JPS62175426A - Antibody and spraying agent containing said substance as active component - Google Patents
Antibody and spraying agent containing said substance as active componentInfo
- Publication number
- JPS62175426A JPS62175426A JP1536086A JP1536086A JPS62175426A JP S62175426 A JPS62175426 A JP S62175426A JP 1536086 A JP1536086 A JP 1536086A JP 1536086 A JP1536086 A JP 1536086A JP S62175426 A JPS62175426 A JP S62175426A
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- virus
- pathogen
- respiratory
- respiratory disease
- 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
- 238000005507 spraying Methods 0.000 title abstract 4
- 239000000126 substance Substances 0.000 title 1
- 244000052769 pathogen Species 0.000 claims abstract description 20
- 208000023504 respiratory system disease Diseases 0.000 claims abstract description 16
- 230000001717 pathogenic effect Effects 0.000 claims abstract description 10
- 108060003951 Immunoglobulin Proteins 0.000 claims abstract description 9
- 102000018358 immunoglobulin Human genes 0.000 claims abstract description 9
- 241000712461 unidentified influenza virus Species 0.000 claims abstract description 9
- 210000003928 nasal cavity Anatomy 0.000 claims abstract description 6
- 241000700605 Viruses Species 0.000 claims description 26
- 235000013601 eggs Nutrition 0.000 claims description 9
- 239000007921 spray Substances 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 4
- 241000204031 Mycoplasma Species 0.000 claims description 4
- 241000606701 Rickettsia Species 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 2
- 239000000443 aerosol Substances 0.000 abstract description 4
- 210000002345 respiratory system Anatomy 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 2
- 230000000069 prophylactic effect Effects 0.000 abstract 2
- 239000010419 fine particle Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 7
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 210000002969 egg yolk Anatomy 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 241000287828 Gallus gallus Species 0.000 description 3
- 235000013330 chicken meat Nutrition 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 206010022000 influenza Diseases 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000003472 neutralizing effect Effects 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 241000219198 Brassica Species 0.000 description 2
- 235000003351 Brassica cretica Nutrition 0.000 description 2
- 235000003343 Brassica rupestris Nutrition 0.000 description 2
- 241001445332 Coxiella <snail> Species 0.000 description 2
- 206010057190 Respiratory tract infections Diseases 0.000 description 2
- QKSKPIVNLNLAAV-UHFFFAOYSA-N bis(2-chloroethyl) sulfide Chemical compound ClCCSCCCl QKSKPIVNLNLAAV-UHFFFAOYSA-N 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000001085 differential centrifugation Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000000428 dust Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 230000035931 haemagglutination Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 235000010460 mustard Nutrition 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 208000020029 respiratory tract infectious disease Diseases 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000238876 Acari Species 0.000 description 1
- 101100234244 Arabidopsis thaliana KDTA gene Proteins 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 241001466953 Echovirus Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 241001500351 Influenzavirus A Species 0.000 description 1
- 241000712079 Measles morbillivirus Species 0.000 description 1
- 241000202934 Mycoplasma pneumoniae Species 0.000 description 1
- 208000002606 Paramyxoviridae Infections Diseases 0.000 description 1
- 208000018569 Respiratory Tract disease Diseases 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 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 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 208000026935 allergic disease Diseases 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
- 210000001099 axilla Anatomy 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- ZINJLDJMHCUBIP-UHFFFAOYSA-N ethametsulfuron-methyl Chemical compound CCOC1=NC(NC)=NC(NC(=O)NS(=O)(=O)C=2C(=CC=CC=2)C(=O)OC)=N1 ZINJLDJMHCUBIP-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 229960003971 influenza vaccine Drugs 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000004264 monolayer culture Methods 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 235000020990 white meat Nutrition 0.000 description 1
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、抗体及びスプレー剤に関し、さらに詳しくは
呼吸器系疾患の病原体に対する抗体及びそれを有効成分
とするスプレー剤に関する。DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to antibodies and sprays, and more particularly to antibodies against pathogens of respiratory diseases and sprays containing the same as an active ingredient.
（従来の技術）
気道感染症等の呼吸器系疾患の病原微生物とシテ、ウィ
ルス、バクテリア、マイコプラズマおよびリケッチア等
が知られており、また、花粉およびダニ等のダスト類も
アレルギー疾患の起因物質としてよく知られている。た
とえば、インフルエンザウィルスは、Ａ％ＢおよびＣ型
に分類されるが、特にＡ型りィルスには、表面抗原の異
なる亜型が突然出現するという特異な現象があり、これ
が約／θ年周期で発生するインフルエンザ大流行の原因
となっており、又、同−亜型内でも毎年のように小変異
を起し、それによシ小渡行をくりかえしている。゛この
ようなインフルエンザ流行に対しては、現在ニワトリ受
精卵で増殖させたウィルスから調製した不活ｉワクチン
を接棹する方法が専ら行なわれている。(Prior art) Microorganisms that cause respiratory tract diseases such as respiratory tract infections, viruses, bacteria, mycoplasma, and rickettsia are known, and dust particles such as pollen and dust mites are also known to be causative agents of allergic diseases. well known. For example, influenza viruses are classified into A% B and C types, but the A type virus in particular has a unique phenomenon in which subtypes with different surface antigens suddenly appear, and this happens at a cycle of approximately /θ years. It is the cause of influenza pandemics, and even within the same subtype, small mutations occur every year, resulting in repeated changes. ``Currently, the only method used to combat such influenza epidemics is inoculation with an inactivated i-vaccine prepared from a virus grown in fertilized chicken eggs.
