JPH0463864B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a non-cariogenic antibody for preventing or suppressing (hereinafter referred to as suppressing) human dental caries induced by Streptococcus miutans and a non-cariogenic composition containing the antibody as an active ingredient. Dental caries is a human and animal disease that is induced on the tooth surface by bacteria in the oral cavity and progressively destroys tooth tissue. Among various caries-causing bacteria,
Streptococcus miutans is the most important. Traditionally, for example, Burgess Manual of Determinative Bacteriology, p. 502 (1974) states that ``Streptococcus miutans is associated with dental caries.Miutans is a microorganism similar to Streptococcus salivarius. However, it has never been studied in detail or compared with Salivarius. However, currently, Salivarius produces fructans from sucrose, while Miutans produces acid production of water-soluble and water-insoluble dextran-like polysaccharides (hereinafter referred to as DPS) from sucrose.
The two are clearly differentiated. As is well known, water-insoluble DPS that produces miutans adheres to the tooth surface and forms dental plaque. Many of the microorganisms in the oral cavity reside in plaque and produce lactic acid, which is said to destroy teeth. Cavity-causing bacteria other than P. miutans also produce lactic acid, but the lactic acid produced by P. miutans is not released from plaque and accumulates directly on the tooth surface. Bratthal et al. investigated the immunological specificity of cell wall antigens of P. miutans and proposed that P. miutans could be classified into seven serotypes from a to g [Bratthall, Odont, Revy., 20:141
(1970) and the same. 20:23 (1970); Perch et al.
Acta.Path.Microbiol.scand.Section B, 82:
357 (1974)]. Next, Makoto Sato serologically investigated the antigen specificity of the P. miutans strain and
), proposed classification into human (H) and rat (R) types, and reported as follows. (b) Most (96.3%) of the human-derived A. myutans were of the human type, and the rest were human. (b) All miutans derived from rats were rat-type. (c) Miutans was not detected in mice or guinea pigs. (d) All hamster and monkey-derived strains were of the human type. The above human, human and rat types are "c, e and f" and "d" according to Bratztal classification, respectively.
and g' and 'a and b' serotypes, 'Kouei Shi, Vol. 28, No. 2, pp. 100-123 (1978)]. Immunological techniques for preventing or at least suppressing dental caries (hereinafter referred to as suppression) are known. For example, British Patent No. 1375866 discloses a method for preventing or at least suppressing dental caries.
The present invention discloses a vaccine for suppressing human dental caries that uses all or part of the bacterial cells of a strain having the same properties as NCTC strain No. 10449 as an antigen. However, it has not yet been reported that human dental caries can be effectively suppressed using a whole bacterial vaccine. Furthermore, it has been reported that when a whole bacterial vaccine of P. miutans was administered to mammals, adverse side effects such as cross-reaction with cardiac muscle antigens and allergic diseases were observed. British Patent No. 1,505,513 discloses an antibody product capable of inhibiting dental caries induced by rat-derived P. aeruginosa. This product is obtained by inactivating cells of specific P. miutans bacteria with heat, immunizing cows with the cells, producing the corresponding antibodies in the animal's body, and collecting the resulting antibodies as milk. It will be done. This product allows for a wide range of antibody administration methods, and is described as having no risk of causing the above-mentioned adverse side effects to humans. However, since the level of activity against human-type bacterial strains is low, it is difficult to put it into practical use for controlling human dental caries induced by P. myutans. The non-cariogenic antibodies disclosed in this British patent are produced by the following method. miutans AHT (serotype a), BHT (serotype b),
Cultures of 10449 (c) and 6715 (d) were grown in dialyzed tryptose medium, the cells were centrifuged from the culture medium, and washed with 0.1M phosphate buffered saline;
Cells were suspended in the same saline solution and inactivated by heating at 60°C for 30 minutes to create an antigen solution containing the four types of cells at a final concentration of 5 x ¹⁰ cells/ml. The immunized, antibody-containing milk is collected from the cow and dried. Antibodies in dried milk are 4 types of miutans
The ability to suppress AHT, BHT, 10449, and 6715 was investigated using the following method. 50 mg in 1 ml of serially diluted (x2) saline solution
of dry milk was added to each antibody solution, and 4 types of formalin-killed S. miutans (2 x 10 ⁸ cells/ml) were added to each antibody solution.
