WO1990004789A1

WO1990004789A1 - Reagent and method for immunological diagnosis of periodontal disease

Info

Publication number: WO1990004789A1
Application number: PCT/JP1989/001063
Authority: WO
Inventors: Kenji Yasuda; Takao Ogawa
Original assignee: Meito Sangyo Kabushiki Kaisha
Priority date: 1988-10-17
Filing date: 1989-10-17
Publication date: 1990-05-03

Abstract

The invention relates to a reagent for detecting cell bodies of Bacteroides gingivalis, which comprises latex particles sensitized with an antibody against a fimbrial antigen of Bacteroides gingivalis, and to a reagent for the diagnosis of periodontal diseases, which comprises latex particles sensitized with a fimbrial antigen of Bacteroides gingivalis. These reagents enable rapid and easy, qualitative or quantitative detection of cell bodies or an antibody thereagainst of Bacteroides gingivalis in a specimen with high sensitivity by bringing them into contact with a specimen such as gingival through fluid, saliva, blood or plaque to check for an agglutination pattern.

Description

Specification

-. Reagents and methods for immunological diagnosis of periodontal disease

Technical field

The present invention relates to a Pacteroides gingivalis greeting reagent comprising latex particles sensitized to an antibody against the pilus antigen of Bactaroides gingivalis, a kit therefor, and a kit therefor. The present invention relates to a method for detecting Bacteroides gingivalis cells in a subject. In addition, the present invention relates to a reagent for diagnosing periodontal disease and a method for diagnosing periodontal disease by measuring an antibody against a pilus antigen of Pacteroides gingivalis in a human subject. More specifically, a diagnostic reagent for periodontal disease consisting of latex particles sensitized to bateroroides' gingivalis pilus antigen, and an antibody against bacteroides gingivalis pilus antigen in a human subject using this reagent. It relates to a method of diagnosing periodontal disease by measuring.

Background art

Bacteroides ♦ gingivalis (hereinafter referred to as gingival is) inhabits a high proportion of the periodontal disease patients' oral cavity, especially in subgingival pockets, and is attracting attention as a causative agent of adult periodontitis. Periodontal disease includes gingivitis with inflammation of the gums, and periodontitis with the formation of periodontal pockets, destruction of the periodontal ligament, and resorption of alveolar bone. Further, periodontitis can be divided into adult periodontitis and localized juvenile periodontitis. Of these, adult periodontitis is the most prevalent, leading to bleeding, tooth sway with drainage, and, in severe cases, tooth loss. However, at present, treatment for periodontitis has not yet been completely established, and adult periodontitis was discovered as early as possible. It is considered that taking preventive measures is of paramount importance. Gram-negative bacilli account for 70-80% of the total bacteria in the above-mentioned localized lesions of adult periodontitis, and it has been reported that a marked increase in gingival is especially observed. I have. So this B. Focusing on gingival is, various methods have been proposed to assist in the diagnosis of adult periodontitis in order to determine the presence or absence of this bacterium and the amount thereof.

For example, plaques (plaque), gingival crevicular fluid, saliva, etc. of adult periodontitis patients are cultured under anaerobic conditions on blood plates to detect black pigment-producing Bacteroides. A method for confirming B. gingival is by examining chemical properties was proposed.

In addition, the enzyme immunoassay is used to examine the presence and degree of antibody titer against ^^ gingival is cell surface antigen in plaque (plaque), gingival crevicular fluid, saliva, and serum. Diagnosis of adult periodontal inflammation has been attempted using as an index that deterioration is associated with an increase in antibody titer. Attempts have also been made to determine the presence or absence of specific bacteria by observing the specimen using a fluorescence microscope. However, any of these methods requires a considerable amount of time and complicated operations and requires expensive special equipment, so it is extremely difficult to spread them widely. Therefore, the development of a quick and simple diagnostic method is desired.

The present inventors have conducted intensive studies to overcome the drawbacks of these conventional methods, and as a result, among various pathogenic factors of B. gingival is, it is important for early colonization especially in adult periodontitis. Focusing on pilus antigens that are present and exhibit strong immunogenicity among the surface cell components of the bacterium, antiserum or monoclonal antibodies against this pilus antigen are prepared, and these alone or in combination are absorbed into latex particles. It has been found that the use of the sensitized latex particles obtained by the wearing makes it possible to quickly and easily determine the presence or absence of B. gingival is and the amount thereof. In particular, the specimen from the subject's oral cavity and the ^ _ ^ gingivalis cell suspension (positive control) are preferably treated with a special treating agent to rapidly remove fimbria from the cells without losing their antigenicity. By extracting a large amount and dissolving the cells themselves, non-specific aggregation between the cells and the latex particles does not occur, and it is found that the presence or absence and the amount of B. gingivalis can be detected with high credibility. And completed the present invention.

Disclosure of the invention

Thus, according to the present invention, there is provided a reagent for detecting ^^ gingivalis cells, comprising latex particles sensitized with an antibody against gingival is pilus antigen. '

Further, according to the present invention, there is provided a kit for detecting B. gingivalis cells, comprising the suspension of latex particles and a treating agent.

Further, according to the present invention, there is provided a method for detecting ^ _ ^ gingivalis cells in a subject, wherein the presence or absence of an agglutination image is determined by bringing the detection reagent into contact with the subject.

According to the reagent and the method of the present invention, in a periodontal lesion of an adult periodontitis patient! ^ The presence or absence of gingival is can be detected quickly with a very simple operation, and the disease can be diagnosed in a short time and the degree of the disease state can be grasped.

Conventionally, a treatment agent is added to a subject such as human plaque (plaque), gingival crevicular fluid, saliva, etc., and B. gingivalis fimbrial antigen in the sample is separated from bacterial cells without losing its antigenicity. At the same time, by dissolving the cells themselves, There is no known method for detecting ^^ gingival is pilus antigen by latex agglutination reaction while preventing non-specific agglutination between bacterial cells and latex particles, and a rapid and simple diagnosis of adult periodontitis is known. Was impossible.

