JPH0657663B2 - Method for producing multifunctional specific antibody - Google Patents
Method for producing multifunctional specific antibodyInfo
- Publication number
- JPH0657663B2 JPH0657663B2 JP63202431A JP20243188A JPH0657663B2 JP H0657663 B2 JPH0657663 B2 JP H0657663B2 JP 63202431 A JP63202431 A JP 63202431A JP 20243188 A JP20243188 A JP 20243188A JP H0657663 B2 JPH0657663 B2 JP H0657663B2
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- pathogenic
- egg yolk
- infectious diarrhea
- diarrhea
- 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.)
- Expired - Fee Related
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は感染性下痢症を予防することを目的とする多機
能特異的抗体に関しより詳しくは感染性下痢症の病原ウ
イルスの１種以上及び病原細菌の１種以上からなる混合
物を抗原として産卵鶏を免疫し、それぞれに対応する抗
体を鶏卵卵黄中の水可溶性蛋白質画分に同時に生産せし
めることを特徴とする多機能特異的抗体の製造方法に関
する。TECHNICAL FIELD The present invention relates to a multifunctional specific antibody for preventing infectious diarrhea, more specifically, one or more pathogenic viruses of infectious diarrhea and A method for producing a multifunctional specific antibody, which comprises immunizing a laying hen with a mixture of one or more pathogenic bacteria as an antigen, and simultaneously producing antibodies corresponding to the hens in a water-soluble protein fraction in egg yolk. Regarding
感染性下痢症は日常遭遇する比較的ありふれた疾患で、
病原ウイルスあるいは病原細菌が消化管内に感染するこ
とにより発生する。しかし免疫力が充分に発達していな
い乳幼児，乳幼若期の家畜及び免疫機能の低下した老化
にとって、消化管感染による下痢症はしばしば深刻な疾
患として問題となる。現在、主として発展途上国で年間
約５００万人の乳幼児が感染性下痢症で死亡しており、
その制圧が緊急な課題として世界保健機構（ＷＨＯ）等
の世界的プロジェクトになっている。Infectious diarrhea is a relatively common disease that we encounter every day,
It occurs when a pathogenic virus or pathogenic bacterium infects the digestive tract. However, diarrhea due to gastrointestinal infection is often a serious problem for infants with poorly developed immunity, livestock at a young age, and aging with impaired immune function. Currently, mainly in developing countries, about 5 million infants die each year from infectious diarrhea,
Its control has become an urgent issue for global projects such as the World Health Organization (WHO).
我々人間を含む動物は体内に侵入してきた異物（細菌，
ウイルス，異種タンパク質等）抗原に対して特異的抗体
を産生し、異物抗原を不活性化あるいは解毒する。特異
的抗体のほとんどは自己体液、特に血液中に産生され
る。これに対し、感染性下痢症はいわば我々の体外であ
る消化管内の壁面細胞へ経口的に侵入してきた病原ウイ
ルス及び病原細菌が付着感染することで発病する。その
ため、感染性下痢症には体内の特異的抗体が有効に利用
され難いといわれている。特に自己免疫力の低い乳幼児
や老人、また乳幼若期の家畜では、特異的抗体の産生も
不充分であるため、感染性下痢症を自己の産する特異的
抗体で予防することは困難である。Animals, including humans, are foreign substances that have invaded the body (bacteria,
Viruses, heterologous proteins, etc.) produce specific antibodies against antigens and inactivate or detoxify foreign antigens. Most specific antibodies are produced in autologous fluids, especially blood. On the other hand, infectious diarrhea is caused by adherent infection of pathogenic viruses and pathogenic bacteria that have orally invaded wall cells in the digestive tract outside our body. Therefore, it is said that specific antibodies in the body are not effectively used for infectious diarrhea. Especially in infants and the elderly with low autoimmune power, and in livestock of young infants, production of specific antibodies is insufficient, so it is difficult to prevent infectious diarrhea with specific antibodies produced by themselves. is there.
しかし最近、この種の感染性下痢症の予防に異種動物に
産生させた特異的抗体の経口的投与が有効であることが
指摘されている［Ｃｕｒｔｉｓ Ｒ． Ｂａｒｔｚら：
Ｔｈｅ Ｊｏｕｒｎａｌ ｏｆ Ｉｎｆｅｃｔｉｏｕｓ
Ｄｉｓｅａｓｅｓ ｖｏｌ． １４２，４３９（１９
８０），Ｒｏｂｅｒｔ Ｈ． Ｙｏｌｋｅｎら：Ｐｅｄ
ｉａｔｒｉｃｓ ｖｏｌ． ８１，２９１（１９８
８）］。However, it has recently been pointed out that oral administration of a specific antibody produced in a heterologous animal is effective in preventing this kind of infectious diarrhea [Curtis R. et al. Bartz et al:
The Journal of Infectious
Diseases vol. 142,439 (19
80), Robert H. et al. Yolken et al .: Ped
iatrics vol. 81,291 (198
8)].
感染性下痢症の病原ウイルス及び病原細菌に対する特異
的抗体を経口的に投与して、該病の感染予防を行うため
には、先ず随時にしかも計画的に該特異的抗体を生産す
ることが可能でなければならない。In order to prevent infection of the disease by orally administering specific antibodies against the pathogenic virus and pathogenic bacterium of infectious diarrhea, it is possible to produce the specific antibody at any time and systematically. Must.
通常、特異的抗体は免疫哺乳動物（ウサギ，ヤギ，ウマ
等）を殺し、その血液より分離する。従って該抗体の大
量生産には必然的に多数の動物を犠牲にしなければなら
ない。免疫に用いる哺乳動物は高価であるのみならず、
その血液中にはウイルス存在の可能性もあって衛生学的
に必ずしも清潔ではなく、その取り扱いに繁雑な操作と
注意を必要とし、また抗体収量も必ずしも良好でない。Usually, the specific antibody kills an immune mammal (rabbit, goat, horse, etc.) and separates it from the blood. Therefore, large numbers of animals must be sacrificed for large-scale production of the antibody. Mammals used for immunization are not only expensive,
There is a possibility that a virus is present in the blood, so it is not always hygienically clean, its handling requires complicated operations and attention, and the antibody yield is not always good.
