JPH05340948A - Transovarial antibody fixed carrier and fixing method - Google Patents
Transovarial antibody fixed carrier and fixing methodInfo
- Publication number
- JPH05340948A JPH05340948A JP4176115A JP17611592A JPH05340948A JP H05340948 A JPH05340948 A JP H05340948A JP 4176115 A JP4176115 A JP 4176115A JP 17611592 A JP17611592 A JP 17611592A JP H05340948 A JPH05340948 A JP H05340948A
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- Japan
- Prior art keywords
- igy
- added
- cellulose
- antibody
- immobilized
- Prior art date
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Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は鶏卵抗体（以下ＩｇＹと
いう）の水不溶性担体への固定化方法およびＩｇＹ固定
化担体に関するものであり、食品，動物飼料，化粧品，
臨床検査薬，研究用試薬，医薬部外品や受動免疫療法等
の分野で，アフィニティ−吸着材，アフィニティ−クロ
マト用担体，バイオセンサー，エンザイムイムノアッセ
イなどに応用が可能なものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for immobilizing an egg egg antibody (hereinafter referred to as IgY) on a water-insoluble carrier and an IgY-immobilized carrier for foods, animal feeds, cosmetics,
It can be applied to affinity-adsorbents, affinity-chromatography carriers, biosensors, enzyme immunoassays, etc. in the fields of clinical test agents, research reagents, quasi drugs, passive immunotherapy and the like.
【０００２】[0002]
【従来の技術】ＩｇＹは抗原感作した親鶏の血液抗体が
鶏卵卵黄中へ移行したものである。鶏卵卵黄より得られ
る特異抗体、即ちＩｇＹを用いた場合、ウサギなどの哺
乳類の血液抗体を用いる従来法に比較して下記のような
利点がある。2. Description of the Related Art IgY is an antigen-sensitized blood antibody of a parent chicken that is transferred to egg yolk. The use of a specific antibody obtained from hen egg yolk, that is, IgY, has the following advantages over the conventional method using a blood antibody of a mammal such as rabbit.
【０００３】抗体調製に採血操作が不要で、採卵とい
う簡単な方法で行える。抗体量として鶏卵１０数個が
ウサギ１頭の全血分に相当する。また、鶏は年間２５０
〜３００個の卵を産むため、特異的抗体の大量生産が可
能である。鶏卵卵黄中には、抗体としてＩｇＹのみが
存在し、種々の抗体クラスが共存する血液と比較して抗
体精製が容易である。鶏の場合大規模養鶏がシステム
化されており、その飼育コストも安価であることから、
安価に抗体が得られる。鶏の場合、鶏病予防を目的と
したワクチネーションがシステム化されており、それを
利用して大量の鶏への免疫が効率よく行える。Blood collection operation is not required for antibody preparation and can be performed by a simple method of egg collection. As the antibody amount, ten or more chicken eggs correspond to the whole blood of one rabbit. In addition, 250 chickens a year
Since ~ 300 eggs are laid, it is possible to mass-produce specific antibodies. Only egg Y is present as an antibody in hen egg yolk, and antibody purification is easier than in blood coexisting with various antibody classes. In the case of chickens, large-scale poultry farming is systematized and the breeding cost is low, so
Antibodies can be obtained at low cost. In the case of chickens, vaccination for the prevention of chicken diseases has been systematized, and it can be used to efficiently immunize a large number of chickens.
【０００４】またＩｇＹは哺乳類の抗体（特にＩｇＧ画
分）とは化学的あるいは物理的に異なる多くの特有な性
質を有している。これらの点に着目して近年食品，動物
飼料，化粧品，臨床検査薬，研究用試薬，医薬部外品な
どの製品や受動免疫療法など広範囲な領域におけるＩｇ
Ｙの応用が近年試みられている。またアフィニティクロ
マトグラフィ用担体やその他の吸着材などへの応用の可
能性も十分にある。IgY has many unique properties which are chemically or physically different from mammalian antibodies (particularly IgG fraction). Focusing on these points, Ig in a wide range of fields such as foods, animal feeds, cosmetics, clinical test agents, research reagents, quasi-drugs and passive immunotherapy in recent years.
The application of Y has been attempted in recent years. In addition, there is a great possibility of application as a carrier for affinity chromatography and other adsorbents.
【０００５】これらの分野でリガンドとしてＩｇＹのよ
うな抗体，酵素，ホルモン，レクチンなど生理活性を有
する種々のタンパク質やペプチドを、場合によってはス
ペーサーを介して担体に固定化を行なう試みについて
は、古くから多くの報告例がある。その大部分はタンパ
ク質を構成しているアミノ酸残基の官能基における反応
によるものである。たとえばリジンのε−アミノ基，Ｎ
末端のアミノ基，システインのスルフヒドリル基，アス
パラギン酸のβ−カルボキシル基，グルタミン酸のγ−
カルボキシル基，Ｃ末端のカルボキシル基，チロシンの
フェノール性水酸基，セリンあるいはトレオニンの水酸
基，アルギニンのグアニジノ基，ヒスチジンのイミダゾ
リル基，トリプトファンのインドリル基，メチオニンの
メチルメルカプト基などである。ところがタンパク質中
特に水酸基，カルボキシル基，アミノ基の含量は多いの
で、これらの官能基を反応させた場合、活性中心部位を
形成しているアミノ酸残基を修飾あるいは変性させる可
能性があり、それに伴い活性を低下あるいは失活させる
という問題がある。In these fields, attempts to immobilize various proteins and peptides having physiological activity such as antibodies such as IgY, enzymes, hormones, and lectins as ligands on a carrier through a spacer in some cases have long been conducted. There are many reports from. Most of them are due to the reaction at the functional groups of amino acid residues constituting proteins. For example, ε-amino group of lysine, N
Terminal amino group, cysteine sulfhydryl group, aspartic acid β-carboxyl group, glutamic acid γ-
Carboxyl groups, C-terminal carboxyl groups, tyrosine phenolic hydroxyl groups, serine or threonine hydroxyl groups, arginine guanidino groups, histidine imidazolyl groups, tryptophan indolyl groups, and methionine methylmercapto groups. However, since the content of hydroxyl groups, carboxyl groups, and amino groups is particularly high in proteins, the reaction of these functional groups may modify or denature the amino acid residue forming the active center site. There is a problem that activity is reduced or deactivated.
【０００６】また一般にＩｇＹのようなタンパク質系の
生理活性物質は、通常の化学合成において頻繁に用いら
れる熱，酸，アルカリ，有機溶媒などの作用により変性
を受けやすい。したがってリガンドである生理活性物質
が固定化前と同等にできる限り近い物理的あるいは化学
的性質を保持できるようにするには、固定化反応条件に
おいて様々な面で制約が多くなる。[0006] In general, protein-based physiologically active substances such as IgY are susceptible to denaturation by the action of heat, acids, alkalis, organic solvents and the like frequently used in ordinary chemical synthesis. Therefore, in order to allow the physiologically active substance that is a ligand to retain the physical or chemical properties that are as close as possible to those before immobilization, there are many restrictions in various immobilization reaction conditions.
【０００７】また固定化されたリガンドが種々の塩濃度
や広いｐＨ範囲においてもしっかりと固定化されリガン
ドの漏出が起らないことや、固定化されたリガンドが目
的とする物質と強固に結合できることなどが条件とな
る。種々の生理活性物質の固定化方法を原理的に大別す
ると担体結合法，架橋法，包括法の３種類であ
る。これらの方法はそれぞれ長所，短所を有しており、
リガンドの種類や目的に応じて使い分けられている。Further, the immobilized ligand is firmly immobilized even in various salt concentrations and a wide pH range, and leakage of the ligand does not occur, and the immobilized ligand can firmly bind to a target substance. Is a condition. In principle, the methods for immobilizing various physiologically active substances are roughly classified into a carrier binding method, a cross-linking method, and an entrapping method. Each of these methods has advantages and disadvantages,
It is used properly according to the type and purpose of the ligand.
【０００８】担体結合法はＩｇＹのようなタンパク質
中の生理活性の発現に可能な限り悪影響を与えない部分
の、特に水酸基，カルボキシル基，アミノ基を選択し
て、担体に共有結合，イオン結合，疎水結合，生化学的
特異結合などを介して固定化するものである。共有結合
による固定化には臭化シアンによる活性化、カルボン酸
のアジド誘導体化、カルボジイミド試薬やウッドワード
試薬Ｋなどによる縮合反応、ジアゾカップリング反応、
グルタルアルデヒドのような２つ以上の反応性に富む官
能基を有する化合物により架橋する方法などがある。こ
れらの方法は結合が強固であり、安定性を増す場合があ
るなどの長所があるが、タンパク質の変性の恐れや、目
的物質との相互作用が起りにくくなる場合があるなどの
短所がある。The carrier binding method is to select covalent bond, ionic bond, or ionic bond to a carrier by selecting a hydroxyl group, a carboxyl group or an amino group, which is a moiety that does not adversely affect the expression of physiological activity in a protein such as IgY as much as possible. It is immobilized through hydrophobic binding, biochemical specific binding, etc. For immobilization by covalent bond, activation with cyanogen bromide, derivatization of carboxylic acid with azide, condensation reaction with carbodiimide reagent or Woodward reagent K, diazo coupling reaction,
There is a method of crosslinking with a compound having two or more highly functional groups such as glutaraldehyde. These methods have advantages such as strong binding and sometimes increased stability, but have disadvantages such as fear of protein denaturation and difficulty in interaction with a target substance.