The agglutination value was measured and the following results were obtained. Antigen used Serotype Titer AHT a 64 BHT b 1024 10449 c <2 6715 d 256 The following composition was prepared using dried milk. (b) Dilute in 1 to 5 times the volume of water and use as a gargle. (b) Adding 25% or more to foods such as sugar-free gum, candy cane, and ice cream. (c) Add 25% or more to regular toothpaste. Next, rats were used as test animals, and when they were fed a cariogenic diet and infected with AHT, BHT, and 6715 in their oral cavities, they were found to have a caries-inhibiting effect. however
10449 was removed from the animal study due to no detectable titer in previous measurements. The antibody of British Patent No. 1505513 suppresses 6715 (human type, serotype d C. myutans) present in the human oral cavity. However, this antibody has no inhibitory activity against human, serotype c M. myutans NCTC 10449 or other human forms of M. myutans. It is well known that 10449 is a representative strain of C. miutans in the human oral cavity. Therefore, it cannot be put to practical use in suppressing dental caries in humans. On the other hand, it is known that oral bacteria of the genus Streptococcus, such as S. miutans, attach to the host's teeth and oral mucosa through pili on the surface layer of the cell wall of the bacteria [Gibbons et al. Ann. Rev.
Microbiol., 29:19-44 (1975)]. The present invention is based on the finding that antigens isolated from the ciliary layer of H. myutans can be used to produce antibodies capable of suppressing H. myutans. The antigens isolated from serotypes c, e, f, and g and the antigen isolated from serotype d (hereinafter referred to as human fimbrial antigen and human fimbrial antigen) were tested for serological specificity. It was possible to classify them according to According to a feature of the invention, serotypes c, e, f or g
A human type pili antigen isolated from the cell surface pili of a Streptococcus miutans strain of serotype d and a human type pili antigen isolated from the cell surface pili of a Streptococcus miutans strain of serotype d. immunizing a mammal with one or more antibodies selected from the following: collecting the resulting antibodies from said mammal; and said isolation being performed at a temperature sufficient to prevent denaturation of the desired antigen. Provided are methods for producing antibodies for preventing or inhibiting human dental caries induced by Streptococcus miutans. According to the following features of the present invention, the present invention provides inhibition of human dental caries induced by Streptococcus miutans, which contains one or more human fimbrial antigens as an active ingredient and contains a pharmaceutically acceptable single substance or excipient. A composition for use is provided. Protein quantification using the Hartree method using bovine serum albumin as a standard for fimbrial antigen isolated from NCTC 10449 (serotype c, human type), which is well known as a typical human caries-inducing microtube, D-
Determination of carbohydrates by the phenol sulfuric acid method using glucose as a standard, and cephaedetus G- with reference to phosphorylase B, calf serum albumin, chicken egg albumin, and chymotrybusinogen A.
100 (manufactured by Pharmacia Huain Chemicals, Sweden). In addition, similar tests were conducted using Imbritz (serotype c), OMZ176 (serotype b), P4 (serotype e), OMZ175 (serotype f), and KIR (serotype g), which were recognized as type strains of P. miutans. . The following results were obtained. (1) This antigen is a type of acidic glycoprotein containing approximately 15-25% protein (e.g., approximately 20%) and carbohydrates 75-85% (e.g., approximately 80%), and has a molecular weight of approximately 6. -9
×10 ⁴ (eg, about 75000). (2) In polyacrylamide gel disk electrophoresis, a characteristic thick band is observed on the cathode side. (3) In the sucrose density gradient ultracentrifugation method, the human fimbrial antigen has a saccharide density of approximately 10-13% and a sucrose specific gravity of approximately
It is present in the fraction with 1.3−1.4. (4) Gel filtration of crude antigen solution [Selfdex G-
100 (Falmacia Huain Chemicals Co., Ltd.
