WO1992021952A1 - Instrument for sampling minute quantity of specimen - Google Patents

Instrument for sampling minute quantity of 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
Prior art date
Application number
PCT/JP1992/000694
Other languages
French (fr)
Japanese (ja)
Inventor
Kenji Yasuda
Shusaburo Hokukoku
Takao Ogawa
Saeko Ito
Original Assignee
Meito Sangyo Kabushiki Kaisha
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
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Application filed by Meito Sangyo Kabushiki Kaisha filed Critical Meito Sangyo Kabushiki Kaisha
Publication of WO1992021952A1 publication Critical patent/WO1992021952A1/en

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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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Abstract

An instrument for sampling a substance to be biologically examined from a specimen, which is characterized by being provided with a sampling part for biologically examined substance composed of a water absorbent material coated with a substantially water-insoluble and water-permeable porous film. By bringing said sampling part of this instrument into contact with the specimen and then with a reagent constituted of a labeled substance specifically bondable to the substance to be examined, the substance to be biologically examined in the specimen can be detected directly, promptly, and easily.

Description

明 細 書  Specification
微量検体採取用具  Small sample collection tool
技術分野  Technical field
本発明は、 生物学的被検物質、 例えば口腔内の歯周病原性細菌又は口 腔内の歯周病原性細菌に対する特異抗体の検出に使用しうる微量検体採 取用具並びにそれを用いる検体中の生物学的被検物質の測定方法及び測 定用キットに関する。  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. The present invention relates to a method for measuring a biological test substance and a kit for measurement.
背景技術  Background art
歯周病は、 歯肉炎と歯周炎に分けられ、 更に、 歯周炎には、 成人性歯 周炎、 限局性若年性歯周炎などがある。 これらの歯周病は、 歯肉の炎症、 出血、 排臁、 歯周ポケッ卜の形成、 歯根膜の破壊、 歯槽骨の吸収、 歯牙 の動揺、 歯牙の喪失にまで至る疾患である。  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.
しかしながら、 現在、 歯周病の治療法は、 まだ完全に確立されていな いのが現状であり、 できるだけ早期に歯周病を発見し、 予防的処置を施 すことが最も重要であると考えられている。 上記の歯周病の病巣局所に は、 種々の細菌が存在するが、 全細菌のうちの 3〜4割がグラム陰性の 嫌気性細菌によって占められていると言われ、 Porphyromonas gingival is (以下 P. gingivalis) ゝ Prevotella intermedia (以下 P. interme dia) 、 Actinobacillus actinomycetemcomitans (以下 A. actinomycet emcomitans) 、 Treponema denticola (以下 T. denticola) 、 Bacteroi des forsythus (以下 B. forsythus) ヽ Capnocytophaga gingivalis (以 下 C. gingivalis) ヽ Fusobacterium nucleatum (以下 F. nucleatum) ヽ Wolinella recta (以下 ^ recta) などが歯周病原性細菌であるとされ ている。 そして、 歯周病の病巣局所では、 これらの歯周病原性細菌の顕 著な増加が認められる。 なお、 Porphyromonas gingivalis^ Prevotella intermedia は、 従来の Bacteroides gingivalis^ Bacteroides inter medius に相当し、 最近の新しい分類 （1 9 9 0年の International Co nference on Periodontal Disease での T. J. M. van Steenbergen によ る） では、 このように呼ばれている。 However, at present, treatment for periodontal disease has not yet been completely established, and it is considered that it is most important to detect periodontal disease as early as possible and to take preventive measures. Have been. Various bacteria are present in the above-mentioned lesions of periodontal disease, but 30-40% of all bacteria are said to be occupied by gram-negative anaerobic bacteria, and Porphyromonas gingival is (hereinafter P gingivalis) ゝ Prevotella intermedia (P. interme dia), Actinobacillus actinomycetemcomitans (A. actinomycet emcomitans), Treponema denticola (T. denticola), Bacteroi des forsythus (B. forsythus) ヽ Capnocytophagagingival gingivalis) ヽ Fusobacterium nucleatum (F. nucleatum) ヽ Wolinella recta (hereinafter ^ recta) are considered to be periodontopathogenic bacteria. Then, at the focal point of periodontal disease, the appearance of these periodontopathic bacteria A significant increase is observed. Note that Porphyromonas gingivalis ^ Prevotella intermedia corresponds to the conventional Bacteroides gingivalis ^ Bacteroides inter medius, and according to a recent new classification (according to TJM van Steenbergen at the International Conference on Periodontal Disease in 1990). So called.
そこで、 これらの歯周病原性細菌に注目し、 歯周病の診断の捕助的手 段として、 その歯周病原性細菌の存否及びその多寡を知るために種々の 方法が提案されている。  Therefore, attention has been paid to these periodontopathic bacteria, and various methods have been proposed as a supplementary means for the diagnosis of periodontal disease in order to know the presence or absence and the amount of the periodontopathic bacteria.
例えば、 歯周病患者の歯肉溝液や歯肉緑下プラーク （歯垢） 等の歯周 ポケッ卜からの検体に棲息する歯周病原性細菌を血液寒天培地で嫌気的 条件下で培養し、 得られた種々のコロニーの詳細な生化学的性状を調べ ることにより、 歯周病原性細菌を検出することが報告されている （Loes che, f. J. , Syed, S. A. , Schmidt, E. and Morrison, E. C. ： J. Perio dont. 5 6、 4 4 7— 4 5 6、 1 9 8 5など参照） 。  For example, 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).
また、 歯周ボケットに棲息する歯周病原性細菌をグラム染色後に顕微 鏡下で調べたり、 暗視野顕微鏡によって調べたり （Listgarten, H. A. et al. ： J. Clin. Periodontol. 5、 1 1 5— 1 3 2、 1 9 7 8参照） 、 歯周病原性細菌に対する抗体と蛍光色素を組み合わせて、 歯周病原性細 菌を検出することも行われている （Zambon, J. J. , Bochacki, V. and Genco, K. J. ： Oral Microbiol. Immunol. 1 , 3 9— 4 4、 1 9 8 6 参照） 。  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).
また、 酵素抗体法 （ELISA法） を用いて、 歯周ポケットに棲息する歯 周病原性細菌を、 それぞれの歯周病原性細菌に対する抗体で検出するこ とも行われている (Zambon, J. J. , Bochacki, V. and Genco, R. J. ： Oral Microbiol. Immunol. 1、 3 9— 4 4、 1 9 8 6参照） 。 In addition, 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).
また、 歯周病原性細菌に非常に特徴的な酵素に着目し、 その酵素活性 を指標にして、 歯周ポケッ卜に棲息する歯周病原性細菌を検出すること も報告されている (Loesche, W. J. ： Oral Microbiol. Immunol. J 5 6 5— 7 0、 1 9 8 6参照） 。  In addition, it has been reported that, by focusing on enzymes that are very characteristic of periodontopathogenic bacteria, the enzyme activity is used as an index to detect periodontopathic bacteria inhabiting periodontal pockets (Loesche, WJ: Oral Microbiol. Immunol. J565-70, 19886).
更に、 最近では、 歯周病原性細菌の遺伝子に注目し、 DNA プローブ法 を用いて、 歯周ポケットに棲息する歯周病原性細菌を検出することも行 われて ヽる (Biotechnica Diagnostics 社) 0 更 ίこ、 PCR (Polymerase chain reaction) 法により、 極微量の菌体をも検出することが可能となFurthermore, recently, researchers have focused on the genes of periodontopathogenic bacteria and used DNA probe methods to detect periodontopathogenic bacteria living in periodontal pockets (Biotechnica Diagnostics) 0 Furthermore, by using the PCR (Polymerase chain reaction) method, it is possible to detect even trace amounts of bacterial cells.
1 0 つてきている。 10 have been created.
一方、 成人性歯周炎患者の歯周病原性細菌に対する抗体価については、 成人性歯周炎患者の歯肉組織での歯周病原性細菌に対する抗体産生細胞 の増加、 つまり、 特異抗体の上昇が、 ELISP0T 法を用いて報告されてい る (Tomohiko Ogawa and Shigeyuki Hamada： Clin. exp. Immunol. 7 i s 6、 1 0 3— 1 1 0、 1 9 8 9参照） 。  On the other hand, regarding the antibody titer against periodontopathogenic bacteria in adult periodontitis patients, the increase in the number of antibody-producing cells against periodontopathogenic bacteria in the gingival tissue of adult periodontitis patients, that is, the increase in specific antibodies It has been reported using the ELISP0T method (see Tomohiko Ogawa and Shigeyuki Hamada: Clin. Exp. Immunol. 7 is 6, 103-110, 1989).
しかしながら、 これらの方法は、 いずれも検出感度が低かったり、 特 異性に問題があったり、 長時間にわたる煩雑な操作と高価で特殊な装置 を必要とするために、 広く普及することは難しく、 迅速かつ簡便な歯周 病診断用試薬及び診断方法の開発が望まれている。  However, all of these methods are difficult to spread widely because of their low detection sensitivity, problems with specificity, and the need for complicated operations over a long period of time and expensive special equipment. The development of simple and convenient reagents for diagnosing periodontal disease and diagnostic methods is desired.
20 本発明者は、 歯周病を含むあらゆる疾患において、 その診断の指標と なる検体中の生物学的被検物質をそれが極微量であっても効率よく採取 することのできる用具、 それを用いて検体中の生物学的被検物質を直接 的かつ迅速、 簡便に検出することのできる測定方法及び測定用キット等 について鋭意研究を行ない、 今回、 本発明を完成するに至った。 発明の開示 20 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
本発明は、 実質的に水不溶性かつ透水性の多孔性フィルムでコーティ ングされた吸水性材料からなる生物学的被検物質採取部分を有すること を特徵とする検体中の生物学的被検物質採取用具を提供するものである。 本発明はまた、 スティック状又は短冊状の吸水性材料からなり、 該吸 水性材料の先端 1〜 5蘭 の長さからなる生物学的被検物質採取部分に 隣接する少なくとも検体と接触する可能性のある部分の表面が防水処理 されていることを特徴とする生物学的被検物質採取用具を提供するもの である。  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.
本発明はさらに、 上記の採取用具の生物学的被検物質採取部分を検体 と接触させた後、 該生物学的被検物質と特異的に結合しうる標識された 物質からなる試薬と接触させ、 該採取部分に結合した標識された試薬を 定性的又は定量的に検出することを特徵とする検体中の生物学的被検物 質の測定方法を提供するものである。  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.
本発明はさらに、 上記の採取用具と、 検体中の検出すべき生物学的被 検物質と特異的に結合しうる標識された物質からなる試薬との組合わせ よりなる検体中の生物学的被検物質の測定用キットを提供するものであ る c  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. Provide kit for measurement of test substance c
図面の簡単な説明  BRIEF DESCRIPTION OF THE FIGURES
図 1は本発明の採取用具の一例の概略断面図である。  FIG. 1 is a schematic cross-sectional view of an example of the sampling tool of the present invention.
発明の詳細な説明  Detailed description of the invention
以下、 本発明についてさらに詳細に説明する。  Hereinafter, the present invention will be described in more detail.
本発明の採取用具は、対象となる検体の種類等に応じて種々の形態を とりうるが、 一般にはスティック状 （先細りとなっていてもよい細い棒 状のもの） や短冊状 （幅の狭いシート状ないしフィルム状のもの） であ ることが望ましい。 また、 該採取用具は、 全体又は少なくともその生物 学的被検物質採取部分が吸水性材料から構成され、 その材質は吸水性の 高いものであれば、 天然、 半合成及び合成のいずれのタイプのものであ つてもよく、 例えば、 高吸水性デンプン系ポリマー、 高吸収性セルロー ス系ポリマー、 デキストラン系ポリマー、 高吸水性合成ポリマー （例え ばポリアクリル酸塩系、 ポリビニルアルコール系、 ポリアクリルァミ ド 系、 ポリオキシエチレン系のポリマー） などが挙げられ、 中でも高吸水 性セルロース系ポリマー及び高吸水性合成ポリマーが好適である。 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). In addition, 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. 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.
具体的には、 歯科分野において 「ペーパーポイント」 として知られて いる紙製スティック （例えば紙製コヨリ） が素材として好適であり、 ま た、 濾紙を幅の狭い短冊状にカツ卜したものも素材として便利に使用す ることができる。  Specifically, a paper stick (for example, paper koyori), which is known as a “paper point” in the dental field, is suitable as a material, and a filter paper cut into a narrow strip is also used as a material. Can be used conveniently.
本発明の一態様によれば、 上記の如き吸収性材料の少なくとも生物学 的被検物質採取部分、 例えば、 スティック状又は短冊状の吸収性材料の 少なくとも先端部分の表面が、 実質的に水不溶性かつ透水性の多孔性フ イルムでコーティングされる。 ςのように吸水性材料の表面を実質的に 水不溶性かつ透水性の多孔性フィルムでコーティングすることにより、 検体中の水分を該フィルムの孔を通過して内部の吸水性材料に吸収させ ると同時に、 検体中の生物学的被検物質を多？ L性フィルムコーテング上 に濃縮されながら吸着、 固定化させることが可能となり、 後の標識され た試薬による検出の際の判定が極めて容易になるという顕著な作用効果 があることが判明した。  According to one aspect of the present invention, 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. By coating the surface of the water-absorbing material with a substantially water-insoluble and water-permeable porous film as shown in ς, the water in the sample passes through the pores of the film and is absorbed by the water-absorbing material inside. At the same time, are there many biological test substances in the sample? It has been found that 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.
従って、 コーティングされる多孔性フィルムの孔径は、 検体中の水分 の透過は許すが、 検出すべき生物学的被検物質は実質的に通過しない程 度の大きさに設定することが望ましく、 例えば、 歯周病原性細菌などの 細菌の検出を目的とする場合には、 コーティングされた多孔性フィルム の平均孔径は一般に 1 0ミクロン以下、 特に 0 · 4 5〜 5. 0ミクロン程 度とすることが望ましい。 Therefore, 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 μm or less, particularly about 0.45 to 5.0 μm.
コーティングするフィルムの材質としては、 実質的に水不溶性のもの であれば特に制限されるものではなく各種のポリマ一を使用することが できるが、 具体的には例えば、 多糖の有機酸又は無機酸エステル （例え ば、 セルロースの詐酸、 プロピオン酸などの低級脂肪酸エステル又は硝 酸エステル） 、 多糖の低級アルキルエーテル誘導体 （例えば、 ェチルセ ルロース、 プロピルセルロース、 ブチルセルロースなど） 、 ポリアミ ド 系樹脂などが挙げられる。 なお、 ポリマーは陽電荷または陰電荷を有し ていてもよい。  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.
