JP2666214B2 - Antibody and method for producing anti-caries agent containing the same as an active ingredient - Google Patents

Description

The present invention relates to a water-insoluble glucan synthase (glucosyltransferase, GTF-1) produced by Streptococcus mutans, a pathogenic bacterium that induces dental caries. The present invention relates to an antibody having an inhibitory activity against C.) and a method for producing a caries preventive agent comprising the antibody as an active ingredient.

(Prior art) It is already known that the attachment process of S. mutans to the tooth surface plays an important role in the development of dental caries.
By controlling mutans' fixation in the oral cavity (adhesion to the tooth surface)
Various attempts have been made to prevent caries.

For example, a method of preventing dental caries using an antibody having immunological activity against S. mutans is known, and is disclosed in British Patent No. 1505513.
The specification describes a method of using breast milk obtained from a cow immunized with S. mutans cells for controlling the oral colonization of S. mutans,
Japanese Patent Application Laid-Open No. 60-38327 discloses an antiserum and / or milk obtained by immunizing a mammal with a fraction such as a cell wall obtained from a culture of S. mutans, a glucosyltransferase inhibitor, a protease and An anti-caries agent is described in combination with one or more synergists selected from the group consisting of dextranase.

(Problems to be solved by the invention) However, the anti-caries effect of the conventional antibody having immunological activity on S. mutans is not always sufficient, and the conventional antibody cannot be obtained from milk of an immunized mammal or its antiserum. Since it is prepared, it has disadvantages such as lack of mass productivity and high production cost, and is often impractical.

The present inventors have conducted various studies to solve such problems in the prior art, and as a result, a method for preparing an antibody using chickens has been described. The use of the enzyme in combination as an antigen has a new finding that it has a sufficient inhibitory effect on the adhesion of S. mutans to the tooth surface and can produce an antibody without the above-mentioned disadvantages. Thus, the present invention has been completed.

In particular, in the production of antibodies using chickens, it is not always possible to obtain a sufficient amount of antibodies specifically reacting with the immunized antigen. For example, when a virus antigen was immunized, a sufficient antibody titer was not obtained.

Therefore, when immunizing chickens, carefully examine the antigen,
Care must be taken to obtain a high antibody titer.

Therefore, the knowledge that an antibody having a sufficiently high antibody titer can be obtained by immunizing a chicken with the water-insoluble glucan synthase produced by S. mutans was first obtained by the present inventors.

An object of the present invention is to show an immune activity against S. mutans,
It has a sufficient inhibitory effect on the adhesion of S. mutans to the tooth surface, is excellent in mass productivity, has low production cost, is excellent in stability, and is useful as an active ingredient of an anti-caries agent and a caries containing antibody. An object of the present invention is to provide a method for producing a prophylactic agent.

(Means for Solving the Problems) An antibody having an inhibitory activity on water-insoluble glucan synthase (hereinafter abbreviated as GTF-I) produced by S. mutans according to the present invention is a serotype having the following physicochemical properties. a, d
Alternatively, it can be obtained as an immunoglobulin produced from eggs produced by chickens immunized with GTF-I produced by S. mutans g.

Action and substrate specificity Acts on sucrose to synthesize water-insoluble glucan.

Molecular weight The molecular weight measured by SDS-polyacrylamide gel electrophoresis is between 155k and 170k daltons.

Immunogen Produces an immunogen in an animal and can generate specific antibodies to the enzyme.

The SDS-polyacrylamide gel electrophoresis (S
DS-PAGE) was performed according to the following procedure.

SDS-PAGE operating conditions; SDS-PAGE is performed according to the method of Laemuli et al.

That is, the crude purified or purified sample described below is individually
100% in 62.5 mM Tris-HCl buffer (pH 6.8) containing SDS, 5% 2-mercaptoethanol and 20% glycerol.
Treat at 3 ° C for 3 minutes.

