when detecting bacteria such as periodontopathogenic bacteria. In general, it is desirable that the average pore diameter of the coated porous film is generally 10 ⁇ m or less, particularly about 0.45 to 5.0 ⁇ m.
  • the material of the film to be coated is not particularly limited as long as it is substantially water-insoluble, and various polymers can be used. Specifically, for example, an organic acid or an inorganic acid of a polysaccharide is used. Esters (eg, lower fatty acid esters or nitric acid esters such as cellulose acid, propionic acid, etc.), lower alkyl ether derivatives of polysaccharides (eg, ethylcellulose, propylcellulose, butylcellulose, etc.), polyamide resins and the like. Can be Incidentally, the polymer may have a positive charge or a negative charge.
  • Esters eg, lower fatty acid esters or nitric acid esters such as cellulose acid, propionic acid, etc.
  • lower alkyl ether derivatives of polysaccharides eg, ethylcellulose, propylcellulose, butylcellulose, etc.
  • polyamide resins e.g, polyamide resins and the like.
  • a method for forming the coating of the porous film of the material relating to the absorbent material a method known per se can be employed.
  • a cellulose-based membrane can be formed by a phase separation method. Specifically, the cell mouth is dissolved in a good solvent such as acetone or methylene chloride, and then a non-good solvent such as ethanol / water having a higher boiling point is added to form a uniformly mixed solution. The solution is cast on a support, and the good solvent is selectively dried and removed using the difference in boiling point between the good solvent and the non-good solvent. Can produce a fine porous membrane.
  • a good solvent such as acetone or methylene chloride
  • a non-good solvent such as ethanol / water having a higher boiling point
  • the porous film coating on the absorbent material can be applied so as to cover at least the entire biological specimen collection portion of the collection device, for example, in the case of a stick or strip of water absorbent material, At least beyond It is applied so as to cover the end part, and the length of the coating part can be at least l mm from the tip, preferably two or more.
  • One way to achieve this is to remove the surface of at least the part of the collection tool that may come into contact with the sample other than the part required to collect the biological test substance (the biological sample collection part).
  • Examples of the method include a water-proof treatment.
  • a small piece of coated water-absorbent material can be secured to a suitable support.
  • Waterproofing is, for example, a coated stick- or strip-shaped water-absorbing material that is in contact with at least the sample adjacent to the biological sample collection part, which is 1-5 mm in length, preferably 2-4 mm in length. Can be applied to the surface of potential parts.
  • a portion of the porous film other than the biological test substance collection portion of the coating is treated with a solvent capable of dissolving the film.
  • FIG. 20 A method of closing the pores of the coating; a polymer substantially insoluble in water and non-water-absorbing (for example, fluororesin, silicone resin, vinyl chloride resin, methacryl resin, polyester resin, celluloid, paraffin, rubber, wood wax) , n method and the like that include applying a waterproof coating of tallow, etc.)
  • a polymer substantially insoluble in water and non-water-absorbing for example, fluororesin, silicone resin, vinyl chloride resin, methacryl resin, polyester resin, celluloid, paraffin, rubber, wood wax
  • n method and the like that include applying a waterproof coating of tallow, etc.
  • the tip of a stick-shaped or strip-shaped water-absorbing material is simply l to 5 mm, preferably 2 to 4 thighs.
  • the collection tool of this embodiment has a substantially water-insoluble and water-permeable porous film coating on the biological sample collection portion, and therefore, the above-mentioned effects achieved by the coating can be reduced.
  • the sample is intensively absorbed only at the localized tip, and the biological test substance is absorbed and fixed at a considerable concentration there. Can be realized.
  • blood components contained in various body fluids for example, blood, saliva, tears, sweat, runny nose, urine, stool, semen, vaginal secretions, pus, sputum, etc. , Serum, various cells, cytokines, hormones, peptides, metabolites, enzymes, bioactive substances, tumor markers, genes (DNA, RNA), Biological test substances such as receptors, antibodies, antigens, microorganisms, and viruses can be efficiently collected by extremely simple operations.
  • the gingival crevicular fluid and saliva can be obtained by inserting the stick-shaped sampling tool of the present invention between the teeth of the subject, between the teeth and the gums, in periodontal pockets, and the like.
  • periodontopathogenic bacteria or their specific antigens, or antibodies against periodontal pathogenic bacteria or their specific antigens can be easily collected.
  • the collection tool can be connected to an appropriate suction source (or vacuum source) if necessary, whereby the biological sample can be collected. In some cases, the collection of analytes can be performed more effectively.
  • Sampling of a biological test substance using the collection tool of the present invention is carried out by contacting the biological test substance collection portion of the collection tool of the present invention with a sample as described above.
  • the biological test substance is concentrated and adsorbed and immobilized on the collection portion as described above.
  • the sampling tool on which the biological test substance has been adsorbed and immobilized in this manner is then brought into contact with a reagent solution consisting of a labeled substance capable of specifically binding to the biological test substance to be detected. Let it.
  • a substance that specifically binds to a biological test substance specifically binds to a biological test substance immunologically or chemically.
  • examples include antibodies, antigens, nucleic acids and the like.
  • antibodies to periodontopathogenic bacteria or their specific antigens, Or specific antigens of periodontopathogenic bacteria specifically binds to a biological test substance immunologically or chemically.