According to the present invention, separation of B. gingival is pilus antigen by a treating agent and latex agglutination reaction using B. gingival is pilus antibody and seizure latex are combined to provide an early adult periodontal periodontal. Rapid and simple diagnosis of flame has become possible.

On the other hand, attention has been paid to antibodies against gingival is, and various methods for measuring the presence or absence of antibodies to this bacterium and the degree of antibody titer have been proposed as a means of diagnosing adult periodontitis. .

For example, gingival crevicular fluid, saliva, blood or serum of adult periodontitis patients are included.The antibody titer against i ix iis is determined by one-way radial immunodiffusion, 0 uchter lony, immunoelectrophoresis. Attempts have been made to measure by passive hemagglutination or ELISA. However, all of these methods are difficult to spread widely because they are less confusing and require complicated operations over a long period of time and expensive special equipment, and the development of rapid and simple diagnostic reagents and methods is easy. Is desired.

The present inventors have conducted intensive studies to overcome the drawbacks of these conventional methods, and as a result, among various pathogenic factors of B. gingival is, especially important in the early colonization of adult periodontitis lesions. Attention is paid to a pilus antigen that exhibits particularly strong immunogenicity among the cell surface antigens of the same bacterium, and a seizure latex sample obtained by adsorbing this pilus antigen to latex particles is used. It is possible to quickly and easily detect antibodies to the pilus antigen of gingival is in human subjects such as gingival crevicular fluid, saliva, blood, and serum, and measure the antibody titer. Yes I found that it was noh.

Thus, according to another aspect of the present invention, there is provided a diagnostic reagent for periodontal disease, comprising a latex particle sensitized with a fimbrial antigen of gingival is.

According to the present invention, there is further provided a method for diagnosing periodontal disease, which comprises contacting the diagnostic reagent with a human subject to determine the presence or absence of an aggregated image.

Using such a reagent of the present invention, an antibody titer against gingival is pilus antigen in a subject such as gingival sulcus, saliva, blood, and serum of a patient with adult periodontitis caused by any gingival is Can be quickly detected with a very simple operation, and can be used in general dental clinics to quickly determine the past and present pathology or treatment of adult periodontitis caused by gingival is. It can be known quickly and easily.

According to the study of the present invention, in particular, gingival is 381, various types of human samples can be obtained by using a diagnostic reagent consisting of latex particles obtained by sensitizing the pilus antigen of 1 strain. It was found that antibodies against pilus antigen of gingival is detected with high sensitivity.

That is, the latex particles sensitized with the pilus antigen of the gingival is 381 strain can adsorb the antibody against the pilus antigen of the gingival is 381 strain, and the antibody can be detected. Antibodies against pili antigens of many other B. gingival is strains were also found to be adsorbed. B. gingival is strains other than the aforementioned 381 strains include:

B. Gingival is R B 2 4 M-2 strains,

B. gingival is D / 2 6 strains, B ^ .gingival is ATC C3 3277 strain,

B-gingival is W8 3 strains

B · gingival is HW2 4 D-1 share,

B. Gingival is R B 22 D-1 strain,

B. gingivalis B H 18/1 0 strains,

B. gingivalis 1 9 A 4 strains,

B. gingivalis HG 379 strains,

B. gingivalis H G405 strain

B. gingival is OMZ 3 1 4 shares

B. gingival is OMZ 40 9 shares

Such strains are also mentioned, and antibodies against the pilus antigens of these strains are also mentioned above. Latex particles sensitized to the pilus antigen of ingivalis 381 strain can be detected with high credibility. it can.

Hereinafter, the present invention will be described in more detail.

《Latex particles and their preparation>

The latex particles used for producing the sensitized latex preparation of the present invention are not particularly limited as long as they are generally used in immunoassay reagents, and any latex particles can be used. For example, polystyrene, carboxylated polystyrene, carboxylated polystyrene having an amino group, polyvinyl toluene, styrene butadiene copolymer, carboxylated styrene butadiene copolymer, styrene-divinylbenzene-copolymer, vinyl toluene Tertiary butyl styrene copolymer, polyester, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, acrylonitrile-butadiene-styrene copolymer, polyvinyl acetate And synthetic polymer latex particles such as polyvinyl chloride, polyvinyl pyrrolidone, and vinyl chloride-acrylate copolymer. Further, these polymer latex particles may be those whose surfaces are treated with a nonionic surfactant or the like. Among these polymer latexes, polystyrene latex is particularly preferred. The particle size of the latex particles can generally be in the range of 0.1 to 10.0 y "m, preferably in the range of 0.1 to 1.0 m. Since は is usually used in the agglutination reaction plate method, it is preferable that the particles be latex particles having a specific gravity of 髙.

<< Dan. Preparation of gingival is pilus antigen >>

The pilus antigen of gingivalis is obtained by cultivating gingivalis under anaerobic conditions, collecting cells after culturing, and isolating the pilus. The case of obtaining pilus antigen of gingivalis38 strain 1 will be described in more detail below. However, the preparation of pilus antigen is not limited to this method, but may be performed by any method capable of separating pilus. If so, that method can also be used.

B .. gingivalis38 To prepare one pilus antigen, first use GAM broth (trade name, manufactured by Nissui Pharmaceutical Co., Ltd.), Brain-Heart-Infusion process (Difco), etc. , Gingivalis38 1 strain inoculated on a medium supplemented with menadione, etc., and cultured at about 37 ° C under anaerobic conditions, a large number of cells can be obtained in about 20 hours or more . This is collected, and the pili are physically peeled off by pitting or the like. Next, the pili are subjected to ammonium sulfate fractionation, ion-exchange chromatography, and gel-perfusion chromatography to obtain a purified fimbrial antigen of the G. gingivalis 381 strain.