【０００９】特開昭５８−５３７５７にはさらに抗体の
炭水化物部位を酸化切断し、アルデヒド基を有する抗体
と側鎖にアミノ基またはヒドラジド基を有する担体と
が、−ＣＨ2−ＮＨ−または−ＣＨ2−ＮＨ−ＮＨ−ＣＯ
−の構造を介して固定化する方法が記載されている。し
かしこの方法においても、担体と抗体の結合が直接的で
あるため、抗体の機能を十分に保持し固定化することが
困難であり、また他のタンパク質の非特異的吸着を防止
できないという問題がある。In JP-A-58-53757, an antibody having an aldehyde group and a carrier having an amino group or a hydrazide group in the side chain are further -CH 2 -NH- or -CH by further oxidizing and cleaving the carbohydrate moiety of the antibody. 2 -NH-NH-CO
A method of immobilizing via the structure of-is described. However, even in this method, since the carrier and the antibody are directly bound to each other, it is difficult to sufficiently retain and immobilize the function of the antibody, and non-specific adsorption of other proteins cannot be prevented. is there.
【００１０】またイオン結合による固定化は操作の簡便
さや再生の可能な点が有利であるが、反応液に用いる緩
衝液の種類，ｐＨ，イオン強度，温度などの影響を受け
やすい。物理的吸着による固定化では結合が一般に十分
な保持能力を得られないことが多い。Immobilization by ionic bonding is advantageous in that it is easy to operate and reproducible, but it is easily affected by the type of buffer solution used in the reaction solution, pH, ionic strength, temperature and the like. Immobilization by physical adsorption generally does not provide sufficient retention capacity for binding.
【００１１】架橋法はグルタルアルデヒド，トルエン
ジイソシアナート，ヘキサメチレンジイソシアナート，
シアヌルクロリドなどの２つ以上の官能基を有する試薬
とリガンドを反応させて分子間で架橋させて巨大分子と
する方法である。この方法は微生物菌体の固定化にはし
ばしば用いられるが、タンパク質系の生理活性物質の固
定化にはあまり有効なものではない。The cross-linking method is glutaraldehyde, toluene diisocyanate, hexamethylene diisocyanate,
This is a method in which a reagent having two or more functional groups such as cyanuric chloride is reacted with a ligand to crosslink between the molecules to form a macromolecule. Although this method is often used for immobilizing microbial cells, it is not very effective for immobilizing protein-based physiologically active substances.
【００１２】包括法は高分子ゲル内にリガンドを閉じ
込める方法である。これにはタンパク質や多糖類のよう
な天然高分子あるいは種々の合成高分子のゲルの内部に
リガンドを閉じ込める格子型，半透膜性の高分子被膜に
よりリガンドを包み込むマイクロカプセル型，リン脂質
のような液体膜にリガンドを包み込むリポソーム型など
がある。また中空子膜内や限外濾過膜で仕切られた空間
中にリガンドを閉じ込める方法もある。これらの方法は
固定化によりリガンドの修飾が起りにくく、自然な状態
を保ったままで固定化が可能である長所があるが、高分
子量の物質の作用を受けにくいことや条件により失活が
起こるなどの欠点がある。The entrapment method is a method of entrapping a ligand in a polymer gel. These include a lattice type in which the ligand is confined in the gel of natural polymers such as proteins and polysaccharides or various synthetic polymers, a microcapsule type in which the ligand is wrapped by a semipermeable polymer coating, and a phospholipid type. There is a liposome type in which a ligand is enclosed in a different liquid film. There is also a method of confining the ligand in the hollow child membrane or in the space partitioned by the ultrafiltration membrane. These methods have the advantage that ligand modification is less likely to occur due to immobilization, and immobilization is possible while maintaining the natural state, but it is less susceptible to the action of high molecular weight substances, and deactivation occurs depending on conditions. There are drawbacks.
【００１３】[0013]
【発明が解決しようとする課題】本発明は上記の従来技
術における種々の欠点を解決し、効率よく、しかもその
生理活性を低下させたり変性させたりする可能性をでき
る限り小さくしたＩｇＹの固定化方法およびＩｇＹ固定
化担体を提供しようとするものである。DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned various drawbacks in the prior art, and efficiently fixes the immobilization of IgY in which the possibility of reducing or denaturing its physiological activity is minimized. The present invention seeks to provide a method and an IgY-immobilized carrier.
【００１４】[0014]
【課題を解決するための手段】上記目的を達成した本発
明による固定化方法は鶏卵抗体の糖鎖のグリコール部位
の酸化開裂により形成されるアルデヒド基と、表面にア
ミノ基を有する水不溶性担体のアミノ基とでシッフ塩基
を形成させた後これを還元することにより、水不溶性担
体に固定化することに要旨を有する。また上記方法によ
れば、鶏卵担体がその糖鎖部分で水不溶性担体に固定さ
れた鶏卵担体が得られる。[Means for Solving the Problems] The immobilization method according to the present invention, which has achieved the above objects, comprises an aldehyde group formed by oxidative cleavage of a glycol moiety of a sugar chain of an egg egg antibody and a water-insoluble carrier having an amino group on the surface. The gist is to immobilize it on a water-insoluble carrier by forming a Schiff base with an amino group and then reducing it. Further, according to the above method, a chicken egg carrier in which the sugar chain portion thereof is fixed to the water-insoluble carrier can be obtained.
【００１５】[0015]
【作用】本発明者等は、効率よく行え、しかも失活の少
ないＩｇＹの固定化方法について種々検討した結果、従
来の方法のようにタンパク質を構成しているアミノ酸残
基中の官能基を反応させるのではなく、タンパク質の糖
鎖中の官能基を反応させれば良いことを見出した。The present inventors have conducted various studies on methods of immobilizing IgY that can be efficiently performed and have little inactivation, and as a result, the functional group in the amino acid residue constituting the protein was reacted as in the conventional method. It was found that the functional group in the sugar chain of the protein should be reacted instead of causing it.
【００１６】ＩｇＹのような糖タンパク質は多くのもの
の存在が知られているが、その糖鎖部分の生化学的な意
義や役割はそれぞれに特有で様々であると考えられてお
り、一般的には熱，各種変性剤，分解酵素（プロテアー
ゼ）などに対する安定性を高めたり防御したりする作用
や、あるいは高次構造の維持に関与する場合が多いとさ
れている。しかしその生理活性の発現に関しては大きく
影響していると考えられる場合はほとんどなく、糖鎖部
分を修飾することによる生理活性の変化に対する影響
は、タンパク質部分を構成しているアミノ酸残基を修飾
する場合と比較すれば小さいものである。特にＩｇＹの
ような抗体の場合には糖鎖部位はその抗原結合部位とは
離れた部分に存在しておりその影響は少ないと言える。[0016] Many glycoproteins such as IgY are known to exist, but the biochemical significance and role of the sugar chain portion are considered to be peculiar and different in general, and generally, Is often involved in the action of enhancing or protecting the stability against heat, various denaturants, degrading enzymes (proteases), etc., or maintaining the higher order structure. However, it is rarely thought that the expression of its physiological activity is greatly affected, and the effect on the change in physiological activity by modifying the sugar chain portion modifies the amino acid residue that constitutes the protein portion. It is smaller than the case. Particularly, in the case of an antibody such as IgY, the sugar chain site is present at a part apart from the antigen binding site, and it can be said that the influence is small.
【００１７】本発明で用いる水不溶性担体は、アミノ基
を有しているか、あるいはアミノ基の導入が容易であ
る、たとえばアルデヒド基，エポキシ基のような官能基
を有しているものであれば特に限定されないが、代表的
なものとしてはポリスチレン、ポリメタクリル酸および
その誘導体、あるいはこれらの共重合体、更にはポリビ
ニルアルコール、スチレン−ジビニルベンゼン共重合体
などの合成高分子化合物、セルロース、キチン、キトサ
ン、アガロースなどの天然高分子化合物またはアシルセ
ルロース、アシルキチンなどの改質天然高分子化合物な
どが挙げられる。例示した高分子化合物のうち、機械的
強度等を考慮すると、適度に架橋したポリスチレン、ポ
リメタクリル酸メチルおよびその誘導体あるいはこれら
の共重合体、セルロース、キチンが望ましい。これらの
中からその目的・用途に応じて適切なゲル強度，粒子
径，細孔径等の特徴を有するものを適宜選択すれば良
い。担体の形態に関しても特に限定されるものではな
く、その用途や目的に応じてビーズ状，繊維状，膜状
（中空糸膜を含む）などいずれにおいても適用できるも
のである。担体におけるアミノ基の含量はある程度多い
方がリガンド導入率の向上に結びつくが、あまり多過ぎ
るとゲル強度が低下するので適当ではない。したがって
０．０１〜０．８０ｍｅｑ／ｇの範囲になるように導入
するのが望ましく、リガンドや目的物質の大きさに応じ
てその値を調整する必要がある。The water-insoluble carrier used in the present invention has an amino group or a functional group such as an aldehyde group or an epoxy group into which an amino group can be easily introduced. Although not particularly limited, as typical ones, polystyrene, polymethacrylic acid and derivatives thereof, or copolymers thereof, polyvinyl alcohol, styrene-synthetic polymer compounds such as divinylbenzene copolymer, cellulose, chitin, Examples thereof include natural polymer compounds such as chitosan and agarose, and modified natural polymer compounds such as acyl cellulose and acyl chitin. Considering mechanical strength and the like, among the exemplified polymer compounds, moderately cross-linked polystyrene, polymethyl methacrylate and its derivatives or their copolymers, cellulose, and chitin are preferable. Among these, those having characteristics such as gel strength, particle diameter, pore diameter, etc. appropriate for the purpose and application may be appropriately selected. The form of the carrier is not particularly limited, and may be in the form of beads, fibers, membranes (including hollow fiber membranes), etc., depending on the use and purpose. If the content of amino groups in the carrier is high to some extent, it will lead to an improvement in the rate of ligand introduction, but if it is too high, the gel strength will decrease, which is not suitable. Therefore, it is desirable to introduce it in the range of 0.01 to 0.80 meq / g, and it is necessary to adjust the value according to the size of the ligand and the target substance.