(made in Sweden)], two peaks appear (absorption at 280 nm was investigated). Human fimbrial antigen is recovered from the first peak. (5) In an isoelectric point fractionation test using the Försterberg method using Carrier Amphorite (manufactured by LKB Products, Sweden) with a pH of 3.5-10.0, the pI value of the human fimbrial antigen was approximately
It was below 3.5. No significant difference was observed in the results of fractionation of human fimbrial antigens from all H. miutans strains. Antibodies obtained from human-type or human-type antigens can be used to suppress type strains;
Practically speaking, it is better to use a combination of molds and molds. Even when used in combination, there is no adverse effect on the storage stability or effectiveness of each antigen. By orally administering antibodies obtained by the method of the present invention to humans, adhesion (infection) of human H. myutans to the surface of bacteria can be suppressed. When the adhesion of C. myutans to teeth is suppressed, the cells of the wild strain of C. myutans aggregate, for example in saliva, and as a result die in a relatively short period of time. Aggregated B. miutans cells can be easily removed by conventional methods such as brushing teeth, using gargles, detergents, and the like. It has been found that continuous oral administration of an effective amount of antibodies corresponding to one or more human fimbrial antigens, for example for several weeks, completely eliminates wild strains of S. miutans from the oral cavity. This antibody can be produced by the following method. The strain of H. miutans used in the present invention may be a wild strain or a mutant strain, but it is advantageous to use a strain with high adhesion ability. For example, a desired strain can be selected based on its high ability to adhere to test tubes. The bacterial strains used in the Examples and Test Examples were Streptococcus miutans mutant K-Dp strain (serotype c, Kaikoken Joyori No. 317) and Streplococcus miutans KH2 (serotype d, Microtechnical Research Institute No. 366). These have properties similar to normal wild strains (parent strains), but differ in that they have significantly higher adhesion ability. These were deposited on March 5, 1982 and July 23, 1983, respectively. Cultivation may be carried out by a conventional method under aerobic conditions, but anaerobic conditions are suitable. The medium used may be a natural medium or a synthetic medium, but a liquid medium is suitable for mass production. The pH of the medium is 5.6-9.0, e.g.
7.0, the culture temperature is 23-39°C, and the culture is usually 24-72 hours. After the completion of the culture, by a conventional method such as centrifugation (8000 rpm, 5-20 minutes),
Isolate the bacterial cells from the culture solution. The isolated bacterial cells are placed in an appropriate hypertonic solution, such as 0.1-1M acetic acid/sodium acetate buffer (PH6.0-8.0), 1M sodium chloride/phosphate buffer (0.01-0.75M), or non-containing buffers. Ionic surfactants, such as Triton-
Suspended in a buffer solution containing an appropriate surfactant such as
(for about 20 minutes) to extract the desired human fimbrial antigen. When observed under an electron microscope, it was found that the human fimbrial antigen could be effectively extracted by the action of hypertonic solution. If desired, ammonium sulfate is added to the culture solution to saturation of 20-70% (eg, 30%) and dissolved by stirring. Next, when the mixed solution is allowed to stand at a low temperature, for example, 4° C., the fimbriae layer settles. Collect the liquid with the supernatant removed and suspend it in a concentrated solution in the same buffer (PH6-8).
Dialyze with the same buffer solution at low temperature (e.g., 4°C) to remove ammonium sulfate and dialysable impurities, collect the dialyzed solution, and centrifuge (e.g., 8000 rpm, 20 minutes).
Then, an extract containing human fimbrial antigen is obtained. The extract containing the human fimbrial antigen thus obtained is subjected to conventional methods such as column fractionation, isoelectric focusing, cold solvent fractional precipitation, membrane concentration, ammonium sulfate salting out, etc. Fractionation and purification is carried out by saccharide density gradient ultracentrifugation alone or in an appropriate combination.