（発明が解決しようとする問題点）
上記の方法は人間の体内で抗体を産生させるものである
が、この予防注射の有効性は満足すべきものではない。(Problems to be Solved by the Invention) Although the above method produces antibodies within the human body, the effectiveness of this preventive injection is not satisfactory.
その原因として、たとえば、流行様の抗原の型がたえず
変異することのほか、一度予防注射をすると、からだの
免疫機構がそれを記憶していて、次の年に別の型のイン
フルエンザワクチンを注射して本、前と同じ抗体をつく
ってしまう（抗原原罪説）こと等がいわれている。さら
にインフルエンザウィルスの増殖が気道表面の粘膜上皮
細胞に限られているため不活化ワクチンで産出される血
中の抗体は、効率の点で難があることも指摘されている
。For example, in addition to the fact that the type of influenza virus that causes epidemics constantly mutates, once a vaccination is given, the body's immune system remembers it, and the next year, a different type of influenza vaccine is given. It is said that this causes the same antibodies as before to be produced (antigen original sin theory). Furthermore, it has been pointed out that since the proliferation of influenza viruses is limited to mucosal epithelial cells on the surface of the respiratory tract, antibodies in the blood produced by inactivated vaccines have a problem in terms of efficiency.
また、上記の種々の病原体勢が引き起こす種々の呼吸器
系疾患についても抗体を用いた有効な予防薬は実用化さ
れていない。Further, effective preventive drugs using antibodies have not been put into practical use for the various respiratory diseases caused by the various pathogenic systems mentioned above.
（問題点を解決するための手段）
本発明者らは、呼吸器系疾患の病原体の抗体であって、
いわゆるスプレー投与に適した抗体を見出すべく種々検
討を行ない、本発明に到達した。(Means for Solving the Problems) The present inventors have developed antibodies against pathogens of respiratory diseases,
We conducted various studies to find antibodies suitable for so-called spray administration, and arrived at the present invention.
すなわち、本発明の要旨は、呼吸器系疾患の病原体を免
疫したメンドリが産生ずる卵より調製された免疫グロブ
リンであって、その呼吸器系疾患の病原体に感染防御能
を有する抗体及びこれを有効成分とするスプレー剤に存
する。That is, the gist of the present invention is an immunoglobulin prepared from eggs produced by hens that have been immunized against pathogens of respiratory diseases, and an antibody that has the ability to protect against the pathogens of respiratory diseases, and an antibody that is effective against the pathogens. It is present in the spray agent as an ingredient.
以下、未発明の詳細な説明する。Hereinafter, a detailed description of the invention will be given.
まず、本発明に係る抗体の製造法の一例について説明す
る。First, an example of the method for producing antibodies according to the present invention will be explained.
すなわち、まず呼吸器系疾患の病原体を用いてメンドリ
を免疫する。この病原体は、感染により呼吸器系疾患を
惹起させるものであシ、ウィルス、バクテリア、マイコ
プラズマおよびリケッチア吟が挙げられる。That is, the hens are first immunized with a respiratory disease pathogen. This pathogen causes respiratory disease through infection, and includes viruses, bacteria, mycoplasma, and rickettsiae.
ウィルスとしては、インフルエンザウィルスＡ％Ｂおよ
びＣ型、パラインフルエンザウィルス／、２、および３
型、Ｒ日つィルス、アデノウィルス、ラインウィルス、
コロナウィルス、ＥＢウィルス、単純ヘルペスウィルス
、麻疹ウィルス、水痘ウィルス、エコーウィルスおよび
コクサラキーウィルス等が挙げられる。Viruses include influenza virus A% B and C, parainfluenza virus/, 2, and 3.
type, R. virus, adenovirus, line virus,
Examples include coronavirus, EB virus, herpes simplex virus, measles virus, varicella virus, echovirus, and coxalaki virus.