  • labeling substance various labeling substances frequently used in the field of immunological measurement or chemical measurement can be used. For example, coloring substances, colored colloid particles, fluorescent dyes,
  • coloring substance examples include an inorganic pigment and an organic pigment.
  • colored colloid particles include metal colloid particles such as gold colloid particles, polymer particles such as colored polymer latex particles, protein-based particles such as colored gelatin particles, colored colloidal silicide particles, and 10 colored pigments.
  • Colloidal alumina particles, colored polysaccharide particles, and the like can be mentioned, and the particle size of these particles is generally in the range of 1 to 10 O nm, preferably 5 to 4 O nm.
  • Such metal colloid particles such as gold colloid particles, typically chloroauric acid (HAuCl ⁇ 2 H 2 0) 1 O mg was dissolved in boiling ultra distilled water 1 0 O ml, immediately after preparation 1% Kuen acid 3 Add an appropriate amount of aqueous sodium solution quickly and boil for 5 to 10 minutes to obtain a clear red liquid. After 2 to 3 minutes, slowly cool to prepare colloidal gold particles.
  • chloroauric acid HuCl ⁇ 2 H 2 0
  • the fluorescent dye for example Furuoresini Sochianeto (FITC), etc. tetrarhodimine isothiocyanate (TRITC) o.
  • the radioisotope e.g. 3 H, 1 4 C, 32 P, such as 1 25 I is included, as is an enzyme, for example Peruokishida Ichize, enzymes to develop color decomposing substrate such as alkaline phosphatase is preferred.
  • alkaline phosphatase can be used as a substrate for 5-bromo-4-monocloth-13-indolyl phosphate or p-nitrotrobutetate. Combination with lazolium chloride can be mentioned.
  • substrates are colorless, transparent and water-soluble, but are decomposed by the action of enzymes, each exhibit a unique color, become water-insoluble, and can be used to detect the presence of labeled substances.
  • Methods for labeling a specific binding substance with such a substance include a physical binding method and a chemical binding method.
  • the former is a method in which the specific binding substance and the labeling substance are physically adsorbed and bonded only by mixing them, and the latter is a method in which the specific binding substance and the functional group of each of the labeling substances are bonded.
  • This method involves direct covalent bonding or indirect covalent bonding between a specific binding substance and a labeling substance using a bivalent reagent such as carbodiimide or glutaraldehyde.
  • the amount of label bound to the collection tool is measured by visual inspection, densitometer, colorimeter, radiation measurement device, etc., and the measurement result is compared with a standard curve created in advance as necessary. This makes it possible to qualitatively or quantitatively detect the amount of the biological test substance in the sample.
  • the collection tool of the present invention used in the above-described measurement system and the reagent comprising the labeled specific binding substance are combined to form a kit, whereby the biological test substance in the sample is analyzed. It can be a measurement kit.
  • a method for measuring a biological test substance in a sample using the collection tool of the present invention is described as a periodontopathogenic bacterium or a specific antigen thereof in a sample such as gingival crevicular fluid, saliva, or plaque treatment solution Or, the case of detecting an antibody or the like against them will be described in more detail.
  • P-gingivalis which is considered to be a periodontopathogenic bacterium, for example, gingivalis 381, gingivalis 1021, gingivalis ATCC 33277, gingi valis EB22D— 1 strain, gingivalis RB 24 M—2 strains, gingivalis RB46D—1 strain, £ ⁇ gingivalis 6/26 strains, gingivalis, 1 strain, ⁇ gingivalis W50 strain, ingivalis f83 strain, gingivalis HI 1 ID—5 strains, P gingivalis Hff24D— 1 strain, P.
  • A. act inomycetemcomi tans N e.g., A. actinomycetemcomitans ATCC 0 29522 strain, A. act inomycetemcomi tans ATCC 29523 strain, A. ac ti noiyce temcomi tans ATCC 29524 strain, A. actinomycetemcomitans Y4 strain, A, actinomycetemcomitans NCTC 9709ans, A.
  • actinoit NCTC 9710 strain A. actinomycetemcomitans 1 strain, A. ac tinomycetemcofflitans 15 strains ⁇ A. actinomycetemcomitans 27 strains ⁇ A. actinomycetemcomitans 29, ⁇ ; ⁇ actinomycetemcomitans 32, 2 ⁇ A. actinomycetemcomitans 39, actinomycetemcomitans 42, 2 ⁇ A. actinomycetemcomitans—67, A. Or, for example, L denticola ATCC 354; or forsythus, for example, B. forsvthus ATCC 430; or ⁇ _gingivalis, for example, C. gingival is ATCC 336, 24; or ⁇ nucleatum, e.g., F. nu cleatum ATCC 2 5 5 8 6; or recta, e.g., W. recta ATCC
  • a medium such as GAM Bouillon, Brain-Heart Infusion Broth supplemented with hemin or menadione, or a medium supplemented with Todd-Hew itt broth supplemented with yeast extract, etc. After culturing for about 20 hours at about 37 ° C under typical conditions, these cells are collected, washed with physiological saline, and lyophilized. The cells thus obtained are bound to a labeling substance or used as an immunizing antigen
  • periodontopathogenic bacteria having pili with specific antigenicity such as P. gingivalis
  • P. gingivalis For example, if GAM bouillon, Brain-Heart In fusion broth, etc. are inoculated into a medium supplemented with hemin and menadione and cultured at about 37 ° C under anaerobic conditions, the amount will increase for about 20 hours or more. A number of cells are obtained. The cells are collected, and the pili are physically separated by pipetting. Next, the pili can be purified through ammonium sulfate fractionation and ion exchange chromatography to obtain P. gingivalis pilus antigen. like this The pilus antigen obtained in this manner is bound to a labeling substance, or used as an immunogen for obtaining a specific antibody.