【００１８】これらの担体に直接ＩｇＹを固定化するこ
とも可能ではあるが、一般的にタンパク質系の物質を結
合させる場合にはたとえば下式にその一例をに示すよう
な両末端にアミノ基を有するポリ（オキシエチレン）
（以下ＰＥＯアミンと言う）を親水性スペーサーとして
結合させた後に、さらにＩｇＹをリガンドとして導入す
る方がより好ましい。 H2NCH2CH2CH2(OCH2CH2)nOCH2CH2CH2NH2 親水性スペーサーを用いる場合にその鎖長についてもリ
ガンドに結合させる目的物質の大きさや性質に応じて適
当に調整する必要があるが、例えばＰＥＯアミンを用い
る場合重合度が１０〜５００の範囲、好ましくは３０〜
４００、さらに好ましくは１００〜３００のものが適し
ている。親水性スペーサーを導入することにより立体障
害が小さくなり、目的物質との結合性を大きく向上させ
ることができる。さらに親水性スペーサーによる排除体
積効果および親水性の向上により、非特異的な吸着を抑
えることができる。Although it is possible to directly immobilize IgY on these carriers, generally, when a protein substance is bound to the carrier, amino groups at both ends as shown in the following formula are shown. Have poly (oxyethylene)
It is more preferable to introduce IgY as a ligand after binding (hereinafter referred to as PEO amine) as a hydrophilic spacer. H 2 NCH 2 CH 2 CH 2 (OCH 2 CH 2 ) n OCH 2 CH 2 CH 2 NH 2 When a hydrophilic spacer is used, its chain length should also be adjusted depending on the size and properties of the target substance to be bound to the ligand. It is necessary to adjust, but when using, for example, PEO amine, the degree of polymerization is in the range of 10 to 500, preferably 30 to
400, more preferably 100-300 are suitable. By introducing a hydrophilic spacer, steric hindrance is reduced, and the bondability with a target substance can be greatly improved. Furthermore, the non-specific adsorption can be suppressed by the excluded volume effect and hydrophilicity improvement by the hydrophilic spacer.
【００１９】一方リガンドであるＩｇＹにおける糖鎖部
分へのアルデヒド基の導入は過ヨウ素酸またはその塩で
酸化して、グリコール部位を開裂してアルデヒド基を形
成させることにより行なうのが好ましい。ＩｇＹにおい
ては哺乳類ＩｇＧと比較してその糖鎖にグルコース残基
が非常に多く存在することが明らかにされており、この
反応の利用は有効である。過ヨウ素酸による酸化反応は
グリコール部位の隣接する水酸基間で生ずるものであ
り、１モルのＩＯ4-がＩＯ3-に還元されことにより２モ
ルのアルデヒド基が形成される。得られる反応物の構造
はグルコシド結合の種類により異なってくるが、下記式
に示すようなものが考えられる。On the other hand, the introduction of the aldehyde group into the sugar chain portion of the ligand IgY is preferably carried out by oxidizing with periodic acid or a salt thereof to cleave the glycol moiety to form an aldehyde group. It has been clarified that IgY has more glucose residues in its sugar chain than that of mammalian IgG, and the use of this reaction is effective. The oxidation reaction with periodate occurs between the adjacent hydroxyl groups of the glycol moiety, and 1 mol of IO 4 - is reduced to IO 3- to form 2 mol of an aldehyde group. The structure of the obtained reaction product varies depending on the type of glucosidic bond, but the following formula is conceivable.
【００２０】[0020]
【化１】 [Chemical 1]
【００２１】過ヨウ素酸の量はＩｇＹと同モル以上の過
剰量を用いて反応させるのが望ましい。反応時のｐＨ値
は３〜６で行なうのが好ましいが、ＩｇＹの安定性や副
反応の発生しやすさの点を考慮してｐＨ４〜５で行なう
のがより好ましい。また反応液には０．１Ｍ程度の酢酸
あるいはクエン酸緩衝液を用いるのが適当である。反応
温度は室温あるいはそれ以下が好ましく、５℃で行なう
のが最も理想的である。反応時間は室温では１時間以内
で十分であるが、５℃では１２〜２４時間程度行なう必
要がある。It is desirable that the periodate is used in an excess amount equal to or higher than that of IgY. The pH value during the reaction is preferably 3 to 6, but more preferably pH 4 to 5 in consideration of the stability of IgY and the likelihood of side reactions. Further, it is suitable to use acetic acid or citrate buffer solution of about 0.1 M as the reaction solution. The reaction temperature is preferably room temperature or lower, and most ideally 5 ° C. A reaction time of 1 hour or less is sufficient at room temperature, but it is necessary to perform the reaction for 12 to 24 hours at 5 ° C.
【００２２】また酸化反応終了後にはエチレングリコー
ル，グリセリン，亜硫酸ナトリウムなどの物質を添加し
て過剰の酸化剤を除去する必要がある。また酸化反応の
際に用いた緩衝液を外液として、透析処理を５℃で１２
時間以上行なうことによりこれらの物質を除去するのが
より好ましい。After the completion of the oxidation reaction, it is necessary to add substances such as ethylene glycol, glycerin and sodium sulfite to remove the excess oxidizing agent. Also, using the buffer solution used in the oxidation reaction as the external solution, perform dialysis treatment at 5 ° C for 12 hours.
It is more preferable to remove these substances by carrying out the treatment for a longer time.
【００２３】こうして得たアルデヒド基の導入されたＩ
ｇＹを、上記のようなＰＥＯアミンの結合した担体とシ
ッフ塩基を形成させて結合する。この反応は室温または
５℃で１０〜３０時間、ｐＨは６〜１０の範囲で行ない
ｐＨ８〜９．５の範囲で行なうのがより望ましい。反応
液としては０．１Ｍ程度の炭酸緩衝液を用いるのが好ま
しい。The thus obtained aldehyde-containing I
gY is bound to a PEO amine bound carrier as described above by forming a Schiff base. This reaction is carried out at room temperature or 5 ° C. for 10 to 30 hours, pH is preferably in the range of 6 to 10, and more preferably pH is in the range of 8 to 9.5. As the reaction solution, it is preferable to use a carbonate buffer solution of about 0.1M.
【００２４】こうして得られたシッフ塩基は安定性に乏
しく、特に酸性あるいはアルカリ性の条件下では分解を
受けやすい。そこで次式に示すように（式中Ｒは担体を
表わす）、還元反応を行なうことにより安定化させるの
が適当である。 ＲＮ＝ＣＨ−ＩｇＹ → ＲＮＨ−ＣＨ2−ＩｇＹ シッフ塩基の還元反応は水素化ホウ素ナトリウム（Ｎａ
ＢＨ4）あるいはシアノ水素化ホウ素ナトリウム（Ｎａ
ＢＨ3ＣＮ）の添加により行なうのが好ましい。反応は
室温または５℃において、ｐＨ８〜９の炭酸緩衝液中で
３〜３０時間行なうのが望ましい。The Schiff base thus obtained has poor stability and is susceptible to decomposition, especially under acidic or alkaline conditions. Therefore, as shown in the following formula (in the formula, R represents a carrier), it is suitable to stabilize by carrying out a reduction reaction. RN = CH-IgY → RNH-CH 2 -IgY The reduction reaction of the Schiff base is carried out by sodium borohydride (Na
BH 4 ) or sodium cyanoborohydride (Na
BH 3 CN) is preferably added. The reaction is preferably carried out at room temperature or 5 ° C. in a carbonate buffer of pH 8-9 for 3 to 30 hours.
【００２５】本発明の固定化方法において、アミノ基を
表面に有する水不溶性担体に、糖鎖にアルデヒド基を導
入したＩｇＹをリガンドとして導入するには、上記のよ
うな方法を基本とすれば特に限定されるのもではない
が、たとえば多孔質セルロースを担体としてＰＥＯアミ
ンを親水性スペーサーとして介してＩｇＹを導入する場
合には、以下の方法が好ましい。In the immobilization method of the present invention, in order to introduce IgY having an aldehyde group introduced into a sugar chain as a ligand into a water-insoluble carrier having an amino group on the surface, the above method is particularly preferable. Although not limited, when the IgY is introduced through a porous cellulose as a carrier and a PEO amine as a hydrophilic spacer, the following method is preferable.