前記吸収性材料にかかる材質の多孔性フィルムのコーティングを形成 する方法としては、 それ自体既知の方法を採用することができ、 例えば、 セルロース系膜は相分離法で製膜することができ、 具体的には、 セル口 ースをアセトンや塩化メチレンなどの良溶媒に溶解し、 更に、 これより も沸点の高いエタノールゃ水などの非良溶媒を加え、 均一に混合した溶 液を作り、 その溶液を支持体の上に流延し、 良溶媒と非良溶媒との沸点 の差を利用して、 良溶媒を選択的に乾燥除去し、 更に、 非良溶媒を乾燥 除去することによって、 均一で微細な多孔質膜を作ることができる。 吸収性材料上の多孔性フィルムコーテングは、 少なくとも採取用具の 生物学的被検物質採取部分全体を覆うようにして施すことができ、 例え ばスティック状又は短冊状の吸水性材料の場合には、 少なくともその先 端部分を覆うように施され、 そのコーテング部分の長さは先端から少な くとも l mm 、 好ましくは 2關以上とすることができる。 As 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. For example, 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. 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.
また、 本発明の採取用具の生物学的被検物質採取部分は必要以上に大 きくする必要はなく、 後の検出操作に支障をきたさない範囲でできるだ Further, it is not necessary to increase the size of the biological test substance collection portion of the collection tool of the present invention more than necessary, as long as it does not hinder the subsequent detection operation.
5 け最小限にとどめることが望ましい。 そのための一手段として、 採取用 具の生物学的披検物質の採取に必要とされる部分 （生物学的被検物質採 取部分） 以外の少なくとも検体と接触する可能性のある部分の表面を防 水処理する方法が挙げられる。 或いはまた別法としてコーティングが施 された吸水性材料の小片を適当な支持具に固定することも可能である。 It is desirable to keep it to a minimum. 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. Alternatively, a small piece of coated water-absorbent material can be secured to a suitable support.
1 0 これにより、 採取具の多孔性フィルムでコーティングされた限られた部 分でのみ、 検体が集中的に吸収されるようになるので、 その部分におけ る生物学的被検物質の吸着、 固定化がより一層効果的に行なわれ、 標識 された試薬による判定がより一層容易になる。 10 This allows the sample to be intensively absorbed only in the limited portion of the collection device coated with the porous film, and the absorption of the biological analyte in that portion, The immobilization is performed more effectively, and the determination using the labeled reagent is further facilitated.
防水処理は例えば、 コーティングしたスティック状又は短冊状の吸水 i s 材料の先端 l〜5 mm 、 好ましくは 2〜4難 の長さからなる生物学的被 検物質採取部分に隣接する少なくとも検体と接触する可能性のある部分 の表面に施すことができる。  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.
防水処理の方法としては、 多孔性フィルムのコーテングの生物学的被 検物質採取部分を除いた部分を該フィルムを溶解しうる溶剤で処理して As a waterproofing method, 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.
20 該コーティングの孔をふさぐ方法；実質的に水に不溶性かつ非吸水性の ポリマー （例えば、 フッ素樹脂、 シリコーン樹脂、 塩化ビニル樹脂、 メ タァクリール樹脂、 ポリエステル樹脂、 セルロイ ド、 パラフィン、 ゴム、 木ろう、 牛脂など） からなる防水コーティングを施す方法等が挙げられ る n 以上に述べた本発明の採取用具の一例を添付の第 1図に示す。 この図 は本発明の採取用具の概略断面図であり、 本図において （1 ) はスティ ック状の吸水性材料 （これは短冊状の吸水性材料であってもよい） であ り、 その先端部分には、 ニトロセルロース、 セルロースアセテート等の 水に実質的に不溶性かつ透水性の多孔性フィルム （2 ) がコーティング されており、 そしてさらに、 先端の所定長さの生物学的被検物質採取部 分 （3 ) を残して、 検体と接触する可能性のある部分には防水処理 （4 ) が施されている。 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.) An example of the above-mentioned sampling tool of the present invention is shown in FIG. This figure is a schematic cross-sectional view of the sampling tool of the present invention. In this figure, (1) is a stick-shaped water-absorbing material (this may be a strip-shaped water-absorbing material). The tip is coated with a water-insoluble and water-permeable porous film (2), such as nitrocellulose and cellulose acetate. Except for part (3), the part that may come into contact with the specimen is waterproofed (4).
また、 本発明の採取用具の別の簡略化した態様として多孔性フィルム のコーティングを施すことなく、 単に、 スティック状又は短冊状の吸水 性材料の先端 l〜5 mm、 好ましくは 2〜4腿 の長さからなる生物学的 被検物質採取部分を残し、 該吸水性材料のそれ以外の部分の少なくとも 検体と接触する可能性のある部分に前述した如く して防水コーティング を施すことにより、 本発明の採取用具を構成することもできる。  Further, as another simplified embodiment of the sampling tool of the present invention, without coating with a porous film, the tip of a stick-shaped or strip-shaped water-absorbing material is simply l to 5 mm, preferably 2 to 4 thighs. By providing a waterproof coating as described above on at least a portion of the water-absorbent material that may come into contact with a sample, leaving a biological sample collection portion having a length, Can also be configured.
この態様の採取用具は、 生物学的披検物質採取部分に、 実質的に水不 溶性かつ透水性の多孔性フィルムのコーティングが存在せず、 従って該 コーティングによって達成される前述した如き作用効果は享受しえない が、 しかし、 この態様の採取用具にあっても、 局限された先端部でのみ 集中的に検体が吸収され、 そこに生物学的披検物質をかなりの濃度で吸 着、 固定化することが可能となる。  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. Although it is not enjoyable, however, even with this type of collection tool, 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.
以上に述べた本発明の採取用具を用いれば、 各種の体液、 例えば、 血 液、 唾液、 涙、 汗、 鼻水、 尿、 便、 ***、 膣分泌液、 膿、 喀痰などに含 まれる血液成分、 血清、 各種細胞、 サイトカイン、 ホルモン、 ペプチド、 代謝産物、 酵素、 生理活性物質、 腫瘍マーカー、 遺伝子 （DNA、 RNA) 、 レセプター、 抗体、 抗原、 微生物、 ウィルス等の生物学的被検物質を、 極めて簡単な操作で効率よく採取することができる。 例えば歯周病に関 していえば、 本発明のスティック状の採取用具を被検者の歯と歯の間、 歯と歯茎の間、 歯周ポケットなどに挿入することによって、 歯肉溝液、 唾液などから、 歯周病原性細菌又はその特異抗原、 或いは歯周病原性細 菌又はその特異抗原に対する抗体を簡単に採取することができる。 また、 検体中の生物学的被検物質を採取する場合に、 必要に応じて、 採取用具を適当な吸引源 （又は真空源） に接続することも可能であり、 それにより、 生物学的被検物質の採取をより効果的に行なうことができ ることもある。 By using the above-described collection tool of the present invention, 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. For example, regarding periodontal disease, 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. For example, periodontopathogenic bacteria or their specific antigens, or antibodies against periodontal pathogenic bacteria or their specific antigens can be easily collected. When collecting a biological test substance in a sample, 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.
次に、 本発明の採取用具を用いる検体中の生物学的被検物質の測定方 法について説明する。  Next, a method for measuring a biological test substance in a sample using the collection tool of the present invention will be described.
本発明の採取用具を用いての生物学的被検物質の採取は、 本発明の採 取用具の生物学的被検物質採取部分を前記の如き検体と接触させること により行なわれる。  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.
本発明の採取用具を検体と接触させると、 前述したように、 その採取 部分に生物学的被検物質が濃縮されて吸着、 固定化される。  When the collection tool of the present invention is brought into contact with a sample, the biological test substance is concentrated and adsorbed and immobilized on the collection portion as described above.
このように生物学的被検物質が吸着、 固定化された採取用具は、 次い で検出すべき生物学的被検物質と特異的に結合しうる標識された物質か らなる試薬溶液と接触させる。  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.
生物学的被検物質と特異的に結合する物質 （以下、 これを 「特異的結 合性物質」 という） としては、 免疫学的又は化学的に生物学的被検物質 と特異的に結合する抗体、 抗原、 核酸などが挙げられる。 例えば、 歯周 病に関していえば、 歯周病原性細菌又はその特異抗原に対する抗体、 あ るいは歯周病原性細菌の特異抗原などが挙げられる。 A substance that specifically binds to a biological test substance (hereinafter referred to as a “specific binding substance”) specifically binds to a biological test substance immunologically or chemically. Examples include antibodies, antigens, nucleic acids and the like. For example, regarding periodontal disease, antibodies to periodontopathogenic bacteria or their specific antigens, Or specific antigens of periodontopathogenic bacteria.
これらの特異的結合物質は標識される。 標識用の物質としては、 免疫 学的測定又は、 化学的測定の分野で屡々使用される各種の標識用物質を 使用することができ、 例えば、 着色物質、 着色コロイド粒子、 蛍光色素、 These specific binding substances are labeled. As the 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,
5 放射性同位元素、 酵素などが挙げられる。 5 Radioisotopes, enzymes, etc.
着色物質としては、 例えば、 無機顔料、 有機顔料などが挙げられる。 着色コロイド粒子としては、 例えば、 金コロイド粒子などの金属コロイ ド粒子、 着色されたポリマーラテックス粒子などのポリマー粒子、 着色 したゼラチン粒子などの蛋白系粒子、 着色したコロイダルシリ力粒子、 1 0 着色したコロイダルアルミナ粒子、 着色した多糖粒子などが挙げられ、 これら粒子の粒径は一般に 1〜1 0 O nm、 好ましくは 5〜4 O nm の範 囲内にあることが望ましい。 このような金属コロイド粒子、 例えば金コ ロイド粒子は、 通常、 塩化金酸 （HAuCl · 2 H20) 1 O mg を沸騰超蒸留 水 1 0 O ml に溶かし、 調製直後の 1 %クェン酸 3ナトリウム水溶液を i s 素早く適当量加え、 5〜1 0分間沸騰すると赤色の透明な液となり、 2 〜 3分後に緩やかに冷却することにより金コロイド粒子を調製すること ができる。 Examples of the coloring substance include an inorganic pigment and an organic pigment. Examples of 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.
また標識用物質としての蛍光色素としては、 例えばフルォレセィンィ ソチアネート （FITC) 、 テトラローダミンイソチアネート （TRITC) な o どが挙げられ、 放射性同位元素としては、 例えば3 H、 1 4 C、 32 P、 1 25 Iなどが包含され、 酵素としては、 例えばペルォキシダ一ゼ、 アルカリ フォスファターゼなどの基質を分解して発色させる酵素が好適である。 特に、 アルカリフォスファターゼは、 その基質として、 5—ブロモ一 4一クロ口一 3—インドリルフォスフェイ ト又は p—二トロブルーテト ラゾリウムクロリ ドとの組み合わせが挙げられる。 これらの基質は無色 透明で水可溶性であるが、 酵素の働きにより分解され、 それぞれ固有の 色を呈し、 水不溶性になり、 標識した物質の存在を知ることができる。 このような物質による特異結合性物質の標識付けの方法には、 物理的 結合法と化学的結合法とがある。 前者は、 特異的結合性物質と標識用物 質を混合するだけで物理的に吸着結合させる方法であり、 後者は、 特異 的結合性物質と標識用物質の各々が有する官能基との間で直接的に共有 結合をするか、 又はカルボジィミ ド、 グルタルアルデヒドなどの二価性 試薬を用い、 特異的結合性物質と標識用物質を間接的に共有結合させる 方法である。 As the fluorescent dye as a labeling substance, for example Furuoresini Sochianeto (FITC), etc. tetrarhodimine isothiocyanate (TRITC) o. Examples of 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. In particular, 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. These 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.
前述した検体と接触させた採取用具を以上に述べた如き標識された特 異結合物質からなる試薬と接触させ、 必要により洗浄して余分の試薬を 除去した後、 用いた標識の種類に応じて、 目視、 デンシトメーター、 比 色計、 放射線測定装置等により採取用具に結合している標識の量を測定 し、 そしてさらにその測定結果を必要に応じて予め作成した標準曲線な どと対比することにより、 検体中の生物学的被検物質の量を定性的又は 定量的に検出することができる。  After contacting the collection tool contacted with the sample described above with a reagent consisting of the labeled specific binding substance as described above, washing as necessary to remove excess reagent, and depending on the type of label used, 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.
以上に述べた測定系で使用される本発明の採取用具と、 標識された特 異結合性物質からなる試薬とは、 組合わせてキット化することにより、 検体中の生物学的被検物質の測定用キットとすることができる。  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.