一方、鶏も侵入異物抗原に対し、自己血液中に特異的抗
体を産生し対応する。しかも上述のように、次世代生命
を異物抗原の攻撃から防御するため、その鶏卵卵黄中に
は該特異的抗体が親鶏血液から移行して存在する。この
事実は、１９６２年にパターソンらによりはじめて見い
出された［Ｐａｔｔｅｒｓｏｎ，Ｒ． ら：Ｊ． Ｉｍ
ｍｕｎｏｌ． ８９，２７２，（１９６２）］。On the other hand, chickens also respond to invading foreign antigens by producing specific antibodies in their own blood. Moreover, as described above, in order to protect the next-generation life from the attack of foreign antigens, the specific antibody is present in the egg yolk of the chicken by being transferred from the parent chicken blood. This fact was first discovered by Patterson et al. In 1962 [Patterson, R. et al. Et al .: J. Im
munol. 89, 272, (1962)].
これ以来、鶏を用いてその卵の卵黄中に種々の抗原に対
する特異的抗体が得られることが報告されている。しか
し、従来の研究報告はいずれも単一抗原を用いて、これ
に対して特異的に結合する抗体（単機能抗体）を鶏卵卵
黄中に生成させ、それを例えば臨床検査試薬に用いるこ
とを目的としているにすぎない。Since then, it has been reported that chickens can be used to obtain specific antibodies against various antigens in the yolk of their eggs. However, all previous research reports have aimed to generate an antibody (monofunctional antibody) that specifically binds to a single antigen in hen egg yolk and use it as a clinical test reagent, for example. I'm just saying.
種々の感染性下痢症は病原ウイルス及び病原細菌の複数
因子によって発症する。従って特異的抗体を用いた経口
的受動免疫により感染性下痢症を予防するには、種々の
病原ウイルス及び病原細菌に対してそれぞれの感染力を
中和する特異的抗体の混合物、即ち、多機能特異的抗体
を調製し利用することが実用である。しかし現在、この
ような感染性下痢症の予防を目的とした多機能特異的抗
体を積極的に調製し応用しようとする試みは、少なくと
も課題分野に関するかぎり、全く行われていない。Various infectious diarrheas are caused by multiple factors of pathogenic viruses and bacteria. Therefore, in order to prevent infectious diarrhea by oral passive immunization using specific antibodies, a mixture of specific antibodies that neutralize the respective infectivity against various pathogenic viruses and bacteria, that is, multifunctional It is practical to prepare and utilize specific antibodies. However, at present, no attempt has been made to positively prepare and apply a multifunctional specific antibody for the purpose of preventing such infectious diarrhea, at least as far as the subject field is concerned.
鶏の場合、一般的に抗原としてのウイルスの株を増すと
それぞれの株に対する抗体価が上昇し難いことが知られ
ている。このことは、感染性下痢症の予防を目的として
の多機能特異的抗体を鶏卵を通じて製造する場合の障害
となっている。In the case of chickens, it is generally known that increasing the number of virus strains as antigens makes it difficult to increase the antibody titer against each strain. This is an obstacle in producing a multifunctional specific antibody through chicken eggs for the purpose of preventing infectious diarrhea.
本発明者の基礎研究により、１）産卵鶏は哺乳動物と異
なり、多種，多量の抗原の免疫注射に耐え、２）しかも
体液に産せられる抗体はかなり急速に鶏卵卵黄中に濃縮
移行し、３）これらの機能は抗原の反復投与で半年以上
もの長期間、同一レベルに維持されることを確認し、か
つ明らかにした。換言すれば、胎生哺乳動物と異なり卵
生鳥類は卵中の次世代生命を保護するため、著量の抗体
を卵に付与する特徴を有し、この原理に基ずいて本発明
は成功したということができる。According to the basic research by the present inventor, 1) laying hens are different from mammals and endure immune injection of various and large amounts of antigens, and 2) antibodies produced in body fluids are concentrated and rapidly transferred to hen egg yolks. 3) It was confirmed and clarified that these functions are maintained at the same level for a long period of more than half a year by repeated administration of the antigen. In other words, unlike fetal mammals, ovarian birds have the characteristic of imparting a significant amount of antibody to eggs in order to protect the next-generation life in eggs, and the present invention was successful based on this principle. You can
本発明者は、感染性下痢症の病因となるウイルス及び細
菌のそれぞれに対して特異的に結合し、それぞれの感染
力を中和する高い抗体価の多機能特異的抗体を工業的に
生産することを目的として鋭意研究を重ねた結果、以下
の事実を見い出し、本発明を完成させた。The present inventor industrially produces a multifunctional specific antibody having a high antibody titer, which specifically binds to each of viruses and bacteria which cause infectious diarrhea and neutralizes the infectivity of each. As a result of earnest research for the purpose, the following facts were found and the present invention was completed.
１）感染性下痢症の病因となる抗原で産卵鶏を免疫し、
その鶏卵卵黄より得た特異的抗体は、同様にウサギを免
疫しその血液から得た特異的抗体とほぼ同等の抗体価を
示した。1) Immunize laying hens with an antigen that causes infectious diarrhea,
The specific antibody obtained from the egg yolk showed an antibody titer almost equal to that of the specific antibody obtained from the blood of a rabbit immunized similarly.
２）追加免疫によるショック死はウサギでは多かった
が、産卵鶏では全くなかった。2) Shock death due to boosting was common in rabbits, but not in laying chickens.
３）１羽のウサギの全血液から得た特異的抗体量は、約
１０個の鶏卵卵黄から得られるそれとほぼ同量であっ
た。3) The amount of specific antibody obtained from the whole blood of one rabbit was almost the same as that obtained from about 10 egg yolks.
４）感染性下痢症の病因となる抗原で産卵鶏を免疫して
も、その産卵率には何ら影響なく免疫鶏１羽当り平均、
年間２５４個の卵を産んだ。4) Even if the laying hens are immunized with the antigens that cause the infectious diarrhea, the laying rate is not affected and the average per immunized chicken is
He laid 254 eggs a year.
５）追加免疫を、ある一定間隔で繰り返せば、鶏卵卵黄
中の特異的抗体の抗体価は、その鶏の産卵期間を通じて
高いレベルで維持された。5) When the booster immunization was repeated at regular intervals, the antibody titer of the specific antibody in the egg yolk was maintained at a high level throughout the egg-laying period of the chicken.