Electrophoresis was performed on a 7.5% separation gel containing 0.1% SDS,
The reaction is performed in a 4% concentrated gel at room temperature at 10 mA for 2 hours. In addition, as a molecular weight marker, ferritin (ferr
itin, 220k Dalton), phosphorylase b (phosphor
ylase b, 94k dalton, bovine serum albumin (bovine)
serus albumin, 67k dalton, catalase (catalas)
e, 60k dalton), ovalbumin (ovalbumin, 43k dalton) and lactase dehydrcgenas
e, 36k Dalton) (both manufactured by Pharmacia), and the band was detected using Coomassie Brilliant Blue (CB
This can be done by staining according to B).

As the GTF-I used to obtain the antibody (hereinafter referred to as anti-GTF-I) obtained according to the present invention, any enzyme having the above-mentioned properties can be used. It can be obtained by purifying from the culture supernatant of .mutans.

That is, S. mutans whose serotype is a, d or g is cultured in an appropriate medium and purified from the culture supernatant.

The g-type bacteria used here include S. mutans 6715 strain, ATC
Known and readily available bacteria such as C 27351 strain can be used.For example, S. mutans 6715 strain is from Osaka University School of Dentistry, ATCC 27351 strain is American Type Culture Collection (American Type Cultures Collec).
(ATCC)]. In addition, known strains may be similarly obtained and used for the a-type bacteria and the d-type bacteria.

As the medium, a medium containing at least glucose can be used. For example, a TTY medium (Trypticass, Tryptos
e, Yeast extract complex medium), BHI (Brain Heart
Infusion) medium, FMC medium and the like can be used.

Further, the culture temperature may be within a range in which cell growth can be obtained and suitable for the production of GTF-I.
From the viewpoint of production of GTF-I, the temperature is usually set to about 37 ° C.

The culturing time varies depending on the culturing conditions such as the culturing temperature and the type of the medium, but may be determined by selecting the time when the optimum yield of GTF-I is reached, and may be about 18 to 24 hours.

Further, other culture conditions may be appropriately selected from the above viewpoint.

 Next, GTF-I is extracted from the cells.

S. mutans GTF-I was obtained by the method of Furuta, T. et al. [Furuta,
T., et al; J, General Microbiology., 131, 235-293 (1
985)], inoculate S. mutans in a BHI dialysis medium, remove cells by centrifugation after growth, add ammonium sulfate to the supernatant, and precipitate a 50% ammonium sulfate fraction from Histidiae.
-After dialyzing against BCI and performing chromatofocusing column chromatography, hydroxyapatite column chromatography is performed to obtain a purified sample.

As an antigen for obtaining an antibody, the bacterial cell culture supernatant or ammonium sulfate concentrated sample obtained by the above procedure, a crudely purified sample after chromatofocusing, or GT as a purified sample
FI or the like can be used.

Next, the method for producing an anti-GTF-I antibody of the present invention will be described in detail.

The anti-GTF-I antibody according to the present invention can be produced from chicken eggs immunized with the above-mentioned GTF-I.

The chicken to be immunized is not particularly limited, but from the viewpoint of mass production of antibodies, it is preferable to use an egg seed such as a white leghorn.

In addition, the immunization method using GTF-I can be performed by a usual method such as subcutaneous injection, intramuscular injection, intraperitoneal administration, or a method such as nasal drop or eye drop. Furthermore, the dose of GTF-I may be appropriately selected so that a desired antibody titer is obtained and the dose does not adversely affect chickens.

Usually, an antibody that specifically reacts with the administered antigen is obtained in chicken eggs (yolk) within a few weeks after the initial immunization.

If necessary, an adjuvant such as FCA (complete Freund's adjuvant) or FIA (incomplete Freund's adjuvant) may be used together with GTF-I.

The anti-GTF-I antibody of the present invention can be prepared from eggs ingested from chickens that have passed one month or more after immunization.

