JPH04355339A - Trace specimem sampling device - Google Patents

Abstract

PURPOSE:To sample even a very small amount of a substance to be inspected biologically by arranging a part, from which the substance to be inspected biologically is sampled, comprising a water absorbing material coated with a non-water soluble and water permeating porous film. CONSTITUTION:The tip part of a stick-shaped (or strip-shaped) water absorbing material 1 is coated with a water permeating porous film 2 unsoluble in water and made of nitrocellulose, cellulose acetate or the like and moreover, a water proofing treatment 4 is applied for a part possibly contacting a sample leaving a specified length of a part 3 from which a substance to be inspected biologically is sampled at the tip. When a sampling device is brought into contact with the sample, the substance to be inspected biologically is concentrated at the part 3 subjected to the sampling to be adsorbed and solidified. Thus, when brought into contact with a sample comprising a substance labeled bondable specifically to the substance to be inspected biologically, the substance to be inspected biologically in the sample is detected directly and quickly.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to a micro-sample collection tool that can be used to detect biological test substances, such as periodontal pathogenic bacteria in the oral cavity or specific antibodies against periodontal pathogenic bacteria in the oral cavity. The present invention relates to a method for measuring a biological test substance in a sample using the method and a kit for the measurement.

0002

[Prior Art and Problems] Periodontal disease is divided into gingivitis and periodontitis, and periodontitis includes adult periodontitis, localized juvenile periodontitis, and the like. These periodontal diseases are diseases that lead to gingival inflammation, bleeding, pus drainage, formation of periodontal pockets, destruction of periodontal ligament, alveolar bone resorption, tooth movement, and tooth loss.

[0003] However, the current treatment methods for periodontal disease are as follows:
At present, periodontal disease has not been completely established, and it is considered most important to discover periodontal disease as early as possible and take preventive measures. Various types of bacteria exist in the above-mentioned periodontal disease lesions, but 3 to 4 of the total bacteria
Porphyromonas gingiva is said to be dominated by Gram-negative anaerobic bacteria.
lis (hereinafter referred to as P. gingivalis), Pre
votella intermedia (hereinafter referred to as P.
intermedia), Actinobacillus
A. actinomycetemcomitans (hereinafter referred to as A. actinomycetemcomitans)
s), Treponema denticola (hereinafter referred to as T. denticola), Bacteroide
s forsythus (hereinafter referred to as B. forsyth
us), Capnocytophaga gingiv
alis (hereinafter referred to as C. gingivalis), Fu
sobacterium nucleatum (hereinafter
F. nucleatum), Wolinella r.
W. ecta (hereinafter referred to as W. recta) and the like are considered to be periodontal pathogenic bacteria. A significant increase in these periodontal pathogenic bacteria is observed in the focal areas of periodontal disease. In addition, Porphyromonas gingiva
lis, Prevotella intermedia
is the conventional Bacteroides gingiv
alis, Bacteroides intermed
ius, and a recent new classification (1990
International Conference
T. on Periodontal Disease. J. M. van Steenbergen), it is called this way.

[0004] Focusing on these periodontal pathogenic bacteria, various methods have been proposed to determine the presence or absence and amount of periodontal pathogenic bacteria as an auxiliary means for diagnosing periodontal disease. .

[0005] For example, periodontal pathogenic bacteria living in specimens from periodontal pockets such as gingival crevicular fluid and subgingival plaque (dental plaque) of patients with periodontal disease are cultured on a blood agar medium under anaerobic conditions. It has been reported that periodontal pathogenic bacteria can be detected by examining the detailed biochemical properties of the various colonies obtained (Loesche, W.J., Syed, S.
．． A. , Schmidt, E. and Morriso
n, E. C. :J. Periodont. 56, 447
-456, 1985, etc.).

[0006] In addition, periodontal pathogenic bacteria living in periodontal pockets can be examined under a microscope after Gram staining or examined using a dark field microscope (Listgarten, M.A.
et al. :J. Clin. Periodontol
．． 5, 115-132, 1978), periodontopathogenic bacteria have also been detected by combining antibodies against periodontopathogenic bacteria with fluorescent dyes (Zambon, J.
．． J. , Bochacki, V. and Genco,
R. J. :Oral Microbiol. Immun
ol. 1, 39-44, 1986).

[0007] In addition, periodontal pathogenic bacteria living in periodontal pockets have been detected using antibodies against the respective periodontal pathogenic bacteria using an enzyme antibody method (ELISA method) (Zambon, Bochack, J.J.
i, V. and Genco, R. J. :Oral M
icrobiol. Immunol. 1, 39-44,
(see 1986).

[0008] It has also been reported that periodontal pathogenic bacteria living in periodontal pockets can be detected by focusing on enzymes that are very characteristic of periodontal pathogenic bacteria and using the enzyme activity as an indicator ( Loesche, W.J.: Oral Micr.
obiol. Immunol. 1, 65-70, 198
(see 6).

[0009] Furthermore, recently, attention has been paid to the genes of periodontal pathogenic bacteria, and DNA probe methods have been used to detect periodontal pathogenic bacteria living in periodontal pockets (Biotechnica Diagnostics).
company). Furthermore, PCR (Polymerase cha
It has become possible to detect even minute amounts of bacterial cells using the in reaction method.

On the other hand, regarding the antibody titer against periodontopathogenic bacteria in adult periodontitis patients, the increase in antibody-producing cells against periodontopathogenic bacteria in the gingival tissue of adult periodontitis patients, that is, the specific antibody reported using the ELISPOT method (Tomohiko Ogawa and
dShigeyuki Hamada: Clin. ex
p. Immunol. 76, 103-110, 1989
reference).

[0011] However, all of these methods have low detection sensitivity, problems with specificity, require long and complicated operations, and require expensive and special equipment, so they are not widely used. However, there is a need for the development of reagents and methods for diagnosing periodontal disease that are quick and simple.

0012

[Means for Solving the Problem] The present inventor has devised a method for efficiently collecting biological test substances in specimens that serve as diagnostic indicators for all diseases including periodontal disease, even if the amount is extremely small. We have conducted extensive research on tools that can be used to detect biological test substances in samples, measurement methods and measurement kits that can be used to directly, quickly, and easily detect biological test substances in specimens, and we have now developed the present invention. It was completed.

[0013] Thus, the present invention provides a method for detecting biological substances in a specimen, characterized in that the biological test substance collection portion is made of a water-absorbing material coated with a substantially water-insoluble and water-permeable porous film. This provides a tool for collecting target test substances.

[0014] The present invention also comprises a stick-like or strip-like water-absorbing material, and the tip of the water-absorbing material is 1 to 5 mm long.
To provide a biological test substance collection tool, characterized in that the surface of at least a part that is adjacent to a biological test substance collection part having a length and that is likely to come into contact with a specimen is waterproofed. It is something.

[0015] The present invention further provides a reagent comprising a labeled substance capable of specifically binding to the biological test substance, after the biological test substance collecting portion of the above-mentioned collection tool is brought into contact with the specimen. The object of the present invention is to provide a method for measuring a biological test substance in a specimen, characterized in that a labeled reagent bound to the collected portion is qualitatively or quantitatively detected.

[0016] The present invention further provides a method for detecting living organisms in a specimen, which comprises a combination of the above-mentioned collection tool and a reagent comprising a labeled substance capable of specifically binding to a biological test substance to be detected in the specimen. The present invention provides a kit for measuring a chemical test substance.

The present invention will be explained in more detail below.

[0018] The collection tool of the present invention can take various forms depending on the type of the target specimen, etc., but generally it is in the form of a stick (a thin rod that may be tapered) or a strip (with a wide width). It is desirable that the material be in the form of a narrow sheet or film. In addition, the collection tool may be made of any type of natural, semi-synthetic or synthetic material as long as the whole or at least the part for collecting the biological test substance is made of a water-absorbing material, and the material has high water absorption. For example, superabsorbent starch-based polymers, superabsorbent cellulose-based polymers, dextran-based polymers, superabsorbent synthetic polymers (e.g., polyacrylate-based, polyvinyl alcohol-based, polyacrylamide-based, polyoxy Among them, highly water-absorbent cellulose-based polymers and highly water-absorbent synthetic polymers are preferred.

Specifically, paper sticks known as "paper points" in the dental field (for example, paper sticks) are suitable as materials, and filter paper cut into narrow strips is also suitable. It can be conveniently used as a material.

According to one aspect of the present invention, the surface of at least the biological test substance collection portion of the absorbent material as described above, for example, at least the tip portion of the stick-like or strip-like absorbent material, is substantially coated with a water-insoluble and water-permeable porous film. By coating the surface of the water-absorbing material with a substantially water-insoluble and water-permeable porous film, the water in the sample can pass through the pores of the film and be absorbed into the water-absorbing material inside. , it is possible to adsorb and immobilize the biological analyte in the sample while concentrating it on the porous film coating, which is remarkable in that it becomes extremely easy to make judgments during subsequent detection using labeled reagents. It was found to be effective.

[0021] Therefore, the pore size of the porous film to be coated should be set to a size that allows the moisture in the specimen to pass through, but does not substantially allow the biological analyte to be detected to pass through. Desirably, for example, when the purpose is to detect bacteria such as periodontal pathogenic bacteria, the average pore diameter of the coated porous film is generally 10 microns or less, particularly about 0.45 to 5.0 microns. is desirable.