  • a periodontopathogenic bacterium for example, A. actinomycetemcoiiiitans, for example, into a medium obtained by adding yeast extract to T odd-Hefitt broth, and culturing at about 37 under anaerobic conditions for about 20 hours, Alternatively, a large number of cells are obtained thereafter.
  • the cells are collected and lyophilized.
  • the obtained freeze-dried cells are suspended in physiological saline, then subjected to autoclaving and heat-extracted.
  • the extract is separated and purified by, for example, ion-exchange chromatography, gel filtration chromatography, etc. to obtain A. actinomycetemcoiiiitans.
  • the polysaccharide antigen thus obtained is bound to a labeling substance or used as an immunogen to obtain a specific antibody.
  • the freeze-dried cells of periodontopathogenic bacteria obtained as described above are immunized to various animals.
  • lyophilized cells of this periodontopathogenic bacterium are injected subcutaneously or intramuscularly into animals such as egrets and goats together with complete Freund's adjuvant and immunized.
  • an antiserum against periodontopathogenic bacteria can be obtained.
  • the obtained antiserum is purified by ammonium sulfate fractionation and ion exchange chromatography according to a conventional method.
  • the mouse is immunized with the freeze-dried cells of periodontopathogenic bacteria obtained as described above, and the spleen cells are taken out.
  • the cells are fused with mouse bone marrow tumor cells using polyethylene glycol or the like, followed by cloning.
  • These hybridomas are transplanted into the abdominal cavity of mice previously treated with pristane, and monoclonal antibodies are obtained from the ascites fluid, or the hybridomas are cultured in a large amount in a serum-free medium, and the monoclonal antibodies are obtained from the culture supernatant.
  • the obtained monoclonal antibody is purified by ammonium sulfate fractionation and ion exchange chromatography according to a conventional method.
  • ⁇ gingivalis 0 example the polysaccharide antigens of fimbrial antigen or ⁇ actinomycetemcomitans immunizing an animal, these specific antigen Usagi with Freund Bok's complete adjuvant, an animal subcutaneously, such as catcher formic
  • an antiserum against the pilus antigen of gingivalis or the polysaccharide antigen of actinomycetemcomitans can be obtained from the blood of these animals according to a method known per se. Further, if necessary, the obtained antiserum is purified by ammonium sulfate fractionation and ion exchange chromatography according to a conventional method.
  • mice are immunized with the P. gingivalis fimbrial antigen or the A. actinomycetemcomitans polysaccharide antigen obtained as described above, and the spleen cells are removed and fused with mouse bone marrow tumor cells using polyethylene glycol or the like. After cloning, a hybridoma that secretes a monoclonal antibody that reacts differently with the pilus antigen of P. gingivalis or the polysaccharide antigen of actinomycetemcomitans is selected.
  • hybridomas are transplanted into the abdominal cavity of mice previously treated with pristane, and monoclonal antibodies are obtained from the ascites fluid, or the hybridomas are cultured in a large amount in a serum-free medium, and the monoclonal antibodies are isolated from the culture supernatant. Can get You. Further, the obtained monoclonal antibody is purified by ammonium sulfate fractionation and ion exchange chromatography according to a conventional method.
  • Antisera or monoclonal antibodies against whole cells of periodontopathogenic bacteria or their specific antigens obtained as described above e.g., fimbrial antigen of P. gingival is, polysaccharide antigen of A. actinomycetemcomi tans, etc.
  • Bind colloidal particles e.g., a method known per se can be used. Generally, the colored colloidal particles are suspended in a carbonate buffer, phosphate buffer, Tris-HCl buffer or glycine buffer having a pH of 7 to 9, and the antibody described above is added thereto, and the mixture is added at room temperature to about 37. Stir at 150 ° C for 15 to 90 minutes and label the antibody with colored colloid particles.
  • Enzymes are added to antiserum or monoclonal antibodies against whole cells of periodontopathogenic bacteria or their specific antigens obtained as described above, for example, P. gingivalis linear antigen, A. actinomycetemcomi tans polysaccharide antigen, etc. Join.
  • the antibody is dissolved in 0.1 M phosphate buffered saline to a concentration of 20 mgZml.
  • an enzyme as a labeling substance for example, 4 O mg of alkaline phosphatase, and dissolve well.
  • a gingival crevicular fluid, saliva or plaque treatment solution of a periodontal disease patient is brought into contact with the collection tool of the present invention, and a sample is collected on the collection tool.
  • the portion of the sample to be sampled is immersed in the above-mentioned colored colloid-labeled antibody solution, and allowed to react for 5 to 15 minutes at room temperature or at about 37 ° C with stirring or stirring. .