If an antibody-sensitized latex is desired, the pilus antigen thus obtained can be used as an immunogen to prepare the antibody as described below. Can be.

On the other hand, if an antigen-triggered latex is desired, the pilus antigen thus obtained can be sensitized to the latex as described above.

<< Preparation of Antibody >>-Immunize an animal with the B. gingival is pilus antigen obtained as described above. For example, this fimbrial antigen is injected subcutaneously or intramuscularly into animals such as egrets and goats together with Freund's complete adjuvant and immunized, and gingival islets are obtained from the blood of the animals according to a method known per se. Antiserum against hair antigens can be obtained.

On the other hand, a mouse is immunized with the pilus antigen, the spleen cells are taken out, the cells are fused with mouse myeloma cells using polyethylene glycol, etc., and after clotting, gingival is ligated to the pilus antigen. Select hybridomas that secrete monoclonal antibodies that react specifically. By culturing the hybridoma, a monoclonal antibody that specifically reacts with B. gingival is pili antiuria can be isolated from the medium.

《Preparation of anti-pilus antibody latex particles》

In order to sensitize the antiserum or monoclonal antibody against the B. gingival is pilus antigen to the latex particles, a physical adsorption method or a chemical bonding method is used according to a method known per se.

For example, in the physical adsorption method, first, purified antiserum or monoclonal antibody against gingival is fimbriae is purified with glycine-buffered saline at 10 to 1000 μg / mQ, preferably 10 to 100 μg / mQ. Dilute to 0 ~ 500 gZmfi.

—Mate latex particles to 5-1 O mgZmfi with glycine buffered saline After being prepared and mixed in the same volume with the above antibody solution, the mixture is adsorbed at 4 to 40 ° C. for 30 minutes to 24 hours with gentle stirring.

In the chemical conjugation method, the purified antiserum or monoclonal antibody against gingivalis fimbria is first diluted with distilled water to 100 to 100 gZmfi, preferably 100 to 500 gZnifi. On the other hand, a latex particle suspension prepared to 1 O mg Ζηιβ with distilled water is used, for example, in an equal amount of an aqueous solution of 0.0 1 to 0.1 Μ 1-1 Ethyl—3-^ -dimethylaminopropyU carbodi iinide hydroch. Mix and react at room temperature for 2 hours, then resuspend the latex particles recovered by centrifugation in distilled water, mix the suspension and the pilus antigen solution in equal amounts, and mix at 4-40 ° C. In step c, the antibody is bound to the latex surface with gentle stirring for 30 minutes to 24 hours.

In both the physical adsorption method and the chemical binding method, sensitization obtained by blocking the antibody-adsorbed or unbound part of the surface layer of latex particles with serum serum albumin (abbreviated as BSA) and then centrifuging. The latex particles should be resuspended in phosphate-buffered saline containing 0.1-1.0% BSA and, if necessary, added with 0.1-0.5% sodium azotized. preferable. << Preparation of pilus antigen-sensitized latex particles >>

In order to sensitize the gingivalis fimbrial antigen obtained as described above to latex particles, a physical adsorption method and a chemical binding method known per se are used. To illustrate the specific method, in the physical adsorption method, first, the purified fimbrial gingivalis fimbrial antigen is for example 1 to 1000 with glycine buffered saline; "g / mQ, preferably 100 ~ 500 gZmfi Dilute.

—Adjust latex particles to 5 mg gZm £ with glycine buffered saline And mixed with an equal volume with the pilus antigen solution, and adsorbed at 40 to 40 ° C. for 30 minutes to 24 hours with gentle stirring.

In the chemical conjugation method, first, the purified fimbrial gingivalis fimbrial antigen is diluted with distilled water to i ~ 100 OigZmfi, preferably 100〜500; A latex particle suspension prepared to 1 O mgZmfi with distilled water is mixed with an equal volume of, for example, an aqueous solution of 0.01 to 0.1 M 1-Ethyl-3— (3-dimethylthylaminopropyl) carbodiimide hydrochloride, and mixed at room temperature for about 2 ml. The latex particles collected by centrifugation are then resuspended in distilled water, and the suspension and the above-mentioned fimbrial solution are mixed in equal amounts.Then, at 4 to 40 ° C for 30 minutes. The pilus antigen is allowed to bind to the latex surface with gentle stirring for ~ 24 hours.

In both the physical adsorption method and the chemical binding method described above, BSA is used to block the portion of the surface of the latex particles that does not adsorb or bind to the pilus antigen, and then sensitized by centrifugation. Latex particles are preferably resuspended in phosphate-buffered saline containing 0.1-1.0% BSA and, if necessary, 0.01-0.5% sodium azotized.

《Subject and its processing>

In the present invention, human される specimens that are expected to be infected with periodontitis, that is, plaque (plaque), gingival crevicular fluid, and saliva of humans that are presumed to contain ^ gingivalis Such a subject is brought into contact with a greeting reagent consisting of latex particles that confused the antibody.In this case, if the subject is used directly as it is, the sensitized latex particles and bacteria other than B. gingivalis Gingival is uncertain. May be fruitful.

Furthermore, the number of bacteria of B. gingival is small in subjects such as plaque, gingival crevicular fluid, saliva, etc. of patients with early adult periodontitis, and the subject is sensitized as it is. Even when reacted with latex, it is sometimes difficult to detect the agglutination reaction because the amount of antigen is small. However, if the subject is subjected to extraction treatment and Z or lysis treatment under conditions that do not cause loss of antigenicity of B. gingival is pili, the substantial proportion of pili adsorbed to latex particles increases, Induces latex agglutination specifically and sensitively even when the amount of microbial cells in plaque (plaque), gingival crevicular fluid, saliva, etc. is low, such as in patients with gingivitis be able to.

Therefore, in the present invention, performing the subject's extraction treatment and Z or bacteriolysis treatment is a very preferable embodiment for increasing the detection sensitivity of the pilus antigen.