The material of the coating film is not particularly limited as long as it is substantially water-insoluble, and various polymers can be used. Specifically, for example, organic acids of polysaccharides or Examples include inorganic acid esters (for example, lower fatty acid esters or nitric acid esters of cellulose such as acetic acid and propionic acid), lower alkyl ether derivatives of polysaccharides (for example, ethyl cellulose, propyl cellulose, butyl cellulose, etc.), and polyamide resins.

[0023] As a method for forming a porous film coating of the material on the absorbent material, a method known per se can be adopted. For example, a cellulose-based membrane can be formed by a phase separation method. Specifically, cellulose is dissolved in a good solvent such as acetone or methylene chloride, and then a bad solvent such as ethanol or water, which has a higher boiling point, is added to create a uniformly mixed solution. By casting on a support and selectively drying and removing the good solvent by utilizing the difference in boiling point between the good solvent and the bad solvent, and further drying and removing the bad solvent, Uniform and fine porous membranes can be created.

The porous film coating on the absorbent material can be applied to cover at least the entire biological analyte collection portion of the collection device, for example in the case of sticks or strips of absorbent material. is applied so as to cover at least the tip thereof, and the length of the coating portion is at least 1 mm, preferably 2 mm from the tip.
It can be more than that.

[0025] Furthermore, the biological test substance sampling portion of the sampling tool of the present invention does not need to be unnecessarily large, and is desirably kept to the minimum possible extent without interfering with subsequent detection operations. As a means of achieving this, waterproofing is applied to the surface of at least the part of the collection tool that is likely to come into contact with the specimen, other than the part required to collect the biological test substance (biological test substance collection part). One method is to do so. Alternatively, it is also possible to secure the coated piece of water-absorbent material to a suitable support. This allows the specimen to be absorbed intensively only in a limited area of the sampling device coated with porous film, which further improves the adsorption and immobilization of the biological test substance in that area. This is effectively carried out, and determination using labeled reagents becomes even easier.

[0026] The waterproofing treatment may be carried out, for example, by contacting at least the sample adjacent to the biological test substance sampling portion, which is comprised of a coated stick-like or strip-like water-absorbing material having a length of 1 to 5 mm, preferably 2 to 4 mm. It can be applied to the surface of possible areas.

[0027] The waterproofing method includes a method in which the coating of a porous film except for the biological test substance sampling area is treated with a solvent capable of dissolving the film to substantially close the pores of the coating; Water-insoluble and non-water-absorbing polymers (e.g., fluorine resins, silicone resins, vinyl chloride resins, methacrylic resins, polyester resins, celluloid, paraffin, rubber, wood wax, beef tallow, etc.)
Examples include a method of applying a waterproof coating consisting of:

An example of the above-mentioned sampling tool of the present invention is shown in the attached FIG. 1. 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); is coated with a substantially water-insoluble and water-permeable porous film (2) made of nitrocellulose, cellulose acetate, etc., and further has a predetermined length of biological test substance sampling portion (3) at the tip.
), the parts that may come into contact with the specimen are waterproofed (4).

[0029] In another simplified embodiment of the sampling tool of the present invention, the tip of a stick-like or strip-like water-absorbing material is simply 1 to 5 mm, preferably 2 to 4 mm, without being coated with a porous film. By leaving the biological test substance collection part consisting of a long length and applying a waterproof coating to at least the other part of the water-absorbing material that is likely to come into contact with the specimen as described above, this method can be used. An inventive collection device may also be constructed.

[0030] In the sampling tool of this embodiment, there is no coating of a substantially water-insoluble and water-permeable porous film on the biological test substance sampling portion, and therefore, the above-mentioned effects achieved by the coating are However, even with this type of sampling tool, the specimen is absorbed intensively only at the localized tip, and the biological test substance is absorbed there at a considerable concentration. can be adsorbed and immobilized.

By using the collection tool of the present invention described above, blood contained in various body fluids, such as blood, saliva, tears, sweat, nasal mucus, urine, feces, semen, vaginal secretions, pus, and sputum, can be collected. Biological test substances such as components, serum, various cells, cytokines, hormones, peptides, metabolites, enzymes, physiologically active substances, tumor markers, genes (DNA, RNA), receptors, antibodies, antigens, microorganisms, viruses, etc. can be collected efficiently with extremely simple operations. For example, regarding periodontal disease, by inserting the stick-shaped collection tool of the present invention into the subject's teeth, teeth and gums, periodontal pockets, etc., gingival crevicular fluid, saliva, etc. Periodontopathogenic bacteria or their specific antigens, or antibodies against periodontopathogenic bacteria or their specific antigens can be easily collected from, for example, periodontopathogenic bacteria or their specific antigens.

[0032] Furthermore, when collecting a biological test substance in a specimen, it is possible to connect the collection tool to an appropriate suction source (or vacuum source) as necessary, thereby
Collection of biological test substances may be more effective.

Next, a method for measuring a biological test substance in a specimen using the collection tool of the present invention will be explained.

[0034] Collection of a biological test substance using the collection tool of the present invention is carried out by bringing the biological test substance collection portion of the collection tool of the present invention into contact with the above-mentioned specimen.

[0035] When the collection tool of the present invention is brought into contact with a specimen,
As described above, the biological test substance is concentrated, adsorbed, and immobilized on the collection portion.

The collection tool on which the biological test substance is adsorbed and immobilized in this way is then brought into contact with a reagent solution consisting of a labeled substance that can specifically bind to the biological test substance to be detected. let

A substance that specifically binds to a biological test substance (hereinafter referred to as a “specific binding substance”) is a substance that specifically binds to a biological test substance immunologically or chemically. Examples include binding antibodies, antigens, nucleic acids, and the like. For example, with regard to periodontal disease, examples include antibodies against periodontopathogenic bacteria or their specific antigens, or specific antigens of periodontopathogenic bacteria.

[0038] These specific binding substances are labeled. As the labeling substance, various labeling substances often used in the fields of immunological measurement or chemical measurement can be used, such as colored substances, colored colloid particles, fluorescent dyes, and radioactive isotopes. , enzymes, etc.

Examples of the coloring substance include inorganic pigments and organic pigments. As colored colloid particles,
For example, metal colloid particles such as whole colloid particles, polymer particles such as colored polymer latex particles, protein particles such as colored gelatin particles, colored colloidal silica particles, colored colloidal alumina particles, colored polysaccharide particles, etc. It is desirable that the particle size of these particles is generally in the range of 1 to 100 nm, preferably 5 to 40 nm. Such metal colloid particles, such as gold colloid particles, are usually made of chloroauric acid (HAu).
10 mg of Cl・2H2O) in 100 ml of boiling ultra-distilled water
, quickly add an appropriate amount of the freshly prepared 1% trisodium citrate aqueous solution, and boil for 5 to 10 minutes to form a red transparent liquid. After 2 to 3 minutes, gently cool to prepare colloidal gold particles. Can be done.

[0040]Fluorescent dyes used as labeling substances include, for example, fluorescein isocyanate (FITC),
Examples of radioisotopes include tetrarhodamine isocyanate (TRITC), and examples of radioactive isotopes include 3H, 14
Enzymes include C, 32P, 125I, etc.
For example, enzymes that decompose substrates and develop color, such as peroxidase and alkaline phosphatase, are suitable.

In particular, alkaline phosphatase may be used in combination with 5-bromo-4-chloro-3-indolylphosphate or p-nitroblue-tetrazolium chloride as its substrate. These substrates are colorless and transparent and water-soluble, but when they are decomposed by the action of enzymes, they each exhibit a unique color and become water-insoluble, allowing the presence of the labeled substance to be detected.

[0042] Methods for labeling a specific binding substance with such a substance include a physical bonding method and a chemical bonding method. The former is a method of physically adsorbing and bonding a specific binding substance and a labeling substance by simply mixing them, while the latter is a method of directly bonding a specific binding substance and a labeling substance with their respective functional groups. or carbodiimide,
This is a method in which a specific binding substance and a labeling substance are indirectly covalently bonded using a bivalent reagent such as glutaraldehyde.

[0043] The collection tool that has been brought into contact with the specimen described above is brought into contact with a reagent made of the labeled specific binding substance as described above, and after washing as necessary to remove excess reagent,
Depending on the type of sign used, visual inspection, densitometer,
By measuring the amount of label bound to the collection tool using a colorimeter, radiation measuring device, etc., and comparing the measurement results with a standard curve prepared in advance as necessary, the biology in the specimen can be determined. The amount of target analyte can be detected qualitatively or quantitatively.

[0044] The collection tool of the present invention used in the above-mentioned measurement system and the reagent consisting of a labeled specific binding substance can be combined into a kit to detect the biological analyte in the specimen. It can be a kit for measuring substances.

[0045] Hereinafter, a method for measuring a biological test substance in a specimen using the sampling tool of the present invention will be described. Detection of antibodies or antibodies against them will be described in more detail.

