JPH01250067A - Detection of streptococcus mutans - Google Patents

Abstract

PURPOSE:To surely detect the presence or absence or quantity of Streptococcus mutans by bringing the monoclone antibody to the Streptococcus mutans or the polyclone antibody obtd. by immunizing this antigen with an animal into reaction with a sample to be inspected. CONSTITUTION:The antigen specific to the Streptococcus mutans and the monoclone antibody to a serum type specific polysaccharide antigen or the polyclone antibody obtd. by immunizing this antigen with a mammal or birds are brought into reaction with the sample to be inspected and whether the antigen corresponding to the antibody exists in the sample to be inspected or not is decided by the presence or absence of an antigen-antibody reaction. Proper bacteria of either (a) type bacteria or (h) type bacteria are selectively used according to purposes for the serum type specific polysaccharide antigen of the Streptococcus mutans and the monoclone antibody and polyclone antibody for this antigen are obtainable according to the conventional practice.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for detecting the presence or absence and amount of Streptococcus mutans in a test sample such as saliva or dental plaque.

[Prior art and problems to be solved by the invention] Conventionally, Streptococcus mutans (Streptococcus mutans)
It is widely known that Streptococcus mutans) plays an extremely important role in inducing experimental caries in humans and animals.

Streptococcus mutans is classified into eight different serotypes (ser) from a to h based on serological specificity.
otope). Among these, type 5 Q* d+ et L g was isolated from the hydrocoel, and in particular, type C seedlings were isolated at a frequency of 70 to 80% of all isolates. Also, according to the classification method based on DNA homology,
Q, el, and f type seedlings exhibit properties that are very similar to each other. On the other hand, d and g type seedlings have strong serological cross-reactivity and can be said to have the same DNA homology. Therefore, Streptococcus mutans isolated from the hydrocolumn can be divided into c, e, and f type W groups and dyg type seedling loops, and for this reason, recently Q, e, and f type seedlings have been It is called Streptococcus mutans in the narrow sense, and dsg type seedlings Streptococcus sobrinus (S treptococcus
cus 5 obrinus).
In addition, in the present invention, Streptococcus mutans is used not in such a narrow sense but in a broad sense).

Thus, Streptococcus mutans is an important bacterium for dental caries, but conventionally.

No practical caries diagnosis method has been proposed that uses Streptococcus mutans as a direct indicator to predict caries activity and caries risk.

Previously, Streptococcus mutans was directly isolated,
Although fixation is possible, this requires special equipment and skill, and is difficult to perform in a general dental clinic.

The present invention was developed in view of the above circumstances, and it is possible to quickly and specifically search for the presence or absence of Streptococcus mutans in a test sample (specimen) such as saliva or dental plaque, and therefore to detect the presence or absence of Streptococcus mutans in a test sample (specimen) such as saliva or dental plaque. Streptococcus can easily and reliably predict caries activity and caries risk.
The purpose of this invention is to provide a method for detecting mutans.

[Means and effects for solving the problem] As a result of repeated studies in order to achieve the above object, the present inventor utilized the serological specificity of Streptococcus mutans to develop its specific antigen and serotype. Using monoclonal antibodies against specific polysaccharide antigens and polyclonal antibodies from animals such as rabbits,
By reacting this with a test sample, if a Streptococcus mutans serotype-specific antigen corresponding to the above-mentioned antibody is present in the test sample, an antigen-antibody reaction such as an agglutination reaction will occur, and therefore, such a reaction will occur. By determining the presence or absence, degree of agglutination, etc., the presence or absence and amount of Streptococcus mutans can be easily detected without using special measuring equipment, and the degree of Streptococcus mutans infection can be determined. This discovery led to the present invention.

The present invention will be explained in more detail below.

The method for detecting Streptococcus mutans of the present invention uses monoclonal antibodies against Streptococcus mutans-specific antigens or serotype-specific polysaccharide antigens, or polyclonal antibodies obtained by immunizing mammals or birds with this antigen. The method is characterized in that it is reacted with a test sample, and it is determined whether or not an antigen corresponding to the antibody is present in the test sample based on the presence or absence of an antigen-antibody reaction.

