JPH01233229A - Antibody and tooth decay preventive containing the same - Google Patents
Antibody and tooth decay preventive containing the sameInfo
- Publication number
- JPH01233229A JPH01233229A JP63058890A JP5889088A JPH01233229A JP H01233229 A JPH01233229 A JP H01233229A JP 63058890 A JP63058890 A JP 63058890A JP 5889088 A JP5889088 A JP 5889088A JP H01233229 A JPH01233229 A JP H01233229A
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- gtf
- mutans
- water
- immunized
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 208000002925 dental caries Diseases 0.000 title claims abstract description 15
- 230000003449 preventive effect Effects 0.000 title description 16
- 235000013601 eggs Nutrition 0.000 claims abstract description 20
- 241000194019 Streptococcus mutans Species 0.000 claims abstract description 15
- 102000004190 Enzymes Human genes 0.000 claims abstract description 12
- 108090000790 Enzymes Proteins 0.000 claims abstract description 12
- 244000052616 bacterial pathogen Species 0.000 claims abstract description 3
- 241000287828 Gallus gallus Species 0.000 claims description 23
- 235000013330 chicken meat Nutrition 0.000 claims description 23
- 230000002401 inhibitory effect Effects 0.000 claims description 12
- 108060003951 Immunoglobulin Proteins 0.000 claims description 7
- 102000018358 immunoglobulin Human genes 0.000 claims description 7
- 108010052221 glucan synthase Proteins 0.000 claims description 6
- 230000001939 inductive effect Effects 0.000 claims 1
- 244000052769 pathogen Species 0.000 claims 1
- 230000001717 pathogenic effect Effects 0.000 claims 1
- 229920001503 Glucan Polymers 0.000 abstract description 17
- 239000000427 antigen Substances 0.000 abstract description 17
- 102000036639 antigens Human genes 0.000 abstract description 17
- 108091007433 antigens Proteins 0.000 abstract description 17
- 230000003053 immunization Effects 0.000 abstract description 16
- 210000002969 egg yolk Anatomy 0.000 abstract description 12
- 238000002649 immunization Methods 0.000 abstract description 12
- 239000004480 active ingredient Substances 0.000 abstract description 5
- 239000008103 glucose Substances 0.000 abstract description 4
- 210000002966 serum Anatomy 0.000 abstract description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 102000003960 Ligases Human genes 0.000 abstract 2
- 108090000364 Ligases Proteins 0.000 abstract 2
- 210000005056 cell body Anatomy 0.000 abstract 1
- 238000000034 method Methods 0.000 description 28
- 230000000694 effects Effects 0.000 description 20
- 239000003795 chemical substances by application Substances 0.000 description 16
- 238000002360 preparation method Methods 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- 235000013345 egg yolk Nutrition 0.000 description 10
- 229940088598 enzyme Drugs 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 10
- 108010000912 Egg Proteins Proteins 0.000 description 9
- 102000002322 Egg Proteins Human genes 0.000 description 9
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 241000124008 Mammalia Species 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 239000008363 phosphate buffer Substances 0.000 description 8
- 239000002244 precipitate Substances 0.000 description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 239000000606 toothpaste Substances 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 5
- 239000012228 culture supernatant Substances 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 239000012521 purified sample Substances 0.000 description 5
- 229940034610 toothpaste Drugs 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 239000004615 ingredient Substances 0.000 description 4
- 235000013336 milk Nutrition 0.000 description 4
- 239000008267 milk Substances 0.000 description 4
- 210000004080 milk Anatomy 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 230000003628 erosive effect Effects 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 239000002324 mouth wash Substances 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 102000016938 Catalase Human genes 0.000 description 2
- 108010053835 Catalase Proteins 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 108010058846 Ovalbumin Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 2
- JUNWLZAGQLJVLR-UHFFFAOYSA-J calcium diphosphate Chemical compound [Ca+2].[Ca+2].[O-]P([O-])(=O)OP([O-])([O-])=O JUNWLZAGQLJVLR-UHFFFAOYSA-J 0.000 description 2
- 229940043256 calcium pyrophosphate Drugs 0.000 description 2
- 239000001768 carboxy methyl cellulose Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000011098 chromatofocusing Methods 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 235000019821 dicalcium diphosphate Nutrition 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000003205 fragrance Substances 0.000 description 2
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229940092253 ovalbumin Drugs 0.000 description 2
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- -1 60 k daltons) Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010006326 Breath odour Diseases 0.000 description 1
- SGHZXLIDFTYFHQ-UHFFFAOYSA-L Brilliant Blue Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 SGHZXLIDFTYFHQ-UHFFFAOYSA-L 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 108010001682 Dextranase Proteins 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- 102000008857 Ferritin Human genes 0.000 description 1
- 108050000784 Ferritin Proteins 0.000 description 1
- 238000008416 Ferritin Methods 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- 229940122959 Glucosyltransferase inhibitor Drugs 0.000 description 1
- 101710197440 Glucosyltransferase-S Proteins 0.000 description 1
- 108010055629 Glucosyltransferases Proteins 0.000 description 1
- 102000000340 Glucosyltransferases Human genes 0.000 description 1
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000004909 Moisturizer Substances 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 101710160102 Outer membrane protein B Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 108010073135 Phosphorylases Proteins 0.000 description 1
- 102000009097 Phosphorylases Human genes 0.000 description 1
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 1
- 235000011613 Pinus brutia Nutrition 0.000 description 1
- 241000018646 Pinus brutia Species 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241000208422 Rhododendron Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000003082 abrasive agent Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 1
- 230000000772 anti-erosive effect Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 235000010216 calcium carbonate Nutrition 0.000 description 1
- FUFJGUQYACFECW-UHFFFAOYSA-L calcium hydrogenphosphate Chemical compound [Ca+2].OP([O-])([O-])=O FUFJGUQYACFECW-UHFFFAOYSA-L 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000004075 cariostatic agent Substances 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 229940112822 chewing gum Drugs 0.000 description 1
- 235000015218 chewing gum Nutrition 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 239000012468 concentrated sample Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 235000019700 dicalcium phosphate Nutrition 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001333 moisturizer Effects 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 229940051866 mouthwash Drugs 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 239000007923 nasal drop Substances 0.000 description 1
- 229940100662 nasal drops Drugs 0.000 description 1
- 239000013588 oral product Substances 0.000 description 1
- 235000015927 pasta Nutrition 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000007505 plaque formation Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- KSAVQLQVUXSOCR-UHFFFAOYSA-M sodium lauroyl sarcosinate Chemical compound [Na+].CCCCCCCCCCCC(=O)N(C)CC([O-])=O KSAVQLQVUXSOCR-UHFFFAOYSA-M 0.000 description 1
- AQMNWCRSESPIJM-UHFFFAOYSA-M sodium metaphosphate Chemical compound [Na+].[O-]P(=O)=O AQMNWCRSESPIJM-UHFFFAOYSA-M 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000010414 supernatant solution Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 108010050327 trypticase-soy broth Proteins 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Birds (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biotechnology (AREA)
- Engineering & Computer Science (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Cosmetics (AREA)
Abstract
Description
【発明の詳細な説明】
本発明は、門蝕を誘発する病原菌であるストレプトコッ
カス争ミュータンス（Ｓｔｒｅ ｔｏｃｏｃｃｕｓ−＠
ｕ ｔ ａ−ｎ ｓ ）が産生ずる水溶性グルカン合
成酵素（グルコシルトランスフェラーゼ、ＧＴＦ−３と
略記する）に対して阻害活性を存する抗体及び該抗体を
有効成分として含む關蝕予防剤に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the use of Stretococcus mutans, a pathogenic bacterium that induces portal erosion.
The present invention relates to an antibody that has an inhibitory activity against water-soluble glucan synthase (glucosyltransferase, abbreviated as GTF-3) produced by U.sub.tans), and a dental caries prevention agent containing the antibody as an active ingredient.
門蝕の発生において、３ 、５ｕｔａｎｓの歯面への付
着過程が重要な役割を果たしていることが既に知られて
おり、Ｓ ０ｍｕｔａｎｓの口腔内への定着（歯面への
付着）を制御して、門蝕を予防しようとする種々の試み
が成されている。It is already known that the process of attachment of S3,5utans to the tooth surface plays an important role in the development of portal caries, and it is known that the process of attachment of S0mutans to the tooth surface is controlled. Various attempts have been made to prevent portal erosion.