(1) Culture of periodontal pathogenic bacteria In order to prepare a periodontal disease diagnostic kit, first, P. P. gingivalis, such as P. gingivalis. P. gingivalis strain 381, P. gingivalis strain 381, P. gingivalis strain 381; gi
P. ngivalis strain 1021, P. ngivalis strain 1021; gingiva
lis ATCC strain 33277, P. gingiv
alis RB22D-1 strain, P. alis. gingival
is strain RB24M-2, P. gingivalis
RB46D-1 strain, P. gingivalis 6
/26 stocks, P. gingivalis W 1 strain, P
．． gingivalis W 50 strains, P. gingivalis W 50 strains, P. gingivalis W. gin
Givalis W 83 strain, P. gingival
is strain HW11D-5, P. gingivalis
HW24D-1 strain, P. gingivalis B
H18/10 strain, P. gingivalis 19A
4 stocks, P. P. gingivalis strain 22B4, P. gingivalis strain 22B4.
P. gingivalis strain 23A4, P. gingivalis strain 23A4. ging
ivalis HG 379 strain, P. gingiva
lis HG 405 strain, P. gingivalis
OMZ strain 314, P. gingivalis O
MZ 369 strain, P. gingivalis OMZ
409 stocks, P. gingivalis LB13D
-3 strain; or P. intermedia, such as P. intermedia ATCC 25261
Stock, P. intermedia ATCC 25611
Stock, P. intermedia OMZ 227 shares,
P. intermedia OMZ 248 strain, P.
intermedia OMZ strain 277, P. in
termmedia OMZ strain 311, P. inte
rmedia OMZ strain 326, P. interme
dia OMZ 327 strain, P. intermedi
a BH 18/23 strain, P. intermedia
BH 20/30 strain, P. intermedia
H187 strain, P. intermedia M 10
7-74 strain; or A. actinomycet
emcomitans, such as A. emcomitans. actinom
ycetemcomitans ATCC 29522
Stock, A. actinomycetemcomitan
s ATCC strain 29523, A. actinomy
cetemcomitans ATCC strain 29524, A. actinomycetemcomitans
Y 4 stocks, A. actinomycetem com
itans NCTC 9709 strain, A. actin
omycetemcomitans NCTC 971
0 shares, A. actinomycetemcomita
ns 1 strain, A. actinomycetem com
itans 15 strains, A. actinomycetem
comitans 27 strains, A. comitans. actinomyc
etemcomitans 29 strains, A. acti
nomycetemcomitans 32 strains, A. nomycetemcomitans.
actinomycetemcomitans 39 strains, A. actinomycetemcomitans
42 stocks, A. actinomycetem com
itans 67 strains, A. actinomycet
emcomitans 75 strains, A. emcomitans. actinom
ycetemcomitans strain 85; or T
．． denticola, such as T. denticola. dentic
ola ATCC 35405; or B. for
sythus, such as B. sythus. forsythus
ATCC 43037; or C. gingiva
lis, for example, C. gingivalis ATCC
33624; or F. nucleatum, such as F. nucleatum ATCC 2558
6; or W. recta, such as W. recta. rec
ta ATCC 33238 etc., for example, GAM
Bouillon, Brain-Heart Infusi
on broth, etc., supplemented with hemin and menadione, or Todd-Hewitt broth
After inoculating the cells into a medium supplemented with yeast extract and cultivating them under anaerobic conditions at about 37°C for about 20 hours, the cells are collected, washed with physiological saline, and freeze-dried. . Either the bacterial cells obtained in this way are bound to a labeling substance, or
Alternatively, it can be used as an immunizing antigen to obtain its specific antibody.

(2) Preparation of fimbriae antigen In order to obtain antibodies against specific antigens of periodontopathogenic bacteria, first, as a preparation of the specific antigen, periodontopathogens having fimbriae with specific antigenicity are prepared. sexual bacteria, such as P. gingivalis, for example, in GAM broth, Brain-Hea
Inoculate into a medium containing hemin and menadione, such as rt Infusion broth, and inoculate it under anaerobic conditions for about 30 minutes.
If cultured at ℃, a large number of bacterial cells can be obtained in about 20 hours or more. Collect the bacteria and physically remove the fimbriae by pipetting. Next, if the pili are purified through ammonium sulfate fractionation and ion exchange chromatography, P. gi
The pili antigen of S. ngivalis is obtained. The fimbrial antigen thus obtained is bound to a labeling substance or used as an immunogen to obtain its specific antibody.

(3) Preparation of polysaccharide antigen Periodontal pathogenic bacteria,
For example, A. actinomycetem commit
ans, for example, Todd-HeWitt b
If a medium prepared by adding yeast extract to roth is inoculated and cultured under anaerobic conditions at about 37°C, a large number of bacterial cells can be obtained in about 20 hours or later. Collect this and
Freeze-dry the cells. After suspending the obtained freeze-dried bacterial cells in physiological saline, heat extraction is performed in an autoclave, and the extract is separated and purified by, for example, ion exchange chromatography or gel filtration chromatography. ．．
actinomycetemcomitans polysaccharide antigen can be obtained. The polysaccharide antigen thus obtained is bound to a labeling substance or used as an immunogen to obtain its specific antibody.

(4) Preparation of antibodies Various animals are immunized with the freeze-dried cells of periodontal pathogenic bacteria obtained as described above. For example, animals such as rabbits and goats are immunized by subcutaneously or intramuscularly injected with freeze-dried cells of this periodontal pathogenic bacteria together with Freund's complete adjuvant, and then the blood of these animals is injected using known methods. Therefore, antiserum against periodontal pathogenic bacteria can be obtained. Furthermore, if necessary, the obtained antiserum is purified by ammonium sulfate fractionation and ion exchange chromatography according to conventional methods.

On the other hand, a mouse is immunized with freeze-dried cells of periodontopathogenic bacteria obtained as described above, and the splenocytes are taken out and cell fused with mouse bone marrow tumor cells using polyethylene glycol or the like to perform cloning. After that, hybridomas secreting monoclonal antibodies that specifically react with periodontal pathogenic bacteria are selected. These hybridomas are transplanted intraperitoneally into mice that have been previously treated with pristane, and monoclonal antibodies are obtained from the ascites, or the hybridomas are cultured in large quantities in serum-free medium.
Monoclonal antibodies can be obtained from the culture supernatant. Furthermore, the obtained monoclonal antibody is purified by ammonium sulfate fractionation and ion exchange chromatography according to conventional methods.

[0051] Furthermore, P.
fimbrial antigen of A. gingivalis or A. gingivalis. act
Animals are immunized with a polysaccharide antigen of P. inomycetemcomitans. For example, after immunizing animals such as rabbits and goats by subcutaneously or intramuscularly injecting these specific antigens with Freund's complete adjuvant, P. gingi
fimbrial antigen of A. valis or A. valis. actinomy
Antisera against polysaccharide antigens of C.cetemcomitans can be obtained. Furthermore, if necessary, the obtained antiserum is purified by ammonium sulfate fractionation and ion exchange chromatography according to conventional methods.

On the other hand, the P.
fimbrial antigen of A. gingivalis or A. gingivalis. act
A mouse was immunized with a polysaccharide antigen of P. inomycetemcomitans, the splenocytes were taken out, the cells were fused with mouse bone marrow tumor cells using polyethylene glycol, etc., and the cells were cloned. gingivalis
fimbrial antigen or A. actinomycetem
Hybridomas secreting monoclonal antibodies that specifically react with the polysaccharide antigen of P. comitans are selected. These hybridomas can be transplanted intraperitoneally into mice that have been previously treated with pristane, and monoclonal antibodies can be obtained from the ascites, or hybridomas can be cultured in large quantities in serum-free medium and monoclonal antibodies can be obtained from the culture supernatant. can. Furthermore, the obtained monoclonal antibody is purified by ammonium sulfate fractionation and ion exchange chromatography according to conventional methods.

(5) Labeling of antibodies with colored colloid particles Whole cells of periodontal pathogenic bacteria obtained as described above or their specific antigens, such as P. gingivalis
fimbrial antigen, A. actinomycetemco
The colored colloidal particles are bound to antiserum or monoclonal antibodies against polysaccharide antigens of P. mitans. For this purpose, methods known per se can be used.

[0054] Generally, colored colloid particles are adjusted to pH
7 to 9 in carbonate buffer, phosphate buffer, Tris-HCl buffer, glycine buffer, etc., add the antibody thereto, stir at room temperature to about 37°C for 15 to 90 minutes,
Label antibodies with colored colloid particles. Next, in order to stabilize and protect the antibody labeled with colored colloidal particles, bovine serum albumin, polyethylene glycol, sucrose, choline chloride, etc.
Stir for 90 minutes. In this way antibodies can be labeled with colored colloidal particles. This is hereinafter referred to as a colored colloid-labeled antibody solution.

(5') Labeling of antibodies with enzymes Whole cells of periodontopathogenic bacteria obtained as described above or their specific antigens, such as P. Pili antigen of A. gingivalis, A. gingivalis. actinomycetemcomitan
An enzyme is attached to an antiserum or monoclonal antibody against a polysaccharide antigen such as s. For this purpose, methods known per se can be used.

[0056] Generally, antibodies are dissolved in 0.1 M phosphate buffered saline at 20 mg/ml. Add enzyme as a labeling substance to 2 ml of this antibody solution,
For example, add 40 mg of alkaline phosphatase and dissolve well. Add 0.1 ml of 1% glutaraldehyde dropwise while stirring gently. After reacting at room temperature for 2 hours, the alkaline phosphatase-labeled antibody is separated and purified from the unbound antibody and alkaline phosphatase by gel filtration. In this way, antibodies can be labeled with alkaline phosphatase. This is hereinafter referred to as an alkaline phosphatase-labeled antibody solution.

