WO2010107120A1 - Anticorps et composition anti-périodontique contenant l'anticorps - Google Patents

Anticorps et composition anti-périodontique contenant l'anticorps Download PDF

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WO2010107120A1
WO2010107120A1 PCT/JP2010/054889 JP2010054889W WO2010107120A1 WO 2010107120 A1 WO2010107120 A1 WO 2010107120A1 JP 2010054889 W JP2010054889 W JP 2010054889W WO 2010107120 A1 WO2010107120 A1 WO 2010107120A1
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antibody
bacteria
periodontal disease
antigen
kinds
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PCT/JP2010/054889
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English (en)
Japanese (ja)
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冬彦 佐藤
貴史 伊井野
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株式会社ファーマフーズ
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Priority to JP2011504899A priority Critical patent/JP4982629B2/ja
Publication of WO2010107120A1 publication Critical patent/WO2010107120A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • the present invention relates to an antibody and an anti-periodontal disease composition containing the antibody, and in particular, a chicken egg antibody that suppresses the formation of a biofilm that becomes a lesion of periodontal disease bacteria and reduces the periodontal disease risk, and the chicken egg antibody. It is related with the anti-periodontal disease composition containing.
  • Periodontal disease is a national disease affecting approximately 80% of adults in Japan, and not only directly causes tooth loss, but also systemic diseases such as heart disease, cerebrovascular disease, pneumonia, lifestyle-related diseases, etc. A strong relationship with is clear.
  • Periodontal disease is an oral infection caused by periodontal disease bacteria attaching to and growing on the tooth surface and gingiva to form a biofilm. Biofilms are said to form and mature as follows.
  • periodontal pathogens such as Porphyromonas gingivalis , Prevotella intermedia , Actinobacillus actinomycetemcomitans , Tannerella forsythesis, Tannerella forsythe ⁇ Dentikora (Treponema denticola) late colonizers such is coaggregation, biofilm through the production of polysaccharides are formed.
  • the balance of the normal bacterial flora in the oral cavity is lost, and a plurality of periodontal pathogens interact with each other to form a biofilm (lesion) having periodontal pathogenicity.
  • Patent Document 1 discloses ciliates or capsules of oral bacteria selected from Bacteroides gingivalis, Bacteroides intermedius, Actinomyces viscosus, Haemophilus actinomycetemcomitans, Fusobacterium nucleatum and Aiconella collodense.
  • An oral composition characterized by containing an egg, egg yolk or a water-soluble yolk fraction obtained from a poultry immunized with an antigen as an antigen.
  • Patent Document 2 contains an antibody obtained by immunizing an animal with a synthetic peptide corresponding to a fragment derived from the amino acid sequence constituting the cilia of Actinobacillus actinomycetemcomitans as an antigen.
  • the composition for oral cavity characterized by this is disclosed.
  • Patent Document 3 discloses a peptide corresponding to a fragment derived from the amino acid sequence of the 72 KDa subunit protein constituting the type II pili of Porphyromonas gingivalis , wherein 5 to 10 consecutive fragments are present.
  • Periodontal disease preventive or periodontal comprising an antibody obtained by immunizing an animal with a peptide or derivative thereof or a salt thereof selected from a fragment group comprising the amino acid residues of An oral composition for treating disease is disclosed.
  • Patent Document 4 discloses an oral composition characterized by containing an antibody obtained by immunizing an animal with a polysaccharide derived from the surface of a bacterial body causing periodontal disease as an antigen.
  • Patent Document 5 includes a composition for oral formation for inhibiting biofilm formation or promoting biofilm removal containing sugar alcohol and / or amino acid
  • Patent Document 6 includes a histidine kinase inhibitor selected from ofloxacin and closantel.
  • An oral biofilm characterized in that it is contained at a concentration of 0.0001 to 10% by mass with respect to the whole product, and contains a phenolic fungicide, a surfactant, and one or more of alcohols.
  • Inhibitory composition Patent Document 7, discloses a coaggregation inhibitor of oral bacteria containing glycine or alanine as an active ingredient.