以下、 本発明の採取用具を用いる検体中の生物学的被検物質の測定方 法を、 歯肉溝液、 唾液又は歯垢処理液等の検体中の歯周病原性細菌もし くはその特異抗原又はそれらに対する抗体等を検出する場合についてさ らに詳細に説明する。 (1) 歯周病原性細菌の培養 Hereinafter, 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. (1) Culture of periodontopathogenic bacteria
歯周病診断用キットを調製するために、 まず、 歯周病原性細菌である とされている P- gingivalis, 例えば、 gingivalis 381株、 gingivalis 1021株、 gingivalis ATCC 33277株、 gingi valis EB 22 D— 1株、 gingivalis RB 24 M— 2株、 gingivalis RB46D— 1株、 £^ gingivalis 6/26株、 gingivalis ， 1株、 ^ gingivalis W 50株、 ingivalis f 83株、 gingivalis HI 1 ID— 5株、 P. gingivalis Hff24D— 1株、 P. gingivalis BHl 8/1 0株、 P, gingivalis 19A4株、 gingivalis 22B4株、 ingi i o valis 23A4株、 L_ gingivalis HG 379株、 gingivalis HG 4  To prepare a kit for diagnosing periodontal disease, first, 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. gingivalis BHl 8/10 strain, P, gingivalis 19A4 strain, gingivalis 22B4 strain, ingi io valis 23A4 strain, L_gingivalis HG 379 strain, gingivalis HG 4 strain
05株、 P. gingivalis OMZ 314株、 ingivalis OMZ 369株、 P. gingivalis OMZ 409株、 ^ gingivalis LB13D— 3株；または、 intermedia. 例えば、 P. intermedia ATCC 25261株、 inter media ATCC 25611株、 P- intermedia OMZ 227株、 P. interraed 05, P. gingivalis OMZ 314, ingivalis OMZ 369, P. gingivalis OMZ 409, ^ gingivalis LB13D-3; or intermedia. For example, P. intermedia ATCC 25261, intermedia ATCC 25611, P- intermedia OMZ 227 strains, P. interraed
I 5 ia OMZ 248株、 P. intermedia OMZ 277株、 P. intermedia OMZ I5ia OMZ 248 strain, P. intermedia OMZ 277 strain, P. intermedia OMZ
311株、 P. intermedia OMZ 326株、 intermedia OMZ 327株、 P. intermedia BH 18 23株、 intermedia BH 20ノ 30株、 ^ intermedia H 187株、 ^ intermedia M 107-74株；または、 A. act inomycetemcomi tans N 例えば、 A. actinomycetemcomitans ATCC 0 29522株、 A. act inomycetemcomi tans ATCC 29523株、 A. ac t i noiyce temcomi tans ATCC 29524株、 A. actinomycetemcomitans Y 4株、 A, actinomycetemcomitans NCTC 9709株、 A. actinoiycet eracomitans NCTC 9710株、 A. actinomycetemcomitans 1株、 A. ac tinomycetemcofflitans 15株ヽ A. actinomycetemcomitans 27株ヽ A. actinomycetemcomitans 2 9株 、 ；^ actinomycetemcomitans 3 2株ヽ A. actinomycetemcomitans 3 9株、 actinomycetemcomitans 4 2株ヽ A. actinomycetemcomitans—6 7株 、 A. actinomycetemcomitans 7 5 株、 actinomycetemcomitans 8 5株；または、 T.. denticola、 例え ば、 L denticola ATCC 3 5 4 0 5 ；または、 forsythus、 例えば、 B. forsvthus ATCC 4 3 0 3 7 ；または、 ^ _ gingivalis、 例えば、 C. gingival is ATCC 3 3 6 2 4 ；または、 ^ nucleatum、 例えば、 F. nu cleatum ATCC 2 5 5 8 6 ;または、 recta, 例えば、 W. recta ATCC311 strains, P. intermedia OMZ 326 strains, intermedia OMZ 327 strains, P. intermedia BH 1823 strains, intermedia BH 20 no 30 strains, ^ intermedia H 187 strains, ^ intermedia M 107-74 strains; or 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 ヽ A. actinomycetemcomitans 27 strains ヽ A. actinomycetemcomitans 29, ^; ^ actinomycetemcomitans 32, 2 ヽ A. actinomycetemcomitans 39, actinomycetemcomitans 42, 2 ヽ 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
3 3 2 3 8などを、 例えば、 GAM ブイョン、 Brain - Heart Infusion ブロスなどにへミン、 メナジオンを添加した培地、 または、 Todd - Hew itt broth に酵母エキスを添加した培地などに接種して、 嫌気的条件下 で約 3 7 °Cで約 2 0時間培養した後、 これらを集菌して、 生理食塩水に て洗浄し、 凍結乾燥菌体にする。 このようにして得られる菌体を標識物 質に結合させるか、 あるいはその特異抗体を得るための免疫抗原として 使用する。 Inoculate 3 3 2 3 8 etc. into 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 for obtaining a specific antibody.
( 2 ) 線毛抗原の調製  (2) Preparation of fimbrial antigen
歯周病原性細菌の特異抗原に対する抗体を得るために、 まず、 特異抗 原の調製として、 特異的抗原性を持つ線毛を有している歯周病原性細菌、 例えば、 P. gingivalis を、 例えば、 GAM ブイヨン、 Brain - Heart In fusion ブロスなどにへミン、 メナジオンを添加した培地に接種して、 嫌気的条件下で約 3 7 °Cで培養すれば、 約 2 0時間またはそれ以後に多 数の菌体が得られる。 これを集菌して、 ピペッティングにより物理的に 線毛を剥離する。 次に、 線毛を硫安分画、 イオン交換クロマトグラフィ 一を経て精製すれば、 P. gingivalis の線毛抗原が得られる。 このよう にして得られる線毛抗原を標識物質に結合させるか、 あるいはその特異 抗体を得るための免疫原として使用する。 In order to obtain antibodies against specific antigens of periodontopathogenic bacteria, first, as preparation of specific antigen, periodontopathogenic bacteria having pili with specific antigenicity, such as 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.
( 3 ) 多糖抗原の調製  (3) Preparation of polysaccharide antigen
歯周病原性細菌、 例えば、 A. actinomycetemcoiiiitans を、 例えば、 T odd - Hefitt broth に酵母エキスを添加した培地に接種して、 嫌気的 条件下で約 3 7でで培養すれば約 2 0時間、 または、 それ以後に多数の 菌体が得られる。 これを集菌して、 凍結乾燥菌体にする。 得られた凍結 乾燥菌体を生理食塩水に懸濁した後、 ォートクレイプにかけ熱抽出し、 その抽出液を例えば、 イオン交換クロマトグラフィー、 ゲル濾過クロマ トグラフィ一などで分離、 精製して、 A. actinomycetemcoiiiitans の多 糖抗原を得ることができる。 このようにして得られる多糖抗原を標識物 質に結合させるか、 あるいはその特異抗体を得るための免疫原として使 用する。  Inoculating 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. Can be obtained. The polysaccharide antigen thus obtained is bound to a labeling substance or used as an immunogen to obtain a specific antibody.
( 4 ) 抗体の調製  (4) Preparation of antibody
上記のごとくして得られる歯周病原性細菌の凍結乾燥菌体を、 種々の 動物に免疫する。 例えば、 この歯周病原性細菌の凍結乾燥菌体をフロイ ントの完全アジュバントと共にゥサギ、 ャギなどの動物の皮下または筋 肉内に注射して免疫した後、 これらの動物の血液からそれ自体既知の方 法に従って歯周病原性細菌に対する抗血清を得ることができる。 更に、 必要に応じて、 得られる抗血清を常法に従い硫安分画、 イオン交換クロ マトグラフィ一で精製する。  The freeze-dried cells of periodontopathogenic bacteria obtained as described above are immunized to various animals. For example, 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. According to the method described above, an antiserum against periodontopathogenic bacteria can be obtained. Further, if necessary, the obtained antiserum is purified by ammonium sulfate fractionation and ion exchange chromatography according to a conventional method.
—方、 上記のごとくして得られる歯周病原性細菌の凍結乾燥菌体をマ ウスに免疫し、 この脾細胞を取り出し、 マウス骨髄腫瘍細胞とポリェチ レングリコール等を用いて細胞融合させ、 クローニングした後、 歯周病 原性細菌に特異的に反応するモノクローナル抗体を分泌するハイプリ ド 一マを選択する。 これらのハイブリ ドーマをあらかじめプリスタン処置 しておいたマウスの腹腔内に移植し、 その腹水よりモノクローナル抗体 を得るか、 またはハイプリ ドーマを無血清培地で大量培養し、 その培養 上清中よりモノクローナル抗体を得ることができる。 更に、 得られるモ ノクローナル抗体を常法に従い、 硫安分画、 イオン交換クロマトグラフ ィ一で精製する。 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. After the periodontal disease Select a hybridoma that secretes a monoclonal antibody that specifically reacts with the protogenic bacterium. 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. Obtainable. Further, the obtained monoclonal antibody is purified by ammonium sulfate fractionation and ion exchange chromatography according to a conventional method.
また、 上記のごとく して得られる ^ gingivalis の線毛抗原又は ^ actinomycetemcomitans の多糖抗原を動物に免疫する 0 例えば、 これ らの特異抗原をフロイン卜の完全アジュバントと共にゥサギ、 ャギなど の動物の皮下又は筋肉内に注射して免疫した後、 これらの動物の血液か らそれ自体既知の方法に従って gingivalis の線毛抗原又は act inomycetemcomitans の多糖抗原に対する抗血清を得ることができる。 更に、 必要に応じて、 得られる抗血清を常法に従い硫安分画、 イオン交 換クロマトグラフィーで精製する。 Further, obtained by as above ^ 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 Alternatively, after immunization by intramuscular injection, 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.
一方、 上記のごとくして得られる P. gingivalis の線毛抗原又は A. actinomycetemcomitans の多糖抗原をマウスに免疫し、 この脾細胞を 取り出し、 マウス骨髄腫瘍細胞とポリエチレングリコール等を用いて細 胞融合させ、 クローニングした後、 P. gingivalis の線毛抗原又は actinomycetemcomitans の多糖抗原に 異的に反応するモノクロ一ナノレ 抗体を分泌するハイプリ ドーマを選択する。 これらのハイプリ ドーマを あらかじめプリスタン処置しておいたマウスの腹腔内に移植し、 その腹 水よりモノクローナル抗体を得るか又はハイプリ ドーマを無血清培地で 大量培養し、 その培養上清中よりモノクローナル抗体を得ることができ る。 更に、 得られたモノクローナル抗体を常法に従い硫安分画、 イオン 交換クロマトグラフィーで精製する。 On the other hand, 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. 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 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.
( 5 ) 着色コロイ ド粒子による抗体の標識  (5) Labeling antibodies with colored colloid particles
上記のごとくして得られる歯周病原性細菌の全菌体又はその特異抗原、 5 例えば、 P. gingival is の線毛抗原、 A. actinomycetemcomi tans の多 糖抗原などに対する抗血清又はモノクローナル抗体に着色コロイド粒子 を結合させる。 そのためにはそれ自体既知の方法を用いることができる。 一般的には、 着色コロイド粒子を pH 7〜9の炭酸緩衝液、 リン酸緩 衝液、 トリス塩酸緩衝液又はグリシン緩衝液などに浮遊させ、 そこに前 t o 記抗体を加え、 室温ないし約 3 7 °Cで 1 5分〜 9 0分間撹拌し、 着色コ ロイド粒子で抗体を標識する。 次に、 着色コロイド粒子で標識した抗体 を安定化、 保護するために、 ゥシ血清アルブミン、 ポリエチレングリコ —ル、 ショ糖、 塩化コリンなどを加え、 更に、 室温ないし約 3 7 °Cで 1 5分〜 9 0分間撹拌する。 このようにして抗体を着色コロイド粒子で標 5 識することができる。 これを以下着色コロイド標識抗体溶液という。  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. For this purpose, 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. Next, in order to stabilize and protect the antibody labeled with the colored colloidal particles, add serum albumin, polyethylene glycol, sucrose, choline chloride, and the like. Stir for minutes to 90 minutes. In this way, the antibody can be labeled with colored colloid particles. This is hereinafter referred to as a colored colloid-labeled antibody solution.
( 5 ' ) 酵素による抗体の標識  (5 ') Labeling of antibody with enzyme
上記のごとく して得られる歯周病原性細菌の全菌体又はその特異抗原、 例え ίま、 P. gingivalis の線 抗原、 A. actinomycetemcomi tans の多 糖抗原などに対する抗血清又はモノクローナル抗体に酵素を結合させる。 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.
0 そのためには、 それ自体既知の方法を用いることができる。 0 For this purpose, a method known per se can be used.
一般的には、 抗体を 2 0 mgZml になるように 0. 1 M リン酸緩衝生 理食塩水に溶解する。 この抗体溶液 2 ml に、 標識用物質としての酵素、 例えば、 アルカリフォスファターゼ 4 O mg を加えてよく溶解する。 静 かに撹拌しながら、 1 %グルタルアルデヒド 0. 1 ml を滴下する。 室温 で 2時間反応させ、 ゲル濾過により、 未結合の抗体及びアルカリフォス ファターゼから、 アル力リフォスファターゼ標識抗体を分離 ·精製する。 このようにして、 抗体をアル力リフォスファターゼで標識することがで きる。 これを以下、 アルカリフォスファターゼ標識抗体溶液という。 Generally, the antibody is dissolved in 0.1 M phosphate buffered saline to a concentration of 20 mgZml. To 2 ml of this antibody solution, add an enzyme as a labeling substance, for example, 4 O mg of alkaline phosphatase, and dissolve well. With gentle stirring, add 0.1 ml of 1% glutaraldehyde dropwise. room temperature For 2 hours. Separate and purify the antibody labeled with alkaline phosphatase from unbound antibody and alkaline phosphatase by gel filtration. In this way, the antibody can be labeled with alkaline phosphatase. This is hereinafter referred to as alkaline phosphatase-labeled antibody solution.
5 ( 6 ) 着色コロイ ド標識抗体による抗原の検出  5 (6) Detection of antigen using colored colloid-labeled antibody
歯周病患者の歯肉溝液、 唾液又は歯垢処理液を本発明の採取用具と接 触させて、 採取用具に検体を採取する。 次に、 この採取用具の生物学的 被検物質採取部分を前述の着色コロイ ド標識抗体溶液に浸潰し、 室温な いし約 3 7 °Cで静置又は撹拌しながら 5〜1 5分間反応させる。 しかる 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. Next, 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. . Scold
1 0 後採取用具を取り出して、 蒸留水で洗浄した後に、 肉眼又はデンシトメ 一ターなどの測定機器を用いて判定することができる。 歯周病患者の検 体が付着していたところが着色コロイ ド粒子の色に染まっていたら、 陽 性であると判定する。 この時、 その色の程度は歯周病原性細菌の存否並 びに多寡と相関しているので菌体の数を定量することもできる。 10 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.
i s 更に、 着色コロイ ド粒子が金コロイ ド粒子の場合、 銀染色により感度 を増すことができる。 i s Furthermore, when the colored colloid particles are gold colloid particles, the sensitivity can be increased by silver staining.
( 6 ' ) アルカリフォスファターゼ標識抗体による抗原の検出 歯周病患者の歯肉溝液、 唾液又は歯垢処理液を本発明の採取用具と接 触させて、 採取用具に検体を採取する。 次に、 この採取用具の生物学的 (6 ′) Detection of Antigen by Alkaline Phosphatase-Labeled Antibody 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. Next, the biological
20 被検物質採取部分を前述のアル力リフォスファターゼ標識抗体溶液に浸 漬し、 室温ないし約 3 7 °Cで、 静置又は撹拌しながら 5〜1 0分間反応 させる。 次に Tween 2 0を含むリン酸緩衝生理食塩水でよく洗浄した 後、 5—プロモー 4一クロ口一3—インドリルフォスフェイ トを含む基 質溶液へ浸漬し、 室温ないし約 3 7 °Cで、 静置又は撹拌しながら 5〜1 0分間反応させる。 しかる後、 採取用具を取り出して、 蒸留水で洗浄し た後に、 肉眼又はデンシトメ一ターなどの測定機器を用いて判定するこ とができる。 歯周病患者の検体が付着していたところが青色に染まって いたら、 陽性であると判定する。 この時、 その色の程度は歯周病原性細 菌の存否並びに多寡と相関しているので菌体の数を定量することもでき る o 20 Immerse the test substance in the above-mentioned solution of the alfa phosphatase-labeled antibody, and allow it to react at room temperature to about 37 ° C for 5 to 10 minutes while standing or stirring. Next, wash well with phosphate buffered saline containing Tween 20 and immerse it in a base solution containing 5-promote 4-monochrome 13-indolyl phosphate, and use at room temperature or about 37 ° C. 5-1 while standing or stirring Incubate for 0 minutes. Thereafter, 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 correlates with the presence or absence of periodontopathogenic bacteria, and the number of cells can be quantified.o
( 7 ) 着色コロイド粒子による抗原の標識  (7) Labeling of antigen with colored colloid particles
上記のごとくして得られる歯周病原性細菌の特異抗原、 例えば、 ^ gingivalis の線毛抗原、 A. actinomycetemcomitans の多糖 ίτι原などに 着色コロイド粒子を結合させるには、 それ自体既知の方法を用いること ができる。  To bind colored colloid particles to the specific antigen of periodontopathogenic bacteria obtained as described above, for example, ^ gingivalis fimbrial antigen, A. actinomycetemcomitans polysaccharide ίτιprogen, use a method known per se be able to.