(6) Detection of antigen using colored colloid-labeled antibodies The gingival crevicular fluid, saliva, or plaque-treated fluid of a patient with periodontal disease is brought into contact with the collection tool of the present invention, and a specimen is collected into the collection tool. Next, the biological test substance collection part of this collection tool is immersed in the above-mentioned colored colloid-labeled antibody solution, and allowed to react for 5 to 15 minutes while standing or stirring at room temperature to about 37°C. After that, the collection tool is taken out and washed with distilled water, and then the determination can be made with the naked eye or with a measuring device such as a densitometer. If the area where the sample from a periodontal disease patient was attached is stained with the color of colored colloid particles,
It is determined to be positive. At this time, since the degree of color correlates with the presence or absence and amount of periodontal pathogenic bacteria, the number of bacterial cells can also be quantified.

Furthermore, when the colored colloidal particles are gold colloidal particles, sensitivity can be increased by silver staining.

(6') Detection of antigen using alkaline phosphatase-labeled antibody Contact the gingival crevicular fluid, saliva, or plaque treatment fluid of a patient with periodontal disease with the collection tool of the present invention, and collect the specimen into the collection tool. . Next, the biological test substance collection part of this collection tool is immersed in the above-mentioned alkaline phosphatase-labeled antibody solution, and allowed to react for 5 to 10 minutes at room temperature to about 37° C. while standing or stirring. Next Tween
After thorough washing with phosphate buffered saline containing 5.
- Immerse in a substrate solution containing bromo-4-chloro-3-indolyl phosphate and react at room temperature to about 37°C for 5 to 10 minutes while standing or stirring. After that,
After taking out the sampling tool and washing it with distilled water, the determination can be made visually or using a measuring device such as a densitometer. If the sample from a patient with periodontal disease is stained blue, it is determined to be positive. At this time, since the degree of color correlates with the presence or absence and amount of periodontal pathogenic bacteria, the number of bacterial cells can also be quantified.

(7) Labeling of antigens with colored colloid particles Specific antigens of periodontal pathogenic bacteria obtained as described above, such as P. gingivalis fimbrial antigen,
A. actinomycetemcomitans
To bind colored colloid particles to polysaccharide antigens, etc.,
Methods known per se can be used.

[0061] Generally, colored colloid particles are adjusted to pH
7 to 9 suspended in carbonate buffer, phosphate buffer, Tris-HCl buffer, glycine buffer, etc., the antigen is added thereto, and stirred at room temperature to about 37°C for 15 to 90 minutes,
Label the antigen with colored colloid particles. Next, in order to stabilize and protect the antigen labeled with colored colloidal particles, bovine serum albumin, polyethylene glycol, sucrose, choline chloride, etc.
Stir for 90 minutes. In this way, antigens can be labeled with colored colloid particles. This is hereinafter referred to as a colored colloid-labeled antigen solution.

(7') Labeling of antigen with enzyme Specific antigen of periodontopathogenic bacteria obtained as above, for example,
P. Pili antigen of A. gingivalis, A. gingivalis. ac
In order to bind an enzyme to a polysaccharide antigen of S. tinomycetemcomitans, a method known per se can be used.

[0063] Generally, the antigen is dissolved in 0.1 M phosphate buffered saline at 20 mg/ml. Add enzyme as a labeling substance to 2 ml of this antigen solution,
For example, add 40 mg of alkaline phosphatase and dissolve well. Add 0.1 ml of 1% glutaraldehyde dropwise while stirring gently. After reacting at room temperature for 2 hours, the alkaline phosphatase-labeled antigen is separated and purified from unbound antigen and alkaline phosphatase by gel filtration. In this way, antigens can be labeled with alkaline phosphatase. This is hereinafter referred to as an alkaline phosphatase-labeled antigen solution.

(8) Detection of antibodies using colored colloid-labeled antigens The gingival crevicular fluid, saliva, or plaque-treated fluid of a patient with periodontal disease is brought into contact with the collection tool of the present invention, and a specimen is collected into the collection tool. Next, the biological test substance collection part of this collection tool is immersed in the above-mentioned colored colloid-labeled antigen solution, and allowed to react for 5 to 10 minutes while standing or stirring at room temperature to about 37°C. After that, the collection tool is taken out and washed with distilled water, and then the determination can be made with the naked eye or with a measuring device such as a densitometer. If the area where the sample from a periodontal disease patient was attached is stained with the color of colored colloid particles,
It is determined to be positive. At this time, since the degree of color is correlated with the presence or absence and amount of antibodies against periodontal pathogenic bacteria, the antibody titer against periodontal pathogenic bacteria can also be quantified.

Furthermore, when the colored colloidal particles are gold colloidal particles, the sensitivity can be increased by silver staining.

(8') Detection of antibodies using alkaline phosphatase-labeled antibodies Bringing gingival crevicular fluid, saliva, or plaque-treated fluid of a patient with periodontal disease into contact with the collection tool of the present invention, and collecting a specimen into the collection tool. . Next, the biological test substance collection part of this collection tool is immersed in the above-mentioned alkaline phosphatase-labeled antigen solution, and allowed to react for 5 to 10 minutes at room temperature to about 37° C. while standing or stirring. Next Tween
After thorough washing with phosphate buffered saline containing 5.
- Immerse in a substrate solution containing bromo-4-chloro-3-indolyl phosphate and react at room temperature to about 37°C for 5 to 10 minutes while standing or stirring. After that,
After taking out the sampling tool and washing it with distilled water, the determination can be made visually or using a measuring device such as a densitometer. If the sample from a patient with periodontal disease is stained blue, it is determined to be positive. At this time, since the degree of color is correlated with the presence or absence and amount of antibodies against periodontal pathogenic bacteria, the antibody titer against periodontal pathogenic bacteria can also be quantified.

(9) Decolorization of stains caused by blood, etc. If the sampling tool becomes stained with blood or pus when collecting a specimen, decolorize it with an oxidizing agent such as hydrogen peroxide, and then remove the colored colloid-labeled antibody. It is preferable to react with a solution or a colored colloid-labeled antigen solution.

According to the present invention described above, the presence or absence of periodontal pathogenic bacteria in the periodontal lesion of a patient with periodontal disease and the amount thereof, or the presence or absence of periodontal pathogenic bacteria in the periodontal lesion of a patient with periodontal disease, can be determined. It is possible to quickly detect the presence or absence of specific antibodies against the disease and the amount thereof using an extremely simple operation, and the disease can be diagnosed and the extent of the pathology can be ascertained within a short period of time. Furthermore, by examining the presence or absence and amount of periodontal pathogenic bacteria in combination with the presence or absence and amount of specific antibodies against periodontal pathogenic bacteria,
The various stages of periodontal disease and the state of healing can be known over time, which can be useful for treatment and prevention of recurrence.

Next, the present invention will be explained in more detail with reference to Preparation Examples, Test Examples, and Examples, but the present invention is not limited thereto.

[0070]

[Example] Preparation Example 1 Preparation of freeze-dried cells of periodontal pathogenic bacteriaP. gingivalis and P. gingivalis. interval
edia, hemin (5 mg/l) and menadione (
1mg/l) added GAM bouillon medium (Nissui Pharmaceutical Co., Ltd.)
Inoculated at 37℃, 80% N2, 10% H2, 10
After mass culturing under anaerobic conditions of % CO2, bacteria were collected by centrifugation. Also, A. actinomycet
emcomitans, Todd-Hewit
t brother (Difco Laboratory
s) with 1% yeast extract added at 37°C, 5%
After mass culturing under anaerobic conditions of CO2, bacteria were collected by centrifugation. After washing these bacterial cells with physiological saline, they were freeze-dried under reduced pressure.

Preparation Example 2P. gingivalis 3
Preparation of pili antigen from P.81 strain and HW24D-1 strain. gingivalis strain 381 and HW2
4D-1 strain was treated with hemin (5 mg/l) and menadione (
1mg/l) added GAM bouillon medium (Nissui Pharmaceutical Co., Ltd.)
Inoculated at 37℃, 80% N2, 10% H2, 10
After mass culturing under anaerobic conditions of % CO2, bacteria were collected by centrifugation. The bacterial cells were mixed with 20 mM Tris-HCl buffer (pH 7.4) + 0.15M NaCl + 1
It was suspended in 0 mM MgCl2, pipetted, and then stirred with a magnetic stirrer to physically detach the fimbriae.

The bacterial cells were removed by centrifugation, the supernatant was fractionated with 40% saturated ammonium sulfate, and the resulting precipitate was dissolved in 20 mM Tris-HCl buffer (pH 8.0) and desalted by dialysis.

Next, DEAE-Sepharose
By ion exchange chromatography using (Pharmacia), a concentration gradient of NaCl was applied and 0.1
The fraction eluted with 5 M NaCl was used as fimbrial antigen.

[0074] Furthermore, this was treated with Sephacryl S
- It was subjected to gel filtration chromatography using 500 (Pharmacia) and confirmed to be a single peak.