【００２６】（１）セルロースの改質 過ヨウ素酸ナトリウムを０．１〜５．０規定、好ましく
は０．５〜１．５規定の硫酸に溶解した溶液に平均粒子
径が２０〜２０００μｍ、平均細孔径が２００〜３００
００Åの粒状多孔質セルロースを添加し、１０〜５０℃
好ましくは２０〜３０℃で、５〜３０時間好ましくは１
０〜２４時間反応させる。上記過ヨウ素酸ナトリウムー
硫酸溶液の過ヨウ素酸ナトリウム濃度は２〜１５重量％
好ましくは４〜１０重量％である。また上記粒状多孔質
セルロースの過ヨウ素酸ナトリウム溶液への浴比は１０
〜３０容量％好ましくは１５〜２５容量％である。この
反応混合物を濾過して生成物を回収し、十分に洗浄し
て、膨潤状態でアルデヒド含量０．１０〜４．００ｍｅ
ｑ／ｇ好ましくは０．２０〜１．００ｍｅｑ／ｇの［ア
ルデヒド］−［セルロース］を得る。この［アルデヒ
ド］−［セルロース］は先に示したような一部のグルコ
ースユニットが開環した構造を有している。(1) Modification of Cellulose A solution of sodium periodate in 0.1 to 5.0 N, preferably 0.5 to 1.5 N sulfuric acid has an average particle size of 20 to 2000 μm and an average particle size. Pore size is 200-300
Add 00 Å granular porous cellulose, 10-50 ℃
Preferably at 20 to 30 ° C., for 5 to 30 hours, preferably 1
React for 0 to 24 hours. The sodium periodate concentration of the sodium periodate-sulfuric acid solution is 2 to 15% by weight.
It is preferably 4 to 10% by weight. The bath ratio of the granular porous cellulose to the sodium periodate solution is 10
-30% by volume, preferably 15-25% by volume. The reaction mixture was filtered to recover the product, washed thoroughly, and swollen to give an aldehyde content of 0.10 to 4.00 me.
q / g [Aldehyde]-[cellulose] of 0.20 to 1.00 meq / g is obtained. This [aldehyde]-[cellulose] has a structure in which some glucose units are ring-opened as described above.
【００２７】（２）スペーサーの導入 重合度が１０〜５００、好ましくは３０〜４００、さら
に好ましくは１００〜３００のＰＥＯアミンをｐＨ９．
５の炭酸緩衝液に溶解させる。これに上記（１）で得た
［アルデヒド］−［セルロース］を添加して攪拌しなが
ら１０〜５０℃好ましくは２０〜３０℃で、５〜３０時
間好ましくは１０〜２４時間反応させる。上記ＰＥＯア
ミンの濃度は０．２〜５．０重量％好ましくは０．４〜
３．０重量％で、この溶液への［アルデヒド］−［セル
ロース］の浴比は３〜２０容量％好ましくは５〜１５容
量％である。この反応混合物を濾過して生成物を回収、
水洗して［ＰＥＯアミン］−［セルロース］を得る。(2) Introduction of Spacer PEO amine having a degree of polymerization of 10 to 500, preferably 30 to 400, and more preferably 100 to 300 is added at pH 9.
Dissolve in 5 carbonate buffer. [Aldehyde]-[cellulose] obtained in the above (1) is added thereto, and the mixture is reacted with stirring at 10 to 50 ° C, preferably 20 to 30 ° C for 5 to 30 hours, preferably 10 to 24 hours. The concentration of the PEO amine is 0.2 to 5.0% by weight, preferably 0.4 to
At 3.0% by weight, the bath ratio of [aldehyde]-[cellulose] to this solution is 3 to 20% by volume, preferably 5 to 15% by volume. The reaction mixture is filtered to recover the product,
It is washed with water to obtain [PEO amine]-[cellulose].
【００２８】（３）ＩｇＹ糖鎖の改質 ヒト低比重リポタンパク質（以下ｈＬＤＬと言う）を完
全アジュバントとともに免疫したレグホンの鶏卵の卵黄
部分からλカラギーナン水溶液に混合して遠心分離して
得られた上清をイオン交換クロマトグラフィおよび塩析
により精製して得た抗ｈＬＤＬ−ＩｇＹを１０〜２０ｍ
ｇ、２０〜５０ｍｌのｐＨ４．５の酢酸緩衝液に溶解し
て、過ヨウ素酸ナトリウム１〜５ｍｇを添加して室温で
攪拌して３０分〜１時間反応させる。さらにエチレング
リコールを０．１Ｍ濃度になるように添加して５℃で５
〜１０時間反応させ、この反応液を上記酢酸緩衝液を外
液として５℃で１２〜２４時間透析を行う。こうして
［アルデヒド］−［ＩｇＹ］溶液を得る。(3) Modification of IgY sugar chain Obtained by mixing human low-density lipoprotein (hereinafter referred to as hLDL) with the complete adjuvant from the yolk part of the egg of leghorn immunized with an aqueous solution of λ carrageenan and centrifuging. 10-20 m of anti-hLDL-IgY obtained by purifying the supernatant by ion exchange chromatography and salting out was obtained.
g, dissolved in 20 to 50 ml of an acetic acid buffer of pH 4.5, added with 1 to 5 mg of sodium periodate, stirred at room temperature and reacted for 30 minutes to 1 hour. Add ethylene glycol to a concentration of 0.1M and add 5 at 5 ℃.
The reaction is allowed to proceed for 10 hours, and the reaction solution is dialyzed at 5 ° C. for 12 to 24 hours using the acetate buffer as an external solution. Thus, an [aldehyde]-[IgY] solution is obtained.
【００２９】（４）ＩｇＹの導入 上記（３）で得た［アルデヒド］−［ＩｇＹ］溶液にｐ
Ｈ９．５の炭酸緩衝液を加えてｐＨを８．０〜９．０に
調整した後に、上記（２）で得た［ＰＥＯアミン］−
［セルロース］３ｇ（膨潤状態）を添加し、５℃で２０
〜３０時間反応させてシッフ塩基を形成させる。この反
応混合物を濾過して生成物を回収し、十分に洗浄して、
膨潤状態でｐＨ９．０の炭酸緩衝液を加え、さらに水素
化ホウ素ナトリウムを０．２〜１ｇ添加して５℃で５〜
１５時間攪拌により還元反応を行う。この反応混合物を
濾過して生成物を回収し、十分に洗浄することにより、
抗ｈＬＤＬ抗体をＰＥＯアミンをスペーサーとして介し
てセルロースに固定化したものを得る。(4) Introduction of IgY p was added to the [aldehyde]-[IgY] solution obtained in (3) above.
After adjusting the pH to 8.0 to 9.0 by adding a carbonate buffer solution of H9.5, [PEO amine] -obtained in (2) above.
[Cellulose] 3 g (swelling state) was added, and the mixture was added at 5 ° C. for 20
Allow to react for ~ 30 hours to form a Schiff base. The reaction mixture was filtered to collect the product, washed thoroughly,
In the swollen state, a carbonate buffer having a pH of 9.0 was added, and 0.2 to 1 g of sodium borohydride was further added to the mixture at 5 ° C. for 5 to 5.
The reduction reaction is carried out by stirring for 15 hours. The reaction mixture was filtered to recover the product and washed thoroughly to
An anti-hLDL antibody immobilized on cellulose via PEO amine as a spacer is obtained.
【００３０】[0030]
【実施例】以下実施例を用いて本発明を説明するが、下
記実施例は本発明を制限するものではなく、前・後記の
趣旨を逸脱しない範囲で変更実施することは全て本発明
の技術的範囲に包含される。EXAMPLES The present invention will be described below with reference to examples, but the following examples are not intended to limit the present invention, and any modification or modification is possible without departing from the spirit of the preceding and the following. It is included in the target range.
【００３１】＜実施例１＞ 抗ｈＬＤＬ−ＩｇＹの固定
化 ｈＬＤＬ（ケミコン社製）を免疫したレグホンの卵黄か
ら抽出および精製して得られた抗ｈＬＤＬ−ＩｇＹの多
孔質セルロースへの固定化を次のようにして実施した。<Example 1> Immobilization of anti-hLDL-IgY Immobilization of anti-hLDL-IgY obtained by extracting and purifying from egg yolk of leghorn immunized with hLDL (Chemicon) was carried out as follows. Was carried out as follows.
【００３２】過ヨウ素酸ナトリウム８００ｍｇを１Ｎ硫
酸２００ｍｌに溶解して、多孔質セルロース（チッソ社
製「セルロファインＣＰＣｍ」）２０ｇを添加し、攪拌
により２０時間反応させた後、反応混合物を濾過して生
成物を回収し、十分に洗浄して［アルデヒド］−［セル
ロース］を得た。アルデヒド含量はオキシム法により定
量を行ない０．５５ｍｅｑ／ｇであった。800 mg of sodium periodate was dissolved in 200 ml of 1N sulfuric acid, 20 g of porous cellulose ("Cellulofine CPCm" manufactured by Chisso Corporation) was added, and the mixture was reacted for 20 hours by stirring, and then the reaction mixture was filtered. The product was recovered and thoroughly washed to obtain [aldehyde]-[cellulose]. The aldehyde content was determined by the oxime method and was 0.55 meq / g.
【００３３】分子量５０００のＰＥＯアミン２ｇをｐＨ
９．５の炭酸緩衝液５０ｍｌに溶解して、上記の［アル
デヒド］−［セルロース］５ｇを加えて攪拌により反応
させて、反応混合物を濾過して生成物を回収し、十分に
洗浄して［ＰＥＯアミン］−［セルロース］を得た。ア
ミノ基含量は平沼産業製「ＣＯＭＴＩＴＥ１０１」を使
用して塩酸による電位差滴定により定量を行なったとこ
ろ０．２０ｍｅｑ／ｇであった。2 g of PEO amine having a molecular weight of 5000 was added to pH.