The antibody titer in the yolk was determined by oxygen immunosorbent assay (BLISA),
It can be measured using a radioimmunoassay or the like,
By measuring the antibody titer at intervals of about two weeks after immunization, the transition of the antibody titer can be tracked.

In the examples described below, changes in the antibody titer were traced by ELISA, and eggs at the stage where the antibody titer was sufficiently increased were collected to prepare the anti-GTF-I antibody of the present invention.

Usually, a high antibody titer can be obtained for about 3 months.

If the antibody titer decreases after immunization, the antibody titer can be increased by performing additional immunization at appropriate intervals.

The anti-GTF-I antibody according to the present invention can be obtained, for example, by extracting and separating immunoglobulin contained in the egg yolk of the chicken immunized as described above. This extraction
As the separation method, for example, various methods capable of extracting and separating immunoglobulins which are generally used, such as a precipitation method using dextran sulfate or polyethylene glycol, an extraction method using propanol or chloroform, and the like can be used.

The anti-GTF-I antibody according to the present invention obtained as described above specifically reacts with GTF-I produced by S. mutans as an antibody. That is, it has an inhibitory activity on the enzyme activity for GTF-I.

The anti-GTF-I antibody according to the present invention, which has an inhibitory activity on GTF-I produced by S. mutans, inhibits S. mutans from adhering to the tooth surface, thereby causing It can control activities and prevent caries.

As described above, the present invention is easier and can be prepared in a large amount, compared to a production method prepared from a normal mammal, and only the IgG fraction of immunoglobulins is obtained by a simple operation of preparing an antibody from egg yolk. And a method for producing an antibody useful as an active ingredient of an agent for preventing dental caries.

These antibody-containing fractions can be blended with a normal caries preventive agent to produce the caries preventive agent according to the present invention.

That is, the caries preventive agent according to the present invention is only oral products such as toothpaste, toothpaste, liquid toothpaste, mouthwash, oral paste, gum massage cream, gargle tablet, troche, chewing gum, canned beverage and the like. Rather, it applies to various foods for that purpose.

The amount of the antibody according to the present invention to be added to the anti-caries agent may be appropriately selected according to the dosage according to the dosage form. For example, an antibody having an antibody titer of 10 ³ or more is about 0.0001 to 10% by weight. be able to.

In addition, as the other components of the anti-caries agent according to the present invention, appropriate components according to the purpose of use, use form, and the like are used.
For example, in the case of toothpaste, abrasives such as calcium carbonate, calcium hydrogen phosphate, calcium pyrophosphate, insoluble sodium metaphosphate, hydrated alumina, and silicic acid, humectants such as glycerin, sorbite, propylene glycol, sodium lauryl sulfate, and lauroyl A foaming agent such as sarcosine sodium and soap powder, a binder such as sodium carboxymethylcellulose and carrageenan, and other suitable components such as a flavor component, a sweetener and a preservative are mixed with water, and the mixture is produced according to a conventional method. Also, in mouthwashes such as mouthwashes and the like, components according to the properties of production are appropriately compounded.

In the present invention, various medicinal ingredients such as a tooth coloring remover, a halitosis preventive, a caries preventive such as fluorine, an antienzyme preventive and the like can be blended.

Therefore, the agent for preventing dental caries according to the present invention effectively suppresses the formation of plaque due to Streptococcus mutans by using the yolk antibody prepared from the immunized egg, and favorably prevents the occurrence of dental caries. Moreover, since the yolk antibody has high safety, the anti-caries agent of the present invention has high safety in use.

(Examples) Hereinafter, the present invention will be described in more detail with reference to Examples. still,
Unless otherwise specified, the percentages shown below indicate weight / volume%.

