JP6482237B2 - Antibodies against tobacco extract - Google Patents
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- JP6482237B2 JP6482237B2 JP2014223550A JP2014223550A JP6482237B2 JP 6482237 B2 JP6482237 B2 JP 6482237B2 JP 2014223550 A JP2014223550 A JP 2014223550A JP 2014223550 A JP2014223550 A JP 2014223550A JP 6482237 B2 JP6482237 B2 JP 6482237B2
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- 241000208125 Nicotiana Species 0.000 title claims description 43
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims description 43
- 239000000284 extract Substances 0.000 title claims description 15
- 241000272534 Struthio camelus Species 0.000 claims description 18
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 claims description 16
- 210000002969 egg yolk Anatomy 0.000 claims description 16
- 229960002715 nicotine Drugs 0.000 claims description 16
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 claims description 16
- 102000002322 Egg Proteins Human genes 0.000 claims description 13
- 108010000912 Egg Proteins Proteins 0.000 claims description 13
- 235000013345 egg yolk Nutrition 0.000 claims description 13
- 239000000427 antigen Substances 0.000 claims description 12
- 108091007433 antigens Proteins 0.000 claims description 12
- 102000036639 antigens Human genes 0.000 claims description 12
- 230000003053 immunization Effects 0.000 claims description 12
- FMMWHPNWAFZXNH-UHFFFAOYSA-N Benz[a]pyrene Chemical compound C1=C2C3=CC=CC=C3C=C(C=C3)C2=C2C3=CC=CC2=C1 FMMWHPNWAFZXNH-UHFFFAOYSA-N 0.000 claims description 8
- 235000013601 eggs Nutrition 0.000 claims description 7
- RTDCJKARQCRONF-UHFFFAOYSA-N N-Nitrosomethylethylamine Chemical compound CCN(C)N=O RTDCJKARQCRONF-UHFFFAOYSA-N 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 239000000551 dentifrice Substances 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims 1
- 230000009747 swallowing Effects 0.000 claims 1
- 239000000126 substance Substances 0.000 description 25
- 230000008499 blood brain barrier function Effects 0.000 description 11
- 241000271567 Struthioniformes Species 0.000 description 10
- 241000287828 Gallus gallus Species 0.000 description 9
- 241000286209 Phasianidae Species 0.000 description 9
- 235000013330 chicken meat Nutrition 0.000 description 9
- 238000002649 immunization Methods 0.000 description 9
- 210000001218 blood-brain barrier Anatomy 0.000 description 7
- 210000004556 brain Anatomy 0.000 description 7
- 241000271566 Aves Species 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 230000000391 smoking effect Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 3
- 230000000711 cancerogenic effect Effects 0.000 description 3
- 231100000315 carcinogenic Toxicity 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 210000002569 neuron Anatomy 0.000 description 3
- 239000000779 smoke Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000000443 aerosol Substances 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 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 1
- 229930195730 Aflatoxin Natural products 0.000 description 1
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 description 1
- 206010012335 Dependence Diseases 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 208000025569 Tobacco Use disease Diseases 0.000 description 1
- 239000005409 aflatoxin Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000019504 cigarettes Nutrition 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- -1 methyl ethyl nitrosamines Chemical class 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 239000000606 toothpaste Substances 0.000 description 1
- 229940034610 toothpaste Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
Description
タバコ抽出液に含まれるニコチン、ベンゾピレン、メチルエチルニトロソアミン、タールを含むタバコ抽出液に含まれる低分子化合物に結合する抗体とその製造方法に関する。 The present invention relates to an antibody that binds to a low-molecular compound contained in a tobacco extract containing nicotine, benzopyrene, methylethylnitrosamine, and tar contained in a tobacco extract and a method for producing the same.