The subject may be treated with a treating agent that does not lose the antigenicity of the gingival is pilus, and preferably does not substantially denature the B. gingival is pilus, and Extracts having a bacteriolytic action are advantageously used.

The treating agent used for such treatment may be any agent as long as it does not lose the antigenicity of gingival is pili as described above, but in general, various surfactants, chaotropic ions and the like are preferably used. used.

The surfactant may be any of anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, steroid surfactants, etc. Among them, nonionic surfactants and steroid surfactants are preferred. In addition, as chaotropic ions, Two hydrochloride, urea and SCN -, I _, C 120 , N 0 3 -, B r -., C (T, Ion such as CH ₃ COO- is used as these chaotropic ions, among others Guanidine hydrochloride and urea are preferred.

Hereinafter, specific examples of the surfactant and the chaotropic ion that can be used as the treatment agent will be described, but the present invention is not limited thereto.

(1) Anionic surfactant: For example, sodium dodecyl sulfate, sodium tetradodecyl sulfate, sodium dodecyl sulfonate, sodium tetradecyl sulfonate, sodium dodecyl benzene sulfonate Lithium, dodecyl—sodium N-sarcosinate, etc.

(2) Cationic surfactants: for example, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, cetyltrimethylammonium bromide, dodecylpyridinium bromide, cetylpyridinium chloride , Tetradecyl ammonium bromide, etc.

(3 :) amphoteric surfactants: for example, palmitoyl lysolecithin, N-dodecyl betaine, stearyl-N-betaine, dodecyl-alanine, and the like.

(4) Nonionic surfactants: polyoxyethylene glycol (7) decyl ether, polyoxyethylene glycol (n) dodecyl ether, polyoxyethylene glycol (10) tridecyl ether, polyoxyethylene glycol ( 11) Tetradecyl ether, polyoxyethylene glycol (n) cetyl ether, polyoxyethylene glycol ( _n ) stearyl ether, polyoxyethylene glycol ( _n ) oleyl ether, polyoxy Ethylene glycol (17) cetyl stearate ether, polyoxyethylene glycol (n) p-t-octynolepheninoleate, polyoxy Polyethylene glycol (n) p-octynolephene dimethyl ether, polyoxyethylene glycol (n) p-nonylphenyl ether, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate Polyoxyethylene glycol (n) sorbitan monoperoxyphosphate, polyoxyethylene glycol (n) sorbitan monopalmitate, polyoxyethylene glycol (n) sorbitan monostearate, polyoxyethylene Glycol (n) sorbitan monooleate, lauryl dimethyl amine oxide, n-octyl-β-D-glycoside, octanoyl- ルー -methinoreganolamide, nonanoyl -methyl glucamide, decanoyl — Ν-Methyldalkami , .Eta. heptyl - S - D - Chiogurukoshi de, n- Okuchiru - D - Chiogurukoshi de, etc..

(5) Steroid surfactants: For example, sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium taurocholate, sodium taurodeoxycholate Tritium, digitonin, 3-[(3-cholamidopropyl) dimethylammonio] 1-1-propanesalphate, 3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxy-1-propanesanole Fate, etc.

(6) chaotropic ions: For _{example, C Ci2 3 COO Na, NaS} CN, NaCfi0 4, Nal, NaBr, NaN 0 3, NaCfi, urea, guanidine hydrochloride, and the like.

As described above, the treatment agent of the present invention does not lose the antigenicity of B. gingivalis pili, preferably does not substantially denature the pili and has a bacteriolytic action on bacteria existing in the oral cavity. What is necessary is just to have it, and a suitable thing can be selected by a relatively simple test. That is, oral bacteria are put into the aqueous solution of the treating agent, and the cells are partially or completely lysed, but the pili are present to the extent that their antigenicity is not lost or exist without being substantially denatured. By examining these facts, it can be determined whether or not it can be used as the treating agent of the present invention.

The treating agent used for the extraction and / or lysis treatment of the body to be greeted in the present invention is generally used by dissolving it in various buffers. For example, in the case of a surfactant, 0.5 to 7% by weight is preferable. Is used at a concentration of 1 to 5% by weight, and in the case of otobicone, it is used at a concentration of 0.1 to 10%, preferably 1 to 8%.

It is desirable that the treatment of the specimen with the treatment agent described above is performed usually at 4 to 40 ° C, preferably at 20 to 37 ° C, for 1 minute to 24 hours, preferably for 5 minutes to 10 minutes.

《Antigen detection ^》

The above-mentioned human subject or the subject subjected to the extraction and lysis treatment is used for examining the presence or amount of pilus antigen in the subject by contacting with the antibody-sensitized latex particles of the present invention. You. At this time, if extraction and lysis are performed, the chemical used in the treatment does not need to be removed, or may be removed.

The anti-piliary antibody-sensitized latex particles are usually used by suspending them in a phosphate buffer. For example, a phosphate buffered saline solution containing 0.1 to 1% by weight of BSA is used, and the suspension is adjusted so that a latex particle contains 0.5 to 1.0% by weight.

The agglutination reaction of latex particles is examined by mixing the suspension thus obtained with the test sample or the liquid of the test sample subjected to the extraction and lysis treatment. Suspension The liquid and the test liquid are mixed so that the concentration of latex particles in the mixed liquid is 0.3 to 1.5% by weight, preferably 0.5 to 1.0% by weight. It is desirable to adjust the respective concentrations of the sample liquid and the sample liquid in advance. If the concentration of the latex particles in the mixed liquid is lower than the above range, aggregation of the latex particles tends to be difficult to detect, which is not desirable. The agglutination reaction can be carried out by dropping the above-mentioned mixed solution on an aggregating plate and leaving it at room temperature for about 1 to 10 minutes, preferably for 2 to 5 minutes. The presence or degree of the aggregated latex particles can be visually observed or measured by an optical method (for example, a method of measuring transmitted light or scattered light using a spectrophotometer).