Preparation Example 3A. actinomycete
mcomitans ATCC strain 29523, Y 4
Preparation of Polysaccharide Antigen from NCTC 9710 Strain A. actinomycetemcomitans
ATCC 29523 strain, Y 4 strain, and NCTC
9710 shares Todd-Hewitt brot
h (Difco Laboratories) was inoculated into a medium supplemented with 1% yeast extract, and incubated at 37°C and 5% C.
After mass culturing under anaerobic conditions of O2, bacteria were collected by centrifugation. The obtained bacterial cells are freeze-dried, suspended in physiological saline, autoclaved, heat extracted, and the extract is, for example, DEAE-Sephadex A-25.
(Pharmacia) and the flow-through fraction was further divided into
It was purified by gel filtration chromatography using acryl S-200 (Pharmacia), and a polysaccharide fraction was collected.

Preparation Example 4 Periodontal pathogenic bacteria P. gingivalis, P.
intermedia and A. actinomyc
whole cells of P. etemcomitans, P. etemcomitans. ging
fimbrial antigens of A. ivalis 381 and HW24D-1 strains, A. actinomycetemcomita
ns ATCC 29523 strain, Y 4 strain and NCT
Preparation of Purified Antiserum Against the Polysaccharide Antigen of P. C. 9710 Strain gingivalis
, P. intermedia and A. actin
omycetemcomitans freeze-dried cells,
P. of Preparation Example 2. A. gingivalis 381 strain and HW24D-1 strain pili antigen, Preparation Example 3.
actinomycetemcomitans ATC
C 29523 strains, Y 4 strains and NCTC 9710
1 mg of each strain of polysaccharide antigen in 1 ml per rabbit.
immunization by subcutaneously injecting it as a water-in-oil emulsion with Freund's complete adjuvant for a total of 3 times at 3-week intervals,
Whole blood was collected on the 7th day after the final booster immunization, and antiserum was separated in a conventional manner. Add 1/2 volume of saturated ammonium sulfate to the total amount of the obtained antiserum while stirring at 4°C to obtain an ammonium sulfate fraction (1/3
saturation). 1 in the sediment obtained by centrifugation.
/3 Saturated ammonium sulfate was added and stirred. This operation was repeated 2 to 3 times, and the finally obtained precipitate was dissolved in phosphate buffer and dialyzed against phosphate buffer to desalt. Next, DEAE −
Sepharose A-25 (Pharmacy
a) by ion exchange chromatography using NaC
A concentration gradient of 0.015 M NaCl was applied, the fraction eluted with 0.015 M NaCl was collected, and 1/3 saturated ammonium sulfate fractionation was performed in the same manner to collect all periodontopathogenic bacteria, P. gin
fimbrial antigen of A. givalis strain 381, A. acti
A purified antiserum against the polysaccharide antigen of S. nomycetemcomitans Y4 strain was obtained.

Preparation Example 5 Periodontal pathogenic bacteria P. gingivalis, P.
intermedia and A. actinomyc
whole cells of P. etemcomitans, P. etemcomitans. ging
fimbrial antigens of A. ivalis 381 and HW24D-1 strains, A. actinomycetemcomita
ns ATCC 29523 strain, Y 4 strain and NCT
Preparation of monoclonal antibody against polysaccharide antigen of P. C. 9710 strain Periodontopathogenic bacteria of Preparation Example 1 gingiv
alis, P. intermedia and A. a
Freeze-dried cells of P. ctinomycetemcomitans, P. ctinomycetemcomitans in Preparation Example 2. gingivalis 3
81 strain and HW24D-1 strain, A. of Preparation Example 3. actinomycetemcomitans
ATCC 29523 shares, Y 4 shares and NCTC
BALB/c mice (female) were immunized by subcutaneously injecting 100 μg of each 9710 strain polysaccharide antigen together with 0.1 ml of Freund's complete adjuvant per mouse as a water-in-oil emulsion three times every three weeks. Three days after the final boost, splenocytes were collected from BALB/c mice (female) and washed three times with Eagle's MEM.

On the other hand, myeloma cells (SP 2/0) derived from BALB/c mice were incubated with Eagle's MEM.
Washed 3 times with Eagl myeloma cells and splenocytes
Mix 1:10 in e's MEM and add 1,200
rpm, 10min. After centrifugation, remove the supernatant, loosen the sediment well, and add 0.5 ml of polyethylene glycol (0.5 ml) + Eagle's MEM (0.5 ml).
l) A mixed solution of +DMSO (0.35 ml) was gently added dropwise to the precipitate at 37° C. and mixed for 1 minute.

Next, 10ml of Eagle's MEM
was added little by little at 1,200 rpm for 10 min. Centrifuge, remove the supernatant, add RPMI-1640 medium containing 10% fetal bovine serum to the pellet, and add 5 x 105
The cells were suspended at cells/ml, and 100 μl each was dispensed into a 96-well microtiter plate. Thereafter, hybridomas were selected using HAT medium and HT medium, and around the 15th day, periodontopathogenic bacteria P. gingivalis, P. interme
dia and A. actinomycetem com
Freeze-dried cells of P. itans, P. itans of Preparation Example 2. gin
fimbrial antigens of S. givalis 381 strain and HW24D-1 strain, A. actinomycete
mcomitans ATCC strain 29523, Y 4
Using a 96-well microtiter plate coated with polysaccharide antigen of strain and NCTC 9710 strain, EL
Using the ISA method, total cells of periodontal pathogenic bacteria, P. gi
pili antigen of A. ngivalis strain 381 and HW24D-1 strain, A. ngivalis strain 381 and HW24D-1 strain. actinomycetemcomi
tans ATCC 29523 strain, Y 4 strain and N
Hybridomas producing antibodies against the polysaccharide antigen of CTC 9710 strain were screened.

The screened hybridomas were cloned by the limiting dilution method. The limiting dilution method is 10%
Add hybridomas to RPMI-1640 medium containing fetal bovine serum at a concentration of 5 cells/ml and BALB/c mouse thymocytes at 5 x 106 cells/ml, and dispense 0.2 ml into a 96-well microtiter plate. 1 periodontal pathogenic bacteria P. ging
ivalis, P. intermedia and A.
freeze-dried cells of P. actinomycetemcomitans, P. actinomycetemcomitans in Preparation Example 2. gingivalis
381 strain and HW24D-1 strain, A. of Preparation Example 3. actinomycetemcomita
ns ATCC 29523 strain, Y 4 strain and NCT
Colonies secreting monoclonal antibodies that react with the polysaccharide antigen of strain C9710 were selected. This operation was repeated twice to complete cloning.

[0081] Next, the hybridoma is transplanted into the peritoneal cavity of a BALB/c mouse that has been previously treated with pristane, and the hybridoma is grown in the ascites fluid to collect the antibody.
The target monoclonal antibody was prepared by culturing the hybridoma in a serum-free medium and collecting the antibody from the culture supernatant.

[0082] 1/3 of the obtained monoclonal antibody
Saturated ammonium sulfate fractionation was performed and purified by DEAE-ion exchange chromatography as well as gel permeation chromatography or Protein A column.

Example 1 Preparation of a biological test substance collection tool A commercially available paper point, for example, Absorbent Paper Points (length 30 mm; Size 15, 25, 35, 45) manufactured by ZIPPERER Co., Ltd. After immersing about 20 mm from the tip in a water-organic solvent mixed solvent solution of nitrocellulose or cellulose acetate,
The solvent was removed by drying and a porous film was coated on the surface. These have pores with a pore size of 10 microns or less. Furthermore, about 2 mm of the tip was left intact, and the rest of the film surface was treated with acetone to close the pores, thereby making that part waterproof (non-water permeable). As a result, a sampling tool as shown in FIG. 1 was prepared.

Example 2 Preparation of colloidal gold labeled antibody solution E. Y Laborat
ory, Inc. 0.07 M carbonate buffer (p
111 μl of H9.0) was added. On the other hand, 0.07 μl of each antiserum obtained in Preparation Example 4 or each monoclonal antibody obtained in Preparation Example 5 was added to 500 μl of a 20 μg/ml antibody solution.
56 μl of M carbonate buffer (pH 9.0) was added. Then, both were mixed and stirred at room temperature for 15 minutes.

Next, 185 μl of 0.007 M carbonate buffer (pH 9.0) containing 10% bovine serum albumin and 1% polyethylene glycol was added, stirred at room temperature for 15 minutes, and the gold colloid-labeled antibody was added. A solution was obtained.

Test Example 1 In vitro detection of periodontal pathogenic bacteria 1) P. Detection of P. gingivalis. gingivalis strain 381 and HW2
4D-1 strain was grown in GAM broth medium (Nissui Pharmaceutical) supplemented with hemin (5 mg/l) and menadione (1 mg/l) at 37°C, 80% N2, 10% H2, 10%
After mass culturing under anaerobic conditions of CO2, bacteria were collected by centrifugation. The cells were suspended in phosphate buffered saline, the absorbance at 550 nm was adjusted to 0.5, and the cell concentration of the cell suspension was adjusted to PETROFF-HAU.
SSER and HELBER Counting c
chamber (Hausser, Scientific
(manufactured by Partnership Horsham). The bacterial cell suspension thus obtained was diluted, and 2 μl of each dilution was adsorbed onto the biological test substance collection tool prepared in Example 1. Therefore,
P. labeled with the colloidal gold particles obtained in Example 2.
gingivalis strain 381 and HW24D-1
The cells were immersed in a monoclonal antibody solution against the pili antigen of the strain and allowed to react. After the reaction was completed, the number of bacterial cells at which coloration of the gold colloid particles was observed was determined. The results are shown in Table 1, and P. P. gingivalis strain 381 and HW24D-1 strain can be detected with a monoclonal antibody solution against fimbrial antigens. gingivalis
The number of bacterial cells of the 381 strain and HW24D-1 strain is 2 x 105
and 1.0×105 pieces.