Example 1 (Method for producing antibody) (a) Preparation of antigen S. mutans strain 6715 (serotype g, obtained from Osaka University Dental School) was cultured in 151 BHI dialysis media at 37 ° C for 18 hours. The culture solution was separated into bacterial cells and culture supernatant by continuous centrifugation. The separated culture supernatant was subjected to 50% saturated ammonium sulfate precipitation,
The resulting precipitate was collected by centrifugation. Next, a solution obtained by dissolving this precipitate in a Histldine-HCI buffer (pH 6.2) was dialyzed against the same buffer, and the precipitate formed in the solution was removed by centrifugation. The supernatant was applied to a 2.5 × 25 cm column of polybuffer PBE94 (Polybuffer PBE94, manufactured by Pharmacia).

The fraction adsorbed on the column was
pH gradient using ffer 74 (Pharmacia)
It was eluted selectively with a 0.1 M NaCl solution.

The GTF activity of each eluted fraction was measured by the method described below, and the protein content was measured by the 280 nm ultraviolet absorption method. As a result, remarkable GTF activity was observed in the fraction having a NaCl concentration of 0.1 M.

Measurement of GTF activity: A sample (10 μl) was treated with 20 mM ( ¹⁴ C-glucose) sucrose [( ¹⁴ C-glucose) sucrose] (0.05 ci /
mol) and 10 μl of 0.2 M phosphate buffer (pH 6.0) containing 20 μM dextran T10, and reacted at 37 ° C. for 1 hour.
The entire amount of the reaction solution was spotted on a filter paper (1.0 × 2.0 cm), washed with methanol, and the amount of radioactivity incorporated in the methanol-insoluble glucan remaining on the filter paper was measured.
The activity was calculated, and the activity of transferring 1 μmol of glucose to glucan per minute was defined as 1 unit (U).

Next, the eluted fractions having a NaCl concentration of 0.1 M were collected and subjected to an ammonium sulfate precipitation treatment of 80% saturation to obtain a precipitate. This is dissolved in 10 mM phosphate buffer (pH 6.0) and dialyzed against the same solution. Further, the precipitate formed in the solution was removed by centrifugation to obtain a supernatant. This supernatant was used as a crude purified product (immune antigen).

The protein concentration and the total amount of protein of the sample were determined by CBB-
G method [Branford, N, M., Anal. Biochem., 72.248, (197
6)], the protein concentration was 0.4 mg / m
l, total protein was 13.2 mg.

Further, the sample was subjected to SDS-PAGE under the above-mentioned operating conditions, and stained with Kumasi Brilliant Blue (CBB). As a result, several bands of low molecular weight, mainly the band at the position of 160 kDalton, were found. was detected.

Further, the GTF-I content in the sample calculated from the result of the above SDS-PAGE was 4.0% by weight.

Separately, activity staining was performed as described below. That is, the sample was subjected to SDS-PAGE in the same manner as described above except that the sample was treated in Tris-HCl buffer at 37 ° C for 30 minutes, and electrophoresis was performed at 4 ° C. It was immersed in a phosphate buffer (pH 6.0) containing 1% sucrose, 0.05% dextran T10 and 0.05% sodium azide for 18 hours. Next, the gel that has been immersed is treated with PAS (Periodic acid
When stained by the Schiff reaction, a remarkable stained band showing glucan formation was detected at a position of 160 kDalton.

(B) Immunization of chickens with antigen 1.0 ml of the purified immunoantigen preparation obtained in (a) (containing 0.4 mg protein as measured by the CBB-G method) and 1.0 ml of FCA (complete Freund's adjuvant) The mixture was mixed 1: 1 to form a W / O emulsion.

After injecting 1.0 ml of the obtained emulsion into the pectoral muscles of chickens for the first immunization, the antibody titer of WSF (described later) obtained from the collected eggs was measured according to the following method, and the change was observed. .

Next, as shown in FIG. 1, it was confirmed that the antibody titer in the yolk began to decrease 8 weeks after the first immunization, and a second immunization was performed in the same manner as the previous time.

Eggs produced by chickens were collected about one month after the secondary immunization.