タバコおよびタバコの煙には多くの化学物質が含まれており、発ガン性物質とされるものもあるとされている。これらの化学物質は、分子量が小さい。そのため、喫煙によって血中に移行すると、脳の血液脳関門（Ｂｌｏｏｄ−Ｂｒａｉｎ Ｂａｒｒｉｅｒ）を通過し、神経細胞にさまざまな生理活性作用を与える。これがさまざまな中毒に発展する。 Tobacco and tobacco smoke contain many chemicals, some of which are considered carcinogenic. These chemical substances have a low molecular weight. Therefore, when it moves into the blood by smoking, it passes through the blood-brain barrier of the brain (Brain-Brain Barrier) and gives various physiologically active actions to nerve cells. This develops into various addictions.
このようなタバコ中の化学物質を検知し、無力化しようとする提案はすでにされている。特許文献１には、アフラトキシン、ベンゾピレンと特異的に結合するモノクロナール抗体で、これらのトキシンを無力化することが開示されている。 Proposals have already been made to detect and neutralize such chemicals in tobacco. Patent Document 1 discloses disabling these toxins with a monoclonal antibody that specifically binds to aflatoxin and benzopyrene.
タバコに含まれる化学物質は、分子量が小さいために血液脳関門を通過する。しかし、これらの化学物質により大きな分子量の物が結合すれば、血液脳関門を通過できず、化学物質の影響を抑制することが可能になる。 Chemical substances contained in tobacco pass through the blood-brain barrier because of their low molecular weight. However, if a substance having a large molecular weight is bound to these chemical substances, it cannot pass through the blood brain barrier, and the influence of the chemical substances can be suppressed.
ここで、タバコの化学物質に結合するものとして抗体は非常に効果的であると考えられる。しかし、特許文献１に開示されているようなモノクロナール抗体では、タバコに含まれる多くの化学物質を対象とすることは難しい。また、モノクロナール抗体を大量に作製するには大規模な設備が必要となる。 Here, antibodies are believed to be very effective as binding to tobacco chemicals. However, with a monoclonal antibody as disclosed in Patent Document 1, it is difficult to target many chemical substances contained in tobacco. In addition, large-scale equipment is required to produce a large amount of monoclonal antibody.
本発明は上記の課題に鑑みて想到されたもので、タバコに含まれる複数の化学物質に結合するタバコ抗体を提供する。 The present invention has been conceived in view of the above problems, to provide a tobacco antibody that binds to a plurality of chemical substances contained in tobacco.
より具体的に本発明に係るタバコ抗体は、タバコ抽出液を抗原として雌ダチョウに免疫し、前記雌ダチョウが産卵した卵の卵黄から得た卵黄抗体であって、ニコチン、ベンゾピレン、メチルエチルニトロソアミンおよびタールに結合することを特徴とする。 Tobacco antibody is according to more specifically the present invention, the tobacco extract was immunized female ostriches as an antigen, a yolk antibody obtained from the egg yolk of eggs the female ostriches were spawning, nicotine, benzopyrene, methyl ethyl nitrosamines And bonded to tar .
本発明に係るタバコ抗体の製造方法は、タバコ抽出液を抗原としてダチョウを含む雌性鳥類に免疫し、産卵された卵の卵黄から抗体（ＩｇＹ）を得る。したがって、均質で大量の抗体を得ることができる。また、得られた抗体はポリクロナール抗体であって、タバコ抽出液中の複数の化学物質に結合することができる。 In the method for producing a tobacco antibody according to the present invention, a female bird containing ostriches is immunized using a tobacco extract as an antigen, and an antibody (IgY) is obtained from the egg yolk of the laid eggs. Therefore, a homogeneous and large amount of antibody can be obtained. The obtained antibody is a polyclonal antibody, and can bind to a plurality of chemical substances in the tobacco extract.
したがって、タバコ中の化学物質を検知し、取り去ることができる。また後述するように本発明に係るタバコ抗体が結合した化学物質は、血液脳関門の通過を抑制されるので、タバコの脳への影響を抑制することが可能となる。 Therefore, chemical substances in tobacco can be detected and removed. As will be described later, since the chemical substance to which the tobacco antibody according to the present invention is bound is inhibited from passing through the blood-brain barrier, the influence of tobacco on the brain can be suppressed.