《Detection of antibody》

In the present invention, a suspension of latex particles sensitized to the pilus antigen of gingival is is obtained as described above, and a human subject such as gingival crevicular fluid, saliva, By mixing or mixing blood or serum and observing or measuring the state of aggregation of the latex particles, the presence or absence or amount of an antibody against the gingival is pilus antigen in the subject can also be examined.

In particular, as described above, using sensitized latex particles obtained by adsorbing piling antigen of gingival is 38 1 strain onto latex particles, various types of gingival H strains of adult gingivitis patients Various antibodies contained in specimens such as gingival crevicular fluid, saliva, blood or serum.Antibodies to ging ival is bacteria can be detected. The advantage is that adult periodontitis can be diagnosed without preparing sensitized latex particles. According to the present invention, sensitizing pilus antigen of ging ival is by using a latex preparation, that is, an example of the latex particles For example, a 0.5% to 1.0% suspension of 0.1% serum albumin / phosphate buffered saline (BSA-PBS) and a patient's gingival crevicular fluid, saliva, blood, and serum, etc. After dropping on a flocculation reaction plate at a ratio of 0: 1 (volume), and then gently rocking at room temperature for about 3 to 5 minutes to react, observe the state of aggregation of latex particles in a bright place or the degree thereof. Alternatively, the diagnosis of adult gingivitis can be performed quickly and easily by evaluating the above-mentioned optical method and measuring the antibody titer against gingival is pilus antigen.

According to the present invention, as described above, before examining an antibody against the pilus antigen of gingivalis, the subject is pretreated to lyse the cells and blood cells present in the subject. It is more preferable to keep it, because non-specific agglutination with latex particles is reduced and measurement sensitivity is increased. In the lysis treatment, a substance having an action of lysing bacterial cells and blood cells is used. Examples of the lysis treatment include the above-described surfactant or chaotropic ion.

The reagent used for the lysis treatment of the cells and blood cells of the subject described above is generally used after being dissolved in various buffers. For example, in the case of a surfactant, 0.5 to 7% by weight, preferably 1% It is used at a concentration of .about.5% by weight, and in the case of a power pick-up, it is used at a concentration of 0.1 to 10M, preferably 1 to 8M.

Lysis treatment of the above-mentioned cells and blood cells is usually 4 to 40. C, preferably at 20 to 37 ° C for 1 minute to 24 hours, preferably 5 minutes to 10 minutes.

Specific modes for carrying out the invention Next, the present invention will be described in more detail with reference to Preparation Examples, Test Examples and Examples, but the present invention is not limited thereto.

Preparation Example 1

Preparation of pilus antigen from B. gingivalis 38 1 strain.

The B. gingivalis strain 381 was cultured in large amounts under anaerobic conditions in GAM broth medium (Nissui Pharmaceutical) and then collected by centrifugation. As bacterial cells 2 0 mM preparative squirrel-HCl buffer (PH 7.4) + 0.1 5 was stirred at M NaCi2 + 1 0 mM MgCj2 suspended in ₂ Bibetti ring after Maguneti Kkusutara peeled off physically pili.

The cells were removed by centrifugation, the supernatant was precipitated with 40% saturated ammonium sulfate, and the obtained precipitate was dissolved in 2 OmM Tris-HCl buffer (PH 8.0) and desalted by dialysis.

Next, a salt concentration gradient was applied by ion-exchange chromatography using DAE-Sepharose (pharraacia), and the fraction eluted with '0.15 M saline was used as pilus antigen.

Further, this was subjected to gel filtration chromatography using Sephacryl S-500 (Pharmacia) to confirm that it was a single peak. Preparation Example 2

Preparation of antiserum to pilus antigen.

The pilus antigen of Preparation Example 1 (1 mg) was subcutaneously injected twice a week every 3 weeks as a water-in-oil emulsion with 1 mi2 of Freund's complete adjuvant per rabbit, and finally immunized. On day 7 after the booster immunization, whole blood was collected, and antiserum was separated as usual.

Preparation Example 3 Preparation of monoclonal antibodies against pilus antigen.

BalbZc mice (early) subcutaneously every 3 weeks using the pilus antigen (100 / ^ g) of Preparation Example 1 together with 0.1 m Freund's complete adjuvant per mouse as a water-in-oil emulsion Immunization was performed by two injections. After the booster, Balb / c mice and spleen cells were collected on the third day and washed three times with Eagle's MEM.

Wash the myeloma cells (SP2Z0) from Balb / c mice three times with Eagle's MEM. Myeloma cells and spleen cells are mixed 1: 1 in Eagle's MEM, centrifuged at 1,20 Orpml for 0 minutes, the supernatant is removed, the pellet is loosened well, and 0.5 mfi polyethylene glycol 400 (1 mfi ) + Eagle's MEM C 1 mj2) + DMS OC0.3 5mi2) was mixed at 37 ° C for 1 minute while gently dropping the mixture on a pellet. Next, Eagle's MEM 1 Omfi was added little by little, and centrifuged at 1,200 rpm for 10 minutes, and the supernatant was removed.

RPMI-1640 containing 10% fetal serum was added to the pellet, suspended in about 5 × 10 ⁵ cells mfi, and dispensed 100 μi2 into a 96-well microtiter plate. Then, ^ 1 Hatcho culture: ¾, hybridomas were selected using HT medium, and around day 15 B. ging was performed by ELISA using a 96-well microtiter plate coated with the fimbrial antigen of Preparation Example 1. A hybridoma producing antibody to the pilus antigen of ivalis 38 1 strain was screened.