ｔ＋ｔ、バクテリアとしては、スタフィロコッカス ア
ウレウス（５ｔａｐｈｙｌｏｃｏｃｃｕａ ａｕｒｅｕ
ｓ、 ）等のグラム陽性球菌、クレプシーラｅｐｐ（Ｋ
ｌｅｂｓｉｅｌｌａ ｓｐｐ ）等の好気性ダラム陰性
桿菌等が挙げられる。t+t, as a bacterium, Staphylococcus aureus
), Gram-positive cocci such as Clepsilla epp (K
Examples include aerobic Durham-negative bacilli such as Lebsiella spp).
さらに、マイコプラズマ、リケッチアとしては、マイコ
プラズマ ニューモニエ（Ｍ７ｃｏｐｌａｓｍａｐｎθ
ｕｍｏｎｉａθ）、コキシーラ プルネテイイ（Ｃｏｘ
ｉｅｌｌａ ’ｂｕｒｎｅｔｉｉ ）等が挙げられる。Furthermore, Mycoplasma and Rickettsia include Mycoplasma pneumoniae (M7coplasmapnθ
umoniaθ), Coxiella prunetheii (Cox
iella 'burnetii).
メンドリの免疫に際しては、上記病原体を精製して用い
るのが通常である。精製は、常法によることができ、た
とえば遠心、吸着、有機溶媒による抽出、電気泳動、ク
ロマトグラフィー等が挙げられるが、分画遠心が最も一
般的である。When immunizing hens, the above-mentioned pathogens are usually purified and used. Purification can be carried out by conventional methods, such as centrifugation, adsorption, extraction with organic solvents, electrophoresis, chromatography, etc., but differential centrifugation is the most common.
免疫は数回に分けて行ない、７００〜１０００μｍ ／
／回程度を、腹股部又は腋下部等に行なうのが一般的
である。免疫後、／ケ月程度経過した後からメンドリが
産生ずる卵を取得する。Immunization was carried out in several doses, 700-1000μm/
It is common to apply this treatment to the abdomen, crotch, armpits, etc. about 1/2 times. Eggs produced by the hens are obtained approximately 1 month after immunization.
この卵より目的とする免疫グロブリンを調製する。たと
えば、黄身を集め、ポリエチレングリコールを用いて抗
体を精製、濃縮することにより特異的に分画することが
できる。この黄身よシ得られた免疫グロブリンは、工ｙ
Ｇである。The desired immunoglobulin is prepared from the eggs. For example, specific fractionation can be achieved by collecting yolks and purifying and concentrating antibodies using polyethylene glycol. The immunoglobulin obtained from this yolk is
It is G.
一方、白身より、たとえば硫酸アンモニウムを用いた濃
縮と セファデックスＧ−１０θ“及び Ｇ−，２００
を用いた分画により得られる免疫グロブリンはＩｆＡで
ある。On the other hand, from the white meat, for example, concentration using ammonium sulfate and Sephadex G-10θ" and G-,200
The immunoglobulin obtained by fractionation using is IfA.
このように調製された抗体は、上記の病原体に対する感
染防御能を有し、とくにスプレー投与に適する。スプレ
ー投与は、鼻腔、酬喉の呼吸気道に適用される。The antibodies thus prepared have the ability to protect against the above-mentioned pathogens and are particularly suitable for spray administration. Spray administration is applied to the respiratory airways of the nasal cavity and throat.
上記投与に適したスプレー剤としては、特に制限されな
いが、微小な浮遊状態の粒子（エアロゾル）として噴霧
する吸入エアロゾル型が好適である。エアロゾルの形成
自体は常法によることができる。粒子の大きさは３〜！
Ｑμｍ程度が一般的であるが、７０〜３０μｍ程度とす
るのが、呼吸気道への沈着効率の点から好ましい。The spray agent suitable for the above administration is not particularly limited, but an inhalation aerosol type that is sprayed as fine suspended particles (aerosol) is suitable. The aerosol itself can be formed by a conventional method. The particle size is 3~!
The thickness is generally about Qμm, but it is preferably about 70 to 30μm from the viewpoint of deposition efficiency in the respiratory airways.
抗体は、通常、蒸留水、生理的食塩水等を用いて溶解す
るのが一般的である。また、必要に応じ各種の安定剤、
たとえばゲラチン等を添加することもできる。Antibodies are generally dissolved using distilled water, physiological saline, or the like. In addition, various stabilizers, as necessary,
For example, gelatin etc. can also be added.
投与量は、気道に吸着した病原体を中オロする量であれ
ばよく、通常、外出前に本抗体の量が／〜７０■程度と
なるように投与すれば十分である。The dose may be any amount that neutralizes pathogens adsorbed to the respiratory tract, and it is usually sufficient to administer the antibody before going out so that the amount of the antibody is about 70 μm.
（実施例） 以下、実施例によりさらに本発明の詳細な説明する。(Example) Hereinafter, the present invention will be further explained in detail with reference to Examples.
