JP5297009B2 - Immune adjuvant aqueous solution - Google Patents
Immune adjuvant aqueous solution Download PDFInfo
- Publication number
- JP5297009B2 JP5297009B2 JP2007192363A JP2007192363A JP5297009B2 JP 5297009 B2 JP5297009 B2 JP 5297009B2 JP 2007192363 A JP2007192363 A JP 2007192363A JP 2007192363 A JP2007192363 A JP 2007192363A JP 5297009 B2 JP5297009 B2 JP 5297009B2
- Authority
- JP
- Japan
- Prior art keywords
- chitosan
- solution
- aqueous solution
- immunoadjuvant
- cationized
- 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
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Abstract
Description
本発明は、免疫反応において抗原に対する抗体の産生能を向上させる効果を持つ免疫アジュバント水溶液に関し、詳しくはカチオン化キトサンを含む免疫アジュバント水溶液に関する。 The present invention relates to an immune adjuvant aqueous solution having an effect of improving the ability to produce an antibody against an antigen in an immune reaction, and more particularly to an immune adjuvant aqueous solution containing cationized chitosan.
免疫とは、生体が有する病気への抵抗力や治癒力であり、分子レベルで見ると、自分自身である「自己」と、外部から侵入した異物「非自己」とを識別し、「非自己」のみを速やかに処理し、そして除去するシステムである。 Immunity is the resistance and healing power of diseases that the living body has, and at the molecular level, it distinguishes itself from “self” and foreign material that has entered from the outside, “non-self”. Is a system that processes and removes only
免疫には、生体に侵入する病原体を非特異的に直ちに除去する先天性免疫反応（自然免疫）と、自然免疫を突破して生体内に侵入した病原体を特異的に除去する獲得免疫反応とがある。獲得免疫反応では、生体が病原体（抗原）と特異的に相互作用する抗体を産生することで病原体に対抗する。このような病原体に感染する前に病原体に対する抵抗力（免疫）を獲得するためには、死菌や弱毒化した病原体などをワクチンとして生体に接種する必要がある。ここで、接種したワクチンによって長期間に渡り、効果的に抗体を産生するためには、抗原とともに接種し抗体産生の手助けをする免疫アジュバントの選択が非常に大きなカギとなる。 Immunity includes an innate immune reaction (innate immunity) that immediately removes pathogens that invade the living body in a non-specific manner and an acquired immune reaction that specifically removes pathogens that break through innate immunity and enter the body. is there. In the acquired immune response, the organism counters the pathogen by producing an antibody that specifically interacts with the pathogen (antigen). In order to acquire resistance (immunity) to a pathogen before infecting with such a pathogen, it is necessary to inoculate a living body with a dead fungus or an attenuated pathogen as a vaccine. Here, in order to produce an antibody effectively over a long period of time with an inoculated vaccine, selection of an immune adjuvant that inoculates with the antigen and assists in the production of the antibody is a very important key.
現在ワクチンの接種方法は、筋肉注射による接種が大半である。この方法は抗原を直接体内に導入して免疫系を刺激するものであり、血中抗体が免疫の主体となるトキソイドワクチン（破傷風など）や弱毒化した生ウイルスワクチン（麻疹など）など、成功例も多い。 Currently, vaccination is mostly by intramuscular injection. This method stimulates the immune system by introducing antigens directly into the body. Successful examples include toxoid vaccines (such as tetanus) and blood vaccines (such as measles) that are attenuated by blood antibodies. There are also many.