  • the post-collection tool After taking out the post-collection tool and washing it with distilled water, it can be determined using the naked eye or a measuring instrument such as a densitometer. If the area where the specimen of the periodontal disease is attached is stained with the color of the colored colloid particles, it is determined to be positive. At this time, the degree of the color correlates with the presence and absence of periodontopathogenic bacteria, and the number of bacterial cells can be quantified.
  • the colored colloid particles are gold colloid particles, the sensitivity can be increased by silver staining.
  • the colored colloidal particles are suspended in a carbonate buffer, phosphate buffer, Tris-HCl buffer or glycine buffer with ⁇ 7 to 9, and the antigen is added thereto, and room temperature to about 37 ° C. Stir for 15 to 90 minutes at C and label the antigen with colored colloid particles.
  • a method known per se can be used to bind an enzyme to a specific antigen of periodontopathogenic bacteria obtained as described above, for example, ⁇ gingivalis fimbrial antigen, actinomycetemcomitans polysaccharide antigen, or the like.
  • a specific antigen of periodontopathogenic bacteria obtained as described above, for example, ⁇ gingivalis fimbrial antigen, actinomycetemcomitans polysaccharide antigen, or the like.
  • 0.1 M phosphate buffered saline is used to bring the antigen to 20 mg / ml. Dissolve in saline.
  • an enzyme as a labeling substance, for example, alkaline phosphatase, and dissolve well. While stirring gently, add 0.1% of 1% glutaraldehyde dropwise.
  • alkaline phosphatase-labeled antigen solution It reacted at room temperature for 2 hours, by gel filtration, from unbound antigen and alkaline phosphatase Fataze, and c in this manner to separate and purify the Al force re phosphatase-labeled antigen, be labeled antigen with Al force Li phosphatase Wear. This is hereinafter referred to as alkaline phosphatase-labeled antigen solution.
  • a gingival crevicular fluid, saliva or plaque treatment solution of a periodontal disease patient is brought into contact with the collection tool of the present invention, and a sample is collected on the collection tool.
  • the part of the sample to be collected is immersed in the above-mentioned colored colloid-labeled antigen solution, and allowed to react for 5 to 10 minutes at room temperature or at about 37 ° C with stirring or stirring. .
  • the determination can be made using a measuring instrument such as the naked eye or a densitometer. If the area where the specimen of the periodontal disease is attached is stained with the color of the colored colloid particles, it is determined to be positive. At this time, since the degree of the color is correlated with the presence or absence and the amount of the antibody against periodontopathogenic bacteria, the antibody titer against periodontopathogenic bacteria can be quantified.
  • the colored colloid particles are gold colloid particles
  • the sensitivity can be increased by silver staining.
  • a gingival crevicular fluid, saliva or plaque treatment solution of a periodontal disease patient is brought into contact with the collection tool of the present invention, and a sample is collected on the collection tool.
  • the biological test substance collection portion of the collection tool is immersed in the above-described alfa-rephosphatase-labeled antigen solution. Soak and react at room temperature to about 37 with standing or stirring for 5 to 10 minutes.
  • the sampling tool is taken out, washed with distilled water, and can be judged with the naked eye or using a measuring instrument such as a densitometer. If the place where the sample of the periodontal disease is attached is stained blue, it is determined to be positive. At this time, the degree of the color is correlated with the presence or absence of the antibody against periodontopathogenic bacteria, and the antibody titer against periodontopathogenic bacteria can be quantified.
  • the sample is stained with blood or pus during sample collection, it may be decolorized with an oxidizing agent such as aqueous hydrogen peroxide and then reacted with a colored colloid-labeled antibody solution or a colored colloid-labeled antigen solution. preferable.
  • an oxidizing agent such as aqueous hydrogen peroxide
  • the presence or absence of periodontopathic bacteria in periodontal lesions of periodontal patients and specific antibodies to periodontopathogenic bacteria in periodontal lesions of patients with more or less periodontal disease can be quickly detected with a very simple operation, and the disease can be diagnosed and the degree of the disease state can be grasped in a short time.
  • the various stages of periodontal disease and the state of healing can be monitored over time. Being able to know this will help with treatment and prevent recurrence.
  • gingivalis and intermedia were inoculated into GAM bouillon medium (Nissui Pharmaceutical) supplemented with hemin (5 mg / 1) and menadione (lmg 1) at 37 ° C, 80% N 2 , 10% H 2 , 10% CO 2
  • GAM bouillon medium Nasui Pharmaceutical
  • menadione lmg 1
  • A. actinomycetemcomitans is a medium in which 1% yeast extract is added to Todd-Hewitt broth (Difco Laboratories), and cultured in large amounts under anaerobic conditions of 37 ° C and 5% CO2, and then collected by centrifugation. did. After washing these cells with physiological saline, they were frozen under reduced pressure. .
  • ⁇ gingivalis 381 strain and HW24D-1 strain were inoculated into a GAM bouillon medium (Hesui Pharmaceutical) supplemented with hemin (5 mgZl) and menadione (lmgZl) at 37 ° C, 80% N 2 , 10% H 2 , after mass cultivation in anaerobic conditions of 1% C0 2, the cells were collected by centrifugation. The cells were suspended in 20 mM Tris-chloride buffer (pH 7.4) +0.15 M NaCl + 1 OmM gCl 2 , pipetted, and stirred with a magnetic stirrer to physically exfoliate the pili.