(C) Antibody production method 13 ml of egg yolk separated from an egg and the same amount of PBS (phosphate buffer, pH 7.4) were mixed, and the resulting mixture was further mixed with the same amount (26 ml) of the mixture. Chloroform was added and the mixture was stirred well.

After completion of the stirring, the mixture was allowed to stand at room temperature for 30 minutes, and then centrifuged at 3,000 rpm for 20 minutes to collect the uppermost transparent fraction. The antibody-containing fraction (Water Soluble Fracti
on: WSF).

This fraction had an antibody titer of 0.7 × 10 ² .

The protein content of WSF (13 ml: prepared from 13 ml of egg yolk) having an antibody titer of 0.7 × 10 ² was measured by the puret method, and the total protein mass in the WSF was determined to be about 2%.
A value of 6 mg was obtained (about 2.0 mg / ml x 13 ml).

(D) Method for measuring antibody titer: The antibody titer was measured by ELISA.

First, 2.5 μg / ml of the purified immunoantigen preparation obtained in Example 2 was used.
Was dissolved in a 50 mM sodium carbonate buffer (pH 9.6) to obtain a 96-well plate [Imron (Im
mulon) 2, manufactured by Dynatech Co., Ltd.], and allowed to stand at 4 ° C. overnight to adsorb the purified antigen contained in the fraction to the plate.

Next, the plate was washed with PBS-T (PBS, p containing 0.05% Tween 20).
H7.4) five times. After washing, plate is washed with 3% BSA
The plate was contacted with PBS (pH 7.4) containing (bovine serum albumin) at 37 ° C. for 1 hour to perform blocking, and then the plate was washed five times with PBS-T.

Here, the two-step dilution of WSF with PBS-T was obtained.
100 μl was added to each well and reacted at 37 ° C. for 1 hour.

After the completion of the reaction, the plate was washed 5 times with PBS-T. Further, 100 μl of a peroxidase-conjugated anti-chicken IgG antibody (protein mass: 1.67 μg / ml) as a secondary antibody was added to the plate.
1 to each well and reacted at 25 ° C for 30 minutes.
Washed 5 times with -T.

Next, 50 ml of 0.2 M disodium phosphate-0.1 M citrate buffer (pH 5.0) was added to each well of the plate to prepare o-
100 µl of a solution in which 20 mg of phenylenediamine and 10 µl of hydrogen peroxide were dissolved was added, and reacted at 25 ° C for 20 minutes. The reaction was stopped by adding 100 μl of a 3N sulfuric acid solution.

After stopping the reaction, the antibody titer was measured by measuring the absorbance (OD ₄₉₂ ) of each well. The antibody titer was determined by the endpoint titer method, and the dilution was determined as the dilution ratio at which the absorbance was 0.2.

(E) Confirmation of GTF-I water-insoluble glucan synthesis inhibitory activity by antibody GTF-produced by S. mutans using antibody obtained in (c)
An activity inhibition experiment of I was performed.

First, WSF having an antibody titer of 0.7 × 10 ² obtained in (c) and an 8-fold dilution thereof were used as test solutions.

Separately, WSF was prepared from eggs of non-immunized chickens (chickens similar to (b)) in the same manner as in (c) to prepare test solutions. In addition, it was confirmed that the WSF did not specifically react with GTF-I.

 As a control, a phosphate buffer solution (PBS) was used.

Next, each of these test solutions was placed in a 13 x 100 mm test tube,
After dispensing 50 µl, 1250 µl of 0.3 mU of GTF-I was further added to each test tube.

After mixing, the mixture was reacted at 37 ° C. for 30 minutes. After the completion of the reaction, 500 μl of 0.2 M phosphate buffer containing 2% sucrose and 40 μM dextran T10 was added to each test tube, and the reaction was carried out again at 37 ° C. for 1 hour. After sonication, the concentration (OD ₅₅₀ ) of the reaction solution was measured, and the water-insoluble glucan synthesis activity was calculated.