以下本発明に係る抗体について説明する。なお、以下の説明は本発明の一実施形態を示すものであり、本発明の趣旨を逸脱しない範囲で、以下の実施形態および実施例は改変されてもよい。 Hereinafter, the antibody according to the present invention will be described. The following description shows one embodiment of the present invention, and the following embodiment and examples may be modified without departing from the spirit of the present invention.
本発明はタバコの抽出液を抗原として用いる。この抽出液中には、多くの化学物質が含まれている。したがって、この抗原から得られる抗体は、複数の化学物質に対して結合することができると考えられる。ここでは、ニコチン、ベンゾピレン、メチルエチルニトロソアミン、タールについて効果が確認された。 The present invention uses tobacco extract as an antigen. Many chemical substances are contained in this extract. Therefore, it is considered that an antibody obtained from this antigen can bind to a plurality of chemical substances. Here, effects were confirmed for nicotine, benzopyrene, methylethylnitrosamine, and tar.
ニコチンは、タバコの依存性を高めるものとして知られている。これはタバコの煙中にも残存し、副流による受動喫煙によっても体内に取り込まれる。また、タールは、ＰＭ２．５と呼ばれるエアロゾル中にも含まれる。ＰＭ２．５は、空気中に浮遊する粒子（エアロゾル）の中で大きさが２．５μｍ以下のものを指す。 Nicotine is known to increase tobacco dependence. This remains in tobacco smoke and is taken into the body by passive smoking caused by sidestreams. Tar is also contained in an aerosol called PM2.5. PM2.5 refers to particles suspended in the air (aerosol) having a size of 2.5 μm or less.
ベンゾピレンは５つのベンゼン環が結合した多環芳香族炭化水素で、発癌性があると言われている。メチルエチルニトロソアミンも発癌性があると言われている。 Benzopyrene is a polycyclic aromatic hydrocarbon bonded with five benzene rings and is said to be carcinogenic. Methylethylnitrosamine is also said to be carcinogenic.
雌性鳥類に対して免疫する工程では、公知の方法を利用することができる。免疫の際は、抗原とともに各種アジュバンドを利用することができる。また、免疫も初回免疫の後、追加免疫してもよい。 A known method can be used in the step of immunizing female birds. In immunization, various adjuvants can be used together with the antigen. Immunization may be boosted after the initial immunization.
利用できる鳥類としては、ウズラ、ニワトリ、ダチョウ等が利用できる。特に、体の大きなダチョウが好適に利用できる。タバコの抽出液は、基本的に毒性を有するので、体型の小さな鳥類は、免疫した際に死亡する場合もあるからである。 Usable birds include quail, chicken and ostrich. In particular, an ostrich with a large body can be suitably used. This is because tobacco extracts are basically toxic, so small birds of small form may die when immunized.
免疫後の鳥類から得られた卵から抗体（ＩｇＹ）を回収する工程では、公知の方法で抗体を回収することができる。回収された抗体は、タバコの抽出液中の化学物質に結合する。この結合によってタバコ中の化学物質を検知し、捕獲することができる。また、タバコ抗体が結合した化学物質は血液脳関門の通過が抑制される。したがって、これらの化学物質の脳への生理活性を抑制することができる。 In the step of recovering antibody (IgY) from eggs obtained from birds after immunization, the antibody can be recovered by a known method. The recovered antibody binds to a chemical substance in the tobacco extract. This binding can detect and capture chemicals in tobacco. In addition, chemical substances bound with tobacco antibodies are inhibited from passing through the blood-brain barrier. Therefore, the physiological activity of these chemical substances on the brain can be suppressed.
得られたタバコ抗体は、タバコのフィルタ、飲み薬、注射薬、ガムや歯磨剤に混入させることができる。また、タバコの葉自体に混入させておいてもよい。タバコ抗体は、いわゆるフィルタ付き巻きたばこのフィルタに含ませておくことで、喫煙時に体内に流れる煙中の化学物質を捕獲することができる。 The obtained tobacco antibody can be mixed in tobacco filters, drinks, injections, gums and dentifrices. Moreover, you may mix with the tobacco leaf itself. By including the tobacco antibody in a so-called filter cigarette filter, it is possible to capture chemical substances in the smoke flowing into the body during smoking.