The screened hybridomas were cloned by limiting dilution. Limiting dilution is i 0% © shea R PM I- 1 6 40 high to medium Buri dormer 5 cells Zrai2, Balb / c mice thymocytes 5 X 1 0 ⁶ cells containing calf serum / πι was added, and 0.2 mi2 was dispensed into a 96-well microtiter plate, and a well of one colony secreting a monoclonal antibody reactive with the pilus antigen of Preparation Example 1 was selected. Repeat this operation twice to complete the cloning / ^ *

Monoclonal antibodies were prepared by culturing hybridomas in a serum-free medium and collecting the antibodies from the culture.

Next, the obtained monoclonal antibody was concentrated after ammonium sulfate salting-out, DEAE ion exchange chromatography, and gel-mouth hyperchromatography, and the antibody subclass was examined by the Ouchterlony method. Incidentally, Usagi anti-mouse _{I g G !, I g G 2} I g G 2b, I g G 3, I gM was used LI TTON BIONETICS Co. preparation. As a result, No.-1 is IgM, No. -2 is IgGNo-3 is IgG! (See Table 1).

Table 1 Subclasses of monoclonal body

Preparation Example 4

Preparation of antibody-sensitized latex particles

Latex particles G2801 of Nippon Synthetic Rubber Co., Ltd. were suspended in glycine-buffered physiological saline to a concentration of 0.5%. Mix 0.1 solution of Monoclonal Antibody No. 1 and 37. After reacting with C for 90 minutes, block with 0.1% serum albumin / phosphate buffered saline (BSA-PBS), and finally suspend in 0.5% BSA-PBS to sensitize antibodies. A latex particle solution was prepared.

Preparation Example 5

Preparation of latex preparation sensitized to pilus antigen of B_. GingivalisS 8 1 strain.

Latex particles G2801 from Nippon Synthetic Rubber Co., Ltd. were suspended in glycine-buffered saline to a concentration of 0.5%. After mixing the hair antigen with 100 ηβ-dalicin-buffered saline solution and reacting at 37 ° C for 90 minutes, 0.1% ゥ serum albumin-phosphate buffered saline (BSA-PBS) And suspended in BS で -PBS at a final concentration of 0.5% to prepare a fibrin antigen-sensitized latex preparation.

Test Example 1 (Reaction of the obtained Egret antiserum and monoclonal antibody with resident bacteria in the oral cavity)

The antigen antibody reaction with the rabbit antiserum and monoclonal antibodies Nos. 1, 2 and 3 against the pilus antigen of Preparation Example 1 and the following oral Bacteroides 21 strain were examined by latex agglutination.

(a) The following strains that are present in the human oral cavity and that cause adult periodontitis belong to B. g i n g i ai is;

B— · g i n g i v a l i s J 8 1 bead,

B. g i n g i v a 1 is RB2 4M- 2 strains,

B. g i n g i v a l, i s 6/2 6 shares,

B. gingivalis ATC C 3 3 2 7 7 strains, B snsva 1 s W8 3 strains,

B l n g l v a 1 s HW2 4 D-1 share,

B S i g v a 1 s R B 2 2 D—1 strain,

B. g ing v a 1 s B H 18Z 10 strain,

B g i n g i v a 1 s 1 9 A 4 strains,

B. g i n g v a 1 s H G 379 strain,

B. ing iva a HG405 strain,

B-g i n g v a OMZ 3 1 4 shares and

B g i n g i v a i s OMZ 4 0 9 shares

(b) The following strains that are present in the human oral cavity but do not cause adult periodontitis

B. endo d o n a 1 i s H G370 strain,

B. nd o d o n a 1 is HG 4 13 strains,

B. endo d o n a 1 is s HG 59 1 strain,

B. 'a s a c c h a ro l y t i c u s N C T C 9 33 7 7-76 strain, B. m e l a n i o g e n i c u s H G 4 65

B. Me l a n i n o g e n i _c u_s H G 557

B. Int e r m e d i u s ATC C 2 5 6 1 1

B. B u c c a e OMZ 330

The latex agglutination reaction was performed by the following method.

Purely culture each of the above 21 strains, collect the cell suspension 4> «ώ, and mix with 4>" β 6Μ, guanidine hydrochloride solution, and stand for 5 minutes at room temperature Gingivali 3 8 1 Sensitized with ゥ rabbit antiserum against pilus antigen and monoclonal antibodies No.-1, No.-2 and No.-3, respectively, obtained in Preparation Examples 2 and 3. Latex particle suspension Was added on the agglutination reaction plate for 3 minutes, and the degree of agglutination was determined according to the following criteria.

Criteria for agglutination;

-: No aggregation is observed.

士: Slight agglomerates are observed at the side 緣.

+: Small aggregates are observed throughout.

+ +: Large aggregates are observed throughout.

+ + +: Only aggregates disappear and cloudiness disappears.

(I, K is agglutination negative, +, +10, +++ is agglutination positive.)

As shown in Table 2, the egret antiserum and monoclonal antibodies No.-1, 2, and 3 reacted with all B. gingivalis, but B. endoaontalis, B-asa c_c ha—r_oly—t_i_C— ii_s, B. me 1 aninogenicus _N B. intermedius and B. buccae <did not react at all, indicating high specificity with B. _ ^ _ i ngivalis.

Table 2 (antigen-antibody reaction between Egret antiserum and monoclonal antibody and oral Bacteroides)

Test example 2

G in g i v a li s pilus antigen detection ffi and B. g i n g i v ali s cells

Plaques (molar plaques) of the molars of healthy subjects (6) and adult periodontitis patients (6) were collected using a scaler, mixed with 4β 6M guanidine hydrochloride solution, and treated for 5 minutes. It was left at room temperature. To this, the suspension obtained in Preparation Example 4 was added. 40 g of a latex particle suspension elicited by the monoclonal antibody No. 1 against gingiva I is pilus antigen was added, and the mixture was mixed for 3 minutes on an agglutination reaction plate before testing. According to the latex agglutination criteria of Example 1, the degree of agglomeration was determined.