Table 1 shows each WSF assuming that the GTF activity when PBS was added was 100%.
The effect was shown.

Example 2 Method for producing GTF-I purified sample Under the same conditions as in Example 1, a supernatant liquid corresponding to the crude purified sample was obtained. The supernatant was used as a 10 mM phosphate buffer (pH 6.0).
And equilibrated hydroxyapatite [Bio Gel HT
P, manufactured by Bio Rad Co.)
cm).

The fraction adsorbed on the column was selectively eluted by a concentration gradient of 0.01 to 0.5 M phosphate buffer (pH 6.0).

The GTF activity and protein content of each fraction were measured in the same manner as described above. As a result, 0.2 M phosphate buffer (pH 6.0)
GTF activity was observed in the fraction obtained by the above method.

Next, an eluted fraction exhibiting high activity with a 0.2 M phosphate buffer (pH 6.0) was collected, and collected with a 10 mM phosphate buffer (pH 6.
0) was dialyzed to obtain a purified enzyme preparation.

Further, the sample was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue (CBB).
A single band was detected at the Dalton position, confirming that the purified enzyme preparation was an enzyme protein having a molecular weight of 160 kDalton.

Further, a purified enzyme preparation in an amount corresponding to 50 mU enzyme activity was added to a 0.1 M phosphate buffer (pH 6.0) containing 1% sucrose and 0.1% sodium azide at a final concentration, and 3 ml of distilled water was added. After adjusting the volume, the mixture was mixed and allowed to undergo an enzyme reaction at 37 ° C. for 18 hours. In addition, the enzyme reaction was stopped by cooling the reaction solution to 4 ° C. with ice.

After completion of the enzymatic reaction, the reaction product was treated by centrifugation at 1500 ng, and separated into a precipitated water-insoluble fraction and a supernatant.

The water-insoluble fraction was washed twice with distilled water and suspended in 3 ml of distilled water to obtain a water-insoluble glucan standard.

Also, add 2.5 times the volume of ethanol to the supernatant,
After standing at 4 ° C. for 1 hour, the resulting precipitate was collected by centrifugation at 1600 ng, dissolved in 3 ml of distilled water, and subjected to the same precipitation treatment again, and the collected precipitate was again added to 3 ml of distilled water. It was dissolved to obtain a water-soluble glucan standard.

Furthermore, when the amount of glucan contained in these samples was quantitatively analyzed by the anthrone method and the type of glucan synthesized by the action of the purified enzyme sample was examined, the purified enzyme sample synthesized water-insoluble glucan. GTF that has the effect of
-I.

In addition, the protein amount of the obtained purified enzyme preparation was measured by the above-mentioned method, and it was 0.48 mg.

Further, an antibody was prepared in the same manner as in Example 1 except that the above-mentioned purified GTF-I immunogen preparation was used as an antigen, and an antibody which specifically reacted with GTF-I was obtained.

Furthermore, the GTF-S activity inhibitory effect of the obtained antibody was tested by the same method, and as a result, similarly excellent effect was recognized.

 Next, examples of the anti-caries agent of the present invention will be described.

(Example 3) Toothpaste manufacturing method Calcium pyrophosphate 42% Glycerin 15% Sorbite 70% 10% Carboxymethylcellulose 12% Saccharin sodium 0.1% Sodium lauryl sulfate 2.0% Fragrance 1.0% Water 100% Obtained antibody-containing fraction (WSF) 0.5
% (With an antibody titer of 0.7 × 10 ³ ).

(Example 4) Production method of maushwash Wash ethanol 22.5% Saccharin sodium 0.05% Lauryl diethanolamide 0.3% Fragrance 1.0% Water Remaining 100% Above components containing antibody (WSF) 0.5 obtained in Example 1
% (With an antibody titer of 0.7 × 10 ³ ).