また、飲み薬や注射薬にすることで、体内に取り込まれたタバコの化学物質が脳内の神経細胞に作用することを抑制することができる。また、ガムや歯磨剤に混入させることで、喫煙後に口腔内に残留した化学物質に結合し、これらの物質が脳内の神経細胞に作用することを抑制する。 In addition, by using a medicine or an injection, it is possible to suppress the action of tobacco chemical substances taken into the body on nerve cells in the brain. Moreover, by mixing in gums and dentifrices, it binds to chemical substances remaining in the oral cavity after smoking, and suppresses these substances from acting on nerve cells in the brain.
さらに、タバコの葉自体に混入させると、フィルタ無し（いわゆる両切りタバコ）を喫煙する場合であっても、化学物質の体内への取り込みを抑制することができる。 Furthermore, when it is mixed in tobacco leaves themselves, it is possible to suppress the uptake of chemical substances into the body even when smoking without a filter (so-called double-cut tobacco).
＜抗原＞
市販のフィルタ無しタバコの１本から、タバコの葉のみを取り出し、１ｍｌのＰＢＳで十分にホモジネートし、４０，０００ＲＰＭで３０分遠心分離後、上澄みを回収した。得られた液をタバコ抽出液とした。
<Antigen>
Only one tobacco leaf was taken out from one commercially available non-filtered tobacco, sufficiently homogenized with 1 ml of PBS, centrifuged at 40,000 RPM for 30 minutes, and the supernatant was collected. The obtained liquid was used as a tobacco extract.
＜抗体の製造方法＞
成熟したメス鳥（ダチョウ、ニワトリ、ウズラ）を用いた。タバコ抽出液（原液）０．２ｍＬをそれぞれフロイントの完全アジュバント０．２ｍＬと混和し、ダチョウ、ニワトリ、ウズラに初回免疫した。各抗原を個別に５羽のダチョウ、５羽のニワトリ、５羽のウズラに接種した。したがって、ダチョウもニワトリもウズラも同量の抗原を接種したことになる。初回免疫後、２週目と４週目に０．２ｍＬの各抗原とフロイントの不完全アジュバントの混和液を、各鳥に追加免疫した。ダチョウは全羽生存したが、鶏は途中で４羽死亡、ウズラも４羽死亡した。
<Method for producing antibody>
Mature female birds (ostrich, chicken, quail) were used. Each 0.2 mL of tobacco extract (stock solution) was mixed with 0.2 mL of Freund's complete adjuvant, and ostrich, chicken and quail were immunized for the first time. Each antigen was individually inoculated into 5 ostriches, 5 chickens and 5 quails. Therefore, ostriches, chickens and quails have been inoculated with the same amount of antigen. After the first immunization, each bird was boosted with 0.2 mL of a mixture of each antigen and Freund's incomplete adjuvant at the second and fourth weeks. All ostriches survived, but four chickens died and four quails died on the way.
初回免疫後８週目に得られた各鳥からの卵の卵黄より卵黄抗体（ＩｇＹ）を精製した。得られた卵黄抗体の反応性をＥＬＩＳＡにより検証した。具体的には、まず、得られた卵の卵黄に５倍量のＴＢＳ（２０ｍＭのＴｒｉｓ−ＨＣｌ、０．１５ＭのＮａＣｌ、０．５％ＮａＮ３）と同量の１０％デキストラン硫酸／ＴＢＳを加え２０分攪拌した。 The egg yolk antibody (IgY) was purified from the egg yolk of each bird obtained 8 weeks after the first immunization. The reactivity of the obtained egg yolk antibody was verified by ELISA. Specifically, first, 5 times the amount of TBS (20 mM Tris-HCl, 0.15 M NaCl, 0.5% NaN 3 ) and the same amount of 10% dextran sulfate / TBS were added to the egg yolk of the obtained egg. The mixture was further stirred for 20 minutes.