実施例／ （インフルエンザウィルス）（精製）
インフルエンザウィルスＷＳＮ（Ｈ／Ｎ／） 及びＡ／
Ｕｄｏｒｎ／ ３０７／７．２（ＨＪ Ｎ、２）を１
０日令のニワ） ＩＪ受精卵各々２００個に接種し、ｚ
４ｔ℃３４ｔ培養後その感染漿尿液を採取した。分画遠
心７回（７，ＱθＯｒｐｍ２０分間、７．００ Ｏｒｐ
ｍ７６時間）及び蔗糖濃度勾配遠心２回（３０〜ｊＱ％
庶泗中３６，０θθｒｐｍ ／時間）により漿尿液中の
ウィルスを精製し、０，００／Ｍ ＫＤＴＡを含むｐＨ
７１％の０，０７Ｍ ト＋）ス緩衝液に浮遊して保存し
た。Example/ (Influenza virus) (purification) Influenza virus WSN (H/N/) and A/
Udorn/ 307/7.2 (HJ N, 2) to 1
0 day old chickens) 200 IJ fertilized eggs each were inoculated,
After culturing at 4t°C for 34t, the infected chorioallantoic fluid was collected. Differential centrifugation 7 times (7,QθOrpm 20 minutes, 7.00 Orp
m76 hours) and sucrose concentration gradient centrifugation twice (30~jQ%
The virus in the chorioallantoic fluid was purified by 36,0θθrpm/hour), and the pH containing 0,00/M KDTA was
It was stored suspended in 71% 0.07M Toss buffer.
（免疫）
／ Ｗ８Ｎ（Ｈ／Ｎ／） ２００，０θｏｍｈＵ／
ｍｌ ９”９／讐Ｊ Ａ／ Ｕ（ｌｏｒｎ／ Ｊ（７７
／ ７Ｊ （Ｈ３Ｎｕ ）ｉｏｏ、ｏｏｏＨｍ／ｘｌ
ｘｒｒｓ３／ｙ１３コントロール
精製ウィルス／、！−を７．５罰の完全フロインドアジ
ュバントとともに、Ｗｌｏとして／♂Ｇ針を用いて５ｔ
月齢の白色レグホンメンドリ３匹の腹腔及び腋下部ｒケ
所に、３回（０日、／？日及び２７日）にわたυそれぞ
れ上記抗原を皮下注射し念（／ ｄｌ ／ ／回）。(Immunity) / W8N (H/N/) 200,0θomhU/
ml 9”9/enemyJ A/U(lorn/J(77
/ 7J (H3Nu) ioo, oooHm/xl
xrrs3/y13 control purified virus/,! - with 7.5% complete Freund's adjuvant as Wlo/5t using a male G needle.
The above antigen was injected subcutaneously into the abdominal cavity and lower axilla of three month-old white leghorn hens three times (day 0, day 2, and day 27).
／回目の注射後／２Ｚ日経過までの間に得た卵を表／に
示した様にグループに分け、その黄身をグループごとに
プールした。各グループとと／θθ罰の黄身を取り／θ
０罰の０．０ ／ Ｍ ＩＪン酸緩衝液（θ、／ Ｍ
ＮａＣｔ、 ｐＨ７，６）と混合し、金属網で濾過した
後、７ｔのポリエチレングリコール（ｐｗａｔ、ｏｏｏ
）を加え、７５分間 ／攪拌した。
コついで、？、０θＯｒｐｍで
７５分間遠心し、上。Eggs obtained up to 2Z days after the /th injection were divided into groups as shown in Table/, and the yolks were pooled for each group. With each group / θθ Take the yolk of punishment / θ
0 punishment of 0.0/M IJ acid buffer (θ,/M
7t of polyethylene glycol (pwat, ooo
) and stirred for 75 minutes.
Next,? , centrifuged for 75 min at 0θOrpm, above.
！
清をガーゼで濾過し、／／ｆのポリエチレングリコール
（ＰＦｉＧ４０θθ ）を加え、さらに７５分間挾押し
、？、θ００ ｒｐｍで７５分間遠心した。! The supernatant was filtered through gauze, polyethylene glycol (PFiG40θθ) was added, and the mixture was further pressed for 75 minutes. , centrifuged at θ00 rpm for 75 minutes.
得られた沈渣を７０ｍｅのリン酸緩衝液（０，７Ｍ Ｈ
ａｃｔ、 ｐＨ７，ｊ ）に溶解し、それに／、％ ｔ
のポリエチレングリコール（ｐｖａｔｏθ０ ）を加え
／！分間掛押し、ついでＪ’、０００ ｒｐｍで７５分
間遠心した。上清を捨てさらにｔｒ、ｏ ｏ 。The obtained precipitate was diluted with 70me phosphate buffer (0.7M H
act, pH 7,j) and dissolved in it/,% t
Add polyethylene glycol (pvatoθ0)/! The mixture was pressed for a minute and then centrifuged at J', 000 rpm for 75 minutes. Discard the supernatant and continue with tr, o o.