  • the cells were removed by centrifugation, the supernatant was fractionated with 40% saturated ammonium sulfate, and the resulting precipitate was dissolved in 20raM Tris-HCl buffer (pH 8.0) and desalted by dialysis. Next, a concentration gradient of NaCl was applied by ion exchange chromatography using DEAE-Sepharose (Pharmacia), and the fraction eluted with 0.15 M NaCl was used as a pilus antigen.
  • A. actinomycetemcomitans ATCC 2953 2 strain, Y4 strain, and NCTC 109710 strain were inoculated into a medium containing 1% yeast extract added to Todd-Hewitt broth (Difco Laboratories). 7, after mass cultivation under anaerobic conditions of 5% C0 2, the cells were collected by centrifugation. The obtained cells are freeze-dried, suspended in physiological saline, autoclaved, heat-extracted, and the extract is extracted using, for example, DEAE-Sephadex A-25 (Pharmacia). Fractionation was carried out by exchange chromatography, and the flow-through fraction was further purified by gel filtration chromatography using S-marker S-200 (Pharmacia), and a polysaccharide fraction was collected.
  • A. actinomycetemcomitans ATCC 295, 2 fimbrial antigens of all cells of the periodontopathogenic bacteria P. gingivalis ⁇ P. intermedia and A. actinomycete mcomitan s, gingivalis 381 and HW24 D-1 Preparation of monoclonal antibodies against polysaccharide antigens of 3 strains, 4 strains of Y4 and NCTC 9710 strain
  • gingivalis 381 and HW24D-1 pilus antigens, ⁇ _actinomycetemcomitans ATCC 2953, AT4, Y4 and NCTC 970 of Preparation Example 3 100 ⁇ g each was added to 0.1 ml ml Freund's complete adjuvant per mouse.
  • a BALBZc mouse female was immunized subcutaneously three times every three weeks as a water-in-oil emulsion together with bunt, and a total of three injections were performed. Washed 3 times with Eagle's MEM.
  • myeloma cells (SP 2/0) derived from BALB / c mice were washed three times with Eagle's ⁇ .
  • the myeloma cells and spleen cells are mixed in Eagle's 1 at 1:10, centrifuged at 1,20 Orpra, 1 Omin. After centrifugation, the supernatant is removed, and the sediment is thoroughly loosened. 5 ml) + Eagle's HEM (0.5 ml) + DHS0 (0.35 ml) were mixed at 37 ° C for 1 minute while gently dropping into the precipitate.
  • Screened hybridomas are cloned by limiting dilution did.
  • the limiting dilution method is as follows: Add 10 cells to 10-16 medium containing 10% fetal serum at a concentration of 5 cells / ml hybridoma and 5 ⁇ 10 6 cells / ml BALB c mouse thymocytes, and add 96 wells. Dispense 0.2 ml each into a microtiter plate, freeze-dried cells of periodontopathogenic bacteria f. Gingivalis, intelmedia and A, actinomycetemcomitans in Preparation Example 1, P.
  • the target monoclonal antibody was prepared by collecting the antibody from E. coli.
  • the obtained monoclonal antibody was subjected to 1/3 saturated ammonium sulfate fractionation, and purified by DEAE-ion exchange chromatography, gel permeation chromatography or Protein A column.
  • gingivalis strain 381 and HW24D-1 strain in GAM bouillon medium Heisei Pharmaceutical supplemented with hemin (5mgZl) and menadione (lmgZl).
  • the cells were collected by centrifugation.
  • the cells are suspended in phosphate-buffered saline, the absorbance at 550 nm is adjusted to 0.5, and the cell concentration of the cell suspension is determined by PETR0FF-HAUSSER and HELBER Counting chamber (Haus ser, Scientific Partnership Horsham;). continue to dilute the cell suspension obtained Te 0 this good Unishi obtained using a 2 1 of each of its dilution
  • the sample was adsorbed on the device for collecting a biological test substance prepared in Example 1.
  • the cells were immersed in a monoclonal antibody solution against the fimbrial antigen of the ⁇ gingivalis strain 381 and the HW24D-1 strain labeled with the gold colloid particles obtained in Example 2 and reacted. After the completion of the reaction, the number of cells in which coloration of the colloidal gold particles was observed was determined. The results are shown in Table 1, which can be detected by a monoclonal antibody solution against the pilus antigen of gingivalis 381 and Hff24 D-1 labeled with gold colloid particles ⁇ Number of cells of gingivalis 381 and HW24D-1 was 10 5 2 X 10 5 and 1.0 X.
  • intermedia ATCC 25611 was grown in GAM broth medium (Nissui Pharmaceutical) supplemented with hemin (5 mg / 1) and menadione (loig / 1) at 37 ° C, 80% N 2 , 10% H 2 , 10% C0 After mass culture under anaerobic conditions of 2 , The cells were collected by centrifugation. The cells were suspended in phosphate buffered saline,
  • the absorbance at 55 Onm was adjusted to 0.5, and the cell concentration of this cell suspension was determined using PETROFF-HAUSSER and HELBER Counting, chamber (Hausser Scientific Partnership Horsham).