It was dissolved in 50 ml of 9.5 carbonate buffer, 5 g of the above [aldehyde]-[cellulose] was added and reacted by stirring, and the reaction mixture was filtered to recover the product, which was thoroughly washed [ PEO amine]-[cellulose] was obtained. The amino group content was 0.20 meq / g as determined by potentiometric titration with hydrochloric acid using "COMMITITE 101" manufactured by Hiranuma Sangyo.
【００３４】一方上記ＩｇＹ４０ｍｇをｐＨ４．５のク
エン酸緩衝液に５０ｍｌに溶解して、過ヨウ素酸ナトリ
ウム５０ｍｇを添加して、攪拌により室温で４５分反応
させた。さらにエチレングリコールを０．１Ｍ濃度にな
るように添加して、５℃で７時間攪拌して反応させ、反
応液を上記緩衝液を外液として５℃で一晩透析処理を行
ない［アルデヒド］−［ＩｇＹ］溶液を得た。On the other hand, 40 mg of the above IgY was dissolved in 50 ml of a citrate buffer solution having a pH of 4.5, 50 mg of sodium periodate was added, and the mixture was reacted at room temperature for 45 minutes by stirring. Further, ethylene glycol was added so as to have a concentration of 0.1 M, and the mixture was reacted by stirring at 5 ° C for 7 hours, and the reaction solution was dialyzed overnight at 5 ° C using the above buffer solution as an external solution [aldehyde]- A [IgY] solution was obtained.
【００３５】上記［アルデヒド］−［ＩｇＹ］液を炭酸
緩衝液を添加してｐＨを９．０に調整し、上記［ＰＥＯ
アミン］−［セルロース］１ｇを加えて、５℃で３０時
間攪拌してシッフ塩基を形成させた。反応混合物を濾過
して生成物を回収し、十分に洗浄してｐＨ８．０の炭酸
緩衝液５０ｍｌ，水素化ホウ素ナトリウム２００ｍｇを
加えて５℃で７時間還元反応を行なった。反応混合物を
濾過して生成物を回収し、十分に洗浄して固定化ＩｇＹ
を得た。A carbonate buffer is added to the above [aldehyde]-[IgY] solution to adjust the pH to 9.0, and the above [PEO] is added.
1 g of amine]-[cellulose] was added and stirred at 5 ° C. for 30 hours to form a Schiff base. The reaction mixture was filtered to collect the product, which was thoroughly washed, 50 ml of a pH 8.0 carbonate buffer solution and 200 mg of sodium borohydride were added, and the reduction reaction was carried out at 5 ° C. for 7 hours. The reaction mixture was filtered to recover the product, washed thoroughly and immobilized with IgY
Got
【００３６】ＩｇＹの固定化量はシッフ塩基反応の残液
をトリクロロ酢酸溶液により沈殿させ、水酸化ナトリウ
ム水溶液に溶解したものをＢＣＡタンパク質定量用試薬
（ピアス社製）により残存量を定量することにより算出
したところ（以下ＴＣＡ−ＢＣＡ法と言う）、２７．９
ｍｇが固定化されていた。The amount of immobilized IgY was determined by precipitating the residual solution of the Schiff base reaction with a trichloroacetic acid solution and dissolving the solution in a sodium hydroxide aqueous solution to quantify the remaining amount with a BCA protein quantification reagent (Pierce). When calculated (hereinafter referred to as TCA-BCA method), 27.9
mg was fixed.
【００３７】上記ＩｇＹを固定化したセルロースゲル１
ｍｌをブタ血清（ＬＤＬ値１５０．５ｍｇ／ｍｌ）２ｍ
ｌと２０ｍｌのバイヤル中で混合して、３０℃で３０分
間振盪することによりインキュベートした後、遠心上清
のＬＤＬ値を「βリポ蛋白Ｃテスト−ワコー」（和光純
薬工業製）により定量した。得られた残存ＬＤＬ値から
ＬＤＬ吸着率を算出した結果を表１に示す。Cellulose gel 1 on which IgY is immobilized
2 ml of pig serum (LDL value 150.5 mg / ml)
After mixing with 1 and 20 ml of vial and incubating by shaking at 30 ° C. for 30 minutes, the LDL value of the centrifugal supernatant was quantified by “β lipoprotein C test-Wako” (manufactured by Wako Pure Chemical Industries). .. Table 1 shows the result of calculating the LDL adsorption rate from the obtained residual LDL value.
【００３８】[0038]
【表１】 [Table 1]
【００３９】＜実施例２＞ 抗ｈＬＤＬ−ＩｇＹの固定
化 テトラエチレンペンタミン１１０ｇをｐＨ９．５の炭酸
緩衝液５０ｍｌに溶解して、実施例１で調製した［アル
デヒド］−［セルロース］１０ｇを加えて攪拌により反
応させて、反応混合物を濾過して生成物を回収し十分に
洗浄して［アミン］−［セルロース］を得た。アミノ基
含量は０．２７ｍｅｑ／ｇであった。<Example 2> Immobilization of anti-hLDL-IgY 110 g of tetraethylenepentamine was dissolved in 50 ml of a carbonate buffer of pH 9.5, and 10 g of [aldehyde]-[cellulose] prepared in Example 1 was added. The mixture was reacted by stirring, and the reaction mixture was filtered to collect the product, which was thoroughly washed to obtain [amine]-[cellulose]. The amino group content was 0.27 meq / g.
【００４０】一方実施例１におけるＩｇＹ４０ｍｇをｐ
Ｈ４．５のクエン酸緩衝液５０ｍｌに溶解して、過ヨウ
素酸ナトリウム５０ｍｇを添加して、攪拌により室温で
４５分反応させた。さらにエチレングリコールを０．１
Ｍ濃度になるように添加して、５℃で７時間攪拌して反
応させ、反応液を上記緩衝液を外液として５℃で一晩透
析処理を行ない［アルデヒド］−［ＩｇＹ］溶液を得
た。On the other hand, 40 mg of IgY in Example 1 was added to p.
It was dissolved in 50 ml of a citrate buffer solution of H4.5, 50 mg of sodium periodate was added, and the mixture was reacted at room temperature for 45 minutes by stirring. Add ethylene glycol to 0.1
The mixture was added so as to have an M concentration and reacted by stirring at 5 ° C for 7 hours, and the reaction solution was dialyzed overnight at 5 ° C using the above buffer solution as an external solution to obtain an [aldehyde]-[IgY] solution. It was
【００４１】上記［アルデヒド］−［ＩｇＹ］液を炭酸
緩衝液を添加してｐＨを９．０に調整し、上記［アミ
ン］−［セルロース］５ｇを加えて、５℃で３０時間攪
拌してシッフ塩基を形成させた。反応混合物を濾過して
生成物を回収し、十分に洗浄してｐＨ８．０の炭酸緩衝
液５０ｍｌ，水素化ホウ素ナトリウム２００ｍｇを加え
て５℃で７時間還元反応を行なった。反応混合物を濾過
して生成物を回収し、十分に洗浄して固定化ＩｇＹを得
た。ＩｇＹの固定化量はシッフ塩基反応の残液のＩｇＹ
量をＴＣＡ−ＢＣＡ法により定量して算出したところ、
３０．５ｍｇが固定化されていた。このＩｇＹ固定化担
体について実施例１と同様にしてＬＤＬ吸着率を求め
た。その結果を表１に示す。A carbonate buffer was added to the above [aldehyde]-[IgY] solution to adjust the pH to 9.0, 5 g of the above [amine]-[cellulose] was added, and the mixture was stirred at 5 ° C. for 30 hours. The Schiff base was formed. The reaction mixture was filtered to collect the product, which was thoroughly washed, 50 ml of a pH 8.0 carbonate buffer solution and 200 mg of sodium borohydride were added, and the reduction reaction was carried out at 5 ° C. for 7 hours. The reaction mixture was filtered to collect the product, which was washed thoroughly to obtain immobilized IgY. The immobilized amount of IgY is IgY of the residual liquid of the Schiff base reaction.
When the amount was quantified and calculated by the TCA-BCA method,
30.5 mg was immobilized. The LDL adsorption rate of this IgY-immobilized carrier was determined in the same manner as in Example 1. The results are shown in Table 1.
【００４２】＜実施例３＞ 抗エンドトキシン−ＩｇＹ
の固定化 ウシアルブミンに結合させてハプテン化したＥ．ｃｏｌ
ｉＪ５由来のリピドＡ（リビ社製）を免疫したレグホン
の卵黄から抽出および精製して得られた抗ｈＬＤＬ−Ｉ
ｇＹの多孔質セルロースへの固定化を次のようにして実
施した。<Example 3> Anti-endotoxin-IgY
Immobilization of E. coli haptenized by binding to bovine albumin. col
i J5 derived lipid A anti hLDL-I a (Ribi Inc.) obtained by extraction and purification from egg yolk leghorn immunized
Immobilization of gY on porous cellulose was carried out as follows.