例えば、Ｓ 、 ｍｕｔａｎｓに対する免疫活性を有す
る抗体を用いた門蝕予防方法が知られており、英国特許
第１５０５５１３号明細書には、Ｓ 、 ｍｕｔａｎｓ
の菌体で免疫した牛から得られた母乳をＳ、 ｍｕｔａ
ｎｓの口腔内定着の＃御に用いる方法が、又特開昭６０
−３８３２７号公報には、Ｓ１口ｔａｎｓの培養から得
た細胞壁等の両分を哺乳動物に免疫することによって得
られた抗血清及び／又は乳と、グルコシルトランスフェ
ラーゼインヒビター、プロテアーゼ及びデキストラナー
ゼからなる群より選ばれる１種以上のシネルギストとを
組み合わせた門蝕予防痢が記載されている。For example, a method for preventing portal erosion using an antibody having immunoactivity against S. mutans is known, and British Patent No. 1505513 describes
Breast milk obtained from cows immunized with S. muta
The method used to control the intraoral fixation of NS was also disclosed in Japanese Patent Application Laid-open No.
Publication No. 38327 discloses that antiserum and/or milk obtained by immunizing a mammal with cell walls obtained from culture of S1 trans, and a glucosyltransferase inhibitor, protease, and dextranase. A preventive method for preventing diarrhea using a combination of one or more synergists selected from the group has been described.
しかしながら、従来のＳ 、 ｍｕｔａｎｓに免疫活性
を有する抗体の門蝕予防効果はｌ・ずしも十分ではなく
、又、従来の抗体は、免疫した哺乳動物の乳やその抗血
清から調製されるので、量産性に欠け、生産コストも高
いなどの欠点を有し、実用的ではない場合が多い。However, the preventive effect of conventional antibodies having immunoactivity against S. mutans is not sufficient, and conventional antibodies are prepared from the milk of immunized mammals or their antisera. , they lack mass productivity, have high production costs, and are often impractical.
本発明者らは、このような従来技術における問題を解決
すべく種々の検討を行った結果、鶏を用いた抗体の調製
方法に、後述の理化学的特性を有する３ 、 ｍｕｔａ
ｎｓの水溶性グルカン合成酵素を抗原として組み合わせ
て用いることによって、Ｓ、 ｔａｕｔａｎｓの歯面へ
の付着に対する十分な阻害効果を有し、上述したような
欠点のない抗体を調製し得るとの新たな知見を得るに到
り、本発明を完成した。The present inventors conducted various studies in order to solve the problems in the conventional technology, and as a result, the present inventors developed a method for preparing antibodies using chickens, which has the physicochemical properties described below.
A novel finding is that by using S. tautans water-soluble glucan synthase in combination as an antigen, it is possible to prepare an antibody that has a sufficient inhibitory effect on the adhesion of S. tautans to the tooth surface and does not have the above-mentioned drawbacks. Having obtained this knowledge, we have completed the present invention.
特に、鶏を用いた抗体の調製においては、必ずしも免疫
した抗原に特異的に反応する抗体が十分量得られるとは
限らない０例えば、ウィルス抗原を免疫した場合、十分
なる抗体価が得られなかった。In particular, when preparing antibodies using chickens, it is not always possible to obtain a sufficient amount of antibodies that specifically react with the immunized antigen. For example, when immunizing with a virus antigen, a sufficient antibody titer may not be obtained. Ta.
従って、鶏に免疫する際には、抗原を十分に吟味し、抗
体価の高いものが得られるように検討しなければならな
い。Therefore, when immunizing chickens, it is necessary to carefully examine the antigen and make sure to obtain one with a high antibody titer.
従って、Ｓ 、 ｍｕｔａｎｓの産生する水溶性グルカ
ンって始めて得られたものである。Therefore, this is the first water-soluble glucan produced by S. mutans.
本発明の目的は、Ｓ 、 ｍｕｔａｎｓに対する免疫活
性を示し、Ｓ 、 ５ｕｔａｎｓの歯面への付着に対す
る十分な阻害効果を存し、量産性にも優れ、生産コスト
が低く、安全性にも優れ、關蝕予防剤の存効成分として
有用な抗体及び該抗体を含む關蝕予防剤を提供すること
にある。The object of the present invention is to exhibit immunoactivity against S. mutans, have a sufficient inhibitory effect on the adhesion of S. The object of the present invention is to provide an antibody that is useful as an active ingredient in a dental caries preventive agent, and a dental caries preventive agent containing the antibody.
本発明のＳ 、 ＋ｍｕｔａｎｓの産生ずる水溶性グル
カン合成酵素（以後ＧＴＦ−３と略称する）に対する阻
害活性を存する抗体は、下記理化学的特性を有する血清
型がｃ、ｅ又はｆであるＳ、 ｍｕｔａｎｓの産生する
ＧＴＦ−５を免疫した鶏が産生ずる卵より調製された免
疫グロブリンとして得ることができる。The antibody of the present invention having inhibitory activity against water-soluble glucan synthase (hereinafter abbreviated as GTF-3) produced by S, +mutans is an antibody of the present invention that has inhibitory activity against water-soluble glucan synthase (hereinafter abbreviated as GTF-3) produced by S, +mutans whose serotype is c, e, or f and has the following physicochemical characteristics. It can be obtained as immunoglobulin prepared from eggs produced by chickens immunized with GTF-5 produced by GTF-5.
０作用及び基質特異性 スクロースに作用し、水溶性のグルカンを合成する。0 action and substrate specificity Acts on sucrose to synthesize water-soluble glucan.
■分子量
５ＤＳ−ポリアクリルアミドゲル電気泳動により測定し
た分子量が、１５０に−１６５にダルトンである。(2) Molecular weight 5DS - The molecular weight measured by polyacrylamide gel electrophoresis is 150 to 165 Daltons.
■免疫原性
動物において免疫原となり、該酵素にたいする特異抗体
を生成させ得る。(2) Immunogenicity Acts as an immunogen in animals and can generate specific antibodies against the enzyme.
なお、上記５ＤＳ−ポリアクリルアミドゲル電気泳動（
ＳＯ５−ＰＡＧＥ）は以下の操作に従って行なったもの
である。In addition, the above 5DS-polyacrylamide gel electrophoresis (
SO5-PAGE) was performed according to the following procedure.
５ＤＳ−ＰＡＧＥ操作条件；
５ＤＳ−ＰＡＧＥはレムリ−（Ｌａｅａ＋＋ｇｌｉ）ら
の方法により行う。5DS-PAGE operating conditions; 5DS-PAGE is performed by the method of Laea++li et al.
すなわち、後述の粗精製または精製標品を個々に２％Ｓ
Ｏ３，５％２−メルカプトエタノール及び２０％グリセ
ロールを含む６２．５ｍＭのトリス塩酸緩衝液（ｐＨ６
，８）中で１００℃、３分間処理する。That is, each of the crude or purified samples described below was added to 2% S.
O3, 62.5 mM Tris-HCl buffer (pH 6) containing 5% 2-mercaptoethanol and 20% glycerol.
, 8) for 3 minutes at 100°C.
電気泳動は、０．１％のＳＤＳを含む７．５％の分離ゲ
ルと、４％の濃縮ゲル中、室温下、１０ｍＡ、２時間の
条件で行う、なお、分子量マーカーとしては、フェリチ
ン（ｆｅｒｒｉｔｉｎ、 ２２０ ｋダルトン）、ホス
フォリラーゼ１）（ｐｈｏｓｐｈｏｒｙｌａｓｅｂ。Electrophoresis was performed in a 7.5% separation gel containing 0.1% SDS and a 4% concentration gel at room temperature for 2 hours at 10 mA. Ferritin was used as a molecular weight marker. , 220 k Daltons), phosphorylase 1) (phosphorylaseb.