一般的には、 着色コロイド粒子を ρΗ 7〜9の炭酸緩衝液、 リン酸緩 衝液、 トリス塩酸緩衝液又はグリシン緩衝液などに浮遊させ、 そこに前 記抗原を加え、 室温ないし約 3 7 °Cで 1 5分〜 9 0分間撹拌し、 着色コ ロイド粒子で抗原を標識する。 次に、 着色コロイド粒子で標識した抗原 を安定化、 保護するために、 ゥシ血清アルブミン、 ポリエチレングリコ ール、 ショ糖、 塩化コリンなどを加え、 更に、 室温ないし約 3 7 で 1 5分〜 9 0分間撹拌する。 このようにして抗原を着色コロイド粒子で標 識することができる。 これを以下着色コロイド標識抗原溶液という。  Generally, the colored colloidal particles are suspended in a carbonate buffer, phosphate buffer, Tris-HCl buffer or glycine buffer with ρΗ7 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. Next, in order to stabilize and protect the antigen labeled with the colored colloid particles, add serum albumin, polyethylene glycol, sucrose, choline chloride, etc., and further add room temperature to about 37 for 15 minutes. Stir for 90 minutes. In this way, the antigen can be labeled with the colored colloid particles. This is hereinafter referred to as a colored colloid-labeled antigen solution.
( 7 ' ) 酵素による抗原の標識  (7 ') Labeling of antigen with enzyme
上記のごとくして得られる歯周病原性細菌の特異抗原、 例えば、 ^ gingivalis の線毛抗原、 に actinomycetemcomitans の多糖抗原などに 酵素を結合させるには、 それ自体既知の方法を用いることができる。 一般的には、 抗原を 2 0 mg/ml になるように 0. 1 M リン酸緩衝生 理食塩水に溶解する。 この抗原溶液 2 ml に、 標識用物質としての酵素、 例えば、 アルカリフォスファタ一ゼ 4 O mg を加えてよく溶解する。 静 かに撹拌しながら、 1 %グルタルアルデヒド 0. l ral を滴下する。 室温 で 2時間反応させ、 ゲル濾過により、 未結合の抗原及びアルカリフォス ファターゼから、 アル力リフォスファターゼ標識抗原を分離 ·精製する c このようにして、 抗原をアル力リフォスファターゼで標識することがで きる。 これを以下、 アルカリフォスファターゼ標識抗原溶液という。 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. Generally, 0.1 M phosphate buffered saline is used to bring the antigen to 20 mg / ml. Dissolve in saline. To 2 ml of this antigen solution, add 4 Omg of an enzyme as a labeling substance, for example, alkaline phosphatase, and dissolve well. While stirring gently, add 0.1% of 1% glutaraldehyde dropwise. 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.
( 8 ) 着色コロイ ド標識抗原による抗体の検出  (8) Detection of antibodies using colored colloid-labeled antigen
歯周病患者の歯肉溝液、 唾液又は歯垢処理液を本発明の採取用具と接 触させて、 採取用具に検体を採取する。 次に、 この採取用具の生物学的 被検物質採取部分を前述の着色コロイ ド標識抗原溶液に浸漬し、 室温な いし約 3 7 °Cで静置又は撹拌しながら 5〜1 0分間反応させる。 しかる 後採取用具を取り出して、 蒸留水で洗浄した後に、 肉眼又はデンシトメ 一ターなどの測定機器を用いて判定することができる。 歯周病患者の検 体が付着していたところが着色コロイド粒子の色に染まっていたら、 陽 性であると判定する。 この時、 その色の程度は歯周病原性細菌に対する 抗体の有無並びに多寡と相関しているので、 菌周病原性細菌に対する抗 体価を定量することもできる。  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. Next, 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. . Then, after removing the sampling tool and washing it with distilled water, 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.
更に、 着色コロイ ド粒子が金コロイ ド粒子の場合、 銀染色により感度 を増すことができる。  Further, when the colored colloid particles are gold colloid particles, the sensitivity can be increased by silver staining.
( 8 ' ) アルカリフォスファタ一ゼ標識抗体による抗体の検出  (8 ') Detection of antibody using alkaline phosphatase labeled antibody
歯周病患者の歯肉溝液、 唾液又は歯垢処理液を本発明の採取用具と接 触させて、 採取用具に検体を採取する。 次に、 この採取用具の生物学的 被検物質採取部分を前述のアル力リフォスファターゼ標識抗原溶液に浸 漬し、 室温ないし約 3 7でで、 静置又は撹拌しながら 5〜1 0分間反応 させる。 次に T een 2 0を含むリン酸緩衝生理食塩水でよく洗浄した 後、 5—ブロモー 4一クロロー 3—ィンドリルフォスフェイトを含む基 質溶液へ浸潰し、 室温ないし約 3 7でで、 静置又は撹拌しながら 5〜1 0分間反応させる。 しかる後、 採取用具を取り出して、 蒸留水で洗浄し た後に、 肉眼又はデンシトメ一ターなどの測定機器を用いて判定するこ とができる。 歯周病患者の検体が付着していたところが青色に染まって いたら、 陽性であると判定する。 この時、 その色の程度は歯周病原性細 菌に対する抗体の有無並びに多寡と相関しているので、 歯周病原性細菌 に対する抗体価を定量することもできる。 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. Next, 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. Next, after washing well with phosphate buffered saline containing Teen 20 and immersing in a base solution containing 5-bromo-4-chloro-3-indolyl phosphate, at room temperature or about 37, The reaction is carried out for 5 to 10 minutes while standing or stirring. Thereafter, 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.
( 9 ) 血液などによる汚れの脱色  (9) Decolorization of dirt by blood etc.
検体採取の際に採取用具が血液や膿などによって汚れた場合には、 過 酸化水素水などの酸化剤で脱色した後に、 着色コロイ ド標識抗体溶液又 は着色コロイド標識抗原溶液と反応させることが好ましい。  If 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.
以上に述べた本発明によれば、 歯周病の患者の歯周病巣部における歯 周病原性細菌の存否並びに多寡又は歯周病の患者の歯周病巣部における 歯周病原性細菌に対する特異抗体の有無並びに多寡を、 極めて簡単な操 作で迅速に検出可能であり、 短時間内に本疾患を診断しまた病態の程度 を把握できる。 しかも、 歯周病原性細菌の存否並びに多寡と、 歯周病原 性細菌に対する特異抗体の有無並びに多寡を組み合わせて調べることに より、歯周病の病態の様々な段階や治癒の状態を時間を追って知ること ができ、 治療や再発防止に役立てることが可能となる。  According to the present invention described above, 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 The presence or absence of the 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. In addition, by examining the presence and absence of periodontopathic bacteria and the presence or absence of specific antibodies against periodontopathic bacteria in combination, 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.
次に、 本発明を調製例、 試験例及び実施例によって更に具体的に説明 するが、 本発明はこれらに限定されるものではない。 実施例 Next, the present invention will be described more specifically with reference to Preparation Examples, Test Examples and Examples, but the present invention is not limited to these. Example
調製例 1 Preparation Example 1
歯周病原性細菌の凍結乾燥菌体の調製  Preparation of freeze-dried cells of periodontopathogenic bacteria
gingivalis と intermediaを、 へミン ( 5 mg/1) 及びメナジ オン （lmg 1) 添加 GAMブイヨン培地 （日水製薬） に接種し、 37°C、 80% N2、 10% H2、 10% CO 2 という嫌気的条件下で大量培養後、 遠心分離により集菌した。 また、 A. actinomycetemcomitans はゝ Todd - Hewitt broth (Difco Laboratories) に 1 %酵母エキスを添加した培 地で、 37°C、 5% CO 2 という嫌気的条件下で大量培養後、 遠心分離 により集菌した。 これらの菌体を生理食塩水にて洗浄した後、 減圧下凍 結 feした。. 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 After mass culture under anaerobic conditions of 2, the cells were collected by centrifugation. 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. .
調製例 2 Preparation Example 2
Ρ^. gingivalis 381株、 及び、 HW24 D— 1株からの線毛抗原の調 製  Preparation of fimbrial antigen from Ρ ^. Gingivalis strain 381 and HW24 D-1 strain
^ gingivalis 381株、 及び、 HW24D— 1株を、 へミン （5mgZl) 及びメナジオン （lmgZl) 添加 GAM ブイヨン培地 （曰水製薬） に接種 し、 37°C、 80% N2、 10% H2、 1 % C02 という嫌気的条件下 で大量培養後、 遠心分離により集菌した。 その菌体を 20 mM トリス塩 酸緩衝液 （pH 7.4) +0.15 M NaCl + 1 OmM gCl2 に懸濁し、 ピ ペッティング後、 マグネティックスターラーで撹拌して、 物理的に線毛 を剥離した。 ^ 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.
遠心分離により菌体を除去し、 その上清を 40%飽和硫安分画し、 得 られた沈渣を 20raM トリス塩酸緩衝液 （pH 8.0) に溶解後、 透析に より脱塩した。 次に、 DEAE - Sepharose (Pharmacia) を用いたイオン交換クロマト グラフィ一により、 NaCl の濃度勾配をかけ、 0. 1 5 M NaCl で溶出 してくる画分を線毛抗原とした。 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.
更に、 これを S印 hacryl S - 5 0 0 (Pharmacia) を用いたゲル濾過 Further, this was subjected to gel filtration using S-mark hacryl S-500 (Pharmacia).
5 クロマトグラフィーにかけ、 単一ピークであることを確認した。 5 Chromatography confirmed a single peak.
調製例 3  Preparation Example 3
A. actinomycetemcomitans ATCC 2 9 5 2 3株、 Y 4株、 及び、 NCTC 9 7 1 0株からの多糖抗原の調製  Preparation of polysaccharide antigen from A. actinomycetemcomitans ATCC 2953, Y4 and NCTC9710
A. actinomycetemcomitans ATCC 2 9 5 2 3株、 Y 4株、 及び、 NCTC 1 0 9 7 1 0株を Todd - Hewitt broth (Difco Laboratories) に 1 %酵 母エキスを添加した培地に接種して、 3 7で、 5 % C02 という嫌気的 条件下で大量培養後、 遠心分離により集菌した。 得られた菌体を凍結乾 燥し、 生理食塩水に懸濁した後、 オートクレイブにかけ、 熱抽出し、 抽 出液を、 例えば、 DEAE - Sephadex A- 2 5 (Pharmacia) を用いたィォ i s ン交換クロマトグラフィーで分画し、 その素通り画分をさらに S印 hacr yl S - 2 0 0 (Pharmacia) を用いたゲル濾過クロマトグラフィ一で精 製し、 多糖画分を分取した。 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.
調製例 4  Preparation Example 4
歯周病原性細菌 P. gingivalis P. intermedia と A. actinomycete 0 mcomitan s の全菌体、 P. gingivalis 3 8 1株及び Hff 2 4 D— 1株の 線毛抗原、 A. act inomycetemcomitans ATCC 2 9 5 2 3株、 Y 4株及 び NCTC 9 7 1 0株の多糖 抗原に対する精製抗血清の調製  Whole cells of periodontopathogenic bacteria P. gingivalis P. intermedia and A. actinomycete 0 mcomitan s, P. gingivalis 38 1 strain and Hff 24 D-1 pilus antigen, A. act inomycetemcomitans ATCC 29 Preparation of purified antiserum against polysaccharide antigens of 52, Y4 and NCTC 9710 strains
調製例 1の菌周病原性細菌 ^ gingivalis^ intermedia と A. ac tinomycetemcomitans の凍結 燥囷体ヽ 調製例 2の P. gingivalis 3 8 1株及び HW 2 4 D- 1株の線毛抗原、 調製例 3の actinomyceteinc omitans ATCC 2 9 5 2 3株、 Y 4株及び NCTC 9 7 1 0株の多糖抗原 の各 l mg をゥサギ 1匹当たり 1 ml のフロイン卜の完全アジュバントと 共に油中水滴型乳剤として、 皮下に 3週間おきに合計 3回注射して免疫 し、 最終追加免疫から 7曰目に全採血し、 常法通り抗血清を分離した。 得られた抗血清の全量の 1/2 容の飽和硫安を 4 °Cで撹拌しながら加え て、 硫安分画 （ 1/3 飽和） を行う。 遠心分離により、 得られた沈渣に 1/3 飽和硫安を加え撹拌した。 この操作を 2〜3回繰り返し、 最終的に 得られた沈渣をリン酸緩衝液に溶解し、 リン酸緩衝液で透析し脱塩した。 次に、 DEAE - Sepharose A - 2 5 (Pharmacia) を用いたイオン交換ク ロマトグラフィ一で、 NaCl の濃度勾配をかけ、 0 . 0 1 5 M NaCl で溶 出してくる画分を回収し、 同様にして 1/3 飽和硫安分画を行い、 歯周 病原性細菌の全菌体、 gingivalis 3 8 1株の線毛抗原、 actinom ycetemcomitans Y 4株の多糖抗原に対する精製抗血清を得た。 Freeze-dried body of the peripathogenic bacterium ^ gingivalis ^ intermedia and A. ac tinomycetemcomitans of Preparation Example 1 P. gingivalis 3 of Preparation Example 2 8 1 mg of the pilus antigen of 1 strain and HW 24 D-1 strain, and 1 mg of the polysaccharide antigen of the actinomyceteinc omitans ATCC 2953 2 strain, Y4 strain and NCTC 9710 strain of Preparation Example 3 The animals were immunized with 1 ml of Freund's complete adjuvant per animal as a water-in-oil emulsion subcutaneously three times every three weeks for a total of three injections. The serum was separated. Add 1/2 volume of saturated ammonium sulfate to the obtained antiserum while stirring at 4 ° C to perform ammonium sulfate fractionation (1/3 saturation). 1/3 saturated ammonium sulfate was added to the obtained sediment by centrifugation, followed by stirring. This operation was repeated two or three times, and the finally obtained precipitate was dissolved in a phosphate buffer, dialyzed against the phosphate buffer, and desalted. Next, by ion exchange chromatography using DEAE-Sepharose A-25 (Pharmacia), a NaCl concentration gradient was applied, and the fraction eluted with 0.015 M NaCl was collected. Then, 1/3 saturated ammonium sulfate fractionation was performed to obtain purified antiserum against all cells of periodontopathogenic bacteria, fimbrial antigen of gingivalis 381 strain, and polysaccharide antigen of actinomycetemcomitans Y4 strain.