— On the other hand, the identification of ginngiVa1is in plaque (plaque) of healthy subjects (6) and adult periodontitis patients (6) was performed as follows.

That is, plaque (plaque) is suspended in a transport medium (RTF: Reduced Transfer F 1 uid), and a CDC medium (C 0 ntr 01 Dasease C enter) containing 繊 heron defibrinated blood and hemin and menadione is used. ) And cultivate for several days under anaerobic conditions. Of the colonies that grew, blackened colonies were selected and separated, and after pure culture, an identification test was performed using RaPD ANASystem (manufactured by McDONNEL ELDODOGLAS). Furthermore, the culture supernatant of those identified as gingiVa1is was subjected to gas chromatography, and the production of phenyldiacid was examined to confirm that it was ^^ gnigVa1is.

As shown in Table 3, no plaque (plaque) of healthy subjects (six) showed aggregation of the latex standard, and all showed negative results. Also, plaque (tooth B. gingivalis was not found in the test for identification of bacteria present in the soil. On the other hand, plaque (plaque) of adult periodontitis patients (6 patients) showed aggregation of the latex sample, and all showed positive results. On the other hand, B. gingi Va1 is was found in the test for identification of bacteria present in plaque (plaque).

B. g i n g i v a l! S Pili antigen in Table 3 (plaque) Plaque No. Latex Plaque

Agglutination C B. ingival

presence of is

Healthy 1-None

"Two rebellion

"3-

"4-nothing

" Five

"6

Adult periodontitis

Patient 7 + Yes

"8 + + Yes

"9 + Yes

"1 0 + Yes

〃 1 1 + + + Yes

〃 1 2 + + Yes Test example 3

Latex agglutination reaction of plaque of adult periodontitis patients and healthy subjects with various treatments

Test Example 3 was conducted in order to examine the effect of various types of treating agents on latex agglutination reaction.

In other words, plaque (plaque) of the molars of adult periodontitis patients No. 11 and healthy subjects No. 1 in Test Example 2 was collected using a scaler, and the concentrations shown in Table 4 below were obtained. It was combined with Treatment 4 and allowed to stand at room temperature for 5 minutes.

To this was added 4 μβ of a suspension of latex particles sensitized with the monoclonal antibody No. — 1 against the Β. Ginivalis fimbrial antigen obtained in Test Example 4, and mixed for 3 minutes on the agglutination reaction plate. Thereafter, the degree of aggregation was determined according to the criteria for latex aggregation in Test Example 1.

Table 4 (latex agglutination reaction when plaque (plaque) of adult periodontitis patients and healthy subjects is treated with various treatment agents) Type of treatment agent Latex agglutination reaction

Concentration

Adult periodontitis patient

1.Polyoxyethylene glue 2% + +

Cole (n) p-1-octyl

Phenyl ether

2.n-octyl- -D- 2.5% + + + —

Glicoside

3.Nonanoyl -N-methyl 2% + + +

Gurucamido

4.n-Hebutyl--D-Cho 2.6% + + +

Gnorecoside

5.n-octyl--D-thio 2.2% + +-glucoside

6.Ne 1 2% + + + ―

1 ώ + + +

8.Urea 8M ++

9.Guanidine hydrochloride 6M ++ 10

10. None + + In the table, 氺 1 is 3-[(3-col amide propyl) dimethyl ammonium]

-1-Propansal Fate,

* 2 is 3-[(3-cholamidopropyl) dimethylammonio]

The results of Test Example 2 are also shown for 2-guanidoxy-1-propane sulfate and guanidine hydrochloride of 9. Test example 1

且- gingivalis3 8 Latex agglutination reaction between Usagi antisera against one strain pilus antigen sensitized latex preparation and bacterial antigens for various port lumen B Acteroides arsenide Bok _0.

The following 21 strains were used.

(a) The following strain belonging to gingival! sa, which is present in the human cavity and causes adult periodontitis;

B.gingivalis ό 8 丄ゝ

B.gingivalis RB24M-2 strain, B.gingivalis 6/26 strain,

B. gingivalis ATCC33277 strain, E. gingivalis W83 strain,

B. gingivalis HW24D-1 strain, B. gingivalis RB22D-1 strain,

B.gingivalis BH18 / 10 strain, B.gingivalis 19A4 strain,

B.gingivalis HG379 strain, E.gingivalis HG405 strain,

B. gingivalis OMZ314 and mutual, gingival is OMZ409

(b) the following strains which are present in the human oral cavity but do not cause adult periodontitis; B melaninoge nicus NG465 bead B_- melaninogenicus HG557 strain, Bintermedins ATCC 25611 strain, Dan.buccae OMZ330 strain,

After culturing the above 21 strains in large amounts by a culture method suitable for each, collect the cells by centrifugation. The cells are treated under aerobic conditions to kill cells, and suspended in phosphate buffered saline (PH 7.4) at 2 mNmfi. Next, immunization was performed by subcutaneously injecting 1 m of killed cells per heron as a water-in-oil emulsion using Freund's complete adjuvant twice every three weeks. After the final boost, blood was collected on day 7 and the antibody titer for each strain was confirmed by a one-way radioimmunodiffusion method and the like, and each antiserum was separated by an ordinary method.

A heron antiserum against the various strains obtained by the above method (4) and the pilus antigen-sensitized latex preparation (40 μΆ) of the gingival ival is 38 1 strain prepared in Preparation Example 5 were placed on an agglutination reaction plate. After mixing well for 3 minutes, the degree of aggregation of latex particles was determined in a bright place.

The latex agglutination was evaluated in five steps according to the following criteria. Table 5 shows the results.

Agglomeration criteria

-: No aggregation is observed.

Soil: Slight agglomeration observed at the edge.

+: Small aggregates are observed throughout.

+ +: Large aggregates are observed throughout.

+ + +: Only aggregates disappear and cloudiness disappears.