９４にダルトン）、牛血清アルブミン（ｂｏｖｉｎｅｓ
ｅｒｕｍ ａｌｂｔ＋＋ｗｉｎ ｓ ６７ ｋダルトン
）、カタラーゼ（ｃａｔａｌａｓｅ、 ６０ ｋダルト
ン）、オブアルブミン（ｏｖａｌｂｕｍｉｎ ｓ ４３
ｋダルトン）及び乳酸脱水素酵素（Ｉａｃｔａｔｅ
ｄｅｈｙｄｒｏｇｅｎａｓｅ 、 ３６ ｋダルトン）
（いずれもファルマシア社製）を用い、バンドの検出は
クマシーブリリアントブル−（ＣＢＢ）による染色によ
って行なうことができる。94 Dalton), bovine serum albumin (bovines)
erum albt++wins 67 k daltons), catalase (catalase, 60 k daltons), ovalbumin (ovalbumin s 43
k Dalton) and lactate dehydrogenase (Iactate
dehydrogenase, 36 k Daltons)
(both manufactured by Pharmacia), and the band can be detected by staining with Coomassie brilliant blue (CBB).
本発明の抗体（以後抗ＧＴＦ−３と称する）を得るため
に用いるＧＴＦ−３としては、上記特性を有する酵素で
あればどる′ようなものでも利用でき、例えば以下のよ
うにしてＳ 、 ＋ｅｕｔａｎｓの培養上清から精製し
て得ることができる。As GTF-3 used to obtain the antibody of the present invention (hereinafter referred to as anti-GTF-3), any enzyme having the above characteristics can be used. For example, S, +eutans can be prepared as follows. can be obtained by purification from the culture supernatant of
即ち、血清型がｃ、ｅ又はｆ型である
Ｓ 、 ｍｕｔａｎｓを適当な培地で培養し、その培養
上清から精製される。That is, S. mutans having serotypes c, e, or f are cultured in an appropriate medium and purified from the culture supernatant.
ここで用いるＣ型苗としては、Ｓ 、 ｍｕｔａｎｓＩ
ｎｇｂｒｉｔｔ株、 Ｍ７８１４８株、 １０４４９株
等の公知で、容易に人手可能な菌を用いることができ、
例えば、Ｓ 、 ｍｕｔａｎＳＭＴ８１４８株＋ Ｉｎ
ｇｂｒｉｔｔ株は大阪大学歯学部から、１０４４９株は
ナショナル コレクションオプ タイプ カルチャーズ
（ＮａｔｉｏｎａｌＣｏｌｌｅｃｔｉｏｎ ｏｆ Ｔｙ
ｐｅ Ｃｕ１ｔｕｒｅｓ （ＮＣＴＣ））から入手でき
る。又、ｅ型閉やｆ型閉についても公知の菌株を同様に
入手して用いればよい。The type C seedlings used here include S, mutansI
Known and easily accessible bacteria such as ngbritt strain, M78148 strain, and 10449 strain can be used.
For example, S, mutan SMT8148 strain + In
gbritt strain was obtained from Osaka University School of Dentistry, and strain 10449 was obtained from the National Collection of Typical Cultures.
pe Cultures (NCTC)). Also, for e-type closure and f-type closure, known strains may be similarly obtained and used.
又、培地としては、少なくともグルコースを含む培地が
利用でき、例えば、ＴＴＹ培地（Ｔｒｙｐｔｉｃａｓｅ
、 ＴｒｙｐｔｏｓｅＳＹｅａｓｔ ｅｘｔｒａｃｔ
の複合培地） 、Ｂ ＨＩ （Ｂｒａｉｎ １ｌｅａ
ｒｔ Ｉｎｆｕｓｉｏｎ）培地、ＦＭＣ培地などを用い
ることが出来る。Further, as a medium, a medium containing at least glucose can be used, for example, TTY medium (Trypticase
, TryptoseSYeast extract
complex medium), BHI (Brain 1lea
rt Infusion medium, FMC medium, etc. can be used.
又、培養温度は、菌体増殖が得られ、且つＧＴＦ−３の
生産に適した範囲内であれば良いが、良好な菌体増殖と
ＧＴＦ−３の生産という点からは、通常３７℃程度とす
るとよい。In addition, the culture temperature may be within a range suitable for bacterial cell growth and GTF-3 production, but in terms of good bacterial cell growth and GTF-3 production, it is usually around 37°C. It is good to say.
又、培養時間は、培養温度、培地の種類等の培養条件に
よって異なるが、ＧＴＦ−３の最適収量に達する時期を
選択して決定すれば良く、１８〜２４時間程度とすれば
よい。Further, the culture time varies depending on culture conditions such as culture temperature and type of medium, but may be determined by selecting the time when the optimum yield of GTF-3 is reached, and may be about 18 to 24 hours.
又、その他の培養条件についても、上記の観点から適宜
選択すれば良い。Further, other culture conditions may be appropriately selected from the above viewpoint.
次に、菌体からＧＴＦ−３を抽出する。Next, GTF-3 is extracted from the bacterial cells.
Ｓ 、 ｌ１ｌｕｔａｎｓのＧＴＦ−Ｓは、５ａｔｏ、
Ｓ、 らの方法（Ｓａｔｏ、 ｓ、＋ ｅｔ ａｌＨ
Ｃａｒｂｏｈｙｄｒ、 Ｒｅ５−＋ １３４＋ ２９３
−３０４ （１９８４） ）に従い、ＢＨＩ透析培地に
３ 、ｍｕｔａｎｓを植菌し、成育後遠心分離により菌
体を除去し、上清に硫酸アンモニウムを加え５０％硫酸
アンモニウム画分の沈澱をＨｉｓｔｉｄｉｎｅ−ＨＣＩ
に透析しクロマトフオーカシングカラムクロマトグラ
フィーを行った後、ヒドロキシアパタイトのカラムクロ
マトグラフィーを行い精製標品を得ることが出来る。GTF-S of S, l1lutans is 5ato,
S, et al.'s method (Sato, S, + et al.
Carbohydr, Re5-+ 134+ 293
-304 (1984)), inoculate BHI dialysis medium with M. mutans, remove the bacterial cells by centrifugation after growth, add ammonium sulfate to the supernatant, and precipitate the 50% ammonium sulfate fraction with Histidine-HCI.
After dialysis and chromatofocusing column chromatography, a purified sample can be obtained by performing hydroxyapatite column chromatography.
抗体を得るための抗原として、以上の操作で得られた菌
体培養上清もしくは硫安濃縮標品、クロマトフオーカシ
ング後の粗精製標品、もしくは、精製標品としてのＧＴ
Ｆ−３等を用いることができる。As an antigen for obtaining antibodies, 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 can be used.
F-3 etc. can be used.
次に、本発明の抗ＧＴＦ−３抗体の調製方法について詳
述する。Next, the method for preparing the anti-GTF-3 antibody of the present invention will be described in detail.
本発明の抗ＧＴＦ−３抗体は、上述のＧＴＦ−８で免疫
された鶏の卵から調製することができる。The anti-GTF-3 antibody of the present invention can be prepared from eggs of chickens immunized with the above-mentioned GTF-8.
免疫される鶏としては、特に制限はないが、抗体の量産
性という点からは、白色レグホーン等の卵用種を用いる
と良い。There are no particular restrictions on the chicken to be immunized, but from the standpoint of mass production of antibodies, egg breeds such as white leghorn are preferably used.
又、ＧＴＦ−３による免疫方法としては、皮下注射、筋
肉注射、腹腔内投与等による通常の方法や、点鼻５点眼
等の方法によって行うことができる。更に、ＧＴＦ−３
の投与量は、所望の抗体価が得られ、且つ鶏に対して悪
影響を与えない量を適宜選択すれば良い。In addition, immunization with GTF-3 can be carried out by conventional methods such as subcutaneous injection, intramuscular injection, intraperitoneal administration, etc., and methods such as nasal and eye drops. Furthermore, GTF-3
The dosage may be appropriately selected so as to obtain the desired antibody titer and not have any adverse effects on the chickens.
通常、初回免疫から数週間で投与抗原に対して特異的に
反応する抗体が鶏卵（卵黄）中に得られる。Usually, antibodies that specifically react with the administered antigen are obtained in chicken eggs (egg yolks) several weeks after the initial immunization.
尚、必要に応じて例えばＦＣＡ（フロイント完全アジュ
バント）、ＦＩＡ（フロイント不完全アジュバント）等
のアジュバントをＧＦＴ−３と共に併用しても良い。Note that, if necessary, an adjuvant such as FCA (complete Freund's adjuvant) or FIA (incomplete Freund's adjuvant) may be used together with GFT-3.