調製例 5 Preparation Example 5
歯周病原性細菌 P. gingivalisゝ P. intermedia と A. actinomycete mcomitan s の全菌体、 gingivalis 3 8 1株及び HW 2 4 D— 1株の 線毛抗原、 A. act inomycetemcomitans ATCC 2 9 5 2 3株、 Y 4株及 び NCTC 9 7 1 0株の多糖 抗原に対するモノクローナル抗体の調製 調製例 1の歯周病原性細菌 ^ gingivalis, intermedia と A. ac tinomycetemcomitans の凍結乾燥菌体、 調製例 2の P. gingivalis 3 8 1株及び HW 2 4 D— 1株の線毛抗原、 調製例 3の ^_ actinomycetemc omitans ATCC 2 9 5 2 3株、 Y 4株及び NCTC 9 7 1 0株の多糖抗原 の各 1 0 0〃g をマウス 1匹当たり 0 . l ml のフロイン卜の完全アジュ バントと共に油中水滴型乳剤として、 BALBZcマウス （雌） の皮下に 3 週間おきに合計 3回注射して免疫し、 最終追加免疫から 3曰目に BALB Zcマウス （雌） から脾細胞を採取し、 Eagle's MEMで 3回洗浄した。 —方、 BALB/cマウス由来のミエ口一マ細胞 （SP 2/0) を Eagle's ΙίΕΜで 3回洗浄した。 ミエローマ細胞と脾細胞を Eagle's ΪΕΜ 中で、 1 ： 1 0に混合し、 1, 20 Orpra、 1 Omin. 遠心分離後、 上清を除去 し沈渣をよくほぐして、 0. 5ml のポリエチレングリコール （0. 5ml) + Eagle' s HEM (0. 5ml) +DHS0 (0. 35ml) の混合溶液を 3 7°C 下で沈渣に静かに滴下しながら 1分間混合した。 A. actinomycetemcomitans ATCC 295, 2 fimbrial antigens of all cells of the periodontopathogenic bacteria P. gingivalis ゝ 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 Preparation of freeze-dried bacterial cells of periodontopathogenic bacteria ^ gingivalis, intermedia and A. ac tinomycetemcomitans of Preparation Example 1, Preparation Example 2 P. gingivalis 381 and HW24D-1 pilus antigens, ^ _actinomycetemcomitans ATCC 2953, AT4, Y4 and NCTC 970 of Preparation Example 3 100 μg 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. On the other hand, myeloma cells (SP 2/0) derived from BALB / c mice were washed three times with Eagle's ΙίΕΜ. 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.
次に、 Eagle's MEM 1 0ml を少しずつ加え、 1， 200rpm、 1 Omin. 遠心分離し、 上清を除去し、 その沈渣に 1 0%ゥシ胎児血清を含む KPM 1- 1640培地を加え、 5 X 105 cells/ml 位になるように浮遊さ せ、 96穴マイクロタイタープレートに 1 00 ずつ分注した。 その 後、 HAT培地、 HT培地によりハイブリ ドーマを選択し、 1 5日目頃に 調製例 1の歯周病原性細菌 ^ gingivalis^ intermedia と acti nomycetemcomitans の凍結乾燥菌体、 調製例 2の ^ gingivalis 38 1株及び Hf 24 D— 1株の線毛抗原、 調製例 3の actinomycetemconi itans ATCC 29523株、 Y 4株及び NCTC 9710株の多糖抗原を コーティングした 96穴マイクロタイタープレートを用いて、 ELISA法 により歯周病原性細菌の全菌体、 P. gingivalis 38 1株及び ^240 — 1株の線毛抗原、 actinomycetemcomitans ATCC 29523株、 Y 4株及び NCTC 9 71 0株の多糖抗原に対する抗体産生ハイプリ ドーマ をスクリーニングした。 Next, add 10 ml of Eagle's MEM little by little, centrifuge at 1,200 rpm, 1 Omin., Remove the supernatant, add KPM 1-1640 medium containing 10% fetal calf serum to the sediment, The cells were suspended at about X 10 5 cells / ml and dispensed 100 times into a 96-well microtiter plate. After that, hybridomas were selected using HAT medium and HT medium, and on the 15th day, freeze-dried cells of periodontopathogenic bacteria ^ gingivalis ^ intermedia and acti nomycetemcomitans in Preparation Example 1 and ^ gingivalis 38 in Preparation Example 2 were prepared. One tooth and one Hf24D-1 pilus antigen, and a 96-well microtiter plate coated with the polysaccharide antigens of the actinomycetemconi itans ATCC 29523, Y4 and NCTC 9710 strains of Preparation Example 3 by ELISA Screening for antibody-producing hybridomas against all cells of peripathogenic bacteria, P. gingivalis 38 1 strain and ^ 240 — 1 strain of pilus antigen, actinomycetemcomitans ATCC 29523, Y4 strain and NCTC 9710 strain of polysaccharide antigen did.
スクリーニングしたハイブリ ドーマは限界稀釈法によりクローニング した。 限界稀釈法は、 1 0 %ゥシ胎児血清を含む 10—1 6 4 0培地 に、 ハイブリ ドーマ 5 cellsZml、 BALB cマウス胸腺細胞 5 X 1 0 6 cells/ml になるように加え、 9 6穴マイクロタイタープレートに 0. 2 ml ずつ分注し、 調製例 1の歯周病原性細菌 f . gingivalis, inte rmedia と A, actinomycetemcomitans の凍結乾燥菌体、 調製例 2の P. gingivalis 3 8 1株及び HW 2 4 D— 1株の線毛抗原、 調製例 3の actinomycetemcomitans ATCC 2 9 5 2 3株、 Y 4株及び NCTC 9 7 1 0株の多糖抗原に反応するモノクローナル抗体を分泌するコロニーを選 び出した。 この操作を 2回繰り返しクローニングを終了した。 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. gingivalis 381 in Preparation Example 2, and A colony secreting a monoclonal antibody reactive with the polysaccharide antigen of the HW24D-1 strain of the pilus antigen, the actinomycetemcomitans ATCC 2953, the Y4 strain, and the NCTC 9710 strain of Preparation Example 3 was selected. Issued. This operation was repeated twice to complete the cloning.
次いで、 あらかじめプリスタン処置された BALBZ cマウスの腹腔内 にハイプリ ドーマを移植し、 その腹水中で増殖させて抗体を採取するか、 又は、 ハイプリ ドーマを無血清培地にて培養し、 その培養上清から抗体 を採取することにより目的のモノクローナル ½体を調製した。  Next, transplant the hybridoma into the abdominal cavity of the BALBZc mouse previously treated with pristane and grow it in the ascites fluid to collect the antibody, or culture the hybridoma in a serum-free medium and culture the supernatant. The target monoclonal antibody was prepared by collecting the antibody from E. coli.
得られたモノクローナル抗体を 1/3 飽和硫安分画を行い、 DEAE—ィ オン交換クロマトグラフィ一ならびにゲル口過クロマトグラフィー又は プロティン Aカラムで精製した。  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.
実施例 1 Example 1
生物学的被検物質採取用具の作成  Preparation of a biological sample collection tool
市販の紙製コヨリ （ペーパーポイント） 、 例えば、 ZIPPERER 社製の Absorbent Paper Points (長さ 3 0關； Size 1 5、 2 5、 3 5、 4 5 ) の先端から約 2 O mni を二トロセルロース又はセルロースァセテ一トの 水一有機溶剤混合溶媒溶液に浸漬した後、 溶剤を乾燥除去して、 その表 面に多孔性フィルムをコートした。 これらは、 1 0ミクロン以下の孔径 の孔を有する。 更に、 先端部分約 2 mm 程はそのまま残し、 それ以外の フィルム表面をァセトン処理してその孔をふさぐことにより、 その部分 を防水性 （非透水性） とした。 これにより第 1図に示す如き採取用具を 作成した。 Around 2 Omni from the tip of commercially available paper-made paper (paper point), for example, ZIPPERER Absorbent Paper Points (length 30 related; Sizes 15, 25, 35, 45), Nitrocellulose Alternatively, after immersion in a mixed solvent solution of cellulose acetate and water / organic solvent, the solvent was removed by drying, and the surface thereof was coated with a porous film. These have pores with a pore size of less than 10 microns. In addition, leave about 2 mm at the tip, The surface of the film was treated with acetone and the pores were closed to make that part waterproof (non-permeable). Thus, a sampling tool as shown in Fig. 1 was prepared.
実施例 2  Example 2
金コロイド標識抗体溶液の調製  Preparation of colloidal gold-labeled antibody solution
E.Y Laboratory, Inc. の金コロイド粒子 （G— 20 ：粒径 2 Onra) 1 ml に、 0.07 M炭酸緩衝液 （pH 9.0) を 111 1加えた。 一方、 調製例 4で得た各抗血清又は調製例 5で得た各モノク口ーナル抗体の 2 0 ^g/ml の抗体溶液 500 βΐ に 0. 0 7Μ炭酸緩衝液 （pH 9.0) を 56 1加えた。 そして、 両者を混合し、 室温で 15分間撹拌した。 次に、 ゥシ血清アルブミンを 1 0%、 ポリエチレングリコールを 1% 含有する 0. 007 »炭酸緩衝液 （PH 9. 0) を 185 ^1 添加し、 室 温で 1 5分間撹拌して、 金コロイド標識抗体溶液を得た。  111 ml of 0.07 M carbonate buffer (pH 9.0) was added to 1 ml of colloidal gold particles (G-20: particle size 2 Onra) from E.Y Laboratory, Inc. On the other hand, add 0.07Μ carbonate buffer (pH 9.0) to 500 βΐ of each antiserum obtained in Preparation Example 4 or each monoclonal antibody obtained in Preparation Example 5 at a concentration of 20 βg / ml. added. Then, both were mixed and stirred at room temperature for 15 minutes. Next, add 0.0007 »carbonate buffer (PH 9.0) containing 10% of serum albumin and 1% of polyethylene glycol to 185 ^ 1 and stir at room temperature for 15 minutes to add gold. A colloid-labeled antibody solution was obtained.
試験例 1 Test example 1
in vitro での歯周病原性細菌の検出  Detection of periodontopathogenic bacteria in vitro
1 ) P. gingivalis の検出  1) Detection of P. gingivalis
gingivalis 381株及び HW24D— 1株を、 へミン （5mgZl ) 及びメナジオン （lmgZl ) 添加 GAM ブイヨン培地 （曰水製薬） で、 gingivalis strain 381 and HW24D-1 strain in GAM bouillon medium (Heisei Pharmaceutical) supplemented with hemin (5mgZl) and menadione (lmgZl).
37°C、 80% N2、 10% H2、 10% C02 という嫌気的条件下で大 量培養後、 遠心分離により集菌した。 この菌体をリン酸緩衝生理食塩水 に懸濁し、 550nm における吸光度を 0.5に合わせ、 この菌体浮遊液 の菌体濃度を PETR0FF - HAUSSER and HELBER Counting chamber (Haus ser, Scientific Partnership Horsham社製;） を用いて求めた 0 このよ うにして得られた菌体浮遊液を稀釈していき、 その各稀釈液の 2 1 を 実施例 1で作成した生物学的被検物質採取用具に吸着させた。 そこで、 実施例 2で得られた金コロイ ド粒子で標識した ^ gingivalis 381 株及び HW24D— 1株の線毛抗原に対するモノクローナル抗体溶液に浸 漬し、 反応させた。 反応終了後、 金コロイド粒子の着色が認められる菌 体数を求めた。 その結果を表 1に示すが、 金コロイ ド粒子で標識した gingivalis 381株及び Hff24 D— 1株の線毛抗原に対するモノクロ ーナル抗体溶液で検出できる ^ gingivalis 381株及び HW24D- 1株の菌体数は 2 X 105 個及び 1.0 X 105 個であった。 After mass cultivation under anaerobic conditions of 37 ° C, 80% N 2 , 10% H 2 and 10% CO 2, 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. Therefore, 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.
表 1 ： P. gingivalisの検出  Table 1: Detection of P. gingivalis
菌体数 （個 Zテスト） 判定  Judgment of bacterial cell count (pcs Z test)
381株 HW24D-1株  381 shares HW24D-1 shares
1.2X107 +++ +++ 1.2X10 7 +++ +++
6.4 106 +++ +++ 6.4 10 6 +++ +++
3.2X106 ++ ++ 3.2X10 6 ++++
1.6X106 ++ ++ 1.6X10 6 ++++
8.0X105 ++ ++ 8.0X10 5 ++++
4.0 105 + + 4.0 10 5 + +
2.0X105 + + 1.0X105 ■ + 2.0X10 5 + + 1.0X10 5 ■ +
5.0X104 5.0X10 4
2) P, intermediaの検出  2) Detection of P and intermedia
intermedia ATCC 25611株を、 へミン （ 5 mg/1) 及びメナジ オン （loig/1) 添加 GAMブイヨン培地 （日水製薬） で、 37°C、 80 % N2、 10% H2、 10% C02 という嫌気的条件下で大量培養後、 遠心分離により集菌した。 この菌体をリン酸緩衝生理食塩水に懸濁し、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,
55 Onmにおける吸光度を 0.5に合わせ、 この菌体浮遊液の菌体濃度 を PETROFF - HAUSSER and HELBER Counting, chamber (Hausser Scien tific Partnership Horsham社製） を用いて求めた。 このようにして得 られた菌体浮遊液を稀釈していき、 その各稀釈液の 2 κ 1を実施例 1で 作成した生物学的被検物質採取用具に吸着させた。 そこで、 実施例 2で 得られた金コロイド粒子で標識した ^ intermedia ATCC 25611株 の全菌体に対するモノクローナル抗体溶液に浸漬し、 反応させた。 反応 終了後、 金コロイド粒子の着色が認められる菌体数を求めた。 その結果 を表 2に示すが、 金コロイド粒子で標識した ^ intermedia ATCC 25 611株の全菌体に対するモノクローナル抗体溶液で検出できる in termedia ATCC 25611株の菌体数は 2 x 105 個であった。 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 2κ1 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 .