一方、粘膜を介してワクチンを接種し、粘膜で局所的に免疫を成立させる方法は、解決すべき課題が多く開発途上にある。感染症の多くは病原体が粘膜表面に接触して粘膜から侵入するか、又はその部分で定着増菌して始まる。このような粘膜表面での感染を防ぐために、生体は粘膜局所で独自に発達した免疫システムを持っている。しかし、ワクチンを経皮接種しても粘膜局所に免疫はほとんど誘導されず、これまで粘膜を介して感染する病原体に対して効果的なワクチン治療を行うことはできなかった。 On the other hand, the method of inoculating a vaccine through the mucous membrane and establishing immunity locally in the mucosa has many problems to be solved and is under development. Many infectious diseases start when a pathogen contacts the mucosal surface and invades from the mucosa, or colonizes and grows at that part. In order to prevent such infection on the mucosal surface, the living body has an immune system uniquely developed in the mucosal region. However, even when the vaccine is administered transdermally, immunity is hardly induced in the mucosal region, and effective vaccine treatment has not been possible for pathogens that infect through the mucosa.
また、粘膜にワクチンを接種すると、免疫はその粘膜に局所的のみならず全身系の免疫をも成立させることが知られており、多くの感染症でいかに粘膜局所での免疫を成立させるかが、感染防御の決め手になると考えられている。 In addition, it is known that immunization establishes not only local but also systemic immunity to the mucosa when vaccination is given to the mucosa, and how to establish immunity locally in many infectious diseases It is considered to be the decisive factor for infection prevention.
このため、粘膜を介して免疫を成立させるようなワクチン及びその接種方法の開発に期待が寄せられ、ワクチンと免疫アジュバントとの併用などについて世界中で鋭意研究されている。しかし、ワクチン接種による粘膜免疫の成立及び免疫の誘導システムに関してはまだ不明の点が多い。また、アジュバントとしての作用を有する物質として知られているものは、アルム、コレラトキシン、フロイントの完全アジュバント、フロイントの不完全アジュバント、オイルアジュバント、サポニン、ジメチルジオクタデシルアンモニウム臭化物、ヘキサデシルアミン、アブリジン、イスコム、細胞壁骨格構成物、リポポリサッカライド、エンドトキシン及びリポソームなどに限られている。しかも、これらの中には毒性が強いものも多数含まれており、ヒトへの接種に認可の下りたものはアルムのみである。 For this reason, the development of a vaccine that establishes immunity through the mucous membrane and the method of inoculation thereof are expected, and research on the combined use of a vaccine and an immune adjuvant has been conducted all over the world. However, there are still many unclear points regarding the establishment of mucosal immunity and the induction system of immunity by vaccination. Also known as substances having an action as an adjuvant are alum, cholera toxin, Freund's complete adjuvant, Freund's incomplete adjuvant, oil adjuvant, saponin, dimethyldioctadecyl ammonium bromide, hexadecylamine, abridine, It is limited to Iscom, cell wall skeleton construct, lipopolysaccharide, endotoxin and liposome. Moreover, many of these are highly toxic, and only alum is approved for human inoculation.
上記したものの他に、近年、キチン及びキトサンが免疫アジュバントとして有用であることが示されている。キトサンは、安全性が高い天然物由来の多糖であり、工業的にはエビ、カニなどの甲殻類から得られるキチンを脱アセチル化することによって生産されている。例えば、特許文献１は、水溶性のキチンオリゴマー及びキトサンオリゴマーを注射剤により免疫アジュバントとして使用することを開示する。特許文献２は、経口接種して使用されるキチンが免疫促進剤（アジュバント）として作用することを開示する。
しかし、このようにキチン、キトサンを免疫アジュバントとして使用する場合、抗原との結合度が高くはなく、大量に接種しなければならないという問題点がある。
In addition to the above, chitin and chitosan have recently been shown to be useful as immune adjuvants. Chitosan is a highly safe polysaccharide derived from natural products, and is industrially produced by deacetylating chitin obtained from crustaceans such as shrimp and crab. For example, Patent Document 1 discloses the use of a water-soluble chitin oligomer and chitosan oligomer as an immune adjuvant by injection.
However, when chitin and chitosan are used as an immunological adjuvant in this way, there is a problem that the degree of binding to the antigen is not high and a large amount must be inoculated.