Here, the serotype-specific polysaccharide antigen of Streptococcus mutans is selected from type A to type H seedlings depending on the purpose, and monoclonal and polyclonal antibodies against this antigen are commonly used. For example, monoclonal antibodies can be prepared by employing a cell fusion method or the like. Further, polyclonal antibodies can be obtained by immunizing mammals or birds with the above antigen, and in this case, rabbits are preferably used as the mammals, and chickens are preferably used as birds. Note that in the present invention, antiserum or milk or egg yolk fractions containing antibodies obtained by immunizing mammals or birds with the antigen as described above may be used as they are.
Alternatively, antibodies may be separated and used from this. in this case,
Isolation and purification of antibodies can be performed by conventional methods.

The present invention uses the above-mentioned monoclonal antibodies and polyclonal antibodies as test agents, and these monoclonal antibodies and polyclonal antibodies are preferably supported on a suitable carrier. Although various carriers can be selected, latex beads are preferably used, and latex beads are preferably used after being sensitized with the above antibody. Latex beads include polystyrene type, carboxy-modified type, and extremely low carboxylic acid type, and any of these can be used in the present invention. Furthermore, although the particle size of the latex beads can be appropriately selected, it is preferable to use particles of 0.1 to 10 lxn. These latex beads are commercially available, such as G2801, VO706, 5FL140- manufactured by Japan Synthetic Rubber Co., Ltd.
L, LB1. manufactured by Sigma, USA. LB3, LB5, LB6,
LB8, LBII, LB16, LB30, SD6, 5D
26.5D91. CLB4, CLB9, etc. can be used.

The method for sensitizing latex beads with the above antibodies is as follows:
A physical adsorption method or a chemical bonding method can be adopted.

The physical adsorption method can be carried out by a conventional method, but in this case, the antibody used for sensitizing the latex beads is 250 to 1
Preferably, it is diluted to about 1,000 times, and the diluent used for diluting the antibody includes glycine buffered saline, glycine buffered saline,
Distilled water or the like is preferably used.

The chemical bonding method can also be carried out according to conventional methods, but the antibody used for sensitization is diluted with a diluted solution such as distilled water at 250%
Preferably, it is diluted to about 1000 times.

The present invention uses the above-mentioned antibodies, particularly latex beads sensitized with the above-mentioned antibodies, as a test agent to react with a test sample such as saliva or dental plaque. If Streptococcus mutans is present, it will cause agglutination, so by determining the presence or absence of antigen-antibody reaction and the degree of agglutination, it is possible to detect the presence of Streptococcus mutans of the serotype to be detected in the test sample. It is possible to judge whether or not it exists, and to what extent it exists.

Here, when detecting Streptococcus mutans in a test sample and generally diagnosing the degree of caries activity and the risk of developing caries in humans, O+ dyer is used as an antibody.
Obtained by using fs G type seedlings as an antigen,
In particular, it is preferable to use those obtained by using C-type seedlings and G-type seedlings as antigens. In this case, antibodies derived from type C seedlings and antibodies derived from type G seedlings are mixed in an appropriate ratio, preferably the former and the latter in a weight ratio of 1:100 to 10.
It is preferable to use a test agent mixed at a ratio of 0:1, and furthermore, it is preferable to use the test sample as it is or lyophilized (live or lyophilized Streptococcus mutans); Accordingly, if any of the Cy dp eo ft G type seedlings is present in the test sample, it can be detected. Note that the test sample can be appropriately diluted with distilled water, phosphate buffered saline, or the like, if necessary.

In addition, if it is necessary to clearly detect the serotype of Streptococcus mutans in the test sample, the above test sample may be subjected to hot water extraction treatment or acid extraction treatment to detect the Streptococcus mutans serotype in the test sample. It is preferable to use the serotype as a hot water extract (Rantolander antigen) or an acid extract (nitrite-extracted antigen, etc.), which allows for more specific detection of serotypes as described above. In addition, even when the abundance of Streptococcus mutans of that serotype is small, it can be detected with high sensitivity. Note that hot water extraction and acid extraction can be performed according to conventional methods.