ｒｐｍで７５分間遠心し、得られる沈直に１０．ｌのθ
、０ ／ ｊ Ｍリンｅ緩衝液（ｐＨＦ、０ ）を加え
た（目的とする抗体の取得）。Centrifuge for 75 minutes at rpm and add 10. θ of l
, 0/j M phosphate buffer (pHF, 0) was added (obtaining the desired antibody).
表 ／。table /.
０ ４ ／％ ／ｒ 、２Ｊ λ７７４７
６ タ？ ／／Ｊ ／２１ ／７％日
ＩＡ・ＩＢＩＯＩ ＩＦＩ
ＧＩＨＩ 工 １１（６）” （９） α１（
６） αＯＩ Ａ １．Ｂ Ｉ ＯＩ
Ｄ Ｉ Ｉｆｔ ＋ＩＡＩＢＩ ＯＩＩ［
ｉｌ Ｉ Ｐ’ ＋ ０
１ １α４ （６１（４３
＊（）中の値は卵の数を示す。0 4 /% /r, 2J λ7747
6 Ta? //J /21 /7%day IA/IBIOI IFI
GIHI Engineering 11(6)” (9) α1(
6) αOI A 1. BIOI
D I Ift +IAIBI OII [
il I P' + 0
1 1 α4 (61 (43 *) The value in parentheses indicates the number of eggs.
〈赤血球凝集抑制（Ｈ工）テスト〉
実施例／で得られた抗体についてＨエテストを行なった
。<Hemagglutination inhibition (H-technique) test> H-technique test was performed on the antibody obtained in Example.
す°な′わち、生理的食塩水を用いて表λに示す各抗体
０．２！＠ｌの一倍階段稀釈液列を作成した。That is, 0.2% of each antibody shown in Table λ using physiological saline! A series of one-fold dilutions of @l was prepared.
ついでこの稀釈抗体にインフルエンザウィルス（ＶＥＩ
Ｎ又はＵｄｏｒｎ ） ０．．２ｊ−を添加し、よく混
和し、３２℃に３θ分間静置し、抗原と抗体を反応させ
た。Next, influenza virus (VEI) was added to this diluted antibody.
N or Udorn) 0. ．． 2j- was added, mixed well, and allowed to stand at 32° C. for 3θ minutes to allow the antigen and antibody to react.
ついでθ、ｊ％ニワトリ赤血球浮−１ＢＱθ、ｊｍ／を
免加し、よく混合し室温に７時間静置後、管底像を読ん
だ。赤血球凝集を完全に抑えた抗体の最高稀釈度の逆数
をＨ工抗体価とする。結果を表コに示す。Next, θ,j% chicken red blood cell float-1BQθ,jm/ was added to the tube, mixed well, and left at room temperature for 7 hours, after which the image of the bottom of the tube was read. The reciprocal of the highest dilution of the antibody that completely suppresses hemagglutination is defined as the H antibody titer. The results are shown in the table below.
すなわち、ＷＳＮ及びＴＴｄｏｒｎウィルスに対する抗
体はそれぞれのウィルスに対し特異的に反応することが
わかる。That is, it can be seen that antibodies against WSN and TTdorn viruses specifically react with each virus.
〈中和テスト〉
実施例／で得られた抗体について中和テストを行なった
。すなわち、生理的食地水を用いて表２に示す各抗体の
１０倍階段稀釈液列を作成した。ついでこの稀釈抗体０
．／！ａｔにインフルエンザウィルス（ＷＳＮ株または
Ｕｄｏｒｎ ｅＰ）液ｏ、ｉ ｒ ｒｔｔｌ （，２，
０００Ｐｌ！Ｕ／ｆｆ／ ）を加え、よく混合し、３７
℃で７時間静置し、ウィルスと抗体を反応させた。つい
で、その０．Ｉｔｕｔずつをコースタ−社製を穴プレー
ト中に周章したＭＤＯＫ（Ｍａｃｌｉｎ −Ｄａｒｂｙ
ｃａｎｉｎｇ Ｋｉｄｎｅｙ ）単層培養細胞コ穴に
接種し、寥湛で／時間型いてウィルスを細胞に吸着させ
た後、／、ｊ％ゲラチンコ、ｊμｔ ／ｒｒｔｌトリプ
シ／および０．６％アカロースを含むＬ／ｊ培地−２、
５’ ｍｅを重層しｊ％ｏｏ２＠度、Ｊ％’Ｃで３日間
培養した。感染細胞で増殖したウィルスは放出され隣接
の細胞に感染を広げて行くが、インフルエンザウィルス
感染細胞は変性死滅するので最初の感染細胞を中心とし
た変性細胞の小集団、すなわちブラックが形成される。<Neutralization test> A neutralization test was conducted on the antibody obtained in Example. That is, a series of 10-fold serial dilutions of each antibody shown in Table 2 was prepared using physiological saline. Then this diluted antibody 0
．． /! Influenza virus (WSN strain or Udorn eP) solution o,ir rttl (,2,
000Pl! Add U/ff/), mix well,
The plate was left standing at ℃ for 7 hours to allow the virus and antibody to react. Then, that 0. MDOK (Maclin-Darby) with Itut stamped on the hole plate made by Coaster Co., Ltd.