６）感染性下痢症ウイルスの１種以上と血液寒天培地を
用い溶血環を指標にして分離した病原性腸内細菌を同時
に含有する下痢症複合抗原で産卵鶏を免疫すれば、単に
感染性下痢症ウイルスの１種以上のみを用いて免疫した
ものよりそれぞれのウイルスに対し、より高い抗体価の
特異的抗体がその鶏卵卵黄中に得られた。また同時に、
用いた病原性腸内細菌に対する特異的抗体も該卵黄中に
得られた。6) Immunization of laying hens with a diarrhea complex antigen that simultaneously contains one or more infectious diarrhea viruses and pathogenic enterobacteria that have been isolated using blood agar as an index using the hemolytic ring as an infectious diarrhea Specific antibodies with higher antibody titers to each virus were obtained in the hen egg yolk than those immunized with only one or more diseased viruses. At the same time,
Specific antibodies against the pathogenic enterobacteria used were also obtained in the yolk.
本発明者は、上述のごとく産卵鶏の抗体生成に対する生
物学的特性を詳細に研究した結果、本発明を完成するに
至った。即ち、本発明は感染性下痢症の病原ウイルスの
１種以上及び病原性腸内細菌の１種以上からなる、感染
性下痢症複合抗原で産卵鶏を免疫し、該免疫鶏の産する
卵の卵黄中に該複合抗原それぞれに対応する特異的抗体
を同時に生成せしめることを特徴とする多機能特異的抗
体の製造方法に関する。The present inventor has completed the present invention as a result of detailed research on the biological characteristics of the laying hen for antibody production as described above. That is, the present invention immunizes a laying hen with an infectious diarrhea complex antigen comprising one or more pathogenic viruses of infectious diarrhea and one or more pathogenic intestinal bacteria, and The present invention relates to a method for producing a multifunctional specific antibody, which comprises simultaneously producing specific antibodies corresponding to the respective complex antigens in egg yolk.
本発明に言う、感染性下痢症の病原ウイルスとはロタウ
イルス，腸性アデノウイルス，ノルオウクウイルス，カ
リシウイルス，アストロウイルス，コロナウイルス，ブ
レダウイルス等、人及び家畜に下痢をおこすウイルスで
ある。他方、病原性腸内細菌とは、血液寒天培地での溶
血環を指標にして下痢症患者の下痢便から分離された細
菌を指し、通常そのほとんどは、人及び家畜に下痢症を
おこす病原性大腸菌，腸炎ビブリオ菌，ウエルシュ菌，
カンピロバクター，ブドウ球菌，サルモネラ菌，ボツリ
ヌス菌，セレウス菌等である。The pathogenic virus of infectious diarrhea as referred to in the present invention is a virus that causes diarrhea in humans and livestock, such as rotavirus, enteric adenovirus, noroku virus, calicivirus, astrovirus, coronavirus, and Bredavirus. . On the other hand, pathogenic enterobacteria refers to bacteria isolated from diarrheal stools of diarrhea patients using the hemolytic ring in blood agar as an index, and most of them are usually pathogenic to cause diarrhea in humans and livestock. Escherichia coli, Vibrio parahaemolyticus, Clostridium perfringens,
These include Campylobacter, Staphylococcus, Salmonella, Botulinum, and Bacillus cereus.
これら感染性下痢症の病原ウイルス及び病原性腸内細菌
をそれぞれ適当な動物細胞及び培地で増殖培養し、常法
によりホルマリン等を用いるそれぞれを不活化した後、
混合して本発明の感染性下痢症複合抗原とする。These infectious diarrhea pathogenic viruses and pathogenic intestinal bacteria are grown and cultivated in appropriate animal cells and media, respectively, and after inactivation of each using formalin etc. by a conventional method,
The mixture is used as the infectious diarrhea complex antigen of the present invention.
該複合抗原を用いた産卵鶏の免疫は常法により行われ
る。即ち、筋肉注射，皮下注射，静脈注射等いずれの方
法でもよいが、本発明の多機能特異的抗体の生産には、
好ましくはそれらの方法の併用によるのがよい。Immunization of laying hens using the complex antigen is performed by a conventional method. That is, any method such as intramuscular injection, subcutaneous injection, and intravenous injection may be used, but for the production of the multifunctional specific antibody of the present invention,
It is preferable to use a combination of these methods.
免疫量は、１羽，１回当りウイルスの場合はタンパク量
として最大５０μｇ、また病原性腸内細菌の場合は細胞
数で最高１×１０９個が可能であるが、産卵率に影響な
く免疫するのにはそれぞれの１／２量とするのが望まし
い。しかし、ウイルスの場合タンパク量で１０μｇ以
下、また細菌の場合１×１０７個以下では高い抗体価が
得られ難い場合があるので、免疫量はこれら以上にする
ことが望ましい。The maximum immunization amount is 50 μg in the case of virus per bird per time, and the maximum number of cells is 1 × 10 9 in the case of pathogenic enterobacteria. In order to achieve this, it is desirable to use 1/2 of each amount. However, it may be difficult to obtain a high antibody titer when the amount of protein is 10 μg or less in the case of virus, and 1 × 10 7 or less in the case of bacteria.
免疫は１週あるいは２週に１回の間隔で合計３回または
それ以上行う。鶏卵卵黄中の抗体価は通常免疫２回目か
ら上昇し、３回目の免疫の約１週間後に最高値に達す
る。この最高値はその後約３ケ月間持続する。その後抗
体価が低下すれば、再度１回免疫すれば、その約１週間
後に抗体価はほぼ前回の最高値まで回復し、さらに約３
ケ月間その最高値が持続する。即ち、鶏を用いることに
より、鶏の産卵期間中継続的に高い抗体価を有する多機
能特異的抗体をその鶏卵卵黄中に生成させることができ
る。Immunization is performed once a week or once every two weeks for a total of three times or more. The antibody titer in egg yolk usually increases from the second immunization and reaches its maximum value about 1 week after the third immunization. This peak has persisted for about three months thereafter. If the antibody titer declines after that, if you immunize once again, the antibody titer will recover to the highest value of about the previous time about 1 week later, and about 3 more
The highest price lasts for months. That is, by using a chicken, a multifunctional specific antibody having a high antibody titer can be continuously produced in the egg yolk of the chicken during the egg-laying period of the chicken.