免疫から１力月以上経過した鶏から採取した卵から本発
明の抗ＧＴＦ−３抗体を調製することができる。The anti-GTF-3 antibody of the present invention can be prepared from eggs collected from chickens that have been immunized for at least one month.
尚、卵黄中の抗体価は、酵素免疫吸着法（ＥＬＩＳＡ）
、 ラジオイムノアッセイ等を用いて測定すること
ができ、免疫後に２週程度の間隔で抗体価を測定するこ
とにより抗体価の推移を追跡することができる。In addition, the antibody titer in egg yolk was determined by enzyme-linked immunosorbent assay (ELISA).
, radioimmunoassay, etc., and the change in antibody titer can be tracked by measuring the antibody titer at intervals of about two weeks after immunization.
後述の実施例においては、ＥＬ［ＳＡでの測定により抗
体価の推移を追跡し、抗体価が十分に上昇した段階の卵
を採取して、本発明の抗ＧＴＦ−３抗体を調製した。In the Examples described below, the anti-GTF-3 antibody of the present invention was prepared by tracking the change in antibody titer by measuring with EL[SA, and collecting eggs at a stage when the antibody titer had sufficiently increased.
又、通常、約３カ月間にわたって高抗体価を得ることが
できる。Moreover, high antibody titers can usually be obtained for about 3 months.
尚、免疫後、抗体価の減少が見られた場合、適当な間隔
で適宜追加免疫することにより抗体価を高くすることが
できる。In addition, if a decrease in the antibody titer is observed after immunization, the antibody titer can be increased by appropriately administering booster immunizations at appropriate intervals.
本発明の高ＧＴＦ−３抗体は、例えば上記のようにして
免疫した鶏の卵黄に含まれる免疫グロブリンを抽出・分
離することによって得ることができる。この抽出・分離
方法としては、例えば、デキストラン硫酸やポリエチレ
ングリコールを用いた沈澱法や、プノパノールやクロロ
ホルムを用いた抽出法等通常用いられている免疫グロブ
リンを抽出・分離できる種々の方法等が利用できる。The high GTF-3 antibody of the present invention can be obtained, for example, by extracting and separating immunoglobulin contained in the egg yolk of a chicken immunized as described above. As this extraction/separation method, various commonly used methods for extracting/separating immunoglobulins can be used, such as a precipitation method using dextran sulfate or polyethylene glycol, or an extraction method using punopanol or chloroform. .
以上のようにして得られた本発明の抗ＧＴＦ−８抗体は
、３ 、＠ｕｔａｎｓの産生するＧＴＦ−３に対して特
異的に抗体として反応する。即ち、ＧＴＦ−８に対して
酵素活性の阻害活性を存する。The anti-GTF-8 antibody of the present invention obtained as described above specifically reacts as an antibody with GTF-3 produced by 3.@utans. That is, it has enzyme activity inhibitory activity against GTF-8.
Ｓ 、 ｍｕｔａｎｓの産生ずるＧＴＦ−３に対して阻
害活性を有する本発明の抗ＧＴＦ−３抗体は、Ｓ 、
ｓ＋ａｔａｎｓの歯面への付着を阻害することによって
、Ｓ 、 ＋ｍｕｔａｎｓの口腔内での活動を制御し、
鵬蝕を予防することができる。The anti-GTF-3 antibody of the present invention having an inhibitory activity against GTF-3 produced by S. mutans includes S. mutans.
By inhibiting the attachment of S+mutans to the tooth surface, the activity of S+mutans in the oral cavity is controlled,
It can prevent corrosion.
以上記載のごとく本発明は、通常の哺乳動物から調製さ
れる製法に比べて、容易に、且つ大量に調製でき、しか
も卵黄から抗体を調製するという前車な操作により免疫
グロブリンのうちｒｇｃ画分だけを特異的に分離精製す
ることができ、醋蝕予防剤の有効成分として有用なる抗
体を提供するものである。As described above, the present invention can be prepared more easily and in large quantities than the usual production method for preparing antibodies from mammals, and moreover, the RGC fraction of immunoglobulin can be prepared by an advanced operation of preparing antibodies from egg yolk. The object of the present invention is to provide an antibody that can be specifically isolated and purified, and which is useful as an active ingredient of an anti-erosive agent.
これらの種々の抗体含有画分は、通常の醋蝕予防剤に配
合し、本発明に係る關蝕予防剤を調製することができる
。These various antibody-containing fractions can be mixed with a conventional dental caries preventive agent to prepare the dental caries preventive agent according to the present invention.
即ち、本発明の門蝕予防剤は練り歯磨き・粉歯磨き・液
状歯磨き等の歯磨き類、マウスウォッシュ、口腔用パス
タ、歯肉マツサージクリーム、うがい用錠剤、トローチ
、チューインガム、缶飲料等口腔内商材だけではなく、
その目的においては種々の食品にも適用されるものであ
る。That is, the dental caries preventive agent of the present invention can be used only in oral products such as toothpastes such as toothpaste, powdered toothpaste, and liquid toothpaste, mouthwash, oral pasta, gingival pine surge cream, gargling tablets, troches, chewing gum, and canned drinks. not,
For that purpose, it is also applicable to various foods.
本発明抗体の醋蝕予防材への配合量は、その投与形態に
応じた投与量に従って適宜選択すれば良く、例えば、１
０’以上の抗体価を有する抗体を０、０００１〜ｌＯ重
量％程度とすることができる。The amount of the antibody of the present invention to be added to the caries preventive material may be appropriately selected according to the dosage form thereof, and for example, 1
The amount of antibodies having an antibody titer of 0' or more can be about 0,0001 to 10% by weight.
尚、本発明の門蝕予防剤の他の成分としては、使用目的
、使用形態等に応じた適当な成分が用いられる０例えば
練り歯磨きの場合では、炭酸カルシウム、燐酸水素カル
シウム、ピロリン酸カルシウム、不溶性メタリン酸ソー
ダ、水化アルミナ。As other components of the preventive agent of the present invention, appropriate components are used depending on the purpose of use, form of use, etc. For example, in the case of toothpaste, calcium carbonate, calcium hydrogen phosphate, calcium pyrophosphate, insoluble Sodium metaphosphate, hydrated alumina.
無水ケイ酸等の研磨剤、グリセリン、ソルビット。Abrasives such as silicic anhydride, glycerin, and sorbitol.
プロピレングリコール等の保湿剤、ラウリル硫酸ナトリ
ウム、ラウロイルサルコシンナトリウム。Moisturizers such as propylene glycol, sodium lauryl sulfate, and sodium lauroyl sarcosine.
石１末等の発泡荊、カルボキシメチルセルロースナトリ
ウム、カラギーナン等のバインダー、さらに適当なる香
料成分、甘味剤、保存剤等の成分を水と混合し、常法に
従って製造する。又、マウスウォッシュ等の口腔洗浄剤
その他においても、製品の性状に応じた成分が適宜配合
される。It is produced according to a conventional method by mixing foamed rhododendrons such as stone powder, a binder such as sodium carboxymethyl cellulose, and carrageenan, and ingredients such as appropriate flavoring ingredients, sweeteners, and preservatives with water. Also, in mouthwashes and other mouthwashes, ingredients are appropriately blended depending on the properties of the product.
尚、本発明においては、歯牙着色除去剤１口臭予防剤、
フッ素等の虫歯予防剤、抗酵素予防剤等の種々の薬効成
分を配合することも可能である。In addition, in the present invention, tooth stain removal agent 1 bad breath prevention agent,
It is also possible to incorporate various medicinal ingredients such as anti-caries agents such as fluorine and anti-enzyme preventive agents.
よって、本発明の鵬蝕予防剤は、前記免疫卵より調製し
た卵黄抗体を用いることにより、ストレプトコッカス・
ミュータンスによるプラークの形成を効果的に抑制し、
ａ！４蝕の発生を良好に防止する。しかも、前記卵黄抗
体は安全性が高いため、本発明の麟蝕予防剤は使用上の
安全性が高いものである。Therefore, the preventive agent of the present invention uses the egg yolk antibody prepared from the immunized eggs to inhibit Streptococcus.
Effectively inhibits plaque formation caused by mutans,
a! 4. Good prevention of eclipse. Furthermore, since the egg yolk antibody is highly safe, the preventive agent of the present invention is highly safe in use.