(1, soil shows aggregation negative, 10, + +, +10 + shows aggregation positive)

Table 5 (Latex agglutination reaction between the pilus antigen-sensitized latex sample of gingival is 388 1 strain and peregrine antiserum against various oral Bacteroides)

As shown in Table 5 above, all rabbit antisera against various strains of human gingivalis that cause human adult periodontitis showed positive agglutination with the tested latex standard.

-On the other hand, human adult periodontitis does not develop. B. endodontalis HG370 strain, HG413 strain, 59i strain, and asaccharolyticusN CTC 9337-76 strain, Dan. The heron antiserum against melaninogenicus HG465, HG555, B. intermedius2 561, and b. Not recognized and judged as negative.

Thus, the pilus antigen-sensitized latex particles of gingivalis of the present invention specifically show agglutination with antisera of various strains of B. gingivalis that cause human adult periodontitis. It was confirmed that.

Test example 5

(Detection of antibodies against various gingivalis strains in samples of healthy subjects and patients with adult periodontitis)

Gingival sulcus and saliva of 6 healthy subjects (who develop adult periodontitis in plaque (plaque) and no gingivalis was detected) and 6 patients with adult periodontitis Agglutination reaction of the pilus antigen-sensitized latex preparation (40 / "β) of the gingiv lis 381 strain obtained in Preparation Example 5 above, and blood and serum (4>« β) respectively. After mixing well for 3 minutes on the plate, the degree of aggregation of latex particles was judged in a bright place The judgment of latex agglutination was in accordance with the criterion of Test Example 4. The results are shown in Table 6. Show. Table 6

各種 The degree of detection and aggregation of antibodies to various gingivalis strains in specimens of normal and adult periodontitis patients.

As shown in Table 6, of the six healthy subjects, four had a negative latex agglutination reaction, while two had a positive coagulation reaction. Healthy individuals who show coagulation positive in this way have not developed human adult periodontitis in the past, but can be diagnosed as having been infected by B. gi ngivalis bacteria.

On the other hand, all six adults with periodontitis had positive latex agglutination reactions. Test example 6

Latex agglutination reaction of gingival sulcus fluid of adult periodontitis patients and healthy subjects with various treatment agents.

Test Example 6 was conducted to examine the effect of various types of treatment agents on latex agglutination reaction. That is, the gingival crevicular fluid of adult periodontitis patients N 0-11 in Test Example 5 and healthy subjects N 0.1 was collected, added to the treatment agent 4 i2 at the concentration shown in Table 7 below, and left at room temperature for 5 minutes. At rest.

To this, add 40 "β to a suspension of latex particles sensitized with the fimbrial antigen of gingivalis38 1 strain gingivalis38 and mix on an agglutination reaction plate for 3 minutes. The degree of agglomeration was determined according to the latex agglutination criteria of 4. The results are shown in Table 7.

Table (Latex agglutination reaction when gingival crevicular fluid of adult periodontitis patients and normal people and various treatment agents were used) Latex agglutination reaction Type of treatment agent Concentration

Adult periodontitis healthy person, polyoxyethylene

Glycol (n) P-1-octinolefeninolee

1 tel 2% ++, 11-octyl -0-glu

Cosid 2-5¾ +++ Nonanoyl methyl

Glucamide 2% +++-, n-Heptyl -J8-D-thio

Glucosides 2.6% +++, n-octyl-D-thio

Glucosides 2.2% ++-, 3- [3-cholamide

Mouth Pisole) Dimethysolean

Monio] -1-propane

Sanoleate 2% +++-, 3-[(3-Callamide

Propyl) dimethyla

Nmonio]-2-Hide

Mouth mouth

Lefeit 2% +++, urea 8M +++

, Guanidine hydrochloride 6M +++

, None + + Industrial applicability

As described above, the use of the reagent and the method of the present invention makes it possible to specifically detect gingival is or an antibody against the gingival is quickly and easily, with high sensitivity, It can be used for early detection of periodontal disease, diagnosis of healing status, etc.

Claims

The scope of the claims

1-Use for detecting Pacteroides gingivalis cells characterized by being composed of latex particles sensitized to antibodies against the fimbrial antigen of Nocteroides gingivalis and Bacteroides gingival is) reagent.

2. The reagent according to claim 1, wherein said antibody is an antiserum or a monoclonal antibody.

3-Kit for detecting Bacteroides gingivalis cells comprising a suspension of latex particles sensitized with antibodies against Pacteroides gingivalis pilus antigen and a treating agent.

4. The kit according to claim 3, wherein the treating agent is a surfactant or chaotropic ion that does not substantially alter the pili of Pacteroides gingivalis.

5-The kit according to claim 4, wherein said treating agent is a treating agent that does not substantially impair the antiurinity of Bacteroides gingivalis fimbriae.

6. A method for detecting Pacteroides gingivalis cells in a subject, wherein the reagent for K detection according to claim 1 is brought into contact with the subject to determine the presence or absence of an agglutination image.

7. The method according to claim 6, wherein the subject is plaque (plaque), gingival crevicular fluid or saliva.

8. The method according to claim 6, wherein a treatment agent that does not substantially lose the antigenicity of the fimbria of Pacteroides' gingiparis is applied to the subject in advance.

9. The method according to claim 6, wherein the treatment is performed using a treatment agent selected from a surfactant and chaotropic ion.

10. A diagnostic reagent for periodontal disease characterized by comprising latex particles sensitized to the pilus antigen of Bacteroides gingivalis.

1 1. The reagent according to claim 1, wherein the Batteroids gingivalis is Bacteroides gingivalis 381 strain.

12. A method for diagnosing periodontal disease, which comprises contacting the diagnostic reagent according to claim 10 with a subject to determine the presence or absence of an aggregated image.

13. The diagnostic method according to claim 12, wherein the subject is gingival crevicular fluid, saliva, blood or serum.