  • JP-A-10-152425 JP-A-9-52822 Japanese Patent Laid-Open No. 8-48695 JP-A-6-40871 JP 2005-29484 A JP 2005-187377 A JP 2005-53851 A
  • antibodies obtained using conventional components of periodontal disease bacteria as antigens have the advantage that they can sterilize and remove specific bacteria without affecting other oral resident bacteria. Since it is necessary to act, once the biofilm is formed, it is difficult to act on the bacteria in the biofilm, and a sufficient prevention / improvement effect cannot be expected.
  • periodontal pathogens in biofilms are known to interact with multiple types of periodontal pathogens, but little is known about the antigenic changes of periodontal pathogens. No antibody has been developed that focuses on the antigenic changes.
  • an object of the present invention is to provide biofilm formation inhibition and removal thereof, an antibody having a high effect on periodontal disease bacteria, and an anti-periodontal disease composition containing the same.
  • the present inventors can pay attention to the interaction of multiple types of periodontal pathogens in biofilms, and obtain a more effective antibody by using a mixed culture of multiple types of periodontal pathogens as an antigen.
  • the present inventors have found that this can be done and have completed the present invention.
  • the antibody of the present invention is obtained from a bird egg immunized with a culture obtained by culturing a mixture of two or more kinds of periodontal disease bacteria as an antigen.
  • the culture is preferably a biofilm-like deposit produced when two or more kinds of periodontal disease bacteria are mixed and cultured.
  • the periodontal disease bacteria may be Porphyromonas gingivalis , Fusobacterium nucleatum , Actinobacillus actinomycetemcomitans , Tanner forsythen, Tanner forsythen ⁇ Dentikora (Treponema denticola), Prevotella intermedia (Prevotella intermedia), is preferably 2 or more kinds of bacteria selected from Streptococcus gordonii (Streptococcus gordonii).
  • the two or more types of periodontal disease bacteria are two or more types of periodontal disease bacteria having different aggregation states depending on whether they are cultured alone or in a mixed culture.
  • the two or more types of periodontal disease bacteria include Fusobacterium nucleatum , Porphyromonas gingivalis , Actinobacillus actinomycetemcomitans , Prevotella interferi Media (Prevotella intermedia), is preferably 1 or more periodontal bacteria selected from four Tan'nera Four Shi Ten cis (Tannerella forsythensis).
  • the surface layer of periodontal pathogens may interact with each other between Fusobacterium nucleatum and the above four kinds of periodontal pathogens, MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Sept. 2002, p.
  • the aggregation state changes in the mixed culture compared to the single culture, and that the antibody may change when the mixed culture is used as an antigen.
  • the aggregation state of the mixed culture changes due to the interaction between the periodontal disease bacteria surface layers.
  • the inventors of the present application in particular, use a mixed culture of P. gingivalis and F. nucleatum as an antigen, so that a very high biofilm formation inhibition rate and coaggregates are obtained. It was found that the adhesion deterrence rate was realized.
  • the inventors of the present application have conducted intensive studies on the antigen preparation method, immunization method, combination of periodontal disease bacteria to be mixed and cultured. As a result, it was revealed that antibodies with higher effectiveness can be obtained. In particular, the results suggesting that there is no particular limitation on the combination of periodontal disease bacteria to be mixed and cultured were obtained.
  • the anti-periodontal disease composition according to the present invention is characterized by containing the antibody as an active ingredient.
  • the present invention by using a culture obtained by mixing and cultivating two or more kinds of periodontal disease bacteria as an antigen, it is possible to provide a more effective antibody than conventional antibodies using a single fungus. . And the anti-periodontal disease composition with a high anti-periodontal disease effect can be provided by using this antibody.
  • periodontopathic bacteria used for the preparation of antigens are Porphyromonas gingivalis , Fusobacterium nucleatum , Actinobacillus actinomycetemcomitans , Tenneraforsi cis (Tannerella forsythensis), Treponema Dentikora (Treponema denticola), Prevotella intermedia (Prevotella intermedia), is preferably 2 or more kinds of bacteria selected from Streptococcus gordonii (Streptococcus gordonii).