Particularly good is the sugar density gradient ultracentrifugation method. In order to prevent denaturation of human fimbrial antigens, fractionation and purification are preferably carried out at a low temperature below room temperature, preferably below 10°C. In the Examples and Test Examples, the active material was processed, for example, at 4°C. The purified human fimbrial antigen solution is treated with an appropriate inactivating agent,
For example, it is treated with formalin (0.2-0.02%) and then dialyzed in a conventional manner to remove the inert agent. The purified human fimbrial antigen solution is, for example, 0.5-
Dilute the protein N to 10-50 μg/ml with 1M phosphate buffered saline (PH approximately 6-8), and add aluminum hydroxide gel as an adjuvant to give a final aluminum dose of 100-500 μg/ml. adsorbs the antigen. Thimerosal 0.05
A preservative such as -0.1 (w/v) may be added.
In this way, an antigen solution for mammalian immunization is obtained. Instead of ammonium sulfate, the same amount of complete Freund's adjuvant may be added to the antigen solution. Immunization is by conventional methods. For example, small animals such as mice, rats, and guinea pigs, and large animals such as rabbits, goats, sheep, cows, and horses can be used. The dose of PLS antigen varies depending on the type of animal and other conditions, but it is usually used as protein N, and for small animals it is 10-
500μg, 100-200μg for large animals (once daily dose)
For example, it is injected subcutaneously into the back of a mammal.
Immunization can be carried out, for example, 2-5 times every 2-5 weeks. If desired, immunization can be carried out by orally administering, for example, 5-10 times the dose and continuing for 3-12 days. For example, in the case of rabbits, a human fimbrial antigen solution (100-200 μg/ml as protein) mixed with an equal amount of Freund's complete adjuvant is administered subcutaneously on the back 2-5 times every 2-5 weeks. 10-14 days after the final immunization, blood is collected from the mammal by conventional techniques, such as cardiac puncture, and plasma containing immunoglobulin is prepared. Plasma can be administered to humans as is, or it can be purified to form antiserum. Plasma or antiserum can be stored at low temperatures for long periods of time and administered to humans without reprocessing. Although it is practical to immunize a mammal with the human-type fimbrial antigen or the human-type fimbrial antigen alone, it is also possible to immunize the animal with a combination of and. The antigen may be obtained from strains of the same serotype or from strains of different serotypes selected from serotypes c, e, f and g. The composition for inhibiting human dental caries according to the present invention contains, as an active ingredient, an antibody against one or more types selected from human fimbrial antigens and human fimbrial antigens. The carrier or excipient of the anti-caries composition according to the invention may be solid, semi-solid or liquid suitable for oral administration. The compositions may thus be, for example, in the form of powders, capsules, granules, tablets, drops, syrups, suspensions or emulsions. Examples of suitable solid carriers are lactose, potato starch, soluble starch, magnesium stearate, clay, diatomaceous earth, and the like. Examples of suitable liquid carriers are water, saline, glycerin, almond oil, lactic acid beverage, juice, and the like. The compositions according to the invention may further contain customary additives such as binders, stabilizers, emulsifiers, suspending agents, lubricants, preservatives, essences. Practical compositions include, for example, antibacterial polishes, mouthwashes, candy, chewing gum, ice creams, and the like. The non-carious compositions can be formed into dosage units such as tablets, ampoules, troches, and troches for administering to humans a fixed amount of an antibody according to the invention. The amount of one or more antibodies contained in each dosage unit varies depending on the type of dosage unit and the purpose of administration, but may be selected so that, for example, a daily dose of 1-10 units of each antibody (see below) can be administered. can. As a result of tests administered to humans and hamsters, for example, by continuously administering 1-4 gel units or more of each antibody per day for several weeks, it was observed that muyutans disappeared from the oral cavity. Pathological tests showed that there were no particular abnormalities even when this antibody was continuously administered in large quantities to hamsters over a long period of time (6-12 months). In this specification, the antibody titer was indicated by two in-gel precipitation titers obtained by in-gel precipitation reaction [Goldman.JCet al.Isolation and
Characterization of glial filaments from
human brain. J. Cell Biol., 78:426 (1978)]. In the following Examples and Test Examples, the cultures were anaerobically cultured at 37°C unless otherwise specified. Commercially available media are specified
Used in PH. Example 1 Streptococcus miutans mutant strain K-
Creation of Dp strain (Feikoken Joyori No. 317). A fresh wild strain of M. miutans (serotype c) isolated from human bacterial plaque was infused into Dots Huyt Broth (Baltimore Laboratories, Inc., USA).