The presence or absence of Streptococcus mutans in a test sample can be detected by the presence or absence of an antigen-antibody reaction, as described above. This can be done by a detection means, but it can also be done by determining the presence or absence of an agglutination reaction within the shell and the degree of agglutination. in this case,
In order to predict the risk of caries in humans, the number of Streptococcus mutans is 104 CF U/m.
It is preferable to prepare the test agent and pre-process the test sample so that a clear agglutination reaction occurs between the test sample and the antibody at a concentration of 1 or more CFU/mQ, more preferably 10 3 CFU/mQ.

〔Effect of the invention〕

According to the present invention, the presence or absence and amount of Streptococcus mutans in a test sample such as saliva or dental plaque can be easily and reliably detected in a short time, and therefore, the degree of caries activity can be determined from the present invention.
The risk of caries can be easily predicted.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples.

[Example 1] Immunoglobulin, sensitized latex beads, and antigen (test sample) were prepared by the following method.

Globulin's 1JL Streptococcus mutans (hereinafter referred to as S.

Rabbits were immunized by intravenously injecting freeze-dried cells of strain 10449 (serotype C) (referred to as P. mutans) suspended in physiological saline at a ratio of 1/cm.

Thereafter, blood was collected using a conventional method, and serum was separated.

The obtained anti-S, mutans serotype-9-serum was
After salting out with 0% saturated ammonium sulfate, 1 of the original serum volume was
Rabbit anti-S, mutans C-type immunoglobulin (hereinafter simply referred to as anti-C-type antibody) was prepared by dissolving the antibody in physiological saline to a volume of /3 and dialyzing against the physiological saline.

We also performed similar immune manipulations on S. mutans6715.
strain (serotype g) and rabbit anti-S, mu
TANSG type immunoglobulin (anti-G type antibody) was prepared.

Dream, latex bead quasi-mouse The latex beads A to D below are suspended in glycine buffered saline to a concentration of 0.5%, and one volume of the above is diluted with the glycine buffered saline. One volume part of the solution was mixed and reacted with stirring at 37° C. for 90 minutes. After the reaction was completed, at 25°C and 3700 rpm for 10
After centrifugation for 1 minute, the resulting sediment was mixed with 0.1% bovine serum albumin (
By carrying out a washing reaction with 2 volumes of phosphate buffered saline containing Difco H), the unadsorbed sites of the latex beads were covered in order to prevent non-specific reactions. Next, the latex beads obtained by centrifugation in the same manner as above were washed with phosphate buffered saline containing bovine serum albumin, and then
．． Sensitized latex beads were suspended in phosphate buffered saline containing bovine serum albumin at a ratio of 5%.

A: Polystyrene type latex beads (G2801 manufactured by Japan Synthetic Rubber Co., Ltd. Particle size 0,876-) B: Carboxy modified type latex beads (vo 706 manufactured by the same company) C: Ultra-low carboxylic acid type latex beads (manufactured by the same company 5
FL140L-3, particle size approximately 1-)D: Extremely low carboxylic acid type latex beads (SFL 140L manufactured by the same company)
-6, particle size approximately 1 pxn, surface charge slightly smaller than latex bead C) (Tone of f 1, Rantz -Randal
l) Antigen S, mutans E 49 (serotype a)
, S, mutans FA-1 (serotype b), S, m
utans 10449 (serotype cLS, mutan
sB13 (serotype d), S, mutans
MT 703R (serotype e), S, mutans
OMZ 175 (serotype f), S, mutan
s 6715 (serotype g) and S.

20 mg of each freeze-dried bacterial cell of P. mutans M Fe28 (serotype h) was suspended in 1 d of physiological saline,
After heating at 21° C. for 20 minutes, the supernatant obtained by centrifugation was used as a Ranto Randal antigen (hereinafter referred to as RR antigen).

2. 10111 g of freeze-dried whole bacterial cell floating antigens each of the above S. mutans cells
was suspended in 1 d of physiological saline and used as a whole bacterial cell floating antigen.

3. Live bacteria floating antigen S, mutans 10449 (serotype C) and S
, mutans 6715 (serotype g) in IQ's Plain Heart Fusion Broth (brain he
art 1nfusion brother.

After culturing at 37°C for 18 hours using D1fco), the bacteria were collected by centrifugation, washed twice with physiological saline, and the saline had a turbidity of 550 nm (○D5..=1.0). It was diluted so that This was diluted to a predetermined level and used as a viable bacterial floating antigen.