(Effects of the Invention) According to the present invention, S. mutans has a sufficient inhibitory effect on the adhesion to the tooth surface, is excellent in mass productivity, has low production cost, is excellent in safety, and is an active ingredient of an anti-caries agent. And a method for producing an anti-caries agent containing the antibody are provided.

In particular, the antibody having an immunological activity against S. mutans according to the present invention has the following advantages as compared with an antibody obtained by immunizing a conventional mammal.

(1) The antibody obtained according to the present invention is obtained in the eggs of immunized chickens, and does not require special or skilled techniques for collecting eggs, handling eggs, and obtaining antibodies from eggs. In addition, since only the IgG class of the immunoglobulin is transferred to the yolk, only IgG can be easily obtained.

On the other hand, in the case where antibodies are obtained by collecting blood from immunized mammals, a sophisticated technique is required for collecting blood, and it is very difficult to separate and purify a large amount of IgG from serum.

(2) The chicken used for the production of the antibody of the present invention is easy to manage, and its management cost is lower than that of a rat, for example.

Moreover, it is difficult to continuously obtain a large amount of blood and milk from mammals, and the method using mammals is not suitable for mass production of antibodies, but chickens continue to lay eggs stably for a long period of time, The antibodies obtained by the present invention can be mass-produced and have low production costs.

(3) The stability of antibodies produced from blood or milk of immunized mammals is not always good, and storage in serum or in a purified state is required at a temperature of about -80 ° C.

The antibody obtained by the present invention has good stability,
It also has good storage properties, and can be stored, for example, in the form of eggs at 4 ° C. for 1-2 months.

[Brief description of the drawings]

FIG. 1 is a graph showing changes in antibody titers of immunized chickens in Example 1.

 ────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hideaki Yokoyama 2-115-2, Sohara-Miyashiro-cho, Kakamigahara-shi, Gifu Examiner Koichi Sesita (56) References Caries and Periodontal Disease-Advances in Research, edited by Shigeyuki Hamada, Nippon Dental Review, 1985, vol. 3, pp. 119-141, Research in Veterinary Science, Vol. 18 (1975). pp. 117-120

Claims (4)

(57) [Claims] 1. A method for producing an antibody against a water-insoluble glucan synthase produced by Streptococcus mutans having a serotype of a, d or g as a cariogenic pathogen, comprising the steps of: B) separating the yolk from the eggs produced by the immunized chicken; c) deriving from the egg yolk against the water-insoluble glucan synthase and against the Streptococcus mutans Extracting an immunoglobulin containing an antibody having immunological activity. 2. In the step (c), the egg yolk and the phosphate buffer are mixed, chloroform is further added, and the obtained mixture is left for a predetermined time, followed by centrifugation to obtain the uppermost transparent layer. The method for producing an antibody according to claim 1, wherein the method is carried out by collecting the fraction as a fraction containing the antibody. 3. A method for producing a caries preventive agent comprising as an active ingredient an antibody against a water-insoluble glucan synthase of Streptococcus mutans whose serotype is a, d or g as a cariogenic causative bacterium; Immunizing a chicken with an insoluble glucan synthase; b) separating yolk from eggs produced by the immunized chicken; c) being induced against the water-insoluble glucan synthase from the egg yolk; Extracting an immunoglobulin containing an antibody having immunological activity against the Streptococcus mutans; and d) formulating a caries preventive agent in the immunoglobulin using an antibody induced against the water-insoluble glucan synthase as an active ingredient. A method for producing a caries preventive agent, comprising the steps of: 4. In the step (c), the egg yolk is mixed with a phosphate buffer, chloroform is further added, and the resulting mixture is left for a predetermined time, followed by centrifugation to obtain a transparent uppermost layer. 4. The method for producing a caries preventive agent according to claim 3, wherein the method is performed by collecting the fraction as a fraction containing the antibody.