Incubate at 37℃ for 24 hours using 20ml of BBL (abbreviated as BBL).
The culture solution was centrifuged (8000r.pm, 20 minutes) to separate the bacterial cells, which were then centrifuged three times (8000r.p.
m., 20 minutes) and washed. Cells were suspended in a similar phosphate buffered saline solution (20 ml) containing 20% nitrogen mustard at a concentration of about 1 million viable bacteria/ml, and incubated until 90% or more of the viable bacteria were killed.
It was kept at ℃. Culture the remaining viable bacteria in Dots Huyt Broth in the same manner as above, and add one loopful of the culture solution.
TYC agar plate medium [STOPPELAAR et al:
Archs. Oral Biol., 12.1199-1201 (1967)] for 48 hours, the culture was allowed to stand at room temperature for 24 hours, and colonies with a high ability to produce water-insoluble DPS were selected. The above method was repeated as desired to obtain a strain with high DPS production ability. The obtained strain was transformed into Streptococcus miutans mutant strain K.
−Dp strain. When this strain was orally administered to hamsters for a long period of time or cultured in various known media, it was found that the strain had extremely high adhesion ability and was genetically stable. Example 2 Production of Streptococcus miutans KH2 (Feikoken Joyori No. 366). According to the method described in Example 1, from a wild strain of serotype d isolated from human oral cavity,
A genetically stable mutant strain was obtained. This bacterium was named Streptococcus miutans KH2. Example 3 Streptococcus miutans mutant strain K-
Production of antibodies using human fimbrial antigen isolated from Dp strain. Streptococcus miutans mutant strain K-
Dp strain (Feikokenkyo No. 317) was grown in a seed medium with the following composition.
Culture was carried out for 24 hours using 500 ml and 15,000 ml of the main medium. Polypopeptone 1.7%, polypopeptone
S0.3%, yeast extract 0.5% (manufactured by Difco, USA),
Dipotassium phosphate 0.25%, sodium chloride 0.5%,
Glucose 0.25% (PH7.0-7.8). At the end of the culture, add ammonium sulfate to the culture solution to make it 33% saturated, stir to dissolve, and heat to 4℃ for 24 hours.
Let it stand for a while. The supernatant of this solution was removed, and the precipitated fraction was collected by centrifugation (8000 rpm, 30 minutes), and this was collected in 0.1M phosphate buffered 1M saline (750ml,
PH8.0) and stirred at 4°C for 72 hours at 1-5 revolutions per minute. Centrifuge the buffer solution (8000r.pm, 30
After removing the bacterial cells, ammonium sulfate was added to the supernatant to make it 60% saturated, the mixture was stirred and dissolved, and the desired antigen was precipitated by standing at 4°C for 48 hours. . The supernatant was removed, the sedimented fraction was centrifuged (8000 rpm, 30 minutes), and the recovered material was suspended in 100 ml of the same phosphate buffered saline.
Place the suspension in a cellophane tube for dialysis and keep at 4°C.
Dialysis was performed for 48 hours using over 5000 ml of the same phosphate buffered saline solution until the ammonium sulfate was removed. The dialyzed fluid was centrifuged (8000 rpm, 30 minutes) and the supernatant was collected. Dilute the supernatant (300 ml) with the same phosphate buffered saline so that the protein N content is 100 μg/ml, and add 200 ml of the diluted solution using a sugar density gradient centrifuge (Hitachi).
65P ultracentrifuge, using zonal rotor RP235T,
When treated with saccharide sugar density 5-30%, 35000 rpm, 18 hours), the desired antigen was found in the fraction with sucrose density around 10-12% (specific gravity approximately 1.31-1.35).