以下実施例により本発明を更に詳細に説明する。 The present invention will be explained in more detail with reference to Examples below.
尚、以下における％表示は、特に、指定されていない場
合には、重量／容量％を示す。In addition, the % display below indicates weight/volume % unless otherwise specified.
実施例１
＋ａｌ抗原の調製
Ｓ 、 ５ｕｔａｎｓ Ｉｎｇｂｒｉｔｔ株（血清型Ｃ
１大阪大学歯学部から入手）を１３ｊｌのＢＨＩｉ３析
培地で３７ｔ、１８時間培養した。培養液を連続遠心に
より菌体と培養上清とに分離した。分離した培養上清に
対して５０％飽和の硫安沈澱処理を行い、得られた沈澱
物を遠心分離法により回収した。次に、この沈澱物をＨ
ｉｓｔｉｄｉｎｅ−ＨＣＩ緩衝液（ｐＨ６，２）に溶解
した溶液を同緩衝液に対して透析し、更に、該溶液中に
生じた沈澱を遠心分離法により除去した後、その上清液
をポリバッファーＰＢＥ９４（Ｐｏｌｙｂｕｆｆｅｒ
ＰＢＥ９４ 、ファルマシア社！りの２．５Ｘ２５ｃｍ
のカラムにかけた。Example 1 Preparation of +al antigen S, 5utans Ingbritt strain (serotype C
1 (obtained from Osaka University School of Dentistry) was cultured in 13jl of BHIi3 analysis medium for 37t for 18 hours. The culture solution was separated into bacterial cells and culture supernatant by continuous centrifugation. The separated culture supernatant was subjected to ammonium sulfate precipitation treatment at 50% saturation, and the resulting precipitate was collected by centrifugation. Next, this precipitate was
A solution dissolved in istidine-HCI buffer (pH 6,2) was dialyzed against the same buffer, and the precipitate formed in the solution was removed by centrifugation, and the supernatant was diluted with polybuffer PBE94. (Polybuffer
PBE94, Pharmacia! Rino 2.5X25cm
column.
カラムに吸着した両分は、バリバフファー７４（Ｐｏｌ
ｙｂｕｆｆｅｒ ７４、ファルマシア社製）を用いたｐ
ｌ＋勾配によって選択的に溶出させた。Both fractions adsorbed on the column were washed with Varibuffer 74 (Pol
ybuffer 74, manufactured by Pharmacia)
It was selectively eluted with a l+ gradient.
溶出された各百分のＧＴＦ活性を以下に記載した方法で
、又タンパク質含量を２８０ｒｓ＋紫外吸収法で測定し
たところ、９Ｎ５．４〜５．０の両分中に顕著なＧＴＦ
活性が認められた。When the GTF activity of each eluted fraction was measured by the method described below, and the protein content was measured by 280rs + ultraviolet absorption method, significant GTF was found in both fractions of 9N5.4 to 5.0.
Activity was observed.
ＧＴＦ活性の測定；
試料１０μ！を、基質としての２０ｓＭ （１４Ｃ−グ
ルコ−スフスクロース（（”Ｃ−ｇｌｕｃｏｓｅ ）ｓ
ｕｃｒｏｓｅ） （０，０５ｃｉ／ｍｏｌ）を含む０．
２Ｍリン酸緩ｊ＃ｉ液（ρ）１６．０）のｌ Ｄｕｌｌ
と混合し、３７℃で１時間反応後、反応液全量を濾紙（
１，ＯＸ ２． Ｏｃｍ）にスポットし、これをメタノ
ールで洗浄後、濾紙上に残ったメタノールに不溶性のグ
ルカン中に取り込まれた放射能量を測定し、ＧＴＦ活性
を算出し、１分間に１μｍＯ１のグルコースをグルカン
に転移させる活性を１単位（Ｕ）とした。Measurement of GTF activity; sample 10μ! and 20sM (14C-glucose)s as a substrate.
ucrose) (0.05 ci/mol).
2M phosphoric acid solution (ρ) 16.0) Dull
After reacting at 37°C for 1 hour, the entire reaction solution was filtered through filter paper (
1, OX 2. After washing this with methanol, the amount of radioactivity incorporated into the methanol-insoluble glucan remaining on the filter paper was measured, the GTF activity was calculated, and 1 μm O1 of glucose was transferred to the glucan per minute. 1 unit (U) of activity.
次に、Ｉ））１５．４〜５．０の溶出画分を集め、これ
に対して、８０％飽和の硫安沈澱処理を行い、沈澱物を
得た。これを１Ｏｎ＋Ｍリン酸緩衝液（ｐＨ６，０）に
溶解し、同溶液に対して透析する。更に、該溶液中に生
じた沈澱を遠心分離法により除去して上清液を得た。こ
の上清液を粗精製標品（免疫抗原）とした。Next, the elution fractions of I)) 15.4 to 5.0 were collected and subjected to ammonium sulfate precipitation treatment at 80% saturation to obtain a precipitate. This is dissolved in 1On+M phosphate buffer (pH 6,0) and dialyzed against the same solution. Furthermore, the precipitate formed in the solution was removed by centrifugation to obtain a supernatant. This supernatant liquid was used as a crudely purified specimen (immunization antigen).
該標品のタンパク質濃度及びタンパク質全量をＣＢＢ−
Ｇ法（Ｂｒａｎｆｏｒｄ、Ｍ、Ｍ、＋ Ａｎａｌ、 Ｂ
ｉｏｃｈｅｍ、。The protein concentration and total protein amount of the sample were determined by CBB-
G method (Branford, M, M, + Anal, B
iochem,.
７２、２４８． （１９７６） ）により測定したとこ
ろ、タンパク１ｔ ｔＨ度が０．４ｍｇ／ｌｅｌ、タン
パク質全量が９．４朔ｇであった。72, 248. (1976) ), the protein 1t tH degree was 0.4 mg/lel, and the total protein amount was 9.4 μg.
更に、該標品を前述の操作条件により５ＤＳ−ＰＡＧＥ
にかけ、クマシ・ブリリアント・ブルー（ＣＢＢ）での
染色を行ったところ、１５５にダルトンの位置のバンド
をメインとする低分子量の数本のバンドが検出された。Furthermore, the specimen was subjected to 5DS-PAGE under the above-mentioned operating conditions.
When the sample was subjected to staining with Kumasi Brilliant Blue (CBB), several bands of low molecular weight, mainly a band at the 155 Dalton position, were detected.
また、上記の５ＤＳ−ＰＡＧＥの結果から算出された該
標品中のＧＴＦ−３含有率は２０重量％であった。Further, the GTF-3 content in the sample calculated from the above 5DS-PAGE results was 20% by weight.
これとは別に、下記の要領で活性染色を行なった。即ち
、該標品のトリス塩酸緩衝液中での処理を３７℃、３０
分で行い、かつ電気泳動を４℃で行う以外は前述と同様
の操作による５ＤＳ−ＰＡＧＥにかけた後、ゲルを３７
℃の１％スクロース及び０．０５％アジ化ナトリウムを
含むリン酸緩衝液（ｐＨ６，０）中に１８時間浸漬した
０次に、浸漬処理したゲルをＰ Ａ Ｓ （Ｐｅｒｉ
ｏｄｊｃａｃｉｄ ５ｃｈｉｆｆ）反応により染色した
ところ１５５にダルトンの位置にグルカン生成を示す顕
著な染色バンドが検出された。Separately, activity staining was performed as described below. That is, the sample was treated in Tris-HCl buffer at 37°C for 30
After 5DS-PAGE in the same manner as described above, except that the electrophoresis was performed at 4°C, the gel was run at 37°C.
The soaked gel was soaked for 18 hours in a phosphate buffer (pH 6,0) containing 1% sucrose and 0.05% sodium azide at
When the sample was stained by odjcacid 5chiff) reaction, a prominent stained band indicating glucan production was detected at the 155 Dalton position.