本発明の感染性下痢症複合抗原を用いて生成される多機
能特異的抗体は、病原性腸内細菌を併用せずに、単に感
染性下痢症の病原ウイルスのみを用いて鶏を免疫して得
られた抗体に比べ、抗体価が高い。これは注目すべき事
実で、同時に使用される病原性腸内細菌がウイルス抗体
生成に対し、何らかの増強作用を有するためであると考
えられる。The multifunctional specific antibody produced using the infectious diarrhea complex antigen of the present invention immunizes a chicken only with the pathogenic virus of infectious diarrhea without using the pathogenic enterobacteria in combination. The antibody titer is higher than that of the obtained antibody. This is a remarkable fact, and it is considered that the pathogenic Enterobacteriaceae used at the same time has some enhancing effect on viral antibody production.
本発明により生産される感染性下痢症予防に有効な多機
能特異的抗体の実際の応用に際しては、該多機能特異的
抗体を含む鶏卵をそのまま食したり、またはその全卵粉
末あるいは卵黄粉末を食用素材として用いたり、また必
要に応じては該多機能特異的抗体を鶏卵卵黄より精製
し、経口的投与に用いることができる。卵黄から該多機
能特異的抗体の調製は、ラムダーカラギナン等の天然多
糖類を卵黄リポタンパク質沈殿剤として用い、その遠心
上精液（卵黄水溶性タンパク質画分）より鶏卵卵黄抗体
を精製する方法（特許出願番号６２−１９４０８３（特
開昭６４−３８０９８号））が簡単で工業的であるが、
製法について特に限定するものではない。In the actual application of the multifunctional specific antibody effective for the prevention of infectious diarrhea produced by the present invention, the chicken egg containing the multifunctional specific antibody is eaten as it is, or the whole egg powder or egg yolk powder is edible. It can be used as a material, or if necessary, the multifunctional specific antibody can be purified from egg yolk and used for oral administration. The preparation of the multifunctional specific antibody from egg yolk uses a natural polysaccharide such as lambda carrageenan as an egg yolk lipoprotein precipitant, and a method of purifying chicken egg yolk antibody from the sperm (yolk yolk water-soluble protein fraction) after centrifugation ( Patent application No. 62-194083 (Japanese Patent Laid-Open No. 64-38098) is simple and industrial,
The manufacturing method is not particularly limited.
以下に実施例を示し本発明をさらに詳細に説明する。Hereinafter, the present invention will be described in more detail with reference to Examples.
実施例１．胎生動物（ウサギ）と卵生動物（産卵鶏）の
粗感染生下痢症複合抗原に対する免疫耐性及び、得られ
る総抗体量の比較 ヒトロタウイルスＷａ株が病因と推定された小児水溶性
下痢便に１％ホルマリン生理食塩水を等量加え一夜放置
後、遠心分離（１，０００ｒｐｍ，５分間）した上清を
ポアーサイズ５μｍの膜で過し、感染性下痢症複合抗
原溶液とした。該複合抗原は、その１ｍｌ当りにヒトロ
タウイルスＷａ株を５．２×１０６ＦＣＦＵ（螢光抗体
腸性フォーカス単位）及び血液寒天培地上で溶血環を指
標に分離される病原性腸内細菌２．３×１０８個を含有
した。該複合抗原溶液をウサギ及び産卵鶏のそれぞれ１
０羽づつに、１羽当り１回，２ｍｌを筋肉注射し免疫区
とした。また、それぞれの２羽づつに０．５％ホルマリ
ン生理食塩液の２ｍｌづつを同様に筋肉注射し、コント
ロールとした。免疫は２週間毎に行い、合計４回免疫し
た。ウサギについては採血を２週間毎に、そして、最終
免疫の２週間後に全採血を行い、常法に従い血清を分離
し、それぞれを同量づつ混合した混合血清を中和抗体価
測定用サンプルとした。一方、産卵鶏については２週間
毎に産した卵の卵黄を全て集め均質化後、その一部をラ
ムダーカラギナン水溶液（１ｍｇ／ｍｌ）で１０倍希釈
し、それを遠心分離（３，０００ｒｐｍ，１０分間）
し、その上清を卵黄１０倍希釈相当液とした。得られた
ウサギ混合血清及び卵黄１０倍希釈相当液を用いて、以
下の方法でヒトロタウイルスＷａ株に対する中和抗体価
を測定した。Example 1. Comparison of immune resistance to crudely infected live diarrhea complex antigen in fetal animals (rabbits) and ovarian animals (laying hens), and comparison of total antibody amounts obtained Human infantile rotavirus Wa strain was suspected to have causative factor 1 After adding an equal amount of% formalin physiological saline and allowing to stand overnight, centrifugation (1,000 rpm, 5 minutes) was carried out and the supernatant was passed through a membrane having a pore size of 5 μm to give an infectious diarrhea complex antigen solution. The complex antigen is a pathogenic enterobacteria which is separated by 5.2 × 10 6 FCFU (fluorescent antibody enteric focus unit) of human rotavirus Wa strain per 1 ml thereof and a hemolytic ring as an index on a blood agar medium. It contained 2.3 × 10 8 . The complex antigen solution was added to each of the rabbit and the hen.
2 mice were injected intramuscularly into each of 0 birds once to give an immune zone. Further, 2 ml of 0.5% formalin physiological saline was similarly intramuscularly injected into each of the two birds to serve as a control. Immunization was performed every 2 weeks, and a total of 4 immunizations were performed. Blood was collected from rabbits every two weeks, and two weeks after the final immunization, and blood was separated according to a conventional method. Mixed sera obtained by mixing equal amounts of each was used as a neutralizing antibody titer measurement sample. . On the other hand, for laying hens, after collecting all the yolks of the eggs laid every two weeks and homogenizing, a part of it was diluted 10 times with an aqueous solution of lambda carrageenan (1 mg / ml) and centrifuged (3,000 rpm, 10 minutes)
Then, the supernatant was used as a 10-fold diluted solution of egg yolk. A neutralizing antibody titer against the human rotavirus Wa strain was measured by the following method using the obtained rabbit mixed serum and a 10-fold diluted egg yolk equivalent solution.