The amount of protein N was about 33-37 μg/ml. Put the obtained extract into a cellophane tube and mix with polyvinyl pyrrolidone until the volume reaches 1/10.
Concentrate at °C. Concentrate in 0.75M phosphate buffer 1M
Saline solution (PH6.2-6.5), protein N content 50-100μ
The antigen solution for mammalian immunization was obtained by diluting the solution to a concentration of g/ml and adding an equal volume of Freund's complete adjuvant to the diluted solution. 1.0 ml of the obtained human fimbrial antigen solution was administered subcutaneously to the back of a rabbit weighing approximately 3 kg in a conventional manner for immunization, which was administered three times at four-week intervals, and blood was collected by cardiac puncture four weeks after the final immunization. , Add ammonium sulfate to 33% saturation, stir and dissolve.
℃ for 48 hours, the precipitated portion was collected by centrifugation (8000 rpm, 20 minutes), placed in a cellophane tube, and dialyzed in purified water at 4℃ until ammonium sulfate disappeared. was concentrated and lyophilized using a conventional method to obtain a human antibody-containing solution (approximately 85 ml). Example 4 Production of antibodies using human fimbrial antigen isolated from Streptococcus miutans KH2. Streptococcus miutans KH2 (Feikoken Joyori No. 366, serotype d) was cultured and treated according to the method described in Example 3 to obtain an antibody-containing solution (approximately 85 ml). Example 5 1 g of butose sugar, 0.05 ml of antibody-containing liquid (human antibody and one or more types of human antibodies), 0.05 ml of soluble starch
A backbone was made by a conventional method using g. Example 6 Syrup was prepared by a conventional method using 0.2 g of CMC sodium, 20 ml of 20% fructose solution, 0.04 g of ethyl paraffin, and 0.1 ml of an antibody-containing solution (human antibody and one or more types of human antibodies). Example 7 A dentifrice was made in the following manner. Calcium hydrogen phosphate fine powder 60%, glycerin
30%, CMC sodium 10%, and paraben (preservative) 0.25%, add antibodies (humanized antibodies and one or more types of humanized antibodies), and adjust the antibody titer (above) to 2 gel units/50g. I adjusted it so that Example 8 A non-cariogenic lactic acid bacteria drink was prepared by the following method. 2000ml of skim milk liquid with solid content of 10% at 110℃
After sterilization for 15 minutes, Lactobacillus casei was transplanted using a conventional method and cultured at 35-37°C for 36 hours. Add 10% fructose solution to the culture, and the number of viable casei bacteria is approximately ^10.8 /
Adjusted to ml. Each antibody (human type, human type, or combination) was added to the mixed solution so that the titer would be 4 units (described above)/50 ml by in-gel precipitation reaction to obtain a non-cariogenic lactic acid bacteria drink. Test Example In the following test, the ability to suppress the adhesion of carious Streptococcus miutans was tested using the humanized antibody produced by the method of the above example and a composition containing the humanized antibody in combination. The number of test animals (hamsters) is 5 female or male in each group unless otherwise specified.
There were a lot of them. Test Example 1 A test was conducted using 21-day-old hamsters. Groups 1 and 2 were test groups, and groups 3 and 4 were control groups. Streptococcus miutans mutant strain K-Dp (Feikoken Joyori No. 317) and Streptococcus miutans KH2 (Feikoken Joyori No. 366) strain were each treated with Tryptocase Soy Broth (manufactured by BBL, USA). , PH7.5−7.8) at 37℃
The cells were cultured for 24 hours. Obtained culture solution (number of viable bacteria approx.
10 ⁸ pieces/ml) using a daily dose of 0.1 ml, the first and third
The K-Dp strain (serotype c) was orally administered to the animals in the group, and the KH2 strain (serotype d) was administered orally to the animals in the second and fourth groups for 5 consecutive days, and colonization was confirmed. after that,
The molar surfaces of the animals of the first and second groups were strongly brushed with a small brush using the dentifrice described in Example 7 (containing the same unit of humanized antibodies and humanized antibodies), 0.1 g per individual. This was done once a day for 14 days. The feed during the test period was a caries-inducing feed diet.