caning Kidney) monolayer culture cells were inoculated into the wells, and the virus was adsorbed to the cells by dipping/time molding. j medium-2,
5'me was overlaid and cultured for 3 days at J%oo2@degree and J%'C. Viruses that multiply in infected cells are released and spread infection to neighboring cells, but as influenza virus-infected cells degenerate and die, a small group of degenerated cells, called black, is formed around the initially infected cells.
このブラックは肉眼でも観察できる。７個のブラックを
作るのに必要な最低ウィルス量として／ＰＦＵ（ｐｌａ
ｑｕｅ ｆｏｒｍｉｎｇ ｕｎｉｔ ）を定幹するが、
これは感染性ウィルス粒子７個に相当する。This black can be observed with the naked eye. /PFU (pla
que forming unit), but
This corresponds to 7 infectious virus particles.
上記３日間培養したプレート上のブラック数を数え、ブ
ラック数を抗体を加えない場合（１００ブラツク）の／
／コ以下に減少させる最高抗体稀釈度の逆数を中和抗体
価とした。結果を表−に示す。すなわち、ＶＥＩＮ及び
Ｕｄｏｒｎウィルスに対する抗体はそれぞれのウィルス
に対し特異的に中和能力を有することがわかる。Count the number of blacks on the plate cultured for 3 days, and calculate the number of blacks (100 blacks) when no antibody is added.
The reciprocal of the highest antibody dilution at which the antibody was diluted to less than 10% was defined as the neutralizing antibody titer. The results are shown in the table. That is, it can be seen that antibodies against VEIN and Udorn viruses have specific neutralizing ability against each virus.
表 −
Ｈｆ 中和
抗体 ＷＳ’ｌＪ Ｕｄｏｒｎ ＷＳＮ
ｔＴｄｏｒｎ（／ＩＩＥＩ！り
（／ｓｔｊ）ｗｓ＊Ｎ ／Ａ ４４ｔ −
Ｂ ／、０．２％ −λｏｏθ く
コｏ。Table - Hf neutralizing antibody WS'lJ Udorn WSN
tTdorn(/IIEI!ri
(/stj)ws*N /A 44t -
B/, 0.2% -λooθ Kukoo.
Ｃ，２０げ −
Ｆ ｊ／コ０Ｏ−
Ｇ ！ｒ／、、２００ ＋
Ｈｊ／ コ００ −
工 ｊ／＠２θＯ−
Ｕｄｏｒｎ 、２Ａ −’ −ウィルス
Ｂ −２！６
０ − ！／２
Ｄ −コ５６０
Ｒ−１０，２％０ ＜２００ ２００００コン
トロール３Ａ −〈、！
００ （−〇ＯＢ −−
Ｃ−−
Ｅ −−
Ｆ −−
ａ −−
（ｉｎ ｖｉｖｏテスト〉
実施例／で得られた抗体を稀釈しないで（／θＯ）又は
蒸留水で／／／θ稀釈（／θ−１）し、０、／＊ｌをノ
・ムスターの鼻腔内にスプレー投与しイルスの有勲を調
べ、ウィルス陽性のノ曳ムスター数を表３に示した。C,20ge-Fj/ko0O-G! r/,, 200 + Hj/ Ko00 - Tech j/@2θO- Udorn, 2A -' - Virus B -2!6 0 -! /2 D-Co560 R-10,2%0 <200 20000 Control 3A -<,!
00 (−〇OB −− C−− E −− F −− a −− (in vivo test) The antibody obtained in Example / was diluted (/θO) or ///θ diluted (/θO) with distilled water. /θ-1), 0, /*l was sprayed into the nasal cavities of No. mustard to examine the effectiveness of the virus, and the number of No. mustard positive for the virus is shown in Table 3.
すなわち、Ｕｄｏｒｎウィルスに対する抗体は特異的に
Ｕｄｏｒｎウィルスの感染を防御することがわかる・
′１
呼ｉ仁 乙：
表 ！
抗体；帰イ；／’ムスター数 肺 鼻腔（ＶＥＩ
Ｎ）
ｊ １０ ｊ ０ ０弘
１０ σ、／Ｒ１！Ｏｊ ０ ０４ ＰＢ８
ｚ ｌ′ａ コ（発明の効果）
本発明に係る抗体は、呼吸器系疾患の病原体による気道
感染を効率的に予防するのに好適なスプレー投与に有効
である。In other words, it can be seen that antibodies against Udorn virus specifically protect against Udorn virus infection. Antibodies; return; /'Muster's number Lungs Nasal cavity (VEI
N) j 10 j 0 0 hiro
10 σ, /R1! Oj 0 04 PB8
(Effects of the Invention) The antibody according to the present invention is effective in spray administration, which is suitable for efficiently preventing respiratory tract infections caused by pathogens of respiratory diseases.