[0087]

[Table 1] Table 1: P.
Detection of P. gingivalis
──────────────────────
Bacterial cell count (pieces/test) Judgment
───
────────
381 strain HW24D-1 strain
──────────────────────
1.2×107
＋＋＋ ＋＋＋
6.4×106
＋＋＋ ＋＋＋
3.2×106
++ ++
1.6×106
++ ++
8.0×105
++ ++
4.0×105
＋ ＋
2.0×105
＋ ＋
1.0×105
- +
5.0×104
− −
──────────────
────────2) P. intermedia
Detection of P. intermedia ATCC 2
5611 strain was treated with hemin (5 mg/l) and menadione (
1mg/l) added GAM bouillon medium (Nissui Pharmaceutical Co., Ltd.)
At 37℃, 80% N2, 10% H
────────────────────
────2. After mass culturing under anaerobic conditions of 10% CO2, bacteria were collected by centrifugation. The cells were suspended in phosphate buffered saline, the absorbance at 550 nm was adjusted to 0.5, and the cell concentration of the cell suspension was determined as PET.
ROFF-HAUSSER and HELBER
Counting, chamber (Hausser
Scientific Partnership H
orsham). The bacterial cell suspension obtained in this way is diluted, and 20% of each dilution is
μl was adsorbed onto the biological test substance collection tool prepared in Example 1. Therefore, P.I. intermedia ATCC
It was immersed in a monoclonal antibody solution against all bacterial cells of the 25611 strain and allowed to react. After the reaction was completed, the number of bacterial cells at which coloration of the gold colloid particles was observed was determined. The results are shown in Table 2. P. inter
media P. can be detected with a monoclonal antibody solution against all bacterial cells of ATCC 25611 strain. inte
The number of bacterial cells of rmedia ATCC 25611 strain is 2×
There were 105 pieces.

[0088]

[Table 2] Table 2
:P. intermedia detection
──────────────
──────
Bacterial cell count (pieces/test) Judgment
──────────
──────────
1.2×107
+++
6.4×106
+++
3.2×106
++
1.6×106
++
8.0×105
++
4.0×10
5 +
2.0×
105 +
1.
0x105
−──────
──────────────3) A. actin
Detection of A. omycetemcomitans A.
actinomycetemcomitans ATC
C 29523 strain, Y 4 strain, and NCTC 971
0 strain is Todd-Hewitt broth (D
ifco Laboratories) with 1% yeast extract added ───
──────────────── in a culture medium at 37℃
After mass culturing under anaerobic conditions of 5% CO2, bacteria were collected by centrifugation. The cells were suspended in phosphate buffered saline, the absorbance at 550 nm was adjusted to 0.5, and the cell concentration of the cell suspension was adjusted to PETROFF -
HAUSSER and HELBER Countin
g chamber (Hausser Scienti
fic Partnership Horsham). The bacterial cell suspension thus obtained was diluted, and 2 μl of each dilution was adsorbed onto the biological test substance collection tool prepared in Example 1. Therefore, A. actinom
ycetemcomitans ATCC 29523 strain, Y 4 strain, and NCTC 9710 strain, and reacted with a monoclonal antibody solution against the polysaccharide antigen. After the reaction was completed, the number of bacterial cells at which coloration of the gold colloid particles was observed was determined. The results are shown in Table 3.A. actinomycetemcomita
ns ATCC 29523 strain, Y 4 strain and NCT
A. C. can be detected with a monoclonal antibody solution against the polysaccharide antigen of strain 9710. actinomycetem
The number of bacterial cells of P. comitans ATCC 29523 strain, Y 4 strain, and NCTC 9710 strain is 2×1.
There were 05 pieces.

[0089]

[Table 3] Table 3:A. ac
Detection of tinomycetemcomitans
──────────────────────
────────── Number of bacterial cells (pieces/test) Judgment
──
────────────────────
ATC
C 29523 shares Y 4 shares NCTC
9710 shares ──────────────
────────────────────
1.2×107
＋＋＋ ＋＋＋ ＋＋
+6.4×106
＋＋＋ ＋＋＋
+++ 3.2
×106 ++
++ ++
1.6×106
++ ++ ++
8.0×105
++ ++
++ 4.0×
105 +
＋ ＋
2.0×105
＋ ＋ ＋
1.0×105
− −
− ──────────────
────────────────────Example 3 Preparation of colloidal gold labeled antigen solution E. Y Laboratory, Inc. of colloidal gold particles (G-20: particle size 20 nm) in 1 ml.
────────────────────
────────────07M carbonate buffer (pH
9.0) was added in an amount of 111 μl. On the other hand, 500 μl of the antigen solution containing 20 μg/ml of each of the fimbrial antigen obtained in Preparation Example 2 or the polysaccharide antigen obtained in Preparation Example 3 was added with 0.0 μg/ml of the antigen solution.
56 μl of 07 M carbonate buffer (pH 9.0) was added. Then, both were mixed and stirred at room temperature for 15 minutes.

Next, 0.007 M containing 10% bovine serum albumin and 1% polyethylene glycol
185 μl of carbonate buffer (pH 9.0) was added and stirred at room temperature for 15 minutes to obtain a colloidal gold labeled antigen solution.

Test Example 2 In vitro detection of specific antibodies against periodontal pathogenic bacteria 1) P. gingi
Detection of antibodies against S. valis Obtained in Preparation Example 4
P. gingivalis strain 381 and HW24
2 μl of each dilution of antiserum against pilus antigen of strain D-1
was adsorbed onto the biological test substance collection tool prepared in Example 1. Therefore, P.I. The cells were immersed in fimbriae antigen solutions of S. gingivalis 381 strain and HW24D-1 strain and allowed to react. After the reaction was completed, the dilution rate of the antibody at which coloring of the colloidal gold particles was observed was determined. The results are shown in Table 4. P. gingivali
P. s 381 strain and HW24D-1 strain that can be detected with pili antigen solutions. The dilution rate of antibodies against the pili antigens of S. gingivalis strain 381 and HW24D-1 was 1/1000 of the serum level.

[0092]

[Table 4] Table 4: P. g
Detection of antibodies against P. indivalis
──────────────────
─────── Antiserum dilution rate determination


────────────────
3
81 stocks HW24D-1 stock
────────────────────
──── 1/
10 +++
+++ 1/
100 ++
++ 1/
1000+
+1/
10000 -
−────────
──────────────────2) A. a
Detection of antibodies against A. ctinomycetemcomitans obtained in Preparation Example 4. actinom
ycetemcomitans ATCC 29523
2 μl of each dilution of antiserum against the polysaccharide antigen of the strain Y4, and the NCTC 9710 strain was adsorbed onto the biological test substance collection tool prepared in Example 1. Therefore, A.
actinomycetemcomitans AT
CC 29523 strain, Y 4 strain and NCTC 971
It was immersed in a polysaccharide antigen solution of 0 strain and allowed to react. After the reaction was completed, the dilution rate of the antibody at which coloration of the colloidal gold particles was observed was determined. The results are shown in Table 5.A. actinomycetem commit
ans ATCC 29523 strain, Y 4 strain and NC
A. which can be detected with polysaccharide antigen solution of TC 9710 strain.
actinomycetemcomitans ATC
The dilution rate of antibodies against polysaccharide antigens of C 29523 strain, Y 4 strain, and NCTC 9710 strain is 1/1 that of the serum label.
It was 000.

[0093]

[Table 5] Table 5:A. actinom
Detection of antibodies against P. ycetemcomitans
────────────────────
──────────── Antiserum dilution rate determination
───
────────────────────
ATCC
29523 shares Y 4 shares NCTC 9
710 shares ──────────────
──────────────────
1/10 +++
＋＋＋ ＋
++ 1/100
++ ++
++ 1
/1000 +
＋ ＋
1/10000
− −
− ──────────────
────────────────────Example 4 Colloidal gold-labeled antibody was used to detect P. gingivalis
──────────────────────────
──────Detection Gingival crevicular fluid from 3 healthy subjects and 6 patients with adult periodontitis was collected using the biological test substance collection tool prepared in Example 1, and then P. labeled with the obtained colloidal gold particles. After being immersed in a monoclonal antibody solution against the pili antigen of S. gingivalis 381 strain and reacting at room temperature for 5 to 15 minutes, the collection tool was taken out and washed with distilled water. Therefore, the test was determined to be positive if the part of the sampling tool to which the gingival crevicular fluid was adsorbed was stained reddish-purple, which is the color of the colloidal gold particles, and negative if it was not stained. Next, in the same manner, P.I. was labeled with the colloidal gold particles obtained in Example 2. It was determined by reacting with a monoclonal antibody solution against the pili antigen of S. gingivalis HW24D-1 strain.

[0094] At the same time, P. gingivalis
Identification was carried out as follows.

That is, gingival crevicular fluid absorbed into a commercially available paper Koyori (Paper Point) was transferred to a transport medium (RTF: Red
Contains defibrated rabbit blood, hemin and menadione.
CDC medium (Control Disease Ce
(inter development) and cultured under anaerobic conditions for several days. Among the growing colonies, select a blackened colony, isolate it, and after pure culture, Rap ID A
NA System (McDONNELL DOUGL
An identification test was carried out using a method (manufactured by AS). Furthermore, P. g
By subjecting the culture supernatant of the species identified as P. indivalis to gas chromatography and examining the production of phenylacetic acid. gingivalis.