表 2 ： P. intermediaの検出  Table 2: Detection of P. intermedia
菌体数 （個 Zテスト） 判定  Judgment of bacterial cell count (pcs Z test)
1.2X107 +++ 1.2X10 7 +++
6.4X106 +++ 6.4X10 6 +++
3.2X106 ++ 3.2X10 6 ++
1.6X106 ++ 1.6X10 6 ++
8.0X105 ++ 8.0X10 5 ++
4.0X105 4.0X10 5
2.0X105 + 2.0X10 5 +
LOxlO5 LOxlO 5
) A. actinomycetemcomitansの検出  A) Detection of actinomycetemcomitans
A. actinomycetemcomitans ATCC 29523株、 Y 4株、 及び、 NCTC 9 7 1 0株は、 Todd - Hewitt broth (Difco Laboratories) に 1 %酵 母エキスを添加した培地で、 3 7 °C、 5 % C02 という嫌気的条件下で 大量培養後、 遠心分離により集菌した。 この菌体をリン截緩衝生理食塩 水に懸濁し、 5 5 0 nm における吸光度を 0 . 5に合わせ、 この菌体浮遊 液の菌体濃度を PETROFF - HAUSSER and HELBER Counting chamber (Ha usser Scienti Xfic Partnership Horsham社製） を用いて求めた。 この ようにして得られた菌体浮遊液を稀釈していき、 その各稀釈液の 2 jt を実施例 1で作成した生物学的被検物質採取用具に吸着させた。 そこで、 実施例 2で得られた金コロイ ド粒子で標識した L actinomycetemcomit ans ATCC 2 9 5 2 3株、 Y 4株及び NCTC 9 7 1 0株の多糖抗原に対 するモノクローナル抗体溶液に浸漬し、 反応させた。 反応終了後、 金コ ロイ ド粒子の着色が認められる菌体数を求めた。 その結果を表 3に示す が、 金コロイ ド粒子で標識した A. actinomycetemcomitans ATCC 2 9 5 2 3株、 Y 4株及び NCTC 9 7 1 0株の多糖抗原に対するモノクロ一 ナル抗体溶液で検出できる A. actinomycetemcomitans ATCC 2 9 5 2 3株 · Υ 4株及び NCTC 9 7 1 0株の菌体数はいずれも 2 x 1 0 5 個で あった。 A. actinomycetemcomitans ATCC 29523, Y4 and NCTC 9 7 1 0 strain, Todd - Hewitt broth (Difco Laboratories ) in the medium containing 1% yeast extract, 3 7 ° C, 5% C0 After mass cultivation in anaerobic conditions of 2, collecting by centrifugation Bacteria. The cells are suspended in a phosphate buffered saline solution, the absorbance at 550 nm is adjusted to 0.5, and the cell concentration of this cell suspension is determined by PETROFF-HAUSSER and HELBER Counting chamber (Hausser Scienti Xfic Partnership Horsham). 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, Υ4 strains and NCTC 9710 strains all had 2 × 10 5 cells.
¾ 3 ： A. actinomycetemcomitans の検出  ¾ 3: Detection of A. actinomycetemcomitans
菌体数 （個ノテスト） 判定  Judgment of bacterial cell count
ATCC 29523株 Y 4株 NCTC 9710株 ATCC 29523 shares Y 4 shares NCTC 9710 shares
1. 2 X 107 +++ +++ +++1.2 X 10 7 +++ +++ +++
6. 4 X 106 +++ +++ +++6.4 X 10 6 +++ +++ +++
3. 2 106 ++ ++ ++ 3.2 10 6 ++ ++ ++
++ ++ ++ 8.0X105 ++ ++ ++ ++ ++ ++ 8.0X10 5 ++ ++ ++
4.0x10s 4.0x10 s
2.0x10s + 2.0x10 s +
LOxlO5 LOxlO 5
実施例 3  Example 3
金コロイド標識抗原溶液の調製  Preparation of Colloidal Gold Labeled Antigen Solution
E.Y Laboratory, Inc. の金コロイ ド粒子 （G— 20 ：粒径 2 Onm) 1 ml に 0.07 M炭酸緩衝液 （pH 9.0) を 111 1 加えた。 一方、 調製例 2で得た線毛抗原又は調製例 3で得た多糖抗原各 20 ig/ml の 抗原溶液 500 ^1 に 0.07 M炭酸緩衝液 （pH 9.0) を 56 yMl 加 えた。 そして、 両者を混合し、 室温で 15分間撹拌した。  111 ml of 0.07 M carbonate buffer (pH 9.0) was added to 1 ml of gold colloid particles (G-20: particle size 2 Onm) of E.Y Laboratory, Inc. On the other hand, 56 yMl of 0.07 M carbonate buffer (pH 9.0) was added to the antigen solution 500 ^ 1 of 20 ig / ml each of the pilus antigen obtained in Preparation Example 2 or the polysaccharide antigen obtained in Preparation Example 3. Then, both were mixed and stirred at room temperature for 15 minutes.
次に、 ゥシ血清アルブミンを 10%及びポリエチレンダリコールを 1 %含有する 0.007 M炭酸緩衝液 （pH 9.0)を 185 添加し、 室温で 15分間撹拌して、 金コロイド標識抗原溶液を得た。  Next, 185 of 0.007 M carbonate buffer (pH 9.0) containing 10% of serum albumin and 1% of polyethylenedaricol was added and stirred at room temperature for 15 minutes to obtain a colloidal gold-labeled antigen solution.
試験例 2 Test example 2
in vitro での歯周病原性細菌に対する特異抗体の検出  Detection of specific antibodies against periodontopathogenic bacteria in vitro
1) P. gingivalis に対する抗体の検出  1) Detection of antibodies against P. gingivalis
調製例 4で得られた gingivalis 381株及び HW24D— 1株の 線毛抗原に対する抗血清の各稀釈液の 2 il を、 実施例 1で作成した生 物学的被検物質採取用具に吸着させた。 そこで、 実施例 3で得られた金 コロイド粒子で標識した gingivalis 381株及び HW24D— 1株 の線毛抗原溶液に浸漬し、 反応させた。 反応終了後、 金コロイド粒子の 着色が認められる抗体の稀釈率を求めた。 その結果を表 4に示すが、 金 コロイド粒子で標識した P. gingivalis 381株及び HW24D— 1株 の線毛抗原溶液で検出できる P. gingivalis 3 8 1株及び HW 2 4 D— 1 株の線毛抗原に対する抗体の稀釈率は血清レベルの 1/1000 であった。 2 il of each dilution of antiserum against pilus antigen of gingivalis strain 381 and HW24D-1 strain obtained in Preparation Example 4 was adsorbed to the device for collecting a biological sample to be prepared in Example 1. . Therefore, gingivalis strain 381 and HW24D-1 strain labeled with the colloidal gold particles obtained in Example 3 were immersed in a fimbrial antigen solution and reacted. After the reaction was completed, the dilution ratio of the antibody in which coloring of the colloidal gold particles was observed was determined. The results are shown in Table 4. The results show that P. gingivalis strain 381 and HW24D-1 strain were labeled with colloidal gold particles. Of the P. gingivalis strain 381 and the HW24D-1 strain, which can be detected with the fimbrial antigen solution, had a dilution of 1/1000 of the serum level.
表 4 : P. gingivalis に対する抗体の検出  Table 4: Detection of antibodies to P. gingivalis
抗血清の稀釈率 判定  Antiserum dilution rate determination
381 株 HW 2 4 D— 1株  381 shares HW 2 4 D— 1 share
1/10 +++ +++  1/10 +++ +++
1/100 ++ ++  1/100 ++ ++
1/1000 + +  1/1000 + +
1/10000  1/10000
2 ) A. actinomycetemcomitans に对する抗体の検出  2) Detection of antibodies against A. actinomycetemcomitans
調製例 4で得られた A. actinomycetemcomitans ATCC 2 9 5 2 3株、 Y 4株、 及び、 NCTC 9 7 1 0株の多糖抗原に対する抗血清の各稀釈液 の 2 / 1 を、 実施例 1で作成した生物学的被検物質採取用具に吸着さ せた。 そこで、 実施例 3で得られた金コロイ ド粒子で標識した L acti nomycetemcomitans ATCC 2 9 5 2 3株、 Y 4株及び NCTC 9 7 1 0株 の多糖抗原溶液に浸潰し、 反応させた。 反応終了後、 金コロイ ド粒子の 着色が認められる抗体の稀釈率を求めた。 その結果を表 5に示すが、 金 コロイ ド粒子で標識した A. actinomycetemcomitans ATCC 2 9 5 2 3 株、 Y 4株及び NCTC 9 7 1 0株の多糖抗原溶液で検出できる ^_ acti nomycetemcomitans ATCC 2 9 5 2 3株、 Y 4株及び NCTC 9 7 1 0株 の多糖抗原に対する抗体の稀釈率は血清ラベルの 1 1 0 0 0であった c  A. actinomycetemcomitans ATCC 295, 23, Y4, and NCTC 970, obtained in Preparation Example 4, 2/1 of each dilution of the antiserum against the polysaccharide antigen were used in Example 1, The sample was adsorbed to the tool for collecting biological test substances. Then, the cells were immersed in a polysaccharide antigen solution of Lactinomycetemcomitans ATCC 2953, Y4 strain and NCTC 9710 strain labeled with gold colloid particles obtained in Example 3 and reacted. After the reaction was completed, the dilution ratio of the antibody in which coloring of the gold colloid particles was observed was determined. The results are shown in Table 5, which can be detected in polysaccharide antigen solutions of A. actinomycetemcomitans ATCC 2953, Y4 and NCTC 9710 labeled with gold colloid particles ^ _ actinomycetemcomitans ATCC 2 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.c
表 5 ： A. actinomycetemcomitans に対する抗体の検出 抗血清の稀釈率 判定  Table 5: Detection of antibodies to A. actinomycetemcomitans Antiserum dilution
ATCC 29523株 Y 4株 NCTC 9710株 1/10 +++ +++ +++ ATCC 29523 shares Y 4 shares NCTC 9710 shares 1/10 +++ +++ +++
1/100 ++ ++ ++  1/100 ++ ++ ++
1/1000 + + +  1/1000 + + +
1/10000 - - . - 1/10000--.-
5 実施例 4 5 Example 4
金コロイド標識抗体による成人性歯周炎患者菌肉溝液中の ^ gingiv alis の 検出  Detection of ^ gingiv alis in the fungal sulcus of adult periodontitis patients by colloidal gold labeled antibody
健常者 3名と成人性歯周炎患者 6名の歯肉溝液を、 実施例 1で作成し た生物学的被検物質採取用具で採取し、 まず、 実施例 2で得た金コロイ 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.
1 0 ド粒子で標識した P. gingivalis 3 8 1株の線毛抗原に対するモノクロ ーナル抗体溶液に浸漬し、 室温で 5〜1 5分間反応させた後、 採取用具 を取り出し、 蒸留水で洗浄した。 そこで、 採取用具の歯肉溝液が吸着し た部分が、 金コロイド粒子の色である赤紫色に染まった場合を陽性、 染 まらなかった場合を陰性と判定した。 次に、 同様にして、 実施例 2で得 i s た金コロイド粒子で標識した P. gingivalis HW 2 4 D— 1株の線毛抗原 に対するモノクローナル抗体溶液と反応させ、 判定した。 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.
また、 同時に、 健常者 3名と成人性歯周炎患者 6名の歯肉溝液中の ^ gingivalis の同定を下記のようにして行った。  At the same time, ^ gingivalis in the gingival crevicular fluid of three healthy subjects and six adult periodontitis patients was identified as follows.
即ち、 市販の紙製コヨリ （ペーパーポイント） に吸収させた歯肉溝液 o を輸送培地 （RTF : Reduced Transfer Fluid) に懸濁して回収し、 ゥサ ギ脱鐵血、 へミン及びメナジオンを含む CDC培地 （Control Disease C enter開発） に播種し、 嫌気条件下で数日間培養した。 生育の見られた コロニーのうち、 黒色化したコロニーを選び、 これを分離し、 純培養後、 Rap ID ANA System (HcDONNELL DOUGLAS社製） により、 同定試験を行 つた。 更に、 P. gingivalis と同定されたものの培養上清をガスクロマ トグラフィーにかけ、 フエニル酢酸の産生を調べることにより、 f n givalis であることの再認識を行った。 That is, the gingival crevicular fluid o absorbed in a commercially available paper kori (paper point) is suspended in a transport medium (RTF: Reduced Transfer Fluid) and collected. (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.
以下にその結果を示す。 表 6に示すように、 健常者 （3名） の歯肉溝 液では、 金コロイ ド粒子による着色は認めず、 すべて陰性の結果を示し 。 また、 歯肉溝液中に存在する細菌の同定試験では ^ gingivalis は認められなかった。 一方、 成人性歯周病患者 （6名） の歯肉溝液では、 6名中 4名に金コロイ ド粒子で標識した gingivalis 3 8 1株の線 毛抗原に対するモノクローナル抗体溶液との反応が見られ、 更に、 金コ ロイ ド粒子で標識した gingivalis HW 2 4 D— 1株の線毛抗原に対す るモノクローナル抗体溶液と反応させるとすべての成人性歯周炎患者で、 金コロイ ド粒子による着色が認められ、 すべて陽性の結果を示した。 ま た、 歯肉溝液中に存在する細菌の同定試験では、 gingivalis を認め た。  The results are shown below. As shown in Table 6, the gingival crevicular fluid of three healthy subjects showed no negative coloration due to gold colloid particles, and all showed negative results. In addition, ^ gingivalis was not detected in the test for identification of bacteria in the gingival crevicular fluid. On the other hand, in 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. In addition, when reacted with a monoclonal antibody solution against the pilus antigen of gingivalis HW24D-1 strain labeled with gold colloid particles, the coloring by gold colloid particles was observed in all adult periodontitis patients. The results were all positive. In a test for identification of bacteria present in the gingival crevicular fluid, gingivalis was observed.
表 6 ：歯肉溝液中の P. gingivalis の検出  Table 6: Detection of P. gingivalis in gingival crevicular fluid
被検者 No. 金コロイ ド標識抗体との反応 培養法による同定  Subject No. Reaction with gold colloid-labeled antibody Identification by culture method
381 株 HW 2 4 D— 1 株  381 shares HW 2 4 D— 1 share
健常者 1 無  Healthy 1
2 無 3 無 1 有 2 ++ ++ 有 3 ++ ++  2 No 3 No 1 Yes 2 ++ ++ Yes 3 ++ ++
4 ++ 有 5 + ++ 有 4 ++ Yes 5 + ++ Yes
6 + + 有  6 + + Yes
実施例 5  Example 5
金コロイド標識抗体による成人性歯周炎患者歯肉溝液中の ^ interm edia の 検出  Detection of ^ interm edia in gingival crevicular fluid of adult periodontitis patients using colloidal gold-labeled antibody
健常者 3名と成人性歯周炎患者 6名の歯肉溝液を、 実施例 1で得た生 物学的被検物質採取用具で採取し、 実施例 2で得た金コロイド粒子で標 識した ^ intermedia ATCC 2 5 6 1 1株の全菌体に対する抗血清溶液 に浸漬し、 反応させた。 室温で 5〜 1 5分間反応させた後、 採取用具を 取り出し、 蒸留水で洗浄した。 そこで、 採取用具の歯肉溝液が吸着した 部分が、 金コロイド粒子の色である赤紫色に染まった場合を陽性、 染ま らなかった場合を陰性と判定した。  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.