（中和抗体価の測定） 調製したウサギ混合血清及び卵黄１０倍希釈相当液につ
いて、生理食塩水を用いそれぞれの希釈系列を作製し
た。その５０μをヒトロタウイルスＷａ株の１００Ｆ
ＣＦＵ（螢光抗体腸性フォーカス単位）を含むウイルス
液５０μと混合し、３７℃，１時間インキュベートし
た後、ＭＡ１０４細胞（サル腎臓細胞）１×１０４細胞
／ウエルへ感染させた。３７℃，２０時間培養した後、
それぞれのＦＣＦＵを測定し、ウイルス１００ＦＣＦＵ
の感染力を５０％阻害するに充分な最大希釈倍率を求め
て、血清中及び鶏卵卵黄中の中和抗体価とした。(Measurement of Neutralizing Antibody Titer) For the prepared rabbit mixed serum and egg yolk 10-fold diluted equivalent solution, each dilution series was prepared using physiological saline. 50μ of that is 100F of human rotavirus Wa strain
After mixing with 50 μl of a virus solution containing CFU (fluorescent antibody enteric focus unit) and incubating at 37 ° C. for 1 hour, 1 × 10 4 cells of MA104 cells (monkey kidney cells) were infected. After culturing at 37 ℃ for 20 hours,
Each FCFU was measured, and virus 100FCFU
The maximum dilution ratio sufficient to inhibit the infectivity of E. coli by 50% was determined and used as the neutralizing antibody titer in serum and egg yolk.
またウサギ混合血清中の抗体量を抗ウサギ抗体ヤギ血清
を用いて、鶏卵卵黄中の抗体量を抗鶏抗体ウサギ血清を
用いて定量的免疫拡散法により、常法通り測定した。Further, the amount of antibody in the rabbit mixed serum was measured by using the anti-rabbit antibody goat serum, and the amount of antibody in the egg yolk was measured by the quantitative immunodiffusion method using the anti-chicken antibody rabbit serum in the usual manner.
（結果） 粗感染性下痢症複合抗原に対するウサギ及び産卵鶏の免
疫耐性の比較 免疫に用いたウサギ１０羽の内、１羽が２回目の免疫後
に、３羽が３回目の免疫後にショック死した。一方、免
疫に用いた産卵鶏については本実験の免疫期間中、ショ
ック死は見られなかった。胎生動物（ウサギ）に比較
し、卵生動物（産卵鶏）はその血中抗体を速やかに卵黄
中へ移行することにより、人為的追加免疫に対する優れ
た耐性を獲得していると推測される。(Results) Comparison of immune resistance of rabbits and laying hens against crude infectious diarrhea complex antigen. Of 10 rabbits used for immunization, one was shock-dead after the second immunization and the third after the third immunization. . On the other hand, no shock death was observed in the laying hens used for immunization during the immunization period of this experiment. It is speculated that, compared with embryos (rabbits), oocytes (laying chickens) acquire superior resistance to artificial booster immunity by rapidly transferring their blood antibodies into the yolk.
ヒトロタウイルスＷａ株に対する中和抗体価の推移 ウサギ混合血清中及び鶏卵卵黄中の中和抗体価の推移を
表１に示した。Transition of Neutralizing Antibody Titer against Human Rotavirus Wa Strain Table 1 shows the transition of neutralizing antibody titer in the mixed serum of rabbit and egg yolk.
鶏卵卵黄中の中和抗体価はウサギ血清中のそれに比較
し、同等であることがわかった。また産卵鶏について
は、その中和抗体価の低下してきた時点で（約３ケ月
毎）再免疫をした結果、その一週間後には鶏卵卵黄中の
中和抗体価が回復することが示された。It was found that the neutralizing antibody titer in chicken egg yolk was equivalent to that in rabbit serum. In addition, the laying hens were re-immunized when the neutralizing antibody titer decreased (about every 3 months), and as a result, it was shown that the neutralizing antibody titer in the egg yolk recovered one week later. .
総抗体量の比較 ウサギ血清１ｍｌ当り１５．６ｍｇの抗体が含まれた。
本実験では、ウサギ全採血において１羽平均５６ｍｌの
血清が得られた（ｎ＝６）。従ってウサギ１羽より得ら
れる抗体量は平均８７３．６ｍｇとなる。一方、鶏卵卵
黄は１個当り平均１０２ｍｇの抗体を含有した。本実験
で免疫鶏の産卵数は平均２５４個／年（ｎ＝１０），コ
ントロール鶏のそれは平均２４５個／年（ｎ＝２）であ
った。従って免疫鶏１羽より得られる抗体量は平均２
５．９０８ｇとなり、これは実にウサギ２９．７羽から
得られる抗体量に相当した。 Comparison of total antibody amount 15.6 mg of antibody was contained per 1 ml of rabbit serum.
In this experiment, one rabbit obtained an average of 56 ml of serum in the whole blood sampling (n = 6). Therefore, the average amount of antibody obtained from one rabbit is 873.6 mg. On the other hand, each egg yolk contained an average of 102 mg of antibody. In this experiment, the average number of eggs laid by the immunized chickens was 254 / year (n = 10), and that of the control chickens was 245 / year (n = 2). Therefore, the amount of antibody obtained from one immunized chicken is 2 on average.
The amount was 5.908 g, which really corresponds to the amount of antibody obtained from 29.7 rabbits.
前述の如く、ヒトロタウイルスＷａ株に対する中和抗体
価は、ウサギ血清と鶏卵卵黄で大差なかったこと、ま
た、感染性下痢症複合抗原に対する産卵鶏の優れた免疫
耐性、また１羽の産卵鶏よりウサギ約３０羽に相当する
抗体量が得られることから、感染性下痢症の病原ウイル
ス，病原細菌に対する多機能特異的抗体の調製に、産卵
鶏を用いその鶏卵卵黄を利用する方がウサギの血液を利
用するより優れたものであることが示された。As described above, the neutralizing antibody titer against the human rotavirus Wa strain was not significantly different between rabbit serum and hen egg yolk, and the excellent immune resistance of the laying hen to infectious diarrhea complex antigen, and one laying hen Since an antibody amount equivalent to about 30 rabbits can be obtained, it is better to use a hen egg yolk for the preparation of multifunctional specific antibodies against pathogenic viruses and pathogenic bacteria of infectious diarrhea. It has been shown to be superior to using blood.