出 願 人 清 水 −史代 理 人
弁理士 長谷用 −ほか／名Applicant Shimizu - Masato Fumiyo
Patent Attorney Hase Yo - Others/Names
Claims (5)
する卵より調製された免疫グロブリンであつて、その呼
吸器系疾患の病原体に感染防御能を有する抗体。(1) An immunoglobulin prepared from eggs produced by hens that have been immunized against a pathogen of a respiratory disease, and an antibody that has the ability to protect against the pathogen of a respiratory disease.
又はリケッチアである特許請求の範囲第１項記載の抗体
。(2) The antibody according to claim 1, wherein the pathogen is a virus, bacteria, mycoplasma, or rickettsia.
求の範囲第１又は２項記載の抗体。(3) The antibody according to claim 1 or 2, wherein the virus is an influenza virus.
求の範囲第１項記載の抗体。(4) The antibody according to claim 1, wherein the immunoglobulin is IgA or IgA.
する卵より調製された免疫グロブリンであつて、その呼
吸器系疾患の病原体に感染防御能を有する抗体を有効成
分とする鼻腔又は咽喉内へのスプレー剤。(5) Immunoglobulin prepared from eggs produced by hens that have been immunized against pathogens of respiratory diseases, and containing antibodies that have the ability to protect against the pathogens of respiratory diseases as an active ingredient for use in the nasal cavity or throat. spray agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1536086A JPS62175426A (en) | 1986-01-27 | 1986-01-27 | Antibody and spraying agent containing said substance as active component |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1536086A JPS62175426A (en) | 1986-01-27 | 1986-01-27 | Antibody and spraying agent containing said substance as active component |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS62175426A true JPS62175426A (en) | 1987-08-01 |
Family
ID=11886631
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1536086A Pending JPS62175426A (en) | 1986-01-27 | 1986-01-27 | Antibody and spraying agent containing said substance as active component |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62175426A (en) |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01265034A (en) * | 1988-04-13 | 1989-10-23 | Taiyo Kagaku Co Ltd | Preventive for viral diarrhea |
| JPH01313438A (en) * | 1988-06-13 | 1989-12-18 | Taiyo Kagaku Co Ltd | Anti-periodontosis composition |
| JPH01313439A (en) * | 1988-06-13 | 1989-12-18 | Taiyo Kagaku Co Ltd | Anti-acne vulgaris bacteria skin drug for external use |
| WO2006022409A1 (en) * | 2004-08-27 | 2006-03-02 | Daikin Industries, Ltd. | Hazardous substance scavenger and application apparatus and application method therefor |
| WO2007034741A1 (en) * | 2005-09-22 | 2007-03-29 | Daikin Industries, Ltd. | Anti-viral agent, and method for treatment of virus-infected cell |
| US8097251B2 (en) | 2001-10-24 | 2012-01-17 | Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw | Functional heavy chain antibodies, fragments thereof, library thereof and methods of production thereof |
| US8188223B2 (en) | 2005-05-18 | 2012-05-29 | Ablynx N.V. | Serum albumin binding proteins |
| US20130183286A1 (en) * | 2003-02-19 | 2013-07-18 | Camas, Inc. | Composition and method for preventing/decreasing respiratory illness |
| US9243065B2 (en) | 2002-11-08 | 2016-01-26 | Ablynx N.V. | Polypeptide constructs including VHH directed against EGFR for intracellular delivery |
| US9320792B2 (en) | 2002-11-08 | 2016-04-26 | Ablynx N.V. | Pulmonary administration of immunoglobulin single variable domains and constructs thereof |
| US9371381B2 (en) | 2002-11-08 | 2016-06-21 | Ablynx, N.V. | Single domain antibodies directed against tumor necrosis factor-alpha and uses therefor |
| JP2016519074A (en) * | 2013-03-14 | 2016-06-30 | コントラフェクト コーポレイション | Compositions and methods based on neutralizing antibodies delivered intranasally for enhanced therapeutic efficiency |
| US10407494B2 (en) | 2003-02-19 | 2019-09-10 | Camas Incorporated | Immunogen adherence and method of making and using same |
| US10639370B2 (en) | 2014-02-04 | 2020-05-05 | Contrafect Corporation | Antibodies useful in passive influenza immunization, and compositions, combinations and methods for use thereof |
| US11246928B2 (en) | 2014-02-04 | 2022-02-15 | Contrafect Corporation | Antibodies useful in passive influenza immunization, and compositions, combinations and methods for use thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS54130995A (en) * | 1978-03-22 | 1979-10-11 | South African Inventions | Improvement relative to immunity sample |