In addition, the viable bacterial floating antigen was seeded on a Mitis salivarius agar plate and the number of viable bacteria was calculated.

The weight after connecting and drying was determined, and the relationship between the number of viable cells and the weight of freeze-dried cells was determined.

4. Nitrite extraction of freeze-dried bacterial cells and live bacterial cells After mixing 4N sodium nitrite (50%) with 50% of glacial acetic acid,
The above-mentioned S. mutans freeze-dried cells and floating antigen 100/jJ2 of live cells were added and extracted for 5 minutes at room temperature. After the reaction, add 4N NaOH200tJA and p
H was neutralized to 6.5 to 7.0 and used as a nitrite-extracted antigen.

Next, using the sensitized latex beads (test agent), react with the antigen solution (test sample) according to the method below, and evaluate the reactivity with S, o+utans by examining the degree of agglutination. The effectiveness of the above test drug was investigated.

3 latex bead agglutination test After mixing sensitized latex bead suspension 30IA and antigen solution 3- on a slide glass,
The mixture was stirred for 0 seconds, and the degree of aggregation at 5, 10, 15, and 20 minutes after the start of the reaction was evaluated in five grades according to the following criteria.

Judgment criteria for aggregation -; No aggregation ± ; Slightly small aggregation observed at the edges +;
Small agglutination is observed throughout ++; Large aggregation appears and white turbidity decreases. +++; Only agglutination occurs and white turbidity disappears. Trial 1: Effect of immunoglobulin concentration
Latex beads A were sensitized as described above by diluting 2,000, 4,000 and 8,000 times with glycine buffered saline.

Sensitized latex beads were obtained.

Next, for anti-C type antibody sensitized latex beads A, C
A sensitized latex bead agglutination test was conducted using the stock solution of the RR antigen of type seedlings and a 27-fold dilution of the RR antigen of type G seedlings for anti-G type antibody sensitized latex beads A.

The results are shown in Tables 1 and 2.

From the results in Tables 1 and 2, it can be seen that when both anti-C type antibody and anti-g type antibody-sensitized latex beads are sensitized with immunoglobulin diluted 500 times, the sensitivity of the agglutination reaction induced by the corresponding antigen is was recognized as the highest.

Table 1 Tex beads sensitizing anti-C type antibody No. 2
Table: Anti-G type antibody sensitized latex beads 2. Effects of differences in latex beads Latex beads A to D were each sensitized using 500-fold diluted immunoglobulin, and these sensitized latex beads were used at the RR antigen stage. Reacted with diluted solution.

The results are shown in Tables 3 and 4.

From the results shown in Tables 3 and 4, it was found that the reactivity of latex beads sensitized with anti-C type antibodies and anti-g type antibodies was hardly affected by the type of latex beads.

3. Influence of serotypes of various antigens Latex beads A sensitized with immunoglobulin diluted 500 times and S belonging to various serotypes.

A sensitized latex bead agglutination test was conducted using the whole floating antigen of S. mutans cells, the nitrite-extracted antigen containing 1.25 μ/mQ of freeze-dried cells, and the stock solution and 26-fold dilution of the RR antigen. Ta. The results are shown in Tables 5-8.

In addition, latex beads A sensitized with immunoglobulin diluted 500 times, and two strains of Streptococcus biogenes (S, pyogenes) as antigens,
Streptococcus sanguis (S, sanguis)
), four strains of Streptococcus salivarius (S.

Streptococcus spp.
1 of 1 strain of S. faecalis
A sensitized latex bead agglutination test was carried out using a whole cell floating antibody suspended in 1 mQ of physiological saline. The results are shown in Table 9.

As seen from the results in Tables 5 to 9, the anti-C type antibody-sensitized latex beads caused a clear agglutination reaction with whole bacterial cell floating antigens of serotypes c, e, and f in about 10 minutes. Furthermore, the anti-g type antibody-sensitized latex beads caused an agglutination reaction with whole bacterial cell floating antigens of serotypes a and g.

Therefore, by using anti-C type antibodies and anti-g type antibodies, especially in combination, it is possible to determine the presence or absence of S, mutans in samples collected from saliva or dental plaque.
It was confirmed that it could be detected regardless of the serotype of S. utans.