次に１ＭのＣａＣｌ２／ＴＢＳを卵黄と同量加え攪拌し、１２時間静置した。その後、１５０００ｒｐｍで２０分遠心し上清を回収した。そして、最終濃度が４０％になるように硫酸アンモニウムを加え４℃で１２時間静置した。 Next, 1M CaCl 2 / TBS was added in the same amount as egg yolk, stirred and allowed to stand for 12 hours. Subsequently, the supernatant was collected by centrifugation at 15000 rpm for 20 minutes. And ammonium sulfate was added so that a final concentration might be 40%, and it left still at 4 degreeC for 12 hours.
１２時間の静置後、１５０００ｒｐｍで２０分遠心し、沈殿物を回収した。最後に、卵黄と同量のＴＢＳに再懸濁し、ＴＢＳにて透析した。以上の方法で、各卵から純度９０％の抗体（ＩｇＹ）が回収できた。 After standing for 12 hours, the mixture was centrifuged at 15000 rpm for 20 minutes to collect the precipitate. Finally, it was resuspended in the same amount of TBS as egg yolk and dialyzed with TBS. By the above method, 90% purity antibody (IgY) could be recovered from each egg.
＜ＥＬＩＳＡ法＞
各卵黄から得られたタバコ抗体のタバコの化学物質に対する反応性はＥＬＩＳＡにより検証した。具体的には、まず９６穴ＥＬＩＳＡプレートの各穴にニコチン、ベンゾピレン、メチルエチルニトロソアミン、タールをそれぞれ別々に５μｇを固層化した（室温で４時間）。
<ELISA method>
The reactivity of tobacco antibodies obtained from each egg yolk to tobacco chemicals was verified by ELISA. Specifically, 5 μg of nicotine, benzopyrene, methylethylnitrosamine and tar were separately solidified in each hole of a 96-well ELISA plate (at room temperature for 4 hours).
その後、ダチョウ抗体（各５羽のダチョウ）、ニワトリ抗体（生存した１羽のニワトリ）、ウズラ抗体（生存した１羽のウズラ）の段階希釈液（原液は２ｍｇ／ｍＬ）を各穴に滴下し、室温で１時間反応させた。洗浄後、各抗体に対するＨＲＰ標識２次抗体を室温で１時間反応させた。十分な洗浄後、ペルオキシダーゼ用発色キット（Ｓ−Ｂｉｏ ＳＵＭＩＬＯＮ）を用いてプレートリーダーにて吸光度（４５０ｎｍ）を測定した。免疫前の各鳥種の卵黄抗体の２倍以上の吸光度値を示す最大希釈倍率をＥＬＩＳＡ値として示した。結果を表１に示す。 Then, serial dilutions of ostrich antibody (5 ostriches each), chicken antibody (one surviving chicken), and quail antibody (one surviving quail) (stock solution is 2 mg / mL) are dropped into each hole. And allowed to react at room temperature for 1 hour. After washing, an HRP-labeled secondary antibody for each antibody was reacted at room temperature for 1 hour. After sufficient washing, absorbance (450 nm) was measured with a plate reader using a peroxidase coloring kit (S-Bio SUMILON). The maximum dilution factor showing the absorbance value of 2 times or more of the yolk antibody of each bird species before immunization was shown as the ELISA value. The results are shown in Table 1.
ダチョウ、ニワトリ、ウズラにおいて、ニコチン、ベンゾピレン、メチルエチルニトロソアミンに対する卵黄抗体が作製されることが判明した。特に、各鳥種類には同量の抗原を免疫したのにもかかわらず、巨大なダチョウが最も反応性が高い抗体が産生されていた。これは、ダチョウを使うことで、少量の抗原でも高感度の抗体が産生できることを示している。 It was found that egg yolk antibodies against nicotine, benzopyrene and methylethylnitrosamine were produced in ostriches, chickens and quails. In particular, despite the immunization of the same amount of antigen for each bird species, giant ostriches produced the most reactive antibodies. This shows that by using ostriches, highly sensitive antibodies can be produced even with a small amount of antigen.