| JPS62215534A (en) * | 1985-11-25 | 1987-09-22 | Fuoobesuto Kk | Specific antibody-containing material from egg, its production and use |
-
1986
- 1986-01-27 JP JP1536086A patent/JPS62175426A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS54130995A (en) * | 1978-03-22 | 1979-10-11 | South African Inventions | Improvement relative to immunity sample |
| JPS62215534A (en) * | 1985-11-25 | 1987-09-22 | Fuoobesuto Kk | Specific antibody-containing material from egg, its production and use |
Cited By (23)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01265034A (en) * | 1988-04-13 | 1989-10-23 | Taiyo Kagaku Co Ltd | Preventive for viral diarrhea |
| JPH01313438A (en) * | 1988-06-13 | 1989-12-18 | Taiyo Kagaku Co Ltd | Anti-periodontosis composition |
| JPH01313439A (en) * | 1988-06-13 | 1989-12-18 | Taiyo Kagaku Co Ltd | Anti-acne vulgaris bacteria skin drug for external use |
| US8097251B2 (en) | 2001-10-24 | 2012-01-17 | Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw | Functional heavy chain antibodies, fragments thereof, library thereof and methods of production thereof |
| US9156905B2 (en) | 2001-10-24 | 2015-10-13 | Vib Vzw | Functional heavy chain antibodies, fragments thereof, library thereof and methods of production thereof |
| US9243065B2 (en) | 2002-11-08 | 2016-01-26 | Ablynx N.V. | Polypeptide constructs including VHH directed against EGFR for intracellular delivery |
| US9371381B2 (en) | 2002-11-08 | 2016-06-21 | Ablynx, N.V. | Single domain antibodies directed against tumor necrosis factor-alpha and uses therefor |
| US9725522B2 (en) | 2002-11-08 | 2017-08-08 | Ablynx N.V. | Pulmonary administration of immunoglobulin single variable domains and constructs thereof |
| US9320792B2 (en) | 2002-11-08 | 2016-04-26 | Ablynx N.V. | Pulmonary administration of immunoglobulin single variable domains and constructs thereof |
| US20130183286A1 (en) * | 2003-02-19 | 2013-07-18 | Camas, Inc. | Composition and method for preventing/decreasing respiratory illness |
| US10407494B2 (en) | 2003-02-19 | 2019-09-10 | Camas Incorporated | Immunogen adherence and method of making and using same |
| US9849175B2 (en) * | 2003-02-19 | 2017-12-26 | Camas Incorporated | Composition and method for preventing/decreasing respiratory illness |
| WO2006022409A1 (en) * | 2004-08-27 | 2006-03-02 | Daikin Industries, Ltd. | Hazardous substance scavenger and application apparatus and application method therefor |
| US8188223B2 (en) | 2005-05-18 | 2012-05-29 | Ablynx N.V. | Serum albumin binding proteins |
| US9067991B2 (en) | 2005-05-18 | 2015-06-30 | Ablynx N.V. | Nanobodies against tumor necrosis factor-alpha |
| US11472871B2 (en) | 2005-05-18 | 2022-10-18 | Ablynx N.V. | Nanobodies against tumor necrosis factor-alpha |
| WO2007034741A1 (en) * | 2005-09-22 | 2007-03-29 | Daikin Industries, Ltd. | Anti-viral agent, and method for treatment of virus-infected cell |
| JP2016519074A (en) * | 2013-03-14 | 2016-06-30 | コントラフェクト コーポレイション | Compositions and methods based on neutralizing antibodies delivered intranasally for enhanced therapeutic efficiency |
| EP3003373A4 (en) * | 2013-03-14 | 2016-10-19 | Contrafect Corp | Composition and methods based on neutralizing antibodies delivered intranasally for enhanced therapeutic efficacy |
| US9718875B2 (en) | 2013-03-14 | 2017-08-01 | Contrafect Corporation | Composition and methods based on neutralizing antibodies delivered intranasally for enhanced therapeutic efficacy |
| US11827693B2 (en) | 2013-03-14 | 2023-11-28 | Contrafect Corporation | Composition and methods based on neutralizing antibodies delivered intranasally for enhanced therapeutic efficacy |
| US10639370B2 (en) | 2014-02-04 | 2020-05-05 | Contrafect Corporation | Antibodies useful in passive influenza immunization, and compositions, combinations and methods for use thereof |
| US11246928B2 (en) | 2014-02-04 | 2022-02-15 | Contrafect Corporation | Antibodies useful in passive influenza immunization, and compositions, combinations and methods for use thereof |
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