【００４３】過ヨウ素酸ナトリウム８００ｍｇを１Ｎ硫
酸２００ｍｌに溶解して、多孔質セルロース（チッソ社
製「セルロファインＧＣＬー１０００ｍ」）２０ｇを添
加し、攪拌により２０時間反応させた後、反応混合物を
濾過して生成物を回収し、十分に洗浄して［アルデヒ
ド］−［セルロース］を得た。アルデヒド含量はオキシ
ム法により定量を行なったところ０．４３ｍｅｑ／ｇで
あった。800 mg of sodium periodate was dissolved in 200 ml of 1N sulfuric acid, 20 g of porous cellulose (“Cellulofine GCL-1000m” manufactured by Chisso Corporation) was added, and the mixture was reacted for 20 hours by stirring, and then the reaction mixture was filtered. The product was recovered and thoroughly washed to obtain [aldehyde]-[cellulose]. The aldehyde content was 0.43 meq / g as determined by the oxime method.
【００４４】分子量１０００のＰＥＯアミン４ｇをｐＨ
９．５の炭酸緩衝液５０ｍｌに溶解して、上記の［アル
デヒド］−［セルロース］５ｇを加えて攪拌により反応
させて、反応混合物を濾過して生成物を回収し、十分に
洗浄して［ＰＥＯアミン］−［セルロース］を得た。ア
ミノ基含量は０．２２ｍｅｑ／ｇであった。4 g of PEO amine having a molecular weight of 1000 was added to pH.
It was dissolved in 50 ml of 9.5 carbonate buffer, 5 g of the above [aldehyde]-[cellulose] was added and reacted by stirring, and the reaction mixture was filtered to recover the product, which was thoroughly washed [ PEO amine]-[cellulose] was obtained. The amino group content was 0.22 meq / g.
【００４５】一方上記ＩｇＹ２０ｍｇをｐＨ４．５のク
エン酸緩衝液５０ｍｌに溶解して、過ヨウ素酸ナトリウ
ム５０ｍｇを添加して、攪拌により室温で４５分反応さ
せた。さらにエチレングリコールを０．１Ｍ濃度になる
ように添加して、５℃で７時間攪拌して反応させ、反応
液を上記緩衝液を外液として５℃で一晩透析を行ない
［アルデヒド］−［ＩｇＹ］溶液を得た。On the other hand, 20 mg of the above IgY was dissolved in 50 ml of a citrate buffer solution having a pH of 4.5, 50 mg of sodium periodate was added, and the mixture was reacted for 45 minutes at room temperature with stirring. Further, ethylene glycol was added to have a concentration of 0.1 M, and the mixture was reacted by stirring at 5 ° C. for 7 hours, and the reaction solution was dialyzed overnight at 5 ° C. with the above buffer solution as an external solution [aldehyde]-[ An IgY] solution was obtained.
【００４６】上記［アルデヒド］−［ＩｇＹ］液を炭酸
緩衝液を添加してｐＨを９．０に調整し、上記［ＰＥＯ
アミン］−［セルロース］１ｇを加えて、５℃で３０時
間攪拌してシッフ塩基を形成させた。反応混合物を濾過
して生成物を回収し、十分に洗浄してｐＨ８．０の炭酸
緩衝液５０ｍｌ，水素化ホウ素ナトリウム２００ｍｇを
加えて５℃で７時間還元反応を行なった。反応混合物を
濾過して生成物を回収し、十分に洗浄して固定化ＩｇＹ
を得た。ＩｇＹの固定化量は実施例１と同様にして算出
し、１７．３ｍｇが固定化されていた。A carbonate buffer was added to the above [aldehyde]-[IgY] solution to adjust the pH to 9.0, and the above [PEO
1 g of amine]-[cellulose] was added and stirred at 5 ° C. for 30 hours to form a Schiff base. The reaction mixture was filtered to collect the product, which was thoroughly washed, 50 ml of a pH 8.0 carbonate buffer solution and 200 mg of sodium borohydride were added, and the reduction reaction was carried out at 5 ° C. for 7 hours. The reaction mixture was filtered to recover the product, washed thoroughly and immobilized with IgY
Got The immobilized amount of IgY was calculated in the same manner as in Example 1, and 17.3 mg was immobilized.
【００４７】上記ＩｇＹを固定化したセルロースを凍結
乾燥した担体１００ｍｇを滅菌した２０ｍｌのバイヤル
中に秤量して、１０ｎｇ／ｍｌのＥ．ｃｏｌｉ０１１
１：Ｂ４由来のリポポリサッカライド（以下ＬＰＳと言
う，Ｄｉｆｃｏ社製）生理食塩水溶液５ｍｌを加えた。
３７℃で２時間振盪しながらインキュベートを行なった
後、その濾液中の残存ＬＰＳ濃度を「トキシノメーター
ＥＴ２０１」（和光純薬工業製）により定量した。その
結果を表２に示す。100 mg of the freeze-dried carrier on which the above-mentioned IgY-immobilized cellulose was weighed in a sterilized 20 ml vial, and 10 ng / ml of E. coli 011
1: 5 ml of physiological saline solution of lipopolysaccharide derived from B4 (hereinafter referred to as LPS, manufactured by Difco) was added.
After incubating at 37 ° C. for 2 hours with shaking, the residual LPS concentration in the filtrate was quantified by “Toxinometer ET201” (manufactured by Wako Pure Chemical Industries). The results are shown in Table 2.
【００４８】[0048]
【表２】 [Table 2]
【００４９】＜実施例４＞ 抗ヒトヘモグロビン−Ｉｇ
Ｙの固定化 ヒトヘモグロビン（バイオザイムラボラトリー社製）を
免疫したレグホンの卵黄から抽出および精製して得られ
た抗ヒトヘモグロビン−ＩｇＹの多孔質セルロースへの
固定化を次のようにして実施した。<Example 4> Anti-human hemoglobin-Ig
Immobilization of Y The immobilization of anti-human hemoglobin-IgY obtained by extracting and purifying from the egg yolk of leghorn immunized with human hemoglobin (manufactured by Biozyme Laboratory) was carried out as follows.
【００５０】過ヨウ素酸ナトリウム８００ｍｇを１Ｎ硫
酸２００ｍｌに溶解して、多孔質セルロース（チッソ社
製「セルロファインＧＣＬ−１０００ｍ」）２０ｇを添
加し、攪拌により２０時間反応させた後、反応混合物を
濾過して生成物を回収し、十分に洗浄して［アルデヒ
ド］−［セルロース］を得た。アルデヒド含量はオキシ
ム法により定量を行ない０．４８ｍｅｑ／ｇであった。800 mg of sodium periodate was dissolved in 200 ml of 1N sulfuric acid, 20 g of porous cellulose ("Cellulofine GCL-1000m" manufactured by Chisso Corporation) was added, and the mixture was reacted for 20 hours by stirring, and then the reaction mixture was filtered. The product was recovered and thoroughly washed to obtain [aldehyde]-[cellulose]. The aldehyde content was 0.48 meq / g as determined by the oxime method.
【００５１】分子量４０００のＰＥＯアミン１ｇをｐＨ
９．５の炭酸緩衝液５０ｍｌに溶解して、上記の［アル
デヒド］−［セルロース］５ｇを加えて攪拌により反応
させて、反応混合物を濾過して生成物を回収し、十分に
洗浄して［ＰＥＯアミン］−［セルロース］を得た。ア
ミノ基含量は０．２２ｍｅｑ／ｇであった。PH of 1 g of PEO amine having a molecular weight of 4000
It was dissolved in 50 ml of 9.5 carbonate buffer, 5 g of the above [aldehyde]-[cellulose] was added and reacted by stirring, and the reaction mixture was filtered to recover the product, which was thoroughly washed [ PEO amine]-[cellulose] was obtained. The amino group content was 0.22 meq / g.
【００５２】一方上記抗体２０ｍｇをｐＨ４．５のクエ
ン酸緩衝液に５０ｍｌに溶解して、過ヨウ素酸ナトリウ
ム５０ｍｇを添加して、攪拌により室温で４５分反応さ
せた。さらにエチレングリコールを０．１Ｍ濃度になる
ように添加して、５℃で７時間攪拌して反応させ、反応
液を上記緩衝液を外液として５℃で一晩透析を行ない
［アルデヒド］−［ＩｇＹ］溶液を得た。On the other hand, 20 mg of the above antibody was dissolved in 50 ml of a citrate buffer solution having a pH of 4.5, 50 mg of sodium periodate was added, and the mixture was reacted for 45 minutes at room temperature with stirring. Further, ethylene glycol was added to have a concentration of 0.1 M, and the mixture was reacted by stirring at 5 ° C. for 7 hours, and the reaction solution was dialyzed overnight at 5 ° C. with the above buffer solution as an external solution [aldehyde]-[ An IgY] solution was obtained.
【００５３】上記［アルデヒド］−［ＩｇＹ］液に炭酸
緩衝液を添加してｐＨを９．０に調整し、上記［ＰＥＯ
アミン］−［セルロース］１ｇを加えて、５℃で３０時
間攪拌してシッフ塩基を形成させた。反応混合物を濾過
して生成物を回収し、十分に洗浄してｐＨ８．０の炭酸
緩衝液５０ｍｌ，水素化ホウ素ナトリウム２００ｍｇを
加えて５℃で７時間還元反応を行なった。反応混合物を
濾過して生成物を回収し、十分に洗浄して固定化抗体を
得た。抗体の固定化量は実施例１と同様にして算出した
ところ、１７．１ｍｇが固定化されていた。A carbonate buffer solution was added to the above-mentioned [aldehyde]-[IgY] solution to adjust the pH to 9.0.