次に、タバコ抽出液を免疫して作製したダチョウ抗体（抗タバコダチョウ抗体）を用いて、ニコチンの脳内拡散防止の効果について確認した。 Next, the effect of preventing diffusion of nicotine in the brain was confirmed using an ostrich antibody (anti-tobacco ostrich antibody) prepared by immunizing a tobacco extract.
血液脳関門のｉｎ ｖｉｔｒｏモデルとしては、ファーマコセル社製ＢＢＢキット（以下単に「ＢＢＢキット」と呼ぶ。）を用いた。 As an in vitro model of the blood-brain barrier, a pharmacocell BBB kit (hereinafter simply referred to as “BBB kit”) was used.
まず、ＢＢＢキットのｗｅｌｌ上層部にニコチン（最終濃度３００μｇ／ｍＬ）と免疫前ダチョウ抗体（最終濃度１ｍｇ／ｍＬ）を３７℃１時間反応させたものを添加した。２日後にｗｅｌｌの下部チャンバー（下層）に透過したニコチン量を液クロマトグラフィーにて測定した。免疫前ダチョウ抗体とは、タバコ抽出液を免疫する前のダチョウが産卵した卵の卵黄から上記に示した方法で得た抗体（ＩｇＹ）である。 First, nicotine (final concentration 300 μg / mL) and preimmune ostrich antibody (final concentration 1 mg / mL) reacted at 37 ° C. for 1 hour were added to the upper layer of the well of the BBB kit. Two days later, the amount of nicotine permeated into the lower chamber (lower layer) of the well was measured by liquid chromatography. The pre-immune ostrich antibody is an antibody (IgY) obtained from the yolk of eggs laid by an ostrich before immunization with a tobacco extract by the method described above.
一方で、ニコチン３００μｇ／ｍＬに抗タバコダチョウ抗体（最終濃度１ｍｇ／ｍＬ）を１時間３７℃反応させたものも別ｗｅｌｌに添加し、同様に下層に透過したニコチン量を測定した。結果を図１に示す。図１は、横軸に免疫前ダチョウ抗体と抗タバコダチョウ抗体を示す。また縦軸は、ＢＢＢキットによるニコチン透過量（μｇ／ｍＬ）を示す。図１を参照して、抗タバコダチョウ抗体はニコチンの脳血管関門の通過を抑制することが判明した。このことは、ニコチンの脳内拡散を防止することが可能であることを示す。 On the other hand, an anti-tobacco ostrich antibody (final concentration 1 mg / mL) reacted with nicotine 300 μg / mL for 1 hour at 37 ° C. was also added to another well, and the amount of nicotine permeated into the lower layer was measured in the same manner. The results are shown in FIG. FIG. 1 shows pre-immune ostrich antibody and anti-tobacco ostrich antibody on the horizontal axis. The vertical axis represents the amount of nicotine permeation (μg / mL) by the BBB kit. Referring to FIG. 1, it was found that the anti-tobacco ostrich antibody suppresses the passage of nicotine through the cerebrovascular barrier. This indicates that nicotine can be prevented from spreading in the brain.
本発明に係る抗タバコダチョウ抗体は、検査キットを始め、タバコのフィルタ、飲み薬、注射薬、ガムや歯磨きとともに利用することができる。 The anti-tobacco ostrich antibody according to the present invention can be used together with a test kit, a tobacco filter, a drink, an injection, a gum and a toothpaste.
Claims (8)
ニコチン、ベンゾピレン、メチルエチルニトロソアミンおよびタールに結合する卵黄抗体。 Immunizing female ostrich with tobacco extract as antigen, egg yolk antibody obtained from egg yolk of egg laid by said female ostrich,
Egg yolk antibody that binds to nicotine, benzopyrene, methylethylnitrosamine and tar .
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