  • Porphyromonas gingivalis P.
  • gingivalis is referred to as "Gingivalis” or “Pg”
  • Fusobacterium nucleatum F. nucleatum
  • Nucleatum Fn
  • Actinobacillus -Actinomycetemcomitans Actinobacillus actinomycetemcomitans
  • actinomycetemcomitans Actinobacillus actinomycetemcomitans
  • ATCC American Type Culture Collection
  • BRC RIKEN BioResource Center
  • the biofilm-like adherent means an insoluble matter having adherence composed of microbial cells, polysaccharides, proteins, etc., produced when two or more kinds of periodontal disease bacteria are mixed and cultured.
  • the mixed culture conditions of the above periodontal disease bacteria are those under which suitable growth and coaggregation can be confirmed when two or more selected periodontal disease bacteria are mixed and cultured (usually anaerobic conditions at about 37 ° C. and about 24-72).
  • suitable growth and coaggregation can be confirmed when two or more selected periodontal disease bacteria are mixed and cultured (usually anaerobic conditions at about 37 ° C. and about 24-72).
  • the following method can be exemplified.
  • each culture solution is mixed at a predetermined ratio (volume). A part of this mixed solution is added to a fresh medium, and statically cultured at 37 ° C. for 24-72 hours under anaerobic conditions.
  • the cells are inactivated by a method such as adding formalin to the culture solution according to a conventional method, and after centrifugation and washing with physiological saline (or PBS), an appropriate amount of physiological saline Suspend in water or PBS.
  • An antigen sample can be prepared by homogenizing this suspension by homomixing and sonication.
  • the bacterial count ratio of the two bacteria is preferably 1: 1 to 100: 1, and more preferably 1: 1 to 30: 1.
  • the antigen preparation prepared as described above may be stored frozen or lyophilized until use for immunization.
  • the chicken used for immunization is not particularly limited, but it is particularly preferable to use egg species such as white leghorn, Rhode Island Red, lateral primus rock, New Hampshire from the viewpoint of mass production of antibodies. .
  • the immunization method is not particularly limited as long as it can immunize animals such as chickens, such as subcutaneous injection, intramuscular injection, and intraperitoneal administration.
  • the inoculation amount of the antigen may be appropriately selected so that the desired antibody titer is obtained and does not adversely affect animals such as chickens.
  • the amount of protein is 0.01 to 10 mg per immunization preferable.
  • booster immunization is recommended every 1 to 4 months for the purpose of maintaining antibody titer.
  • the antigen may be repeatedly inoculated about 3 to 5 times once a week after the first immunization to before the booster immunization.
  • the antibody titer in chicken eggs can be measured using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, microtiter method, etc., and the antibody titer is measured by measuring the antibody titer at intervals of about 2 weeks after immunization. The transition of the price can be tracked. Usually, when one month or more has passed since the first immunization, a desired specific antibody having a sufficient antibody titer is transferred and accumulated in the yolk of a chicken egg.
  • ELISA enzyme-linked immunosorbent assay
  • the egg yolk antibody of the present invention can be obtained as egg yolk liquid or powdered egg yolk powder by separating egg yolk from chicken eggs immunized as described above. Further, immunoglobulin (IgY) may be extracted and purified from egg yolk by a known method.
  • immunoglobulin IgY
  • Preparation of egg yolk antibody from egg yolk may be performed according to known methods used for immunoglobulin extraction and separation, such as dextran sulfate and polyethylene glycol (PEG), agar, carrageenan, farseleran, pectin, xanthan gum, alginic acid, Methods for precipitating lipoproteins using alginates, alginic acid derivatives, etc., and separating and purifying them from the supernatant (for example, Journal of Immunological Methods, 46, P63-68 (1981), Immunological Communication, 9 (5), P475-493 (1980), JP-A-63-215699, JP-A-64-38098), extraction methods using propanol, chloroform and the like.