また、 同時に、 健常者 3名と成人性歯周炎患者 6名の歯肉溝液中の intermedia の同定を実施例 4の P. gingivalis の検出と同様にして 行った。  At the same time, the identification of intermedia in the gingival sulcus fluid of 3 healthy subjects and 6 adult periodontitis patients was performed in the same manner as in the detection of P. gingivalis in Example 4.
即ち、市販の紙製コヨリ （ペーパーポイント） に吸収させた歯肉溝液 を輸送培地 （ETF : Reduced Transfer Fluid) に懸濁して回収し、 ゥサ ギ脱繊血、 へミン及びメナジオンを含む CDC培地 （Control Disease C enter開発） に播種し、 嫌気条件下で数日間培養した。 生育の見られた コロニーのうち、 黒色化したコロニーを選び、 これを分離し、 純培養後、 Rap ID ANA System (HcDONNELL DOUGLAS社製） により、 同定試験を行 つた。  That is, 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).
以下にその結果を示す。 表 7に示すように、 健常者 （3名） の歯肉溝 液では、 金コロイド粒子による着色は認めず、 すべて陰性の結果を示し た。 また、 歯肉溝液中に存在する細菌の同定試験では、 P. intermedia 認められなかった。 一方、 成人性歯周病患者 （6名） の歯肉溝液では、 金コロイ ド粒子による着色が認められ、 すべて陽性の結果を示した。 ま た、 歯肉溝液中に存在する細菌の同定試験では、 P. intermedia を認め o The results are shown below. As shown in Table 7, the gingival crevicular fluid of three healthy subjects showed no negative coloration due to colloidal gold particles, and all showed negative results. Was. In addition, P. intermedia was not detected in the identification test of bacteria present in the gingival crevicular fluid. On the other hand, the gingival crevicular fluid of adult periodontal disease patients (6 patients) was colored by gold colloid particles, and all showed positive results. In addition, the identification test for bacteria present in the gingival crevicular fluid revealed P. intermedia.o
表 7 ：歯肉溝液中の intermedia の検出  Table 7: Detection of intermedia in gingival crevicular fluid
被検者 No. 金コロイ ド標識抗体との反応 培養法による同定 健常者 1  Subject No. Reaction with gold colloid-labeled antibody Identification by culture method Healthy subject 1
2 R¾  2 R¾
3  Three
患 者 1 ++ 有  Patient 1 ++ Yes
2 有 3 + 有 4 ++ 有 5 ++ 有 6 ++  2 Yes 3 + Yes 4 ++ Yes 5 ++ Yes 6 ++
実施例 6 Example 6
金コロイ ド標識抗体による限局性若年性歯周炎患者歯肉溝液中の actinoniy cetemcomitans の検出  Detection of actinoniy cetemcomitans in the gingival crevicular fluid of patients with localized juvenile periodontitis using gold colloid-labeled antibody
健常者 3名と限局性若年性歯周炎患者 6名の歯肉溝液を、 実施例 1で 得た生物学的被検物質採取用具で採取し、 実施例 2で得た金コロイ ド粒 子で標識した に actinomycetemcomitans ATCC 2 9 5 2 3株、 Y 4株 及び NCTC 9 7 1 0株の多糖抗原に対するモノクローナル抗体溶液を混 合後、 浸漬し、 反応させた。 室温で 5 1 5分間反応させた後、 採取用 具を取り出し、 蒸留水で浼浄した。 そこで、 採取用具の歯肉溝液が吸着 した部分が、 金コロイド粒子の色である赤紫色に染まった場合を陽性、 染まらなかった場合を陰性と判定した。 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. After labeling with, 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.
また、 同時に、 健常者 3名と限局性若年性歯周炎患者 6名の歯肉溝液 5 中の A.  At the same time, A. in the gingival crevicular fluid 5 of 3 healthy subjects and 6 patients with localized juvenile periodontitis.
actinomycetemcomitans の同定を実施例 4の ^ gingivalis の検出と 同様にして行った。  Identification of actinomycetemcomitans was performed in the same manner as in the detection of ^ gingivalis in Example 4.
即ち、 市販の紙製コヨリ （ペーパーポイント） に吸収させた歯肉溝液 を輸送培地 （RTF : Reduced Transfer Fluid) に懸濁して回収し、 ゥサ i n ギ脱繊血、 へミン及びメナジオンを含む CDC培地 （Control Disease C enter開発） に播種し、 嫌気条件下で数日間培養した。 生育の見られた コロニーを選び、 これを分離し、 純培養後、 Rap ID ANA System (McDON NELL DOUGLAS社製） により、 同定試験を行った。  That is, the gingival sulcus fluid absorbed in a commercially available paper kori (paper point) is suspended in a transport medium (RTF: Reduced Transfer Fluid) and collected. 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).
以下にその結果を示す。 表 8に示すように、 健常者 （3名） の歯肉溝 5 液では、 金コロイド粒子による着色は認めず、 すべて陰性の結果を示し た。 また、 歯肉溝液中に存在する細菌の同定試験では、 L actinomycet emcomitans は認められなかった。 一方、 限局性若年性歯周病患者 （6 名） の歯肉溝液では、 金コロイド粒子による着色が認められ、 すべて陽 性の結果を示した。 また、 歯肉溝液中に存在する細菌の同定試験では、0 A. actinomycetemcomitans ¾rsSめた o  The results are shown below. As shown in Table 8, no coloration by colloidal gold particles was observed in the five gingival sulcus solutions of healthy subjects (three), and all showed negative results. In addition, Lactinomycet emcomitans was not detected in the identification test for bacteria present in the gingival crevicular fluid. On the other hand, the gingival crevicular fluid of patients with localized juvenile periodontal disease (6 patients) was colored by colloidal gold particles, and all showed positive results. In addition, in the identification test of bacteria present in the gingival crevicular fluid, 0 A. actinomycetemcomitans ¾rsS
表 8 ··歯肉溝液中の に actinomycetemcomitans の検出 被検者 No. 金コロイド標識抗体との反応 培養法による同定 健常者 1 - 無  Table 8Detection of actinomycetemcomitans in gingival crevicular fluid Subject No. Reaction with colloidal gold-labeled antibody Identification by culture method Healthy subjects 1-None
2 無 3 無 2 nothing 3 nothing
患 者 1 + 無  Patient 1 + none
2 + 有  2 + Yes
3 + 有  3 + Yes
4 有  4 Yes
5 有  5 Yes
6 有  6 Yes
実施例 7  Example 7
金コロイ ド標識線毛抗原による成人性歯周病患者歯肉溝液中の ^ i t o ngivalis に対する抗体の検出  Detection of ^ itongivalis antibody in gingival crevicular fluid of adult periodontal disease patients by gold colloid-labeled fimbrial antigen
健常者 3名と成人性歯周炎患者 6名の歯肉溝液を、 実施例 1で得た生 物学的被検物質採取用具で採取し、 実施例 3で得た金コロイ ド粒子で標 識した ^ gingivalis 3 8 1株及び HW 2 4 D— 1株の線毛抗原溶液を 混合後、 浸漬し、 反応させた。 室温で 5〜1 5分間反応させた後、 採取 1 5 用具を取り出し、 蒸留水で洗浄した。 そこで、 採取用具の歯肉溝液が吸 着した部分が、 金コロイド粒子の色である赤紫色に染まった場合を陽性、 染まらなかった場合を陰性と判定した。  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.
また、 同時に、 健常者 3名と成人性歯周炎患者 6名の歯肉溝液中の ^ gingivalis の線毛抗原に対する抗体の検出を ELISA 法を用いて、 下 0 §己のようにして行った。  At the same time, antibodies to ^ gingivalis fimbrial antigen in the gingival sulcus of three healthy subjects and six patients with adult periodontitis were detected using ELISA as described below. .
まず、 炭酸緩衝液 （pH 9 . 0 ) に溶解した gingivalis 3 8 1株、 及び、 HW 2 4 D— 1株の線毛抗原を、 ELISA 用プレートの各ゥエルに 1 μ ずつ分注し、 4 °Cで一晚放置した。 翌日、 Tween 2 0を含むリン酸 緩衝生理食塩水 （PBST) で洗浄した後、 1 0 %ャギ血清を含む PBST で、 室温、 1時間放置し、 ブロッキングをした。 次に、 市販の紙製コヨリ (ペーパーポイント） にあらかじめ採取しておいた歯肉溝液を、 PBST に溶かし出して回収したものを加えた。 3 7 °Cで 1時間反応させた後、 アルカリフォスファターゼ標識抗ヒト I g (G + M + A) を加え、 3 7 5 でで 1時間反応させた。 次に、 PBST でよく洗浄した後に、 p— Nitroph enylphosphate を含む基質溶液を加え、 3 7 °Cで 3 0分間反応させた。 そこで、 吸光度を測定し、 その発色の程度により、 被検体中の P. ging ivalis の線毛抗原に対する抗体の検出をした。 First, fibrin antigens of gingivalis 381 and HW24D-1 dissolved in carbonate buffer (pH 9.0) were dispensed at 1 μl / 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. for 1 hour, alkaline phosphatase-labeled anti-human Ig (G + M + A) was added, and the reaction was carried out at 375 for 1 hour. Next, after washing well with PBST, a substrate solution containing p-nitrophenyl phosphate was added, and reacted at 37 ° C for 30 minutes. Therefore, the absorbance was measured, and an antibody against the fimbrial antigen of P. gingivalis in the specimen was detected based on the degree of color development.
以下にその結果を示す。 表 9に示すように、 健常者 （3名） の歯肉溝 1 0 液では、 金コロイド粒子による着色は認めず、 すべて陰性の結果を示し た。 また、 ELISA法による歯肉溝液中に存在する gingivalis の線毛 抗原に対する抗体の検出では、 L_ gingivalis の線毛抗原に対する抗 体は認められなかった。 一方、 成人性歯周炎患者 （6名） の歯肉溝液で は、 金コロイド粒子による着色が認められ、 すべて陽性の結果を示した。 i s また、 ELISA 法による歯肉溝液中に存在する ^ gingivalis の線毛抗 原に対する抗体の検出では、 L_ gin i alis の線毛抗原に対する抗体を 認めた。  The results are shown below. As shown in Table 9, the gingival sulcus solution of healthy subjects (3) showed no negative coloration due to colloidal gold particles, and all showed negative results. In addition, in 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. On the other hand, 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.
表 9 ：歯肉溝液中の ^ gingivalis に対する抗体の検出 被検者 No. 金コロイド標識線毛抗原との反応 ELISA 法 Table 9: Detection of antibodies against ^ gingivalis in gingival crevicular fluid Subject No. Reaction with colloidal gold-labeled fimbrial antigen ELISA method
0 健常者 1 - - 0 Healthy 1--
2 -2-
3 - - 患 者 1 ++ ++ 3--Patient 1 ++ ++
2 + + ++ ++ 2 + + ++ ++
+ ++  + ++
++  ++
実施例 8  Example 8
金コロイド標識多糖抗原による限局性若年性歯周炎患者歯肉溝液中の acti nomycetemcomitans に对する ί>ι対の検出  Colloidal gold-labeled polysaccharide antigen detects actinomycetemcomitans in the gingival crevicular fluid of patients with localized juvenile periodontitis
健常者 3名と限局性若年性歯周炎患者 6名の歯肉溝液を、 実施例 1で 得た生物学的被検物質採取用具で採取し、 実施例 3で得た金コロイ ド粒 l o 子で標識した A. actinomycetemcomitans ATCC 2 9 5 2 3株、 Y 4株 及び NCTC 9 7 1 0株の多糖.抗原溶液を混合後、 浸漬し、 反応させた。 室温で 5〜1 5分間反応させた後、 採取用具を取り出し、 蒸留水で洗浄 した。 そこで、 採取用具の歯肉溝液が吸着した部分が、 金コロイ ド粒子 の色である赤紫色に染まった場合を陽性、 染まらなかった場合を陰性と 5 判定した。  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.
また、 同時に、 健常者 3名と限局性若年性歯周炎患者 6名の歯肉溝液 中の A. actinomycetemcomitans の多糖抗原に対する抗体の検出を ELI SA 法を用いて、 下記のようにして行った。  At the same time, the detection of antibodies against the polysaccharide antigen of A. actinomycetemcomitans in the gingival sulcus of three healthy subjects and six patients with localized juvenile periodontitis was performed using the ELI SA method as follows. .
まず、 炭酸緩衝液 （pH 9 . 0 ) に溶解した actinomycetemcomitan 0 s ATCC 2 9 5 2 3株、 Y 4株及び NCTC 9 7 1 0株の多糖抗原を、 ELI SA用プレー卜の各ゥエルに 1 ずつ分注し、 4 °Cで一晚放置した。 翌日、 PBST で洗浄いた後、 1 0 %ャギ血清を含む PBST で、 室温、 1 時間放置し、 ブロッキングをした。 次に、 市販の紙製コヨリ （ペーパー ポイント） にあらかじめ採取しておいた歯肉溝液を、 PBST に溶かし出 して回収したものを加えた。 3 7 で 1時間反応させた後、 アルカリフ ォスファターゼ標識抗ヒト I g (G +M + A) を加え、 3 7 °Cで 1時間 反応させた。 次に、 PBSTでよく洗浄した後に、 p— Mtrophenylphosph ate を含む基質溶液を加え、 3 7 °Cで 3 0分間反応させた。 そこで、 吸 5 光度を測定し、 その発色の程度により、 披検体中の actinomycetemc omitans に対する抗体の検出をした。 First, 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 next day, after washing with PBST, the plate was left standing at room temperature for 1 hour with PBST containing 10% goat serum for blocking. Next, 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. After the reaction at 37 for 1 hour, alkaline phosphatase-labeled anti-human Ig (G + M + A) was added, and the reaction was carried out at 37 ° C for 1 hour. Next, after washing well with PBST, a substrate solution containing p-Mtrophenylphosphate was added and reacted at 37 ° C for 30 minutes. Therefore, the absorbance was measured and the antibody against actinomycetemc omitans in the specimen was detected based on the degree of color development.