  • the cell suspension obtained in this manner was diluted, and 2 ⁇ 1 of each dilution was adsorbed to the device for collecting a biological test substance prepared in Example 1. Therefore, the cells were immersed in a monoclonal antibody solution against all the cells of ⁇ intermedia ATCC 25611 labeled with colloidal gold particles obtained in Example 2 and reacted. After the reaction was completed, the number of bacterial cells in which coloring of the colloidal gold particles was observed was determined. The results are shown in Table 2. The number of cells of the intermedia ATCC 25611 strain detected by a monoclonal antibody solution against all the cells of ⁇ intermedia ATCC 25 611 strain labeled with colloidal gold particles was 2 ⁇ 10 5 .
  • the cell suspension thus obtained was diluted, and 2 jt of each dilution was adsorbed to the device for collecting a biological test substance prepared in Example 1. Therefore, the cells were immersed in a monoclonal antibody solution against polysaccharide antigens of L actinomycetemcomitans ATCC 2953, Y4 and NCTC 9710 labeled with gold colloid particles obtained in Example 2, and Reacted. After the reaction was completed, the number of bacterial cells in which coloring of the gold colloid particles was observed was determined. The results are shown in Table 3, which shows that A.
  • actinomycetemcomitans labeled with gold colloid particles can be detected with a monoclonal antibody solution against polysaccharide antigens of ATCC 2953, Y4 and NCTC 9710 strains.
  • Actinomycetemcomitans ATCC 2953 2 strains, ⁇ 4 strains and NCTC 9710 strains all had 2 ⁇ 10 5 cells.
  • ATCC 29523 shares Y 4 shares NCTC 9710 shares
  • the dilution rate of the antibody to the polysaccharide antigen of the 9523 strain, the Y4 strain, and the NCTC 9710 strain was 1100 on the serum label.
  • ATCC 29523 shares Y 4 shares NCTC 9710 shares 1/10 +++ +++ +++
  • the gingival crevicular fluid of three healthy subjects and six patients with adult periodontitis was collected using the biological test substance collection tool prepared in Example 1, and the gold colloid obtained in Example 2 was used first.
  • the cells were immersed in a monoclonal antibody solution against the fimbrial antigen of P. gingivalis strain 38 labeled with 10 particles and allowed to react at room temperature for 5 to 15 minutes. Then, the collection tool was removed and washed with distilled water. Therefore, the part where the gingival crevicular fluid of the sampling tool was adsorbed was judged to be positive if it was stained with red-purple, which is the color of the colloidal gold particles, and negative if it was not stained. Next, in the same manner, a reaction was performed with a monoclonal antibody solution against the fimbrial antigen of the P. gingivalis HW24D-1 strain labeled with the colloidal gold particles obtained in Example 2 and subjected to determination.
  • ⁇ gingivalis in the gingival crevicular fluid of three healthy subjects and six adult periodontitis patients was identified as follows.
  • the gingival crevicular fluid o absorbed in a commercially available paper kori is suspended in a transport medium (RTF: Reduced Transfer Fluid) and collected.
  • RTF Reduced Transfer Fluid
  • Developed by Control Disease C enter and cultured for several days under anaerobic conditions. From among the colonies that grew, blackened colonies were selected, separated, purified, and then subjected to an identification test using the Rap ID ANA System (HcDONNELL DOUGLAS). I got it. Furthermore, the culture supernatant of those identified as P. gingivalis was subjected to gas chromatography to examine the production of phenylacetic acid, thereby re-recognizing fngivalis.
  • the gingival crevicular fluid of three healthy subjects showed no negative coloration due to gold colloid particles, and all showed negative results.
  • ⁇ gingivalis was not detected in the test for identification of bacteria in the gingival crevicular fluid.
  • the gingival crevicular fluid of adult periodontal disease patients (6) 4 out of 6 patients showed a reaction with a monoclonal antibody solution against the pilus antigen of gingivalis 381 strain labeled with gold colloid particles.
  • Gingival sulcus fluid of three healthy subjects and six patients with adult periodontitis was collected using the biological sample collection tool obtained in Example 1, and labeled with colloidal gold particles obtained in Example 2.
  • the resulting ⁇ intermedia ATCC 25611 strain was immersed in an antiserum solution against all the cells and reacted. After reacting at room temperature for 5 to 15 minutes, the sampling tool was removed and washed with distilled water. Therefore, the part where the gingival crevicular fluid of the sampling tool was adsorbed was judged to be positive if it was stained with reddish purple, which is the color of the colloidal gold particles, and negative if it was not stained.
  • the gingival sulcus fluid absorbed in a commercially available paper koyori (paper point) is suspended in a transport medium (ETF: Reduced Transfer Fluid) and collected. (Developed by Control Disease C enter) and cultured for several days under anaerobic conditions. Among the colonies that grew, blackened colonies were selected, separated, purely cultured, and then subjected to an identification test using a Rap ID ANA System (manufactured by HcDONNELL DOUGLAS).
  • the gingival crevicular fluid of three healthy subjects showed no negative coloration due to colloidal gold particles, and all showed negative results.
  • P. intermedia was not detected in the identification test of bacteria present in the gingival crevicular fluid.
  • the gingival crevicular fluid of adult periodontal disease patients (6 patients) was colored by gold colloid particles, and all showed positive results.
  • the identification test for bacteria present in the gingival crevicular fluid revealed P. intermedia.o
  • Gingival sulcus fluid of three healthy subjects and six patients with localized juvenile periodontitis was collected using the biological test substance collection tool obtained in Example 1, and the gold colloid particles obtained in Example 2 were collected.