  • PEG polyethylene glycol
  • agar agar
  • carrageenan farseleran
  • pectin xanthan gum
  • alginic acid alginic acid
  • egg yolk fluid is egg yolk water-soluble protein powder obtained by pulverizing egg yolk water-soluble protein using carrageenan or the like, or egg yolk water-soluble protein is ion-exchange chromatography, hydrophobic chromatography, affinity It can be prepared in various forms such as a purified chicken egg antibody purified by a known protein purification method such as chromatography, gel filtration, sodium sulfate salting out, ammonium chloride salting out.
  • the purity of the egg antibody of the various prepared samples obtained as described above is calculated based on the weight of the egg egg antibody relative to the weight of the powder.
  • the form of egg yolk powder usually 1 to 2% of the egg antibody, and in the form of egg yolk water-soluble protein powder It is usually 8-12%, and in the form of purified chicken egg antibody, it is usually 95% or more.
  • egg yolk juice, egg yolk powder, egg yolk water-soluble protein powder, purified antibody, etc. containing the above-described chicken egg antibody can be used as an anti-periodontal disease composition as they are.
  • the dosage form can be appropriately selected from powder / granular form, liquid form, capsule form, tablet form, jelly form and the like.
  • the anti-periodontal disease composition of the present invention comprises a tooth color remover, a halitosis preventive agent, a caries preventive agent such as fluorine, a medicinal component such as an anti-enzyme preventive agent, an excipient, an emulsifier, a saccharide, and a vitamin as necessary.
  • Other ingredients such as flavours, perfumes and the like can be included.
  • the anti-periodontal disease composition of the present invention includes toothpastes such as toothpaste, toothpaste and liquid toothpaste, dental materials, mouthwash, intraoral pasta, gingival massage cream, gargle tablets, troches, chewing gum, canned beverages, etc. Not only oral materials but also foods such as chocolate, candy, yogurt, cheese, ice cream, prepared milk, baby food, pet food and the like.
  • toothpastes such as toothpaste, toothpaste and liquid toothpaste, dental materials, mouthwash, intraoral pasta, gingival massage cream, gargle tablets, troches, chewing gum, canned beverages, etc.
  • oral materials not only oral materials but also foods such as chocolate, candy, yogurt, cheese, ice cream, prepared milk, baby food, pet food and the like.
  • a polishing agent such as calcium carbonate, calcium hydrogen phosphate, calcium pyrophosphate, insoluble sodium metaphosphate, alumina, anhydrous silicic acid, humectant such as glycerin, sorbit, propylene glycol, sodium laurate sulfate, lauroyl sarcosine
  • a foaming agent such as sodium and soap powder, a binder such as sodium carboxymethylcellulose and carrageenan, and other components such as perfume ingredients, sweeteners, preservatives and colorants may be mixed with water and produced according to a conventional method.
  • Formalin was added to the obtained mixed culture solution to 0.5% (v / v), and the cells were left at 37 ° C. for 24 hours to inactivate the cells.
  • the mixed culture solution was centrifuged to collect the insoluble fraction, washed three times with physiological saline, and then the insoluble fraction was dispersed in physiological saline and then homogenized.
  • This solution was subjected to microscopic observation under Gram staining to determine the number of bacteria and appropriately diluted to obtain antigen G3 (Gingivalis: 5.0 ⁇ 10 9 cells / mL, Nucleatum: 1.3 ⁇ 10 9 cells / mL).
  • gingivalis was cultivated and treated alone in the same manner as described above, and the bacterial concentration was adjusted to be equivalent to the number of mixed culture antigen G3 (5.0 ⁇ 10 9 cells / mL).
  • the nucleatum is cultured and treated alone, and the bacterial concentration is adjusted to be the same as that of the mixed culture antigen G3 (1.3 ⁇ 10 9 cells / mL) to obtain the antigen G2. It was.
  • antigen G1 and G2 are mixed and adjusted to be equivalent to the number of the above mixed culture antigen G3 (Gingivalis: 5.0 ⁇ 10 9 cells / mL, Nucleatum: 1.3 ⁇ 10 9 cells / mL) And antigen G4 was obtained.
  • FIG. 1 is image data showing the state of each antigen obtained as described above.