2000 (manufactured by Funabashi Farm) and were given ad libitum with deionized water. During the 60-day test period, feed was collected from the molar surface of each individual at appropriate times and placed on TYC agar plates (Stoppelaar et al, 15 ml, PH 7.4) and Mitis salivarius agar plates (15 ml, PH 7.4,
Bacterial colonization on the tooth surface was investigated by culturing at 37°C for 72 hours using Difco (manufactured by Difco, USA). 1st
In the oral cavity of the animals of Group 2 and Group 2 (of the test group), the concentration of the bacterial strain gradually decreases after administration of the antibody according to the invention, within about 2-4 weeks in some animals and about 4-4 weeks in the remaining animals. The bacteria disappeared within 7 weeks. In the control groups (groups 3 and 4), no decrease in the bacterial concentration in the oral cavity was observed. After the test period, each test animal was anesthetized with pentabarbital, the jaws were removed and autoclaved (120-125°C, 1-2 minutes), the soft tissue was removed, thoroughly washed with water, and dried. It was used as a bacterial specimen. The morbidity rate and caries rate were calculated from the number of molars in which caries was induced among the total molars in each test group.
Shown in Table 1.

[Table] Human type strains other than serotype c, that is, well-known standard strains P-4 (type e) and OMZ175 (type f)
When we conducted a test similar to the above using 1. and KIR (type g) separately, it was found that this human antibody suppressed the adhesion ability of these strains in the same way as for type c strain. Ta. Test Example 2 Changes in the concentration of Streptococcus miutans strains in the flora of the adult oral cavity due to administration of the humanized antibody described in Example 7 and the humanized antibody-containing bactericidal polish were investigated using the following method. Bacterial plaque was collected from the oral cavity three times each from 10 adults (5 males, 3 females) and cultured using the method described in Test Example 1. As a result of examining the degree of plaque and tartar deposition, the presence of Streptococcus miutans (serotype c) was found in the oral cavity of 10 patients. All 10 subjects brushed their teeth in any manner using the above toothpaste every day after breakfast and dinner. We collected dental plaque from each person at least once a week, cultured it in the same manner as above, and examined changes in the number of B. myutans bacteria. As a result, the number of M. myutans in the oral cavity was found to be approximately 2-3 weeks later for 3 people. In the remaining 7 patients, little or no detection was observed after about 4-7 weeks. The OHI value dyed with erythrosin is
In all cases, the levels were significantly lower than before the start of administration. Test Example 3 The variation in the miutance concentration in the oral cavity of an adult due to the administration of the humanized antibody having the same unit as described in Example 5 and Vacucar containing the humanized antibody was investigated according to the method described in Test Example 2. The subjects were 20 adult women. Of these, 19 were serotype c and the remaining one was serotype d. One Vatukaru was administered to each person every day at bedtime and kept in the oral cavity for as long as possible. After about 2 weeks, no myutans were detected in the oral cavity of 5 people. The concentration of myutans in the oral cavity of the remaining subjects gradually decreased, and a small amount of myutans was detected in the oral cavity about 4 weeks after the start of administration. The OHI value before the start of the test was significantly lowered by administration of Vatukar. When Baccar was used, the residence time of antibodies in the oral cavity was longer than in the case of dentifrice, so it was recognized that an inhibitory effect equal to or greater than the administration of dentifrice could be obtained.

Claims (1)

[Scope of Claims] 1. Antigens are isolated from pili by a step of dispersing one or more strains of the group consisting of human and human Streptococcus miutans in a hypertonic buffer containing sodium chloride; Streptococcus spp. consists of the steps of immunizing a mammal with an antigen, producing the corresponding antibody in the mammal's body, and collecting antiserum from the mammal.
A method for producing an antibody for preventing or inhibiting human dental caries induced by P. myutans. 2. The production method according to claim 1, wherein the antibody has the ability to inhibit dental caries in hamsters.