山）抗原の鶏への免疫
＋ａ＋で得た粗精製免疫抗原標品１．ＯｍＡ（ＣＢＢ−
Ｇ法で測定した場合の０．４ｍｇタンパク質量を含む）
とＦＣＡ （フロイント完全アジュバント）］、Ｏｍｊ
！を１：１混合してＷ２Ｏ型のエマルジョンとした。Mountain) Immunization of antigen to chickens + crudely purified immune antigen preparation obtained by a+ 1. OmA(CBB-
Contains 0.4mg protein amount when measured by G method)
and FCA (Complete Freund's Adjuvant)], Omj
! were mixed 1:1 to form a W2O type emulsion.
得られたエマルシランを鶏の胸筋にｌ、Ｑ＋ａｆｆｉ’
つ注射し、初回免疫を行った後、下記の方法に従って、
採取した卵から得たＷＳＦ（後述）の抗体価を測定し、
その推移を観察した。Apply the obtained emulsilan to the breast muscle of the chicken.
After the first immunization, follow the method below.
Measure the antibody titer of WSF (described later) obtained from the collected eggs,
We observed its progress.
次に、第１図に示すように初回免疫後８週後に卵黄中の
抗体価が下がり始めたのを′１１１１認して、前回と同
様にして２次免疫を行った。Next, as shown in FIG. 1, it was noticed that the antibody titer in the egg yolk started to decrease 8 weeks after the first immunization, and a second immunization was performed in the same manner as the previous time.
２次免疫終了後、約１ケ月経過した“後から鶏が産生ず
る卵を集卵した。Eggs produced by the chickens were collected approximately one month after the completion of the secondary immunization.
（ｃ）抗体の調製
卵から分離した卵黄１３ｍｊ！とこれと同量のＰＢＳ（
リン酸緩衝液、ｐＨ７，４）を混合し、得られた混合液
に更に混合液と同！（２６ｍ６）のクロロホルムを加え
て、これをよく撹拌した。(c) Preparation of antibodies Egg yolk isolated from eggs 13mj! and the same amount of PBS (
Phosphate buffer, pH 7,4) and add the same mixture to the resulting mixture! (26 m6) of chloroform was added and this was stirred well.
撹拌終了後、混合液を室温下で３０分間放置した後、こ
れを３．００ Ｏｒｐｍ 、２０分間の遠心分離にかけ
、最上層の透明画分を回収し、抗体含有画分（Ｗａｔｅ
ｒ ５ｏｌｕｂｌｅ Ｆｒａｃｔｏｎ：Ｗ Ｓ Ｆ ）
とした。After stirring, the mixture was allowed to stand at room temperature for 30 minutes, and then centrifuged at 3.00 Orpm for 20 minutes to collect the top layer, the transparent fraction, and the antibody-containing fraction (Wate).
r 5olable Fracton: W S F )
And so.
この両分は、２．ｌＸ１０’の抗体価を有していた。These two parts are 2. It had an antibody titer of 1X10'.
尚、該２．１Ｘ１０’の抗体価を示すＷＳＦ（１３閘ｊ
’ ； Ｉ ３ｖｇ１．の卵黄から調製）のたんばく質
含有ン；度をビニ−レフト法により測定し、該ＷＳＦ中
の全たんばく質量を求めたところ約２６ｍｇという値を
得た（約２．０ｍｇ／１ＩＩ６ Ｘ ｌ ３＋ａｊ！
） 。In addition, WSF (13 blocks) showing the antibody titer of 2.1 x 10'
'; I 3vg1. The protein content of the WSF (prepared from the egg yolk of 3+aj!
).
ｔｄ）抗体価の測定方法； 抗体価の測定は、ＥＬ［ＳＡによって行った。td) Method for measuring antibody titer; The antibody titer was measured by EL[SA.
まず、実施例２で得た精製免疫抗原標品を１０μｇ／ｒ
ａｇとなるように５０ｍＭ炭酸ナトリウム緩衝液（ｐＨ
９，６）に溶解させて得られた溶液を、９６穴プレート
〔イムロン（Ｉｍｍｕｌｏｎ ） ２．グイナテック社
製〕の各ウェルに１００μＥずつ入れ、４℃で一晩放置
し、該両分に含まれる精製抗原を含むＰＢＳ （ｐＨ７
，４）と３７℃で１時間接触させて、ブロッキングを行
った後、ＰＢＳ−Ｔでプレートを５回洗浄した。First, the purified immune antigen preparation obtained in Example 2 was added at 10 μg/r.
50mM sodium carbonate buffer (pH
The solution obtained by dissolving in 9,6) was placed in a 96-well plate [Immulon 2. 100 μE was added to each well of a tube (manufactured by Guinatec), left overnight at 4°C, and PBS (pH 7) containing the purified antigen contained in both aliquots was added.
, 4) at 37° C. for 1 hour to perform blocking, and then the plate was washed 5 times with PBS-T.
ここで、先に得たＷＳＦのＰＢＳ−Ｔによる２段階稀釈
液の１００μｌを各ウェルに加え、３７℃で１時間反応
させた。Here, 100 μl of the previously obtained two-step dilution of WSF in PBS-T was added to each well and reacted at 37° C. for 1 hour.
反応終了後、プレートをＰＢＳ−Ｔで５回洗浄し、更に
、プレートに２次抗体としてのペルオキシダーゼ結合抗
ニワトリＩｇＧ抗体（たんばく質量１．６７μｇ／ｍｊ
りの１００１ＩＪを各ウェルに加え、２５℃で３０分間
反応させた後、ＰＢＳ−Ｔで５回洗浄した。After the reaction, the plate was washed 5 times with PBS-T, and a peroxidase-conjugated anti-chicken IgG antibody (protein mass 1.67 μg/mj) was added to the plate as a secondary antibody.
1001IJ was added to each well, reacted at 25°C for 30 minutes, and then washed 5 times with PBS-T.
次に、プレートの各ウェルに０．２ｈリン酸２ナトリウ
ム−０，１Ｍクエン酸緩衝液（ｐＨ１５，０） ５０
ｍｊ！に基質であるＯ−フ二二レンジアミン２０ｍｇお
よび過酸化水素１０μｌを溶解した溶液を１００μｌ加
え、２５℃で２０分間反応させた。Next, add 50 ml of disodium phosphate-0.1 M citrate buffer (pH 15.0) to each well of the plate for 0.2 h.
mj! 100 μl of a solution containing 20 mg of the substrate O-phenyl diamine and 10 μl of hydrogen peroxide was added to the mixture, and the mixture was reacted at 25° C. for 20 minutes.
反応停止は、３Ｎ硫酸溶液１００μ！加えることで行っ
た。To stop the reaction, use 100μ of 3N sulfuric acid solution! I did it by adding.
反応停止後、各ウェルの吸光度（ＯＤｌ、ｔ）を測定す
ることによって抗体価を測定した。抗体価はエンドポイ
ントタイター法により求め、吸光度が０、２となる稀釈
倍率とした。After stopping the reaction, the antibody titer was measured by measuring the absorbance (ODl, t) of each well. The antibody titer was determined by the end point titer method, and the dilution ratio was set to give an absorbance of 0 or 2.
（ｅｌ抗体によるＧＴＦ−３の水溶性グルカン合成阻害
活性のｆ！認
ｔｃ＋で得た抗体を用いてＳ 、 ｓ＋ｕｔａｎｓの産
生ずるＧＴＦ−３の活性阻害実験を行った。(f! Recognition of water-soluble glucan synthesis inhibitory activity of GTF-3 by el antibody) Using the antibody obtained with tc+, an experiment was conducted to inhibit the activity of GTF-3 produced by S, s+utans.
まず、（Ｃ１で得た２、１Ｘ１０’の抗体価を有するＷ
ＳＦ及びその８倍稀釈液を試験溶液とした。First, (W with an antibody titer of 2,1X10' obtained in C1)
SF and its 8-fold dilution were used as test solutions.
これとは別に、免疫をしていない鶏（（ｂ）と同様の鶏
）の卵から１０１と同様にしてＷＳＦを調製し試験溶液
とした。尚、該ＷｓＦがＧＴＦ−３に対して特異的に反
応しないことを確認した。Separately, WSF was prepared from eggs of non-immunized chickens (the same chickens as in (b)) in the same manner as in 101 and used as a test solution. In addition, it was confirmed that the WsF did not specifically react with GTF-3.
また、対照としてはリン＃Ｉｌｌ街液（ＰＢＳ）を用い
た。In addition, phosphorus #Ill street solution (PBS) was used as a control.