  • No. 1 is Gimbalis alone (G1)
  • No. 2 is Nucleatum alone (G2)
  • No. 3 is a mixed culture of both (G3)
  • No. 4 is a single culture of Gimbalis and Nucleatum (G4) It is a thing.
  • Each image shows a state after each antigen is dispersed by ultrasonic treatment and allowed to stand for 30 minutes. It is clearly observed that a change in the state of the antigen is caused by the mixed culture.
  • FIG. 2 is image data showing an electron micrograph of each antigen. Each image data was imaged using an electron microscope (manufactured by Hitachi High-Technologies Corporation, TM-1000).
  • No. 1 is Gimbalis alone (G1)
  • No. 2 is Nucleatum alone (G2)
  • No. 3 is a mixed culture of both (G3)
  • No. 4 is a single culture of Gimbalis and Nucleatum (G4) It is a thing. Aggregation between Gimbalis and Nucleatum is clearly different between No. 4 in which each single culture is mixed and No. 3 which is a mixed culture. It can be presumed that the cohesiveness is different from the mixture of the single culture solution when mixed culture is performed due to the interaction between the surface layers of both bacteria.
  • Antigens G1 to G4 prepared above are mixed in equal amounts with Freund's complete adjuvant, and 1 ml each is injected into the thigh muscle of a hen (Boris Brown) for the first immunization. went. Similarly, the second immunization was performed 9 weeks after the first immunization. In this example, the change in antibody titer was followed by ELISA, and eggs at the stage where the antibody titer was sufficiently increased were collected and used for the preparation of each antibody.
  • IgY control IgY prepared in the same manner from unimmunized chicken eggs was used.
  • the plate is lightly washed four times with distilled water, the biofilm formed on the plate is stained with crystal violet (hereinafter referred to as CV), ethanol is added, and the dye is extracted from the colored biofilm.
  • the extract was measured for absorbance (OD570) using a spectrophotometer. Then, the absorbance value of the positive control to which no chicken egg antibody was added was the biofilm formation inhibition rate of 0%, the absorbance value of the negative control to which neither the chicken egg antibody nor periodontal bacteria were added was the biofilm formation inhibition rate of 100%, The biofilm formation inhibition rate was determined from the absorbance value. The results are shown in FIG.
  • antibody P3 obtained using antigen G3 obtained by mixing and culturing two bacteria especially antibodies P1 and P2 obtained using a single fungus as an antigen at a low concentration (2 ⁇ g / mL) It can also be seen that the biofilm formation inhibition rate is higher than that of antibody P4 obtained using a mixture of these antigens.
  • antibody P3 obtained using antigen G3 obtained by mixing and culturing two bacteria is obtained using antibodies P1 and P2 obtained using a single bacterium as an antigen, or a mixture of these antigens. It can be seen that the coaggregation adhesion inhibition rate to the collagen-coated plate is higher than that of the obtained antibody P4.
  • the antibody P3 obtained using the antigen obtained by mixing and culturing the two bacteria effectively suppresses adhesion of the coaggregate to the collagen, which is the initial stage of biofilm formation, It can be seen that the biofilm formation itself is also effectively suppressed.
  • the anti-biofilm formation inhibition rate and the co-aggregate adhesion inhibition rate of the antibody obtained due to the different aggregation properties of at least two periodontal disease bacteria in the mixed culture antigen solution If periodontitis is improved, the periodontal disease bacteria that can be used are not limited to two bacteria, Gindivarius bacteria and Nucleatum bacteria, and the possibility that other periodontal disease bacteria may be used can be predicted.
  • the biofilm-like bacterial cell adhering mixture cultured in the cell culture dish was collected using a scraper, the supernatant was discarded by centrifugation, washed twice with PBS, and finally 20 ml of PBS. After adding and suspending, the suspension is sonicated in ice-cold water for 10 minutes (Sonics & Materials Inc., Ultrasonic processor, Model: VC130, Amplitude: 50%, Timer: 10 min, Pulser: 5 sec), and antigen G9 was obtained.