1 g of amine]-[cellulose] was added and stirred at 5 ° C. for 30 hours to form a Schiff base. The reaction mixture was filtered to collect the product, which was thoroughly washed, 50 ml of a pH 8.0 carbonate buffer solution and 200 mg of sodium borohydride were added, and the reduction reaction was carried out at 5 ° C. for 7 hours. The reaction mixture was filtered to collect the product, which was thoroughly washed to obtain the immobilized antibody. The amount of immobilized antibody was calculated in the same manner as in Example 1, and as a result, 17.1 mg was immobilized.
【００５４】上記ＩｇＹを固定化したセルロース１ｍｌ
を２０ｍｌのバイヤル中に取り、２００ｍｇ／ｄｌのヒ
トヘモグロビン水溶液を加えて５時間振盪後遠心分離を
行ないその上清のヒトヘモグロビン濃度をＴＭＢ（テト
ラメチルベンジジン）法により定量した。その結果を表
３に示す。1 ml of the above-mentioned IgY-immobilized cellulose
Was taken in 20 ml of vial, 200 mg / dl of human hemoglobin aqueous solution was added, and the mixture was shaken for 5 hours and then centrifuged. The concentration of human hemoglobin in the supernatant was quantified by the TMB (tetramethylbenzidine) method. The results are shown in Table 3.
【００５５】[0055]
【表３】 [Table 3]
【００５６】＜比較例１＞ 抗ｈＬＤＬ−ＩｇＹの固定
化 両末端にカルボキシル基を有している分子量５０００の
ポリ（オキシエチレン）（以下ＰＥＯ酸と言う）２３ｇ
をｐＨ４．５のクエン酸緩衝液に溶解して氷冷した後、
１ｍｇの１−エチル−３−（３ージメチルアミノプロピ
ル）−カルボジイミド（以下ＥＤＣと言う）を添加して
３０分間攪拌した。その後実施例１において調製した
［アミン］−［セルロース］５ｇを加えて室温で２０時
間攪拌して反応させた。反応物を濾過して生成物を回収
し、十分に洗浄して［ＰＥＯ酸］−［セルロース］を得
た。<Comparative Example 1> Immobilization of anti-hLDL-IgY 23 g of poly (oxyethylene) having a carboxyl group at both ends and a molecular weight of 5000 (hereinafter referred to as PEO acid)
Was dissolved in a citrate buffer solution having a pH of 4.5 and ice-cooled,
1 mg of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (hereinafter referred to as EDC) was added and stirred for 30 minutes. After that, 5 g of [amine]-[cellulose] prepared in Example 1 was added and reacted at room temperature for 20 hours with stirring. The reaction product was filtered to collect the product, which was thoroughly washed to obtain [PEO acid]-[cellulose].
【００５７】実施例１におけるＩｇＹ４０ｍｇをｐＨ
４．５のクエン酸緩衝液に溶解して氷冷した後、１ｍｇ
のＥＤＣを添加して３０分間攪拌した。その後上記［Ｐ
ＥＯ酸］−［セルロース］を加えて室温で攪拌して反応
させた。反応物を濾過して生成物を回収し、十分に洗浄
して固定化ＩｇＹを得た。ＩｇＹの固定化量はシッフ塩
基反応の残液のＩｇＹ量をＴＣＡ−ＢＣＡ法により定量
して算出したところ、２７．７ｍｇが固定化されてい
た。このＩｇＹ固定化担体について実施例１と同様にし
てＬＤＬ吸着率を求めた。その結果を表１に示す。40 mg of IgY in Example 1 was adjusted to pH.
1 mg after dissolving in 4.5 citrate buffer and cooling on ice
EDC was added and stirred for 30 minutes. After that, [P
EO acid]-[cellulose] was added and reacted at room temperature with stirring. The reaction product was filtered to collect the product, which was thoroughly washed to obtain immobilized IgY. The amount of immobilized IgY was calculated by quantifying the amount of IgY in the residual solution of the Schiff base reaction by the TCA-BCA method, and as a result, 27.7 mg was immobilized. The LDL adsorption rate of this IgY-immobilized carrier was determined in the same manner as in Example 1. The results are shown in Table 1.
【００５８】＜比較例２＞ 抗ｈＬＤＬ−ＩｇＹの固定
化 分子量５０００のＰＥＯアミン２ｇをｐＨ９．５の炭酸
緩衝液５０ｍｌに溶解して、実施例１における［アルデ
ヒド］−［セルロース］５ｇを加えて攪拌により反応さ
せて、反応混合物を濾過して生成物を回収し、十分に洗
浄して［ＰＥＯアミン］−［セルロース］を得た。アミ
ノ基含量はアミノ基含量は塩酸による電位差滴定）によ
り定量を行ない、０．２１ｍｅｑ／ｇであった。<Comparative Example 2> Immobilization of anti-hLDL-IgY 2 g of PEO amine having a molecular weight of 5000 was dissolved in 50 ml of a carbonate buffer having a pH of 9.5, and 5 g of [aldehyde]-[cellulose] in Example 1 was added. After reacting by stirring, the reaction mixture was filtered to collect the product, which was thoroughly washed to obtain [PEO amine]-[cellulose]. The amino group content was determined by potentiometric titration with hydrochloric acid) and found to be 0.21 meq / g.
【００５９】上記［ＰＥＯアミン］−［セルロース］を
ｐＨ９．５の炭酸緩衝液５０ｍｌに懸濁して２５％グル
タルアルデヒド水溶液２ｍｌを加えて室温で２０時間攪
拌により反応させた。反応混合物を濾過して生成物を回
収し、十分に洗浄して［ＰＥＯアルデヒド］−［セルロ
ース］を得た。アルデヒド含量はオキシム法により定量
を行ない０．３８ｍｅｑ／ｇであった。The above [PEO amine]-[cellulose] was suspended in 50 ml of a carbonate buffer solution having a pH of 9.5, 2 ml of a 25% aqueous glutaraldehyde solution was added, and the mixture was reacted at room temperature for 20 hours with stirring. The reaction mixture was filtered to collect the product, which was thoroughly washed to obtain [PEO aldehyde]-[cellulose]. The aldehyde content was 0.38 meq / g as determined by the oxime method.
【００６０】実施例１におけるＩｇＹ４０ｍｇをｐＨ
９．５の炭酸緩衝液５０ｍｌに溶解して、上記［ＰＥＯ
アルデヒド］−［セルロース］を加え、室温で２０時間
攪拌により反応させた。反応混合物を濾過して生成物を
回収し、十分に洗浄して、ｐＨ９．０の炭酸緩衝液５０
ｍｌに懸濁させ、ＮａＢＨ4１ｇを添加して室温で１０
時間攪拌により反応させた。反応混合物を濾過して生成
物を回収し、十分に洗浄して固定化ＩｇＹを得た。Ｉｇ
Ｙの固定化量はシッフ塩基反応の残液のＩｇＹ量をＴＣ
Ａ−ＢＣＡ法により定量して算出し、２２．０ｍｇが固
定化されていた。このＩｇＹ固定化担体について実施例
１と同様にしてＬＤＬ吸着率を求めた。その結果を表１
に示す。40 mg of IgY in Example 1 was adjusted to pH.
Dissolve in 50 ml of 9.5 carbonate buffer and add [PEO]
Aldehyde]-[cellulose] was added and reacted at room temperature for 20 hours with stirring. The reaction mixture was filtered to recover the product, washed thoroughly and washed with 50 mM carbonate buffer, pH 9.0.
Suspend in ml, add 1 g of NaBH 4 and add at room temperature for 10
The reaction was allowed to stir for hours. The reaction mixture was filtered to collect the product, which was washed thoroughly to obtain immobilized IgY. Ig
The amount of immobilized Y was determined by the amount of IgY in the residual solution of the Schiff base reaction being TC
It was quantified and calculated by the A-BCA method, and 22.0 mg was immobilized. The LDL adsorption rate of this IgY-immobilized carrier was determined in the same manner as in Example 1. The results are shown in Table 1.
Shown in.
【００６１】＜比較例３＞ 抗エンドトキシン−ＩｇＹ
の固定化 テトラエチレンペンタミン１１０ｇをｐＨ９．５の炭酸
緩衝液５０ｍｌに溶解して、実施例３で調製した［アル
デヒド］−［セルロース］１０ｇを加えて攪拌により反
応させて、反応混合物を濾過して生成物を回収し十分に
洗浄して［アミン］−［セルロース］を得た。アミノ基
含量は塩酸による電位差滴定により定量を行ない、０．
３１ｍｅｑ／ｇであった。Comparative Example 3 Anti-endotoxin-IgY
Immobilization of tetraethylenepentamine (110 g) was dissolved in 50 ml of carbonate buffer (pH 9.5), 10 g of [aldehyde]-[cellulose] prepared in Example 3 was added and reacted by stirring, and the reaction mixture was filtered. The product was collected and thoroughly washed to obtain [amine]-[cellulose]. Amino group content was determined by potentiometric titration with hydrochloric acid,
It was 31 meq / g.
【００６２】分子量１０００のＰＥＯ酸２３ｇをｐＨ
４．５のクエン酸緩衝液に溶解して氷冷した後、１ｍｇ
のＥＤＣを添加して３０分間攪拌した。その後上記［ア
ミン］−［セルロース］５ｇを加えて室温で２０時間攪
拌して反応させた。反応物を濾過して生成物を回収し、
十分に洗浄して［ＰＥＯ酸］−［セルロース］を得た。23 g of PEO acid having a molecular weight of 1000 was added to pH.