次に、これらの各試験溶液をディスポーザブルリプレー
ト（三光純薬社製）のウェルに５μ１分注した後、更ｋ
ｍｏ、３＊ＵノＧＴ Ｆ−３５ＩＩ ｌをツレぞれのウ
ェルに加えた。Next, after dispensing 5 μl of each of these test solutions into the wells of a disposable replate (manufactured by Sanko Pure Chemical Industries),
Mo, 3*U of GT F-35II was added to each well.
混合した後、３０分間、３７℃で反応させた。After mixing, the mixture was allowed to react at 37° C. for 30 minutes.
反応終了後、更に、各ウェルに前述と同様に２０１１Ｉ
Ｍの〔１４Ｃ−グルコ−、ス〕スクロースを１０μを加
え再び３７℃で１時間反応後、反応液全量を濾紙にスポ
ットし、これをメタノールで洗浄後、濾紙上に残ったメ
タノールに不溶性のグルカン中に取り込まれた放射能量
を測定し、ＧＴＦ活性を算出した。After the reaction is complete, add 2011I to each well in the same manner as above.
Add 10μ of [14C-gluco,su]sucrose of M and react again at 37°C for 1 hour. The entire reaction solution was spotted on a filter paper and washed with methanol. The amount of radioactivity incorporated into the cells was measured, and GTF activity was calculated.
表１に、ＰＢＳを加えた時のＧＴＦ活性を１００％とし
て各ＷＳＦの効果を示した。Table 1 shows the effects of each WSF, taking the GTF activity when PBS was added as 100%.
実施例２ ＧＴＦ−３精製標品の調製１５１のＢＨＩ
透析培地を使用する以外は、実２施例１と同様の条件で
、粗精製標品に対応する上清液を得た。Example 2 Preparation of GTF-3 Purification Standard BHI of 151
Example 2 A supernatant solution corresponding to a crudely purified sample was obtained under the same conditions as in Example 1 except that a dialysis medium was used.
その上清液を１（ｌａＭリン酸緩衝液（ｐＨ６，０）で
、平衡化したヒドロキシアパタイト（Ｂｉｏ Ｇｅｌ
ＩＩＴＰ 。The supernatant was added to hydroxyapatite (Bio Gel) equilibrated with 1 (laM phosphate buffer (pH 6,0)).
IITP.
バイオラッド（Ｂｉｏ−Ｒａｄ）社製〕のカラム（１，
０Ｘ１３ｃｍ）にかけた。Column (1, manufactured by Bio-Rad)
0x13cm).
カラムに吸着した百分は、０．０１〜０．５６リン酸緩
衝液（ｐＨ１６，０）の濃度勾配によって選択的に溶出
させた。The percentage adsorbed on the column was selectively eluted by a concentration gradient of 0.01-0.56 phosphate buffer (pH 16,0).
各両分の、ＧＴＦ活性及びタンパク質含量を上述と同様
の方法で測定した結果、０．２Ｍのリン酸緩衝液（ａｌ
ｌ６．０）による両分中にＧＴＦ活性が認められた。The GTF activity and protein content of each portion were measured using the same method as described above.
GTF activity was observed in both cases.
次に、０．２Ｍリン酸緩衝液（ｐＨ６，０）による高活
性を示す溶出画分を集め、それを１０＋ｗＭのリン酸緩
衝液（ａｌｌ ６．０ ＞に対して透析し、精製酵素標
品を得た。Next, the eluted fractions showing high activity with 0.2M phosphate buffer (pH 6.0) were collected and dialyzed against 10+wM phosphate buffer (all 6.0) to prepare purified enzyme preparations. I got it.
更に、該標品を５ＤＳ−ＰＡＧＥにかけ、クマシー・ブ
リリアント・ブルー（ＣＢＢ）での染色を行ったところ
、１５５にダルトンの位置に単一のバンドが検出され、
該精製酵素標品が分子量１５５にダルトンの酵素タンパ
ク質であることが確認された。Furthermore, when the sample was subjected to 5DS-PAGE and stained with Coomassie brilliant blue (CBB), a single band was detected at the Dalton position of 155.
The purified enzyme preparation was confirmed to be an enzyme protein with a molecular weight of 155 Daltons.
更に、５０＋ｓＵ酵素活性量に相当する量の精製酵素標
品を最終濃度で・１％スクロース及び０．１％のアジ化
ナトリウムを含む０．１Ｈのリン酸緩衝液（ａｌｌ ６
．０ ）に加え、蒸留水で３ｍｌに容量を調製した後、
混合し、３７℃、１８時間酵素反応を行わせた。なお、
酵素反応は、反応溶液を水冷して４℃にすることにより
停止させた。Furthermore, an amount of the purified enzyme preparation corresponding to 50+sU enzyme activity was added to the final concentration in 0.1H phosphate buffer containing 1% sucrose and 0.1% sodium azide (all 6
．． 0) and adjusted the volume to 3 ml with distilled water,
The mixture was mixed and the enzyme reaction was carried out at 37°C for 18 hours. In addition,
The enzyme reaction was stopped by cooling the reaction solution with water to 4°C.
酵素反応終了後、反応生成物を１６００゜ｇの遠心分離
で処理し、沈澱した水不溶性画分と上′清液に分けた。After the enzymatic reaction was completed, the reaction product was centrifuged at 1600°g and separated into a precipitated water-insoluble fraction and a supernatant.
水不溶性画分は、蒸留水で２度洗浄し、３ｍａの蒸留水
に懸濁して、水不溶性グルカン標品とした。The water-insoluble fraction was washed twice with distilled water and suspended in 3 ma distilled water to obtain a water-insoluble glucan sample.
また、上ｔｎ液にその２．５倍容量のエタノールを加え
、４℃で１時間放置後、生じた沈澱を１６００、 ｇの
遠心分離で回収し、それを３ａＩｉｌの蒸留水に溶解さ
せ、更に同様の沈澱処理を再度行って回収された沈澱を
再び３ｔａｌの蒸留水に溶解し水溶性グルカン標品とし
た。In addition, 2.5 times the volume of ethanol was added to the upper TN solution, and after leaving it at 4°C for 1 hour, the resulting precipitate was collected by centrifugation at 1,600g, dissolved in 3aIil of distilled water, and further The same precipitation treatment was performed again, and the recovered precipitate was dissolved again in 3 tal distilled water to obtain a water-soluble glucan sample.
更に１、これらの標品に含まれるグルカンの量をアンス
ロン法により定量分析した結果、水不溶性グルカン標品
中には０．０５Ｂ／ｍｌ、水溶性グルカン標品中には２
．６ｍｇ／ｌｌｌの値を得た。従ワて該精製酵素標品の
作用によって合成されるグルカンの種類を調べたところ
、該精製酵素標品は水溶性グルカンを合成する作用を有
するＧＴＦ−３であることが明らかとなった。Furthermore, as a result of quantitative analysis of the amount of glucan contained in these preparations using the Anthrone method, the amount of glucan contained in the water-insoluble glucan preparation was 0.05B/ml, and the amount of glucan contained in the water-soluble glucan preparation was 2.
．． A value of 6 mg/lll was obtained. When the type of glucan synthesized by the action of the purified enzyme preparation was investigated, it was revealed that the purified enzyme preparation was GTF-3, which has the ability to synthesize water-soluble glucan.
なお、得られた該精製酵素標品のタンパク質量を上述の
方法で測定したところ、５．６Ｂであつた。In addition, when the protein amount of the obtained purified enzyme preparation was measured by the above-mentioned method, it was 5.6B.
更に、上記ＧＴＦ−３精製免疫抗原標品を抗原として用
いる以外は実施例１と同様にして抗体の調製を行ったと
ころ、同様にＧＴＦ−３と特異的に反応する抗体が得ら
れた。Furthermore, an antibody was prepared in the same manner as in Example 1 except that the purified GTF-3 immune antigen preparation was used as the antigen, and an antibody that specifically reacted with GTF-3 was obtained in the same manner.
更に、得られた抗体のＧＴＦ−３活性阻害効果を同様の
方法で試験した結果、同様に優れた効果が認められた。Furthermore, as a result of testing the GTF-3 activity inhibiting effect of the obtained antibody using the same method, similarly excellent effects were observed.
次に本発明の醋蝕予防剤の実施例を記載する。Next, Examples of the dental caries preventive agent of the present invention will be described.