  • Antigen G5 was obtained in the same manner as antigen G1 of Example 1 except that TSB medium was used and sonication was performed as an inactivation treatment.
  • Antigen G6 was obtained in the same manner as antigen G2 of Example 1, except that TSB medium was used and sonication was performed as an inactivation treatment.
  • Antigen G7 was obtained in the same manner as antigen G3 of Example 1 except that TSB medium was used and sonication was performed as an inactivation treatment.
  • Antigen G8 was obtained in the same manner as antigen G4 in Example 1, except that TSB medium was used and sonication was performed as an inactivation treatment.
  • FIG. 5 shows that in the case of antigen G9, peaks appear around 30 KDa and 28 KDa, and the protein composition is clearly different from antigen G6 (F.n alone) and antigen G5 (P.g alone).
  • Clinical isolate crossover test A “clinical isolate” is a strain that is actually isolated from the oral cavity of a human and is said to be a strain with high biofilm-forming ability. Therefore, it can be said that an actual practical effect can be confirmed if the effectiveness of the antibody studied by the present inventors can be confirmed against clinical isolates.
  • ST1, S-10, etc. of clinical isolates of Pg and Fusobacterium are control numbers and do not specify the structure of the strain.
  • the following preparation was performed to prepare an antigen solution used for ELISA.
  • the clinical isolates and standard strains used were anaerobically cultured (37 ° C.) using a TSB liquid medium containing 5 ⁇ g / mL hemin and 1 ⁇ g / mL vitamin K3.
  • the culture broth was collected by centrifugation and washed 3 times with PBS. Thereafter, the bacteria were suspended in 0.1 M carbonate buffer (pH 9.6) containing a protease inhibitor and stored at ⁇ 80 ° C. until disrupted. The thawed bacterial suspension was subjected to ultrasonic treatment (30 seconds ⁇ 10 times, on ice) to obtain a bacterial cell disruption solution. Centrifuge supernatant of the disrupted solution is diluted with 0.1 M carbonate buffer (pH 9.6) containing protease inhibitors so that the protein concentration is 500 ⁇ g / mL, and then the antigen solution for solid phase (clinical isolate / Standard strain) was prepared and stored at -30 ° C.
  • the test was performed at a protein concentration of 50 ⁇ g / mL and an antibody concentration of 5 ⁇ g / mL in the antigen solution for immobilization.
  • the antibodies P7 to P9 were each dissolved in a blocking solution and then centrifuged, and 100 ⁇ L of each supernatant was poured into each well as a primary antibody solution and incubated at 37 ° C. for 1 hour to perform the primary antibody treatment. . Thereafter, each well was washed 3 times with PBST and transferred to secondary antibody treatment.
  • anti-chicken IgY [IgG] H + L (AP label, ZYMED LABORATORIES) was used. 100 ⁇ L of a solution obtained by diluting the secondary antibody with a blocking solution 2,000-fold was poured into each well and incubated at 37 ° C. for 1 hour. After washing each well 3 times with PBST, 100 ⁇ L of coloring solution (1 mg / mL Disodium p-nitrophenyl phosphate) Hexahydrate in 10% diethanolamine-HCl buffer (pH 9.8)) was poured into each well. After incubating at room temperature for about 15 minutes, the reaction was stopped by adding 50 ⁇ L of 5N NaOH and subjected to absorbance measurement (405 nm) using a plate reader.
  • FIG. 6 shows the results of applying the ELISA method to the standard strain and clinical isolate of P. g.
  • gray indicates antibody P8
  • black indicates antibody P9
  • white indicates antibody P7.
  • the high affinity of antibodies P7 and P9 for various clinical isolates compared to antibody P8 indicates the usefulness of the antibody obtained in the present invention.
  • FIG. 7 shows the result of applying the ELISA method to the standard strain and clinical isolate of the genus Fusobacterium .
  • gray indicates antibody P8
  • black indicates antibody P9
  • white indicates antibody P7.
  • the high affinity of antibodies P7 and P9 for various clinical isolates compared to antibody P8 indicates the usefulness of the antibody obtained in the present invention.