実施例２．鶏卵卵黄中に生産される下痢症病原ウイルス
抗体の抗体価におよぼす免疫条件としての単及び複合抗
原投与の影響 ＭＡ１０４細胞（サル腎臓細胞）で培養したヒトロタウ
イルスＷａ株及び２９３細胞（胎児腎，形質転換細胞）
で培養した腸性アデノウイルス４１を、ウイルスタンパ
ク量としてそれぞれ２０μｇづつ、及び小児水溶性下痢
便より分離した病原性大腸菌の細胞２×１０８個を含有
する感染性下痢症複合抗原液の１ｍｌを産卵鶏１羽毎に
筋肉注射して免疫した。初回免疫後、２週間毎に同量の
該複合抗原を追加免疫し合計３回免疫した。最終免疫後
３ケ月間に免疫鶏が産した卵より卵黄を分離して均質化
後、この卵黄液をラムダーカラギナン水溶液（１ｍｇ／
ｍｌ）で１０倍希釈し、それを遠心分離（３，０００ｒ
ｐｍ，１０分間）し、その上清を卵黄１０培希釈相当液
として抗体価を測定した。Example 2. Effect of single and multiple antigen administration as immunizing conditions on antibody titer of diarrhea pathogenic virus antibody produced in chicken egg yolk Human rotavirus Wa strain and 293 cell (fetal kidney, cultured in MA104 cells (monkey kidney cells) Transformed cells)
1 ml of infectious diarrhea complex antigen solution containing 20 μg each of the enteric adenovirus 41 cultivated in 1) as a viral protein amount and 2 × 10 8 cells of pathogenic Escherichia coli isolated from pediatric water-soluble diarrhea stool Each laying chicken was intramuscularly injected for immunization. After the first immunization, the same amount of the complex antigen was boosted every 2 weeks for a total of 3 immunizations. The yolk was separated from the egg laid by the immunized chicken within 3 months after the final immunization and homogenized, and this yolk liquid was added to an aqueous solution of lambda carrageenan (1 mg /
diluted 10 times with (ml) and centrifuged it (3,000 r
pm, 10 minutes), and the supernatant was used as a liquid equivalent to 10-fold diluted egg yolk to measure the antibody titer.
なお、比較試験として、それぞれのウイルスをタンパク
量として２０μｇ、同時に含む溶液及び別々に含む溶液
のそれぞれ１ｍｌで産卵鶏を上述同一の条件で免疫し、
同様の方法により最終免疫後３ケ月間に得られた卵の卵
黄より卵黄１０倍希釈相当液を調製した。As a comparative test, 20 μg of each virus as a protein amount, 1 ml each of a solution containing simultaneously and a solution containing separately was immunized under the same conditions as above.
By a similar method, a 10-fold diluted yolk solution was prepared from the yolk of the egg obtained 3 months after the final immunization.
対象としては、産卵鶏１羽当り、１ｍｌの生理食塩水を
同様条件で投与し、同様の方法で卵黄１０倍希釈相当液
を調製した。As a subject, 1 ml of physiological saline was administered per laying chicken under the same conditions, and a 10-fold diluted egg yolk-equivalent solution was prepared by the same method.
（ウイルスに対する中和抗体価の測定） 実施例１に示される中和抗体価の測定方法に従う。ただ
し腸性アデノウイルス４１に対しては、その感染細胞と
して２９３細胞を用いた。(Measurement of Neutralizing Antibody Titer Against Virus) The method for measuring neutralizing antibody titer shown in Example 1 is followed. However, for enteric adenovirus 41, 293 cells were used as the infected cells.
（病原性大腸菌に対する凝集抗体価の測定） 免疫に用いた菌液を生理食塩水で希釈し６０００ｎｍの
吸光度（濁度）１．０の懸濁液を調製した。この菌懸濁
液５０μと同じく生理食塩水で種々の倍率に希釈して
卵黄１０倍希釈相当液の５０μとを混合し一夜放置し
た後、マイクロウエル底面の菌の凝集の有無を調べた。
凝集抗体価は菌を凝集させる鶏卵卵黄の最大希釈倍率で
表した。(Measurement of agglutinating antibody titer against pathogenic E. coli) The bacterial solution used for immunization was diluted with physiological saline to prepare a suspension having an absorbance (turbidity) of 1.0 at 6000 nm. After 50 μL of this bacterial suspension was diluted with physiological saline to various magnifications and mixed with 50 μL of a 10-fold diluted egg yolk solution and left overnight, the presence or absence of aggregation of bacteria on the bottom surface of the microwell was examined.
The agglutinating antibody titer was represented by the maximum dilution ratio of chicken egg yolk that aggregates the bacteria.
（結果） 表２に示すように、ヒトロタウイルスＷａ株、腸性アデ
ノウイルス４１及び病原性大腸菌の混合物を抗原として
免疫して得られる鶏卵の抗体は、これら２種のウイルス
のみを同時に、またはそれらを個別的に抗原として免疫
して得られる鶏卵の抗体よりも、各ウイルスに対する中
和抗体価においてかなり大であることが見い出された。
もちろん、同効果をもたらすに使用した抗原には病原性
大腸菌が含有されたので同細菌に対する凝集抗体価も充
分認められた。従って、同抗体は、感染性下痢症の特異
的抗体として、ウイルス性下痢症の予防はもちろん病原
性細菌による下痢症の予防にも有効と考えられる。 (Results) As shown in Table 2, the antibody of chicken eggs obtained by immunizing with a mixture of human rotavirus Wa strain, enteric adenovirus 41 and pathogenic Escherichia coli as an antigen is the same as these two viruses at the same time, or It was found that the neutralizing antibody titer against each virus was considerably higher than that of the chicken egg antibody obtained by immunizing them individually as an antigen.
Of course, since the antigen used to bring about the same effect contained pathogenic Escherichia coli, a sufficient titer of agglutinating antibody against the bacterium was also observed. Therefore, the antibody is considered to be effective as a specific antibody for infectious diarrhea not only in preventing viral diarrhea but also in preventing diarrhea caused by pathogenic bacteria.