  • a monoclonal antibody solution against a polysaccharide antigen of actinomycetemcomitans ATCC2953 strain, Y4 strain and NCTC9710 strain was mixed, immersed and reacted. After reacting for 5 minutes at room temperature, for collection The utensils were taken out and cleaned with distilled water. Therefore, it was determined that the portion of the sampling tool to which the gingival sulcus was adsorbed was colored red-purple, which is the color of the colloidal gold particles, as positive.
  • the gingival sulcus fluid absorbed in a commercially available paper kori is suspended in a transport medium (RTF: Reduced Transfer Fluid) and collected.
  • RTF Reduced Transfer Fluid
  • the cells were seeded on a medium (developed by Control Disease C enter) and cultured for several days under anaerobic conditions. A colony where growth was observed was selected, isolated, and subjected to pure culture.
  • An identification test was performed using a Rap ID ANA System (manufactured by McDON NELL DOUGLAS).
  • the gingival crevicular fluid of three healthy subjects and six patients with adult periodontitis was collected using the biological sample collection tool obtained in Example 1, and labeled with gold colloid particles obtained in Example 3.
  • the pilus antigen solutions of the ⁇ gingivalis strain 381 and the HW24D-1 strain were mixed, immersed and reacted. After reacting at room temperature for 5 to 15 minutes, the sampling device was removed and washed with distilled water. Therefore, the sample was determined to be positive when the portion of the sampling tool adsorbed by the gingival crevicular fluid was colored red-purple, which is the color of the colloidal gold particles, and negative when not stained.
  • fibrin antigens of gingivalis 381 and HW24D-1 dissolved in carbonate buffer (pH 9.0) were dispensed at 1 ⁇ l / well into each well of an ELISA plate. It was left at 100 ° C for 10 minutes. The next day, the cells were washed with phosphate buffered saline (PBST) containing Tween 20 and then washed with PBST containing 10% goat serum. It was left at room temperature for 1 hour to perform blocking. Next, gingival crevicular fluid previously collected in a commercially available paper gyri (paper point) was dissolved in PBST and collected. After reacting at 37 ° C.
  • PBST phosphate buffered saline
  • the gingival sulcus solution of healthy subjects (3) showed no negative coloration due to colloidal gold particles, and all showed negative results.
  • the detection of an antibody against gingivalis pilus antigen present in the gingival crevicular fluid by ELISA no antibody against L_gingivalis pilus antigen was found.
  • the gingival crevicular fluid of adult periodontitis patients (6 patients) was colored by colloidal gold particles, and all showed positive results.
  • the detection of antibodies against ⁇ gingivalis fimbrial antigen present in the gingival crevicular fluid by ELISA revealed antibodies against the L_ginialis pilus antigen.
  • the gingival crevicular fluid of three healthy subjects and six patients with localized juvenile periodontitis was collected using the biological test substance collection tool obtained in Example 1, and the gold colloid particles obtained in Example 3 were collected.
  • a polysaccharide antigen solution of A. actinomycetemcomitans ATCC 2953, Y4 strain and NCTC 9710 strain labeled with a pup was mixed, immersed and reacted. After reacting at room temperature for 5 to 15 minutes, the sampling tool was removed and washed with distilled water. Therefore, the part where the gingival crevicular fluid of the sampling tool was adsorbed was judged to be positive if it was stained in red-purple, the color of gold colloid particles, and negative if it was not stained.
  • the polysaccharide antigens of actinomycetemcomitan 0 s ATCC 2953, Y4 and NCTC 9710 dissolved in carbonate buffer (pH 9.0) were added to each well of the ELISA plate.
  • the mixture was dispensed at a time and left at 4 ° C for 10 minutes.
  • the plate was left standing at room temperature for 1 hour with PBST containing 10% goat serum for blocking.
  • the gingival crevicular fluid which was previously collected on a commercially available paper twister (paper point), was dissolved in PBST. Then, what was collected was added.
  • Each antiserum obtained in Preparation Example 4 or each monoclonal antibody obtained in Preparation Example 5 is dissolved in 0.1 M phosphate buffered saline so as to be 2 Oral.
  • phosphate buffered saline aqueous phosphate buffered saline
  • 4 Omg of alkaline phosphatase (EIA Grade: 01-221) manufactured by ZYMED, and dissolve well.
  • 0.1 ml of 1% glutaraldehyde dropwise and react at room temperature for 2 hours.
  • an equal amount of saturated ammonium sulfate is added and stirred.
  • the precipitate obtained by centrifugation is dissolved in a minimum amount of phosphate buffered saline and dialyzed against phosphate buffered saline.
  • the mixture was subjected to gel filtration using S-marked hadex G-200 to obtain an Al-force phosphatase-labeled antibody.
  • Gingival sulcus fluid of three healthy subjects and six patients with adult periodontitis was collected using the biological test substance collection tool created in Example 1, and first labeled with alkaline phosphatase obtained in Example 9.