  • the antibody P9 obtained by using the antigen G9 obtained by mixing and culturing the two bacteria on the cell culture dish is compared with the antibody P8 obtained by using the antigen G8 mixed with the single bacteria. It can be seen that the biofilm formation inhibition rate is high.
  • nucleatum F.n
  • actinomycetemcomitans A.a
  • Aa bacteria and Fn bacteria were each cultured in a conventional manner using TSB medium. Add each culture solution to TSB medium and mix it so that the turbidity (OD600) of the Aa bacteria culture solution is 0.06 and the turbidity (OD600) of the Fn culture solution is 0.04.
  • a liquid was prepared. This mixture is allowed to stand at 37 ° C for 24 hours under anaerobic conditions, and then the cells are collected by centrifugation. The supernatant is discarded, washed twice with PBS, and finally 20 mL of PBS is added. After suspension, this suspension was subjected to sonication in the same manner as in Example 2 to obtain antigen G11.
  • the A.a bacterium was cultured and treated alone in the same manner as described above to obtain the antigen G10.
  • the P10 to P12 antibodies and the unimmunized control antibody were added at 100 ⁇ g / ml, respectively, and 200 ⁇ l was dispensed into each well of a 96-well flat bottom transparent plate.
  • the plate was shaken for 30 seconds for the first 30 seconds using a 96-well microphone plate reader (ARVO MX, Perkin Elmer) with shaking function, and then turbid for a total of 30 times every 1 minute and 20 seconds (40 minutes in total). Degree (OD630) was measured. When coaggregation of periodontal disease bacteria occurs, the turbidity (OD630) decreases, so the coaggregation inhibitory effect was measured by observing the decrease in turbidity (OD630). The results are shown in FIG.
  • the antibody P11 obtained using the mixed culture antigen (G11) of Aa bacteria and Fn bacteria is the antibody P10 obtained using the Aa bacteria single antigen (G10), and a mixture of Aa bacteria and Fn bacteria.
  • the turbidity (OD630) decreased more slowly and was found to have a high ability to inhibit coaggregation. This suggests the possibility that antibodies obtained using an antigen obtained by mixing and cultivating two or more kinds of other periodontal disease bacteria may exhibit a high ability to inhibit biofilm formation.
  • the present invention can be applied to, for example, an anti-periodontal disease composition excellent in the effect of suppressing the formation of a biofilm that becomes a lesion of periodontal disease.

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Abstract

L'invention porte sur un anticorps qui est efficace pour inhiber la formation d'un biofilm et retirer celui-ci et a un effet élevé à l'encontre de bactéries périodontiques; et sur une composition anti-périodontique qui contient ledit anticorps. Un anticorps d'œuf de poule qui est obtenu à partir d'un œuf aviaire ayant été immunisé par un antigène qui est une culture obtenue par une culture mixte d'au moins deux sortes de bactéries périodontiques, est utilisé comme composition anti-périodontique. Les bactéries périodontiques comprennent de préférence au moins deux sortes de bactéries choisies parmi Porphyromonas gingivalis, Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Tannerella forsythensis, Treponema denticola, Prevotella intermedia et Streptococcus gordonii.
PCT/JP2010/054889 2009-03-20 2010-03-19 Anticorps et composition anti-périodontique contenant l'anticorps WO2010107120A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
JP2019004888A (ja) * 2016-07-11 2019-01-17 三菱ケミカル株式会社 口腔内検査方法
JP7181688B2 (ja) 2015-08-05 2022-12-01 日油株式会社 バイオフィルム形成予防剤および口腔用組成物

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JPH10152425A (ja) * 1998-01-05 1998-06-09 Lion Corp 口腔用組成物

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JP2019004888A (ja) * 2016-07-11 2019-01-17 三菱ケミカル株式会社 口腔内検査方法
JP2020141686A (ja) * 2016-07-11 2020-09-10 三菱ケミカル株式会社 口腔内検査方法
JP7122337B2 (ja) 2016-07-11 2022-08-19 株式会社ジーシー 口腔内検査方法

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