On the other hand, when using a nitrite-extracted antigen or RR antigen obtained by extracting whole bacterial cells, the specificity with anti-C type antibodies and anti-type antibodies increases, and therefore
It has been found that it is preferable to use RR antigen or nitrite antigen in order to detect even serotypes of S. mutans.

In addition, the results in Table 9 show that anti-C type and anti-g type antibodies are S,
It has been shown that a weak cross-agglutination reaction occurs against the whole bacterial cell floating antigen of S. pyogenes 19618, but S.
When using the RR antibody of P. pyogenes 19618 or the nitrite-extracted antibody, such cross-reactivity does not occur.

Table 7 Tex beads sensitizing anti-C type antibody No. 8
Table 4. Effects of anti-g type antibody sensitized latex beads 4 and the concentration of various antigens. A sensitized latex bead agglutination test was conducted using bacterial floating antigens.

The results are shown in Tables 10-13.

Anti-C type antibodies and anti-g antibodies as shown in Table 10.11
Both types of antibody-sensitized latex beads are 2.5μ/m
An agglutination reaction occurred up to the whole bacterial cell floating antigen of serotype C at a concentration of
It was observed that an agglutination reaction occurred up to a concentration of 23%, and the sensitivity was improved by 23 times.

Furthermore, from the results in Table 12.13, it was found that the use of nitrite-extracted antigens improved sensitivity.

[Example 2] Latex beads sensitized by the physical adsorption method used in Example 1 [latex beads sensitized with anti-C type antibody and anti-g type antibody diluted with glycine buffered saline (hereinafter referred to as G
(described as anti-C type or anti-g type antibody sensitized latex beads by BS dilution)], anti-g type antibody sensitized latex beads by chemical bonding method, and distilled water diluted d-absorbing anti-C type antibody sensitized latex beads. The effects were compared.

The results are shown in Tables 14-16.

Chemical 8 "Legal experience - Preparation of latex beads Latex beads A were suspended in distilled water to a concentration of 0.5%, and 1 volume part of the suspension contained 0.1 mol WSC.
(water 5olable carba+aide
, 1-ethyl-3-(3-dimethylaminopropyl)
-carbodiimide-hydrochloride) solution was added.

After 2 hours of incubation at 37°C, 7000 r at 25°C.
Centrifuged at pm for 10 minutes. The precipitate was washed with 2 volumes of distilled water, and 1 volume of immunoglobulin (anti-G type antibody) diluted with distilled water was added to latex beads obtained by centrifugation in the same manner as above, and cultured at 37°C for 2 hours. After centrifugation as above, 0.1% bovine serum albumin and 0.05% Na
The precipitate was washed with 2 volumes of phosphate buffered saline containing N, centrifuged in the same manner as above, and the precipitate was washed with 2 volumes of phosphate buffered saline containing 0.1% bovine serum albumin and 0.05% NaN. Sensitized latex beads were suspended in 1 volume of physiological saline.

Latex beads A were suspended in distilled water to a concentration of 0.5%, 1 volume of immunoglobulin (anti-C type antibody) diluted with distilled water was added to 1 volume of the suspension, and cultured at 37°C for 2 hours. did. After centrifugation at 7000 rpm for 10 min at 25°C, 0.1% bovine serum albumin and 0.05
The precipitate was washed with 2 volumes of phosphate buffered saline containing 0.1% bovine serum albumin and 0.05% NaN, and centrifuged in the same manner as above. Sensitized latex beads were suspended in 1 volume of physiological saline.

The results in Tables 14 and 15 show that latex beads sensitized by the chemical bonding method have the same effect as latex beads sensitized by the physical adsorption method, and that immunoglobulin-sensitized latex beads diluted with distilled water It was found that the sensitivity was high.

Applicant: Laion Co., Ltd. Agent: Patent Attorney Takashi Kojima

Claims (1)

[Claims] 1. A monoclonal antibody against Streptococcus mutans or a polyclonal antibody obtained by immunizing an animal with this antigen is reacted with a test sample to determine whether an antigen corresponding to the above antibody is present in the test sample. A method for detecting Streptococcus mutans, characterized by determining whether or not there is an antigen-antibody reaction.