本発明により産卵鶏を用いて鶏卵中に抗ウイルス抗体を
生成させるに際して、抗原として全く起源の異なる病原
性腸内細菌を混合して免疫することにより、１種以上の
ウイルスに対する中和抗体価を明らかに上昇せしめるこ
とが可能となった。According to the present invention, when antiviral antibodies are produced in hen eggs using laying hens, pathogenic enterobacteria of completely different origins are mixed and immunized as antigens to obtain neutralizing antibody titers against one or more viruses. It became possible to raise clearly.
前記のように従来より鶏を免疫する場合、抗原としての
ウイルスの株を増すと、抗体価の上昇は困難であるとさ
れてきたが、本発明においては意外にも逆に抗体価のか
なりな上昇を認め、これは本発明において初めて見出さ
れた知見であり、本発明の効果の一つである。As described above, when chickens are conventionally immunized, it has been considered difficult to increase the antibody titer by increasing the number of virus strains as antigens. An increase was observed, which is the first finding found in the present invention and is one of the effects of the present invention.
この事実は鶏卵を利用する他の、種々の抗原に対する特
異的抗体の生産にも当然適用できる有効な方法と考えら
れる。This fact is considered to be an effective method that can be naturally applied to the production of specific antibodies against various antigens using chicken eggs.
また本発明の他の効果は、感染性下痢症をおこす種々の
病原ウイルスのみならず種々の病原細菌を混合ないしは
物理的に固定化して免疫することにより、それらの感染
力を中和する特異的抗体の混合物、即ち、多機能特異的
抗体を鶏卵卵黄中に生成せしめ得ることが明かとなった
点にある。また鶏卵を利用することにより、併せて工業
的スケールでこの種の感染性下痢症予防を目的とした該
下痢症病原ウイルス及び病原細菌に対する特異的抗体の
製造が可能と考えられる。Another effect of the present invention is to specifically immobilize not only various pathogenic viruses that cause infectious diarrhea but also various pathogenic bacteria by immobilizing them by mixing or physically immobilizing them. It has been revealed that a mixture of antibodies, i.e. multifunctional specific antibodies, can be generated in hen egg yolk. Moreover, it is considered possible to produce specific antibodies against the diarrhea pathogenic virus and pathogenic bacteria for the purpose of preventing this kind of infectious diarrhea on an industrial scale by utilizing chicken eggs.
また鶏卵は我々人類が有史以前から食してきた食品で、
特にその蛋白栄養学的価値は牛乳のそれにも勝ることが
知られている。特に日本では卵の生食も一般化してお
り、この場合は卵黄中の抗体はそのまま食品として食さ
れる。Eggs are a food that we humans have eaten since prehistoric times.
In particular, its protein nutritional value is known to exceed that of milk. Particularly in Japan, the raw diet of eggs is also common, and in this case, the antibody in the yolk is eaten as it is as food.
本発明は鶏卵中に感染性下痢症の予防に有効な多機能特
異的抗体を生産し、それを卵黄のまま、あるいは必要に
応じて分離精製して経口投与することにより、簡便かつ
安全に該下痢症の予防が可能となる利点を有する。The present invention produces a multifunctional specific antibody effective in the prevention of infectious diarrhea in chicken eggs, and it is simply and safely administered as an egg yolk or by orally administering after separating and purifying as necessary. It has an advantage that diarrhea can be prevented.
本発明は感染性下痢症を経口的受動免疫で予防すること
を目的として行われた研究成果の一部であるが、現在広
く世界的に問題となっている感染性下痢症の征圧に貢献
できる一手段を提供するものであると考えられる。The present invention is a part of the results of research conducted for the purpose of preventing infectious diarrhea by oral passive immunization, and contributes to the control of infectious diarrhea, which is currently a worldwide problem. It is considered to provide one possible means.
Claims (1)
び病原性腸内細菌の１種以上からなる、感染性下痢症複
合抗原で産卵鶏を免疫し、該免疫鶏の産する卵の卵黄中
に該複合抗原それぞれに対応する特異的抗体を同時に生
成せしめることを特徴とする多機能特異的抗体の製造方
法。1. Immunizing a laying hen with an infectious diarrhea complex antigen comprising at least one pathogenic virus for infectious diarrhea and at least one pathogenic intestinal bacterium, and A method for producing a multifunctional specific antibody, which comprises simultaneously producing specific antibodies corresponding to the respective complex antigens in egg yolk.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63202431A JPH0657663B2 (en) | 1988-08-12 | 1988-08-12 | Method for producing multifunctional specific antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63202431A JPH0657663B2 (en) | 1988-08-12 | 1988-08-12 | Method for producing multifunctional specific antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0253737A JPH0253737A (en) | 1990-02-22 |
| JPH0657663B2 true JPH0657663B2 (en) | 1994-08-03 |
Family
ID=16457404
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63202431A Expired - Fee Related JPH0657663B2 (en) | 1988-08-12 | 1988-08-12 | Method for producing multifunctional specific antibody |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0657663B2 (en) |
Families Citing this family (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5772999A (en) * | 1996-07-30 | 1998-06-30 | Dcv Biologics, L.P. | Method of preventing, countering, or reducing NSAID-induced gastrointestinal damage by administering milk or egg products from hyperimmunized animals |
| KR100280332B1 (en) * | 1997-12-31 | 2001-02-01 | 김정우 | Production of egg yolk antibody(anti-K99) against fimbrial antigen of E. coli strain K99 |
| KR100280333B1 (en) * | 1997-12-31 | 2001-02-01 | 김정우 | Production of egg yolk antibody(anti-K987P) against fimbrial antigen of E. coli strain K987P |
| KR100280331B1 (en) * | 1997-12-31 | 2001-02-01 | 김정우 | Production of egg yolk antibody(anti-K88) against fimbrial antigen of E. coli strain K88 |
| CZ285883B6 (en) * | 1998-01-19 | 1999-11-17 | Medipharm Cz, S. R. O. | Peroral preparation for preventing and therapy of infectious gastroenteritis of calves |
| KR100267747B1 (en) * | 1998-04-03 | 2000-10-16 | 이재진 | Complex immunological preparation containing egg-yolk antibodies, for prevention and treatment of porcine diarrhea caused by enterotoxigenic escherichia coli or porcine epidemic diarrhea virus |
| KR100267746B1 (en) * | 1998-04-03 | 2000-10-16 | 대한민국 | Oral immunological preparation containing egg-yolk antibodies, for prevention and treatment of porcine diarrhea caused by enterotoxigenic escherichia coli |