The results are shown below. As shown in Table 6, no coloring due to colloidal gold particles was observed in the gingival crevicular fluid of healthy subjects (3 people), and all results were negative. In addition, in an identification test for bacteria present in gingival crevicular fluid, P. gin
Givalis was not observed. On the other hand, in the gingival crevicular fluid of adult periodontal disease patients (6 patients), 4 out of 6 patients received P.I. gingivalis 3
A reaction was observed with a monoclonal antibody solution against fimbriae antigen of P. 81 strain, and furthermore, a reaction was observed with the monoclonal antibody solution against pili antigen of P.
When reacted with a monoclonal antibody solution against fimbrial antigen of S. gingivalis HW24D-1 strain, coloration by colloidal gold particles was observed in all adult periodontitis patients, and all showed positive results. In addition, in an identification test for bacteria present in gingival crevicular fluid, P. gingiva
I recognized lis.

[0097]

[Table 6] Table 6: P in gingival crevicular fluid
．． Detection of S. gingivalis ───────
──────────────────────────
─ Subject No. Reaction with gold colloid labeled antibody Identification by culture method
──────────────────
381 shares HW
24D-1 stock ────────────────
────────────────── Healthy person
1 - -
Nothing
2
− −
Nothing
3
− −
None Patient 1
- +
Yes
2 ++
++ Yes 3
++ ++
Yes
4-
++
Yes
5 +
++ Yes
6
＋ ＋
Yes
────────────────────
─────────────Example 5 Colloidal gold-labeled antibody was used to detect P. in gingival crevicular fluid of adult periodontitis patients. Detection of intermedia ────────────────────────
──────────3 healthy subjects and 6 patients with adult periodontitis
P. gingival crevicular fluid was collected using the biological test substance collection tool obtained in Example 1, and labeled with the colloidal gold particles obtained in Example 2. intermedia ATCC 25611
It was immersed in an antiserum solution against all the bacterial cells of the strain and allowed to react. After reacting for 5-15 minutes at room temperature, the collection tool was removed and washed with distilled water. Therefore, if the part of the sampling tool to which gingival crevicular fluid was adsorbed was stained reddish-purple, which is the color of the colloidal gold particles, it was judged to be positive, and if it was not stained, it was judged to be negative.

[0098] At the same time, P. intermedia
The identification of P. in Example 4 was carried out. The detection was carried out in the same manner as the detection of P. gingivalis.

That is, gingival crevicular fluid absorbed into a commercially available paper Koyori (Paper Point) was transferred to a transport medium (RTF: Red
Contains defibrated rabbit blood, hemin and menadione.
CDC medium (Control Disease Ce
(inter development) and cultured under anaerobic conditions for several days. Among the growing colonies, select a blackened colony, isolate it, and after pure culture, Rap ID A
NA System (McDONNELL DOUGL
An identification test was carried out using a method (manufactured by AS).

[0100] The results are shown below. As shown in Table 7, no coloring due to colloidal gold particles was observed in the gingival crevicular fluid of healthy subjects (3 people), and all results were negative. In addition, in an identification test for bacteria present in gingival crevicular fluid, P. int
ermedia was not recognized. On the other hand, in the gingival crevicular fluid of adult periodontal disease patients (6 patients), coloring due to colloidal gold particles was observed, and all showed positive results. In addition, in an identification test for bacteria present in gingival crevicular fluid, P. int
ermedia was recognized.

[0101]

[Table 7] Table 7: P. in gingival crevicular fluid. Detection of intermedia ──────
──────────────────────────
─ Subject No. Reaction with gold colloid labeled antibody Identification by culture method ──────────
──────────────────────
Healthy person 1 -
Nothing
2-
Nothing
3
−
No patient 1
++
Yes 2
+
Yes 3
+
Yes 4
++
Yes 5
++
Yes
6 ++
Yes ─────
──────────────────────────
-Example 6 A.A. in gingival crevicular fluid of patients with localized juvenile periodontitis using colloidal gold-labeled antibodies. actinomy cetem
Detection of comitans ────────────
────────────────────Gingival crevicular fluid from 3 healthy subjects and 6 patients with localized juvenile periodontitis was collected in Example 1.
A. The sample was collected using the biological test substance collection tool obtained in Example 2 and labeled with the colloidal gold particles obtained in Example 2. actinomy
After mixing monoclonal antibody solutions against polysaccharide antigens of S. cetemcomitans ATCC 29523 strain, Y4 strain, and NCTC 9710 strain, it was immersed and reacted. After reacting for 5-15 minutes at room temperature, the collection tool was removed and washed with distilled water. Therefore, if the part of the sampling tool to which gingival crevicular fluid was adsorbed was stained reddish-purple, which is the color of the colloidal gold particles, it was judged to be positive, and if it was not stained, it was judged to be negative.

[0102] At the same time, A. actinomy
The identification of P. cetemcomitans was carried out in Example 4.
．． The detection was carried out in the same manner as the detection of P. gingivalis.

That is, gingival crevicular fluid absorbed into a commercially available paper Koyori (Paper Point) was transferred to a transport medium (RTF: Red
Contains defibrated rabbit blood, hemin and menadione.
CDC medium (Control Disease Ce
(inter development) and cultured under anaerobic conditions for several days. Colonies that showed growth were selected, isolated, and pure cultured, followed by Rap ID ANA System (McD
Identification test was conducted using ONNELL DOUGLAS Co., Ltd.).

The results are shown below. As shown in Table 8, no coloring due to colloidal gold particles was observed in the gingival crevicular fluids of healthy subjects (3 people), and all results were negative. In addition, in an identification test for bacteria present in gingival crevicular fluid, A. act
inomycetemcomitans was not observed. On the other hand, in the gingival crevicular fluid of 6 patients with localized juvenile periodontal disease, coloration due to colloidal gold particles was observed, and all the results were positive. In addition, in an identification test for bacteria present in gingival crevicular fluid, A.
actinomycetemcomitans was observed.

[0105]

[Table 8] Table 8: A. in gingival crevicular fluid. actinomy
Detection of cetemcomitans ──────────────────────
──────────── Subject No.
Reaction with gold colloid labeled antibody Identification by culture method ──────────────────────
──────────── Healthy person 1
−
Nothing
2-
Nothing
3
−
No patient 1
+
Nothing 2
+
Yes 3
+
Yes
4 +
Yes
5
+
Yes 6
+
Yes ────────────
──────────────────────Example 7
P. in the gingival crevicular fluid of adult periodontal disease patients using colloidal gold-labeled fimbrial antigen. Detection of antibodies against S. gingivalis ──────────────────────
──────────── Gingival crevicular fluid from 3 healthy subjects and 6 patients with adult periodontitis was collected using the biological test substance collection tool obtained in Example 1. P. labeled with colloidal gold particles obtained in step 3. gingivalis strain 381 and HW
After mixing the fimbriae antigen solution of the 24D-1 strain, it was immersed and reacted. After reacting for 5-15 minutes at room temperature, the collection tool was removed and washed with distilled water. Therefore, the test was determined to be positive if the part of the sampling tool to which the gingival crevicular fluid was adsorbed was stained reddish-purple, which is the color of the colloidal gold particles, and negative if it was not stained.

[0106] At the same time, P. gingivalis
Antibodies against fimbrial antigens were detected using ELISA method as described below.

First, P.I. was dissolved in carbonate buffer (pH 9.0). gingivalis strain 381, and
1 μg of fimbriae antigen of HW24D-1 strain was dispensed into each well of an ELISA plate and left overnight at 4°C. The next day, wash with phosphate buffered saline (PBST) containing Tween 20, followed by 10% goat serum.
Blocking was performed with PBST at room temperature for 1 hour. Next, gingival crevicular fluid, which had been collected in advance using a commercially available paper point, was dissolved in PBST and the collected fluid was added. After reacting at 37°C for 1 hour, alkaline phosphatase-labeled anti-human Ig (
G+M+A) was added and reacted at 37°C for 1 hour. Next, after thoroughly washing with PBST, p-Nitroph
Add a substrate solution containing enylphosphate,
The reaction was carried out at 37°C for 30 minutes. Therefore, the absorbance is measured and the degree of color development is determined to determine whether P. Antibodies against the pili antigen of S. gingivalis were detected.

The results are shown below. As shown in Table 9, no coloring due to colloidal gold particles was observed in the gingival crevicular fluids of healthy subjects (3 people), and all results were negative. Also, E
P. present in gingival crevicular fluid by LISA method. gin
Detection of antibodies against fimbrial antigens of S. pivalis
P. No antibodies against the pili antigen of S. gingivalis were observed. On the other hand, in the gingival crevicular fluid of adult periodontitis patients (6 patients), coloring due to colloidal gold particles was observed, and all the results were positive. In addition, the presence of P. gingival
For the detection of antibodies against pili antigens of P. is gi
Antibodies against the pili antigen of S. ngivalis were observed.