  • ⁇ Gingivalis 38 1 strain and ⁇ gingival! S HW 24 D- 1 strain were immersed in a monoclonal antibody solution against the pilus antigen, allowed to react at room temperature for 5 to 15 minutes, and then the collection tool was taken out. Washing was performed with phosphate buffered saline containing 20. Next, it was immersed in a substrate solution containing 5-promote 4-monocloth-3-indolyl phosphate, and reacted for 5 to 15 minutes. Therefore, the part of the sampling tool where the gingival crevicular fluid is adsorbed turns blue. Stain was judged as positive, and non-stain was judged as negative.
  • ⁇ gingivalis in the gingival crevicular fluid of three healthy subjects and six adult periodontitis patients was identified as follows.
  • gingival sulcus fluid 5 absorbed in a commercially available paper kori is suspended in a transport medium (RTF: Reduced Transfer Fluid) and collected.
  • RTF Reduced Transfer Fluid
  • the cells were seeded on a medium (developed by Control Disease C enter) and cultured for several days under anaerobic conditions. From among the colonies that grew, blackened colonies were selected, separated, purely cultured, and subjected to an identification test using the Rap ID ANA System (manufactured by McDONNELL DOUGLAS). Furthermore, the culture supernatant of ⁇ gingivalis was subjected to gas chromatography to examine the production of phenylsulfuric acid, thereby re-recognizing P. gin givalis.
  • the collection tool of the present invention is useful for detecting a biological test substance, for example, a periodontopathogenic bacterium in the oral cavity or a specific antibody against the periodontopathogenic bacterium in the oral cavity, For example, it is suitable as an auxiliary tool for diagnosis of periodontal disease.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Instrument servant à échantillonner une substance provenant d'un spécimen et devant subir un examen biologique. Il est caractérisé en ce qu'il est pourvu d'un élément d'échantillonnage de ladite substance constitué d'un matériau absorbant l'eau revêtu d'un film poreux sensiblement non hydrosoluble et perméable à l'eau. On met ledit élément d'échantillonnage de l'instrument en contact avec le spécimen puis avec un réactif constitué d'une substance marquée qui est apte à se lier de manière spécifique à la substance à examiner. Ceci permet la détection directe, rapide et sans difficultés de la substance devant subir l'examen biologique dans le spécimen.
PCT/JP1992/000694 1991-06-01 1992-05-28 Instrument servant a echantillonner une quantite minuscule d'un specimen WO1992021952A1 (fr)

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JP3/157724 1991-06-01
JP3157724A JPH04355339A (ja) 1991-06-01 1991-06-01 微量検体採取用具

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JPH0797395A (ja) * 1993-09-28 1995-04-11 Kyowa Medex Co Ltd ポルフイロモナス・ジンジバリス線毛蛋白質の配列を含有するペプチド類及びその用途
DK1696236T3 (da) * 1998-03-30 2014-08-18 Orasure Technologies Inc Opsamlingsindretning til analyse af mundvæsker
JP4603557B2 (ja) * 2007-01-15 2010-12-22 有限会社 ミクロデント 定量規格化培養方法
JP2017058154A (ja) * 2015-09-14 2017-03-23 学校法人 日本歯科大学 歯肉溝滲出液の採取用具
CN116113826A (zh) 2020-09-11 2023-05-12 富士胶片株式会社 浓缩器件、被检体液的浓缩方法、被检体液的检查方法、及检查试剂盒
CN116057378A (zh) 2020-09-11 2023-05-02 富士胶片株式会社 浓缩器件、被检体液的浓缩方法、被检体液的检查方法、及检查试剂盒
CN116171378A (zh) 2020-09-11 2023-05-26 富士胶片株式会社 被检体液的浓缩方法、及被检体液的检查方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6073463A (ja) * 1983-09-30 1985-04-25 Lion Corp 歯周病診断法
JPS61142016U (fr) * 1985-02-22 1986-09-02
JPS61180017U (fr) * 1985-04-27 1986-11-10
JPS63246668A (ja) * 1987-03-31 1988-10-13 Kyoto Ikagaku Kenkyusho:Kk 糞便中の潜血検出方法
JPH01152237U (fr) * 1988-04-13 1989-10-20
JPH02107970A (ja) * 1988-10-17 1990-04-19 Meito Sangyo Kk バクテロイデス・ジンジバリス菌体の検出用試薬、そのためのキット及び方法
JPH0263456U (fr) * 1988-10-31 1990-05-11
JPH02141665A (ja) * 1988-11-24 1990-05-31 Godo Shiyusei Kk 糞便中のヘモグロビンの検出方法

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Publication number Priority date Publication date Assignee Title
JPS6073463A (ja) * 1983-09-30 1985-04-25 Lion Corp 歯周病診断法
JPS61142016U (fr) * 1985-02-22 1986-09-02
JPS61180017U (fr) * 1985-04-27 1986-11-10
JPS63246668A (ja) * 1987-03-31 1988-10-13 Kyoto Ikagaku Kenkyusho:Kk 糞便中の潜血検出方法
JPH01152237U (fr) * 1988-04-13 1989-10-20
JPH02107970A (ja) * 1988-10-17 1990-04-19 Meito Sangyo Kk バクテロイデス・ジンジバリス菌体の検出用試薬、そのためのキット及び方法
JPH0263456U (fr) * 1988-10-31 1990-05-11
JPH02141665A (ja) * 1988-11-24 1990-05-31 Godo Shiyusei Kk 糞便中のヘモグロビンの検出方法

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