（実施例３） 練り歯磨き
ピロリン酸カルシウム ４２％グリセリン
１５％ソルビット７０％
１０％カルボキシメチルセルロース １
２％サンカリンナトリウム ０．１％ラウ
リル硫酸ナトリウム ２．０％香 料
１．０ ％
水 残
量１００ ％
以上の成分に実施例１で得た抗体含有画分（ＷＳＦ）０
．５％（抗体価２．１ｘｌＯ’のもの）を配合する。(Example 3) Toothpaste calcium pyrophosphate 42% glycerin 15% sorbitol 70%
10% carboxymethyl cellulose 1
2% Sankarin Sodium 0.1% Sodium Lauryl Sulfate 2.0% Fragrance
1.0%
The antibody-containing fraction (WSF) obtained in Example 1 was added to the component with a residual water content of 100% or more.
．． 5% (antibody titer 2.1xlO').
（実施例４） マウシュウォソシュ
エタノール ２２．５％サンカリン
ナトリウム ０．０５％ラウリルジェタノール
７ミド ０．３％香 料
１．０ ％水
残 量１００
％
以上の成分に実施例１で得た抗体含を画分（ＷＳＦ）０
．５％（抗体価２．ｌＸ１０’のもの）を配合する。(Example 4) Moushwasosh ethanol 22.5% Sankarin sodium 0.05% Laurylgetanol 7mide 0.3% Fragrance
1.0% water
100 remaining
The fraction (WSF) containing the antibody obtained in Example 1 was 0% or more.
．． 5% (antibody titer 2.1 x 10').
（発明の効果）
本発明により、Ｓ 、 ｍｕｔａｎｓの歯面への付着に
対する十分な阻害効果を有し、量産性にも優れ、生産コ
ストが低く、安全性にも優れ、う蝕子防剤の有効成分と
して有用な抗体及び該抗体を含むう蝕予防剤が提供され
た。(Effects of the Invention) The present invention has a sufficient inhibitory effect on the adhesion of S. mutans to the tooth surface, is excellent in mass production, has low production costs, is excellent in safety, and is a caries preventive agent. An antibody useful as an active ingredient and a caries preventive agent containing the antibody have been provided.
特に、本発明のＳ 、 ｍｕｔａｎｓに対する免疫活性
を有する抗体は、従来の哺乳動物を免疫して得る抗体と
比較して以下のような利点を有する。In particular, the antibody having immunological activity against S. mutans of the present invention has the following advantages compared to conventional antibodies obtained by immunizing mammals.
（１）本発明の抗体は、免疫した鶏の卵生に得られ、採
卵、卵の取り扱いおよび卯からの抗体の取得に特別な、
あるいは熟練した技術を必要としない。(1) The antibody of the present invention is obtained from the eggs of immunized chickens, and special methods are used for egg collection, egg handling, and antibody acquisition from rabbits.
Or it doesn't require any skill.
しかも、卵黄には免疫グロブリンのうちＩｇＧクラスし
か移行しないので、ＩｇＧのみを容易に得ることができ
る。Furthermore, since only the IgG class of immunoglobulins is transferred to egg yolk, only IgG can be easily obtained.
これに対して、免疫した哺乳動物がら採血により抗体を
得る場合には、採血に熟練した技術が必要とされ、しか
も血清がら大量のＩｇＧを分離・精製することは非常に
困難である。On the other hand, when antibodies are obtained by blood collection from immunized mammals, skilled blood collection techniques are required, and it is extremely difficult to separate and purify large amounts of IgG from serum.
（２）本発明の抗体の調製に用いられる鶏は、管理が容
易であり、例えばラット等と比較してもその管理費用が
安い。(2) The chickens used for preparing the antibodies of the present invention are easy to manage, and their management costs are low compared to, for example, rats.
しかも、哺乳動物から継続的に多量の血液や乳を得るこ
とは困難であり、哺乳動物を用いる方法は抗体の量産に
は適さないが、鶏は長期間にゎたって安定して卵を生み
続けるので、本発明の抗体は量産可能であり、かつ生産
コストが低い。Moreover, it is difficult to continuously obtain large amounts of blood and milk from mammals, and methods using mammals are not suitable for mass production of antibodies, but chickens can continue to produce eggs stably over a long period of time. Therefore, the antibody of the present invention can be mass-produced and the production cost is low.
（３）免疫した哺乳動物の血液や乳から調製した抗体の
安定性は必ずしも良好でなく、血清中で、あるいは安定
性は必ずしも良好でなく、血清中で、あるいは精製した
状態でも一８０℃程度の温度条件下での保存が必要とさ
れる。(3) The stability of antibodies prepared from the blood or milk of immunized mammals is not necessarily good, and even in serum or in a purified state, the stability is not necessarily good, and even in serum or in a purified state, it is about -80°C It is necessary to store the product under the following temperature conditions.
本発明の抗体は、良好な安定性を有し、また保存性も良
く、例えば卵の状態で４℃で１〜２箇月間保存できる。The antibody of the present invention has good stability and good storage stability, and can be stored, for example, in egg form at 4°C for 1 to 2 months.
最後に、鶏を用いた抗体を調製する場合、必ずしも免疫
した抗原に特異的に反応する抗体が十分量得られるとは
限らない。例えば、ウィルス抗原を免疫しても、十分な
抗体価が得られながった。Finally, when preparing antibodies using chickens, it is not always possible to obtain a sufficient amount of antibodies that specifically react with the immunized antigen. For example, even when immunized with a viral antigen, a sufficient antibody titer could not be obtained.
従って、Ｓ 、 ｍｕｔａｎｓの菌体表層蛋白抗原で鶏
を免疫しても、実用的レベルの抗体価を有する抗体が得
られるかどうかは全く予想されないものであり、本発明
者らによって始めて達成されたものである。Therefore, even if chickens are immunized with S. mutans bacterial surface protein antigen, it is completely unpredictable whether antibodies with a practical level of antibody titer will be obtained, and this was achieved for the first time by the present inventors. It is something.
第１図は、実施例１において、免疫した鶏の抗体価の推
移を示すグラフである。FIG. 1 is a graph showing the change in antibody titer of immunized chickens in Example 1.
Claims (2)
ｆであるストレプトコッカス・ミュータンスの産生する
水溶性グルカン合成酵素を免疫した鶏が産生する卵より
調製された免疫グロブリンであって、前記酵素に対して
阻害活性を有する抗体。(1) The serotype of the pathogenic bacteria that causes caries is c, e, or
An immunoglobulin prepared from eggs produced by chickens immunized with water-soluble glucan synthase produced by Streptococcus mutans, which is f., and an antibody having inhibitory activity against the enzyme.
ｆであるストレプトコッカス・ミュータンスの産生する
水溶性グルカン合成酵素を免疫した鶏が産生する卵より
調製された免疫グロブリンであって、前記酵素に対して
阻害活性を有する抗体を有効成分として含む齲蝕予防剤
。(2) The serotype of the caries-inducing pathogen is c, e, or
An immunoglobulin prepared from eggs produced by chickens immunized with water-soluble glucan synthase produced by Streptococcus mutans, which is f. agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63058890A JPH01233229A (en) | 1988-03-12 | 1988-03-12 | Antibody and tooth decay preventive containing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63058890A JPH01233229A (en) | 1988-03-12 | 1988-03-12 | Antibody and tooth decay preventive containing the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01233229A true JPH01233229A (en) | 1989-09-19 |
Family
ID=13097377
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63058890A Pending JPH01233229A (en) | 1988-03-12 | 1988-03-12 | Antibody and tooth decay preventive containing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01233229A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0253458A (en) * | 1988-08-18 | 1990-02-22 | Lion Corp | Food |
| JP2007290988A (en) * | 2006-04-24 | 2007-11-08 | Kao Corp | Bacterial plaque formation inhibitor |
-
1988
- 1988-03-12 JP JP63058890A patent/JPH01233229A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0253458A (en) * | 1988-08-18 | 1990-02-22 | Lion Corp | Food |
| JP2689511B2 (en) * | 1988-08-18 | 1997-12-10 | ライオン株式会社 | Food |
| JP2007290988A (en) * | 2006-04-24 | 2007-11-08 | Kao Corp | Bacterial plaque formation inhibitor |
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