| KR100337383B1 (en) * | 2000-12-26 | 2002-05-22 | 김정완 | Egg yolk antibody against rotavirus |
| KR20030026654A (en) * | 2001-09-26 | 2003-04-03 | 주식회사 씨트리 | An antigen for production of an antibody against human rotavirus, and a preparing method and a use thereof |
| KR100542483B1 (en) * | 2001-11-30 | 2006-01-16 | 주식회사 에그 바이오택 | Egg production method containing complex special immune protein against Escherichia coli, rotavirus, and astrovirus, the pathogen of infant diarrhea, yolk powder and formula powder containing complex special immune protein isolated from the eggs |
| KR100471116B1 (en) * | 2001-12-12 | 2005-03-08 | 주식회사 단바이오텍 | Soluble Protein For Prevention and Treatment Of Enterotoxigenic E. coli Infection, Eggs Containing The Same And Method For Producing Thereof |
| KR100471117B1 (en) * | 2001-12-12 | 2005-03-08 | 주식회사 단바이오텍 | Soluble Protein For Prevention and Treatment Of Rotavirus Infection, Eggs Containing The Same And Method For Producing Thereof |
| KR100542484B1 (en) * | 2001-12-28 | 2006-01-16 | 주식회사 에그 바이오택 | Method for producing eggs containing E. coli, Salmonella enteritidis, Salmonella typhimurium, Helicobacter pylori, Rotavirus special immune protein and egg yolk, egg yolk powder or water-soluble complex special immune protein powder Ice cream and yogurt |
| KR20040005540A (en) * | 2002-07-10 | 2004-01-16 | 백반석 | The method for production of specific yolk antibody containing anti-helicobacter pylori IgY,anti-helicobacter pylori enzyme(urease, proteases, lipase, phosph olipase)IgY,anti-E.coli(EHEC),anti-salmonella enteritidis IgY,anti-staphylococcus aureus IgY. |
| KR20040005539A (en) * | 2002-07-10 | 2004-01-16 | 백반석 | The method for separation and production of water-soluble specific protein(IgY) using electrolytic water and saccharide |
| KR20040018589A (en) * | 2002-08-23 | 2004-03-04 | 주식회사 한국야쿠르트 | ANTI-ROTAVIRUS and IgY/EGG CONTAINING ANTI-ROTAVIRUS, and METHOD OF PREPARING IgY/EGG CONTAINING ANTI-ROTAVIRUS |
| CN1318449C (en) * | 2003-05-13 | 2007-05-30 | 雅臣药业集团（远东）有限公司 | Application of anti-atypical pneumonia (SARS) specific IgY in the preparation of preparations for preventing and treating atypical pneumonia |
| CN113845588B (en) * | 2021-05-26 | 2023-06-09 | 西南民族大学 | Preparation method and application of yolk antibody for resisting porcine rotavirus |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0753669B2 (en) * | 1985-11-25 | 1995-06-07 | 株式会社ゲン・コーポレーション | Material containing specific antibody from hen's egg, production method and use thereof |
-
1988
- 1988-08-12 JP JP63202431A patent/JPH0657663B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0253737A (en) | 1990-02-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2698778B2 (en) | Passive immunization of mammals using avian antibodies | |
| AU729502B2 (en) | Oral administration of chicken yolk antibodies to treat disease | |
| JPH0657663B2 (en) | Method for producing multifunctional specific antibody | |
| AU600240B2 (en) | Specific antibody-containing substance from eggs and method of production and use thereof | |
| US5772999A (en) | Method of preventing, countering, or reducing NSAID-induced gastrointestinal damage by administering milk or egg products from hyperimmunized animals | |
| Wiedemann et al. | Chicken egg antibodies for prophylaxis and therapy of infectious intestinal diseases: V. in vivo studies on protective effects against Escherichia coli diarrhea in pigs | |
| US20020044942A1 (en) | Transfer factor composition and process for producing same | |
| KR910005409B1 (en) | Process for preparing specific antibody containing substance from eggs | |
| JP2022513337A (en) | Compositions and Methods for Treating Intestinal Infectious Diseases and Acute Diarrhea in Animals | |
| Akita et al. | Neutralization of enterotoxigenic escherichia coli heat‐labile toxin by chicken egg yolk immunoglobulin Y and its antigen‐binding fragments | |
| CN100412092C (en) | Bacillus coli resisting chicken yolk antibody, its preparation and use | |
| EP1589995B1 (en) | Use of avian antibodies | |
| KR100267746B1 (en) | Oral immunological preparation containing egg-yolk antibodies, for prevention and treatment of porcine diarrhea caused by enterotoxigenic escherichia coli | |
| CN101735320A (en) | Yolk immunoglobulin for preventing and treating cryptosporidiosis andersoni | |
| CN114073766A (en) | Helicobacter pylori epitope peptide and application thereof | |
| CN112625126A (en) | anti-Hafnia alvei yolk antibody and preparation method and application thereof | |
| JPH01265034A (en) | Preventive for viral diarrhea | |
| JPH05501565A (en) | Methods of using vaccine conjugates, vaccine preparations and articles of manufacture | |
| JPH0542901B2 (en) | ||
| RU2683027C2 (en) | Polyvalent combined immunizing and/or therapeutic preparation for use in bacterial infections or food poisoning, particularly salmonellosis, method for production of this preparation, its use and vaccine comprising this preparation | |
| JPH05255113A (en) | Specific antibody and shrimp infectious disease-controlling composition compounded with the same | |
| CN102770452A (en) | An IgY for a PA-MSHA bacterial strain, and preparation method and application thereof | |
| JPH0665101A (en) | Method for controlling viral disease in salmon/trout using specific antibody-bearing egg | |
| DHAR | IMMUNO-STIMULATORY EFFECT OF LEVAMISOLE HYDRO CHLORIDE AND TRICLABENDAZOLE ON THE IMMUNE RESPONSE OF PESTE DES PETITS RUMINANTS (PPR) VACCINATION IN GOATS | |
| KR19990079333A (en) | Oral immunization for yolk antibody for the prevention and treatment of swine pandemic diarrhea |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20080803 Year of fee payment: 14 |
|
| LAPS | Cancellation because of no payment of annual fees |