[0109]

[Table 9] Table 9: P. in gingival crevicular fluid. gi
Detection of antibodies against S. ngivalis ────
──────────────────────────
── Subject No. Reaction with gold colloid labeled pili antigen ELISA method ────
──────────────────────────
── Healthy person 1
−
-2
−
-3
−
- disease
person 1 ++
++
2
+
+3
+
+ 4
++
++
5 +
++
6
＋ ＋
+ ────────────────────
──────────Example 8 A. actinomycetemco
Detection of resistance to P. mitans Gingival crevicular fluid from 3 healthy subjects and 6 patients with localized juvenile periodontitis was collected using the biological test substance collection tool obtained in Example 1, and the biological test substance obtained in Example 3 was collected. A. labeled with colloidal gold particles. actinomycet
emcomitans ATCC29523 strain, Y 4
After mixing the polysaccharide antigen solutions of the strain and NCTC 9710 strain, they were immersed and reacted. After reacting for 5-15 minutes at room temperature, the collection tool was removed and washed with distilled water. Therefore,
It was judged as positive when the part of the collection tool to which gingival crevicular fluid was adsorbed was stained reddish-purple, which is the color of colloidal gold particles, and negative when it was not stained.

[0110] At the same time, A. actinom
Detection of antibodies against polysaccharide antigens of P. ycetemcomitans was carried out using ELISA method as described below.

First, A.I. was dissolved in carbonate buffer (pH 9.0). actinomycetemcomita
ns ATCC29523 strain, Y4 strain and NCTC
1 μg of the 9710 strain polysaccharide antigen was dispensed into each well of an ELISA plate and left overnight at 4°C. The next day, after washing with PBST, the plate was left standing at room temperature for 1 hour for blocking with PBST containing 10% goat serum. Next, gingival crevicular fluid, which had been previously collected in a commercially available paper Koyori (Paper Point), was dissolved in PBST and recovered. After reacting at 37°C for 1 hour, alkaline phosphatase-labeled anti-human Ig (
G+M+A) was added and reacted at 37°C for 1 hour. Next, after thoroughly washing with PBST, p-Nitroph
Add a substrate solution containing enylphosphate,
The reaction was carried out at 37°C for 30 minutes. Therefore, the absorbance is measured and the degree of color development is determined to determine whether the A. acti
Antibodies against P. nomycetemcomitans were detected.

The results are shown below. As shown in Table 10, no coloring due to colloidal gold particles was observed in the gingival crevicular fluid of healthy subjects (3 people), and all results were negative. Also,
A. present in gingival crevicular fluid by ELISA method. a
In the detection of antibodies against polysaccharide antigens of A. ctinomycetemcomitans, A. actinomyc
No antibodies against the polysaccharide antigen of C. etemcomitans were observed. On the other hand, patients with localized juvenile periodontitis (6
In the gingival crevicular fluid, coloration due to colloidal gold particles was observed, and all results were positive. Also, ELISA
A. present in gingival crevicular fluid by method. actinom
In the detection of antibodies against polysaccharide antigens of A. ycetemcomitans, A. actinomycetemco
Antibodies against the polysaccharide antigen of S. mitans were observed.

[0113]

[Table 10] Table 10: A. actinom
Detection of antibodies against P. ycetemcomitans
────────────────────────
──────────── Subject No.
Reaction with colloidal gold labeled polysaccharide antigen ELISA
Law ──────────────────────
────────────── Healthy person 1
−
−
2
−
-3
−
- Patient 1
+
+
2 +
++
3
++
++ 4
+
+
5 +
+
6
+
+ ──────────────────
────────────────── Example 9 Each antiserum obtained in alkaline phosphatase-labeled antibody Preparation Example 4 or each monoclonal antibody obtained in Preparation Example 5 was incubated at 20 m
Dissolve in 0.1 M phosphate buffered saline to give a total volume of 0.1 M phosphate buffered saline. Add alkaline phosphatase (EIA Grade: 01) manufactured by ZYMED to 2 ml of this antibody solution.
-2201) and dissolve well. Add 0.1 ml of 1% glutaraldehyde dropwise while stirring gently, and allow to react at room temperature for 2 hours. Next, add an equal amount of saturated ammonium sulfate and stir. The precipitate obtained by centrifugation is dissolved in a minimum amount of phosphate buffered saline and dialyzed against phosphate buffered saline. After dialysis, Sephadex G-
200 to obtain an alkaline phosphatase-labeled antibody.

Example 10 P. in gingival crevicular fluid of adult periodontitis patients induced by alkaline phosphatase-labeled antigen. gi
Detection of S. ngivalis Gingival crevicular fluid from 3 healthy subjects and 6 patients with adult periodontitis was collected using the biological test substance collection tool prepared in Example 1. phatase-labeled P. gingival
is strain 381 and P. gingivalis HW
After being immersed in a monoclonal antibody solution against the fimbriae antigen of the 24D-1 strain and reacting at room temperature for 5 to 15 minutes, the collection tool was taken out and washed with phosphate buffered saline containing Tween 20. Next, 5-bromo-4-chloro-3
- immersion in a substrate solution containing indolyl phosphite;
The reaction was allowed to take place for 5-15 minutes. Therefore, if the part of the sampling tool to which gingival crevicular fluid was adsorbed was stained blue, it was judged to be positive, and if it was not stained, it was judged to be negative.

[0115] At the same time, P. gingivalis
Identification was carried out as follows.

That is, gingival crevicular fluid absorbed into a commercially available paper Koyori (Paper Point) was transferred to a transport medium (RTF: Red
Contains defibrated rabbit blood, hemin and menadione.
CDC medium (Control Disease Ce
(inter development) and cultured under anaerobic conditions for several days. Among the growing colonies, select a blackened colony, isolate it, and after pure culture, Rap ID A
NA System (McDONNELL DOUGL
An identification test was carried out using a method (manufactured by AS). Furthermore, P. g
By subjecting the culture supernatant of the species identified as P. indivalis to gas chromatography and examining the production of phenylacetic acid. gingivalis.

The results are shown below. As shown in Table 11, no coloring was observed in the gingival crevicular fluid of healthy subjects (3 people), and all the results were negative. In addition, in an identification test for bacteria present in gingival crevicular fluid, P. gingivalis was not observed. On the other hand, in the gingival crevicular fluid of adult periodontal disease patients (6 patients), P. gingival
A reaction with a monoclonal antibody solution against the pili antigen of is was observed, and all showed positive results. In addition, in an identification test for bacteria present in gingival crevicular fluid, P. ging
Ivalis was approved.

[0118]

[Table 11] Table 11: P. in gingival crevicular fluid. Detection of S. gingivalis ───
──────────────────────────
─── Subject No. Alkaline phosphatase Identification by culture method
Reaction with labeled antigen
──────────────────────────
────── Healthy person 1
−
Nothing 2
−
None 3
−
None patient 1 ++
Yes
2 ++
Yes
3
+ Yes 4
+
Yes 5
+
Yes 6
++
Yes ──────────
──────────────────────

[Brief explanation of the drawing]

FIG. 1 is a schematic cross-sectional view of an example of a collection tool of the present invention.

[Explanation of symbols]

1 Absorbent material 2 Porous film coating 3 Biological test substance collection part 4 Waterproofing part

Claims (14)

[Claims] 1. A biological analyte in a specimen, comprising a biological analyte collection portion made of a water-absorbing material coated with a substantially water-insoluble and water-permeable porous film. Collection tools. 2. The sampling tool according to claim 1, wherein at least the tip portion is made of a stick-like or strip-like water-absorbing material coated with a substantially water-insoluble and water-permeable porous film. 3. Claim 1 or 2, wherein the surface of at least a portion of the water-absorbing material other than the biological test substance collection portion that is likely to come into contact with the specimen is waterproofed.
Collection equipment as described. 4. The collection tool according to claim 1, wherein the water-absorbing material is made of a super-absorbent cellulose polymer or a super-absorbent synthetic polymer. 5. The sampling tool according to claim 1, wherein the porous film has an average pore diameter of 10 microns or less. 6. The collection tool according to claim 1, wherein the porous film is made of an organic or inorganic acid ester of a polysaccharide or a lower alkyl ether derivative of a polysaccharide. [Claim 7] It is made of a stick-like or strip-like water-absorbing material, and the tip of the water-absorbing material has a length of 1 to 5 mm and has a possibility of coming into contact with at least the specimen adjacent to the biological test substance collection part. A biological test substance collection tool, characterized in that a certain part of the surface is waterproofed. 8. Collection according to any one of claims 1 to 7, wherein the specimen is gingival crevicular fluid, saliva, or dental plaque treatment fluid, and the biological test substance is periodontal pathogenic bacteria or antibodies against them. Tools. 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 specimen. 1. A method for measuring a biological test substance in a sample, the method comprising: contacting the sample with a reagent consisting of a labeled substance, and qualitatively or quantitatively detecting the labeled reagent bound to the sample. 10. The method according to claim 8, wherein the specimen is gingival crevicular fluid, saliva, or dental plaque treatment fluid, and the biological test substance is periodontal pathogenic bacteria or antibodies against them. 11. The method according to claim 10, wherein the labeled reagent is cells of periodontal pathogenic bacteria or parts thereof, or antibodies against them, labeled with colored colloidal particles or enzymes. 12. The method according to claim 11, wherein the colored colloid particles are colloidal gold. 13. The method according to claim 11, wherein the enzyme is alkaline phosphatase and the substrate is 5-bromo-4-chloro-3-indolyl phosphate. 14. A combination of 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 specimen. A kit for measuring biological test substances in specimens.