以下にその結果を示す。 表 1 0に示すように、 健常者 （3名） の歯肉 溝液では、 金コロイド粒子による着色は認めず、 すべて陰性の結果を示 した。 また、 ELISA法による歯肉溝液中に存在する A. actinomycetemc 1 0 omitans の多糖抗原に対する抗体の検出では、 A. actinomycetemcomita ns の多糖抗原に対する抗体は認められなかった。 一方、 限局性若年性 歯周炎患者 （6名） の歯肉溝液では、 金コロイド粒子による着色が認め られ、 すべて陽性の結果を示した。 また、 ELISA法による歯肉溝液中に 存在する actinoraycetemcomitans の多糖抗原に対する抗体の検出で i s は、 A. act inomycetemcomi tans の多糖抗原に対する抗体を認めた。  The results are shown below. As shown in Table 10, in the gingival sulcus of healthy subjects (3), no coloration was observed with colloidal gold particles, and all showed negative results. Further, in the detection of an antibody against the polysaccharide antigen of A. actinomycetemc 10 omitans present in the gingival crevicular fluid by ELISA, no antibody against the polysaccharide antigen of A. actinomycetemcomitans was found. On the other hand, in the gingival crevicular fluid of patients with localized juvenile periodontitis (6 patients), coloring by colloidal gold particles was observed, and all showed positive results. In addition, in the detection of an antibody against the polysaccharide antigen of actinoraycetemcomitans present in the gingival crevicular fluid by ELISA, is was found to be an antibody against the polysaccharide antigen of A. actinomycetemcomitans.
表 1 0 ：歯肉溝液中の A. act inomycetemcomi tans に対する抗体の検出 被検者 No. 金コロイ ド標識多糖抗原との反応 ELISA 法 健常者 1 - - Table 10: Detection of antibodies against A. act inomycetemcomi tans in gingival crevicular fluid Subject No. Reaction with gold colloid-labeled polysaccharide antigen ELISA method Healthy subjects 1--
2 - - 0 3 ― 2--0 3 ―
患 者 1 + +  Patient 1 + +
2 + ++  2 + ++
3 ++ ++  3 ++++
4 + + 実施例 9 4 ++ Example 9
アル力リフォスファターゼ標識抗体  Al-force phosphatase-labeled antibody
調製例 4で得た各抗血清又は調製例 5で得た各モノクロナ一ル抗体を 2 O ral になるように 0 . 1 M リン酸緩衝生理食塩水に溶解する。 この 抗体溶液 2 ml に、 ZYMED社製のアルカリフォスファターゼ （EIA Grade ： 0 1 - 2 2 0 1 ) 4 O mg を加えて良く溶解する。 静かに撹拌しなが ら 1 %グルタルアルデヒドを 0 . l ml 滴下し、 室温で 2時間反応させる。 次に、 飽和硫安を等量加えて撹拌する。 遠心分離により得られた沈澱を 最少量のリン酸緩衝生理食塩水に溶解し、 リン酸緩衝生理食塩水に透析 する。 透析終了後、 S印 hadex G — 2 0 0を用いてゲル濾過し、 アル力 リフォスファターゼ標識抗体を得た。  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. To 2 ml of this antibody solution, add 4 Omg of alkaline phosphatase (EIA Grade: 01-221) manufactured by ZYMED, and dissolve well. While stirring gently, add 0.1 ml of 1% glutaraldehyde dropwise and react at room temperature for 2 hours. Next, 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. After completion of the dialysis, the mixture was subjected to gel filtration using S-marked hadex G-200 to obtain an Al-force phosphatase-labeled antibody.
実施例 1 0 Example 10
アル力リフォスファターゼ標識抗原による成人性歯周炎患者歯肉溝液 中の P. gingivalis の検出  Detection of P. gingivalis in the gingival crevicular fluid of adult periodontitis patients by using an anti-lipophosphatase-labeled antigen
健常者 3名と成人性歯周炎患者 6名の歯肉溝液を、 実施例 1で作成し た生物学的被検物質採取用具で採取し、 まず、 実施例 9で得たアルカリ フォスファターゼで標識した ^ gingivalis 3 8 1株と ^ gingival! s HW 2 4 D— 1株の線毛抗原に対するモノクロナール抗体溶液に浸漬し、 室温で 5〜1 5分間反応させた後、 採取用具を取り出し、 Tween 2 0を 含むリン酸緩衝生理食塩水で洗浄した。 次に、 5—プロモー 4一クロ口 —3—インドリルフォスフェイ トを含む基質溶液に浸潰し、 5〜1 5分 間反応させた。 そこで、 採取用具の歯肉溝液が吸着した部分が、 青色に 染まった場合を陽性、 染まらなかった場合を陰性と判定した。 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.
また、 同時に、 健常者 3名と成人性歯周炎患者 6名の歯肉溝液中の ^ gingivalis の同定を下記のようにして行った。  At the same time, ^ gingivalis in the gingival crevicular fluid of three healthy subjects and six adult periodontitis patients was identified as follows.
即ち、 市販の紙製コヨリ （ペーパーポイント） に吸収させた歯肉溝液 5 を輸送培地 （RTF : Reduced Transfer Fluid) に懸濁して回収し、 ゥサ ギ脱繊血、 へミン及びメナジオンを含む CDC培地 （Control Disease C enter開発） に播種し、 嫌気条件下で数日間培養した。 生育の見られた コロニーのうち、 黒色化したコロニーを選び、 これを分離し、 純培養後、 Rap ID ANA System (McDONNELL DOUGLAS社製） により、 同定試験を行 1 0 つた。 更に、 ^ gingivalis と同定されたものの培養上清をガスクロマ トグラフィ一にかけ、 フエニル酔酸の産生を調べることにより、 P. gin givalis であることの再認識を行った。  That is, gingival sulcus fluid 5 absorbed in a commercially available paper kori (paper point) is suspended in a transport medium (RTF: Reduced Transfer Fluid) and collected. 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.
以下にその結果を示す。 表 1 1に示すように、 健常者 （3名） の歯肉 溝液では、 着色は認めず、 すべて陰性の結果を示した。 また、 歯肉溝液 i s 中に存在する細菌の同定試験では、 P. gingivalis は認められなかった。  The results are shown below. As shown in Table 11, the gingival crevicular fluid of healthy subjects (3) showed no coloration and all showed negative results. In addition, P. gingivalis was not detected in the identification test for bacteria present in the gingival crevicular fluid is.
一方、 成人性歯周病患者 （6名） の歯肉溝液では、 6名中 6名に  On the other hand, the gingival crevicular fluid of adult periodontal disease patients (6)
ngivalis の線毛抗原に対するモノクローナル抗体溶液との反応が見ら れ、 すべて陽性の結果を示した。 また、 歯肉溝液中に存在する細菌の同 定試験では、 ^ gingivalis を認めた。 The reaction with the monoclonal antibody solution against the pilus antigen of ngivalis was observed, and all showed positive results. In addition, in the same test for bacteria present in the gingival crevicular fluid, ^ gingivalis was observed.
0 表 1 1 ：歯肉溝液中の ^ gingivalis の検出  0 Table 11: Detection of ^ gingivalis in gingival crevicular fluid
被検者 No. アルカリフォスファタ一ゼ 培養法による同定 標識抗原との反応  Subject No. Alkaline phosphatase Identification by culture method Reaction with labeled antigen
健常者 1 - 無  Healthy 1-None
2 無 3 2 nothing Three
患 者 1 ++  Patient 1 ++
2 ++ 有 3 + 有 4 + 有 5 + 有 6 ++ 有 産業上の利用可能性  2 ++ Yes 3 + Yes 4 + Yes 5 + Yes 6 ++ Yes Industrial applicability
以上述べたように、 本発明の採取用具は、 生物学的被検物質、 例えば 口腔内の歯周病原性細菌又は口腔内の歯周病原性細菌に対する特異抗体 を検出するのに有用であり、 例えば、 歯周病の診断の補助的手段として 適している。  As described above, 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.

Claims

請 求 の 範 囲 The scope of the claims
1 . 実質的に水不溶性かつ透水性の多孔性フィルムでコーティングさ れた吸水性材料からなる生物学的披検物質採取部分を有することを特徵 とする検体中の生物学的被検物質採取用具。  1. A tool for collecting 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. .
2. 少なくとも先端部分が実質的に水不溶性かつ透水性の多孔性フィ ルムでコ一ティングされたスティック状又は短冊状の吸水性材料からな る請求の範囲第 1項記載の採取用具。  2. The collection tool according to claim 1, wherein at least the tip portion is made of a stick-shaped or strip-shaped water-absorbing material coated with a substantially water-insoluble and water-permeable porous film.
3. 該吸水性材料の生物学的披検物質採取部分以外の部分のうち少な くとも検体と接触する可能性のある部分の表面が防水処理されている請 求の範囲第 1又は 2項記載の採取用具。  3. Claim 1 or 2 in the claim, wherein at least the part of the water-absorbent material other than the biological test substance collection part that may come into contact with the specimen is waterproofed. Fishing tools.
. 吸水性材料が高吸水性セルロース系ポリマー又は高吸水性合成ポ リマ一よりなるものである請求の範囲第 1〜3項のいずれかに記載の採 取用具。  The sampling tool according to any one of claims 1 to 3, wherein the water-absorbing material is made of a superabsorbent cellulosic polymer or a superabsorbent synthetic polymer.
5. 多孔性フィルムが 1 0ミクロン以下の平均孔径を有するものであ る請求の範囲第 1〜4項のいずれかに記載の採取用具。  5. The collection tool according to any one of claims 1 to 4, wherein the porous film has an average pore size of 10 microns or less.
6. 多孔性フィルムが多糖の有機酸もしくは無機酸ェステル又は多糖 の低級アルキルエーテル誘導体からなる請求の範囲第 1〜 5項のいずれ かに記載の採取用具。  6. The collection tool according to any one of claims 1 to 5, wherein the porous film comprises a polysaccharide organic or inorganic acid ester or a polysaccharide lower alkyl ether derivative.
7. スティック状又は短冊状の吸水性材料からなり、 該吸水性材料の 先端 1〜5 mmの長さからなる生物学的な披検物質採取部分に隣接する 少なくとも検体と接触する可能性のある部分の表面が防水処理されてい ることを特徴とする生物学的被検物質採取用具。  7. It is made of a stick-shaped or strip-shaped water-absorbing material, and is likely to come into contact with at least the sample adjacent to the biological sample collection part having a length of 1 to 5 mm at the tip of the water-absorbing material. A biological sample collecting tool characterized in that the surface of the portion is waterproofed.
8. 検体が歯肉溝液、 唾液又は歯垢処理液であり、 生物学的被検物質 が歯周病原性細菌又はそれに対する抗体である請求の範囲第 1〜 7項の いずれかに記載の採取用具。 8. The method according to claims 1 to 7, wherein the specimen is gingival crevicular fluid, saliva or plaque treatment liquid, and the biological test substance is periodontopathogenic bacteria or an antibody thereto. A collection tool according to any of the above.
9. 請求の範囲第 1〜 7項のいずれかに記載の採取用具の生物学的被 検物質採取部分を検体と接触させた後、 該生物学的被検物質と特異的に 結合しうる標識された物質からなる試薬と接触させ、 該採取部分に結合 9. A label capable of specifically binding to the biological test substance after the biological test substance collection portion of the collection tool according to any one of claims 1 to 7 is brought into contact with the sample. Contact with the reagent consisting of
5 した標識された試薬を定性的又は定量的に検出することを特徴とする検 体中の生物学的被検物質の測定方法。 5. A method for measuring a biological test substance in a sample, comprising qualitatively or quantitatively detecting the labeled reagent.
1 0. 検体が歯肉溝液、 唾液又は歯垢処理液であり、 生物学的被検物 質が歯周病原性細菌又はそれに対する抗体である請求の範囲第 8項記載 の方法。  10. The method according to claim 8, wherein the specimen is gingival crevicular fluid, saliva, or plaque treatment liquid, and the biological test substance is a periodontopathic bacterium or an antibody thereto.
1 0 1 1 . 標識された試薬が、 着色コロイ ド粒子又は酵素で標識された、 歯周病原性細菌の菌体もしくはその一部又はそれらに対する抗体である 請求の範囲第 1 0項記載の方法。  10. The method according to claim 10, wherein the labeled reagent is a cell of periodontopathogenic bacteria, a part thereof, or an antibody thereto, which is labeled with colored colloid particles or an enzyme. .
1 2. 着色コロイド粒子が金コロイ ドである請求の範囲第 1 1項記載 の方法。  12. The method according to claim 11, wherein the colored colloid particles are gold colloid.
i s 1 3. 酵素がアルカリフォスファターゼであり、 その基質が 5—プロ モ一 4—クロ口一 3—インドリルフォスフェイ トである請求の範囲第 1 1項記載の方法。 The method according to claim 11, wherein the enzyme is alkaline phosphatase, and the substrate is 5-promo-4-cloclo-3-indolyl phosphate.
1 4. 請求の範囲第 1〜7項のいずれかに記載の採取用具と、 検体中 の検出すべき生物学的被検物質と特異的に結合しうる標識された物質か 2 0 らなる試薬との組合わせよりなる検体中の生物学的被検物質の測定用キ ッ卜。  1 4. The collection tool according to any one of claims 1 to 7 and a reagent comprising a labeled substance capable of specifically binding to a biological test substance to be detected in a sample. A kit for measuring a biological test substance in a sample, the kit comprising:
PCT/JP1992/000694 1991-06-01 1992-05-28 Instrument for sampling minute quantity of specimen WO1992021952A1 (en)

Applications Claiming Priority (2)

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JP3/157724 1991-06-01
JP3157724A JPH04355339A (en) 1991-06-01 1991-06-01 Trace specimem sampling device

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JPH0797395A (en) * 1993-09-28 1995-04-11 Kyowa Medex Co Ltd Peptides containing sequence of porphyromonas gingivalis cilium protein and its use
ATE328278T1 (en) * 1998-03-30 2006-06-15 Orasure Technologies Inc COLLECTION APPARATUS FOR ONE-STEP TESTING OF ORAL FLUID
JP4603557B2 (en) * 2007-01-15 2010-12-22 有限会社 ミクロデント Quantitative standardized culture method
JP2017058154A (en) * 2015-09-14 2017-03-23 学校法人 日本歯科大学 Tool for collecting gingival crevice exudate
EP4212845A4 (en) 2020-09-11 2024-02-21 Fujifilm Corp Concentration device, liquid specimen concentration method, liquid specimen inspection method, and inspection kit
JPWO2022054516A1 (en) 2020-09-11 2022-03-17
EP4212876A4 (en) 2020-09-11 2024-02-28 Fujifilm Corp Liquid specimen concentration method, and liquid specimen inspection method

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