1 mg after dissolving in 4.5 citrate buffer and cooling on ice
EDC was added and stirred for 30 minutes. After that, 5 g of the above [amine]-[cellulose] was added and reacted at room temperature for 20 hours with stirring. The reaction is filtered to recover the product,
It was thoroughly washed to obtain [PEO acid]-[cellulose].
【００６３】実施例３におけるＩｇＹ４０ｍｇをｐＨ
４．５のクエン酸緩衝液に溶解して氷冷した後、１ｍｇ
のＥＤＣを添加して３０分間攪拌した。その後上記［Ｐ
ＥＯ酸］−［セルロース］を加えて室温で攪拌して反応
させた。反応物を濾過して生成物を回収し、十分に洗浄
して固定化ＩｇＹを得た。ＩｇＹの固定化量はシッフ塩
基反応の残液のＩｇＹ量をＴＣＡ−ＢＣＡ法により定量
して算出し、２８．４ｍｇが固定化されていた。このＩ
ｇＹ固定化担体について実施例３と同様にしてＬＰＳ吸
着量を定量した。その結果を表２に示す。40 mg of IgY in Example 3 was adjusted to pH.
1 mg after dissolving in 4.5 citrate buffer and cooling on ice
EDC was added and stirred for 30 minutes. After that, [P
EO acid]-[cellulose] was added and reacted at room temperature with stirring. The reaction product was filtered to collect the product, which was thoroughly washed to obtain immobilized IgY. The immobilized amount of IgY was calculated by quantifying the amount of IgY in the residual liquid of the Schiff base reaction by the TCA-BCA method, and 28.4 mg was immobilized. This I
The amount of LPS adsorbed on the gY-immobilized carrier was quantified in the same manner as in Example 3. The results are shown in Table 2.
【００６４】＜比較例４＞ 抗ヘモグロビン−ＩｇＹの
固定化 テトラエチレンペンタミン１１０ｇをｐＨ９．５の炭酸
緩衝液５０ｍｌに溶解して、実施例４で調製した［アル
デヒド］−［セルロース］１０ｇを加えて攪拌により反
応させて、反応混合物を濾過して生成物を回収し十分に
洗浄して［アミン］−［セルロース］を得た。アミノ基
含量は塩酸による電位差滴定により定量を行ない、０．
３１ｍｅｑ／ｇであった。<Comparative Example 4> Immobilization of anti-hemoglobin-IgY 110 g of tetraethylenepentamine was dissolved in 50 ml of a carbonate buffer of pH 9.5, and 10 g of [aldehyde]-[cellulose] prepared in Example 4 was added. The mixture was reacted by stirring, and the reaction mixture was filtered to collect the product, which was thoroughly washed to obtain [amine]-[cellulose]. Amino group content was determined by potentiometric titration with hydrochloric acid,
It was 31 meq / g.
【００６５】分子量４０００のＰＥＯ酸２３ｇをｐＨ
４．５のクエン酸緩衝液に溶解して氷冷した後、１ｍｇ
のＥＤＣを添加して３０分間攪拌した。その後上記［ア
ミン］−［セルロース］５ｇを加えて室温で２０時間攪
拌して反応させた。反応物を濾過して生成物を回収し、
十分に洗浄して［ＰＥＯ酸］−［セルロース］を得た。23 g of PEO acid having a molecular weight of 4000 was adjusted to pH.
1 mg after dissolving in 4.5 citrate buffer and cooling on ice
EDC was added and stirred for 30 minutes. After that, 5 g of the above [amine]-[cellulose] was added and reacted at room temperature for 20 hours with stirring. The reaction is filtered to recover the product,
It was thoroughly washed to obtain [PEO acid]-[cellulose].
【００６６】実施例３におけるＩｇＹ４０ｍｇをｐＨ
４．５のクエン酸緩衝液に溶解して氷冷した後、１ｍｇ
のＥＤＣを添加して３０分間攪拌した。その後上記［Ｐ
ＥＯ酸］−［セルロース］を加えて室温で攪拌して反応
させた。反応物を濾過して生成物を回収し、十分に洗浄
して固定化ＩｇＹを得た。ＩｇＹの固定化量はシッフ塩
基反応の残液のＩｇＹ量をＴＣＡ−ＢＣＡ法により定量
して算出し、２７．２ｍｇが固定化されていた。このＩ
ｇＹ固定化担体について実施例４と同様にしてヒトヘモ
グロビン吸着量を定量した。その結果を表３に示す。40 mg of IgY in Example 3 was adjusted to pH.
1 mg after dissolving in 4.5 citrate buffer and cooling on ice
EDC was added and stirred for 30 minutes. After that, [P
EO acid]-[cellulose] was added and reacted at room temperature with stirring. The reaction product was filtered to collect the product, which was thoroughly washed to obtain immobilized IgY. The immobilized amount of IgY was calculated by quantifying the amount of IgY in the residual solution of the Schiff base reaction by the TCA-BCA method, and 27.2 mg was immobilized. This I
The amount of human hemoglobin adsorbed on the gY-immobilized carrier was quantified in the same manner as in Example 4. The results are shown in Table 3.
【００６７】[0067]
【発明の効果】本発明により、ＩｇＹをその活性を失活
させたりあるいは変性させたりすることなく水不溶性担
体に固定化することが可能となり、固定化前に近い活性
を維持したＩｇＹ固定化担体を提供することが可能とな
った。本発明は吸着材，アフィニティクロマト用担体，
エンザイムイムノアッセイ，バイオセンサーなど広範囲
な領域において有用なものである。INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to immobilize IgY on a water-insoluble carrier without deactivating or denaturing its activity, and an IgY-immobilized carrier which maintains an activity close to that before immobilization. It has become possible to provide. The present invention relates to an adsorbent, a carrier for affinity chromatography,
It is useful in a wide range of fields such as enzyme immunoassays and biosensors.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｇ０１Ｎ 33/544 Ｚ 9015−2Ｊ (72)発明者 世古 政弘 滋賀県大津市堅田２丁目１番１号 東洋紡 績株式会社総合研究所内 (72)発明者 田中 昌和 滋賀県大津市堅田２丁目１番１号 東洋紡 績株式会社総合研究所内 (72)発明者 金 武祚 三重県四日市市赤堀新町９番５号 太陽化 学株式会社内 (72)発明者 藤木 優 三重県四日市市赤堀新町９番５号 太陽化 学株式会社内 (72)発明者 八田 一 三重県四日市市赤堀新町９番５号 太陽化 学株式会社内─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification number Reference number within the agency FI Technical display location G01N 33/544 Z 9015-2J (72) Inventor Masahiro Seko 2-1-1 Katata, Otsu City, Shiga Prefecture No. Toyobo Co., Ltd. Research Institute (72) Inventor Masakazu Tanaka 2-1-1 Katata, Otsu City, Shiga Prefecture Toyobo Co., Ltd. Research Institute (72) Inventor Kim Takehisa 9-5 Akahori Shinmachi, Yokkaichi City, Mie No. Taiyo Kagaku Co., Ltd. (72) Inventor Yu Fujiki 9-5 Akabori-shinmachi, Yokkaichi-shi, Mie Prefecture Sun Kagaku Co., Ltd. (72) Inventor Hajime Hatta 9-5 Akahori-shinmachi, Yokkaichi-shi, Mie Sun Kagaku Within the corporation
Claims (2)
開裂により形成されるアルデヒド基と、表面にアミノ基
を有する水不溶性担体のアミノ基とでシッフ塩基を形成
させた後これを還元することにより、水不溶性担体に固
定化することを特徴とする鶏卵抗体の固定化方法。1. A Schiff base is formed between an aldehyde group formed by oxidative cleavage of a glycol moiety of a sugar chain of an hen egg antibody and an amino group of a water-insoluble carrier having an amino group on the surface, and then the Schiff base is reduced. A method for immobilizing an egg egg antibody, which comprises immobilizing the antibody on a water-insoluble carrier according to
たものであり、鶏卵抗体がその糖鎖の部位において水不
溶性担体に固定されたものであることを特徴とする鶏卵
抗体固定化担体。2. A chicken egg antibody-immobilized carrier immobilized by the method according to claim 1, wherein the chicken egg antibody is immobilized on a water-insoluble carrier at the sugar chain site.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4176115A JPH05340948A (en) | 1992-06-09 | 1992-06-09 | Transovarial antibody fixed carrier and fixing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4176115A JPH05340948A (en) | 1992-06-09 | 1992-06-09 | Transovarial antibody fixed carrier and fixing method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH05340948A true JPH05340948A (en) | 1993-12-24 |
Family
ID=16007947
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4176115A Withdrawn JPH05340948A (en) | 1992-06-09 | 1992-06-09 | Transovarial antibody fixed carrier and fixing method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH05340948A (en) |
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| WO2002026256A1 (en) * | 2000-09-28 | 2002-04-04 | Shibayagi Co., Ltd. | Excessive antibody production inhibitors, process for producing the same and method of using the same |
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1992
- 1992-06-09 JP JP4176115A patent/JPH05340948A/en not_active Withdrawn
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2002026256A1 (en) * | 2000-09-28 | 2002-04-04 | Shibayagi Co., Ltd. | Excessive antibody production inhibitors, process for producing the same and method of using the same |
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