キトサン特有の性質として、製膜性、抗菌性、保水性及び凝集能などの機能が知られており、機能性高分子として各方面で実用されている。 Functions such as film-forming properties, antibacterial properties, water retention and aggregating ability are known as properties unique to chitosan and are practically used in various fields as functional polymers.
例えば、特許文献３に開示する方法によれば、キトサンの加水分解によりキトサンオリゴ糖が製造され、これは医薬品、食品、化粧品分野などへ利用されている。また、医療分野においてキチン、キトサンは、特許文献４が開示するように、感染防御物質としての応用が期待されている。
For example, according to the method disclosed in
さらに、キトサンの用途を拡大するため、各種キトサン誘導体が製造されている。中でも、カチオン化キトサン誘導体は、特許文献５が開示するように水溶性が高く、特許文献６が開示するように、免疫賦活剤としての応用も模索されている。
従って、本発明の目的は、上述した従来技術の問題点を解決し、生体への安全性が高く、目的部位に局所的に免疫を誘導できる免疫アジュバント水溶液、免疫アジュバント分散液及びその接種方法を提供することである。 Therefore, an object of the present invention is to solve the above-mentioned problems of the prior art , an immune adjuvant aqueous solution, an immune adjuvant dispersion liquid and an inoculation method thereof that are highly safe to the living body and can induce immunity locally at the target site. Is to provide.
上記目的は以下の本発明によって達成される。すなわち、本発明は、２，３−エポキシプロピルトリメチルアンモニウムクロライドによりカチオン基が導入され、カチオン化度が１〜３であるカチオン化キトサンを、水溶性の有機酸を含む有機酸水溶液に溶解させてカチオン化キトサン水溶液とした後、上記カチオン化キトサン水溶液のｐＨを７．０超、９．０以下に調整したことを特徴とする免疫アジュバント水溶液を提供する。 The above object is achieved by the present invention described below. That is, in the present invention, cationized chitosan having a cationic group introduced by 2,3-epoxypropyltrimethylammonium chloride and having a cationization degree of 1 to 3 is dissolved in an organic acid aqueous solution containing a water-soluble organic acid. Provided is an aqueous immunized adjuvant solution obtained by preparing a cationized chitosan aqueous solution and then adjusting the pH of the cationized chitosan aqueous solution to more than 7.0 and not more than 9.0.
本発明の免疫アジュバント水溶液においては、カチオン化キトサンは、カチオン化キトサンの脱アセチル化度が３０〜１００％であることが好ましい。 In immunoadjuvant solution of the present invention, cationized chitosan is preferably deacetylation degree of mosquitoes thione chitosan is 30% to 100%.
本発明はさらに、上記免疫アジュバント水溶液に、さらにキトサンを分散して含む免疫アジュバント分散液、及び、上記免疫アジュバント水溶液を、霧状としてヒト以外の動物の肺内に噴霧することを特徴とする免疫アジュバントの接種方法を提供する。 The present invention further relates to an immune adjuvant dispersion comprising chitosan further dispersed in the above-mentioned immune adjuvant aqueous solution, and the immune adjuvant aqueous solution sprayed as a mist into the lungs of animals other than humans. Methods for inoculating adjuvants are provided.
本発明によれば、生体への安全性が高く、主に全身系免疫に関与するＩｇＧのみならず、粘膜などで感染防御に関わり、抗ウイルス作用を有する分泌型ＩｇＡの産生能にも優れた免疫アジュバント水溶液、免疫アジュバント分散液及び当該免疫アジュバントの接種方法が提供される。 According to the present invention, safety to the living body is high, and not only IgG mainly involved in systemic immunity but also infection protection in mucous membranes, etc., and excellent ability to produce secretory IgA having antiviral action An immune adjuvant aqueous solution, an immune adjuvant dispersion, and a method for inoculating the immune adjuvant are provided.
次に好ましい実施の形態を挙げて本発明をさらに詳細に説明する。
なお、本発明中において「部」又は「％」とあるのは、特に断りのない限り質量基準である。
まず、本発明の免疫アジュバントを構成するカチオン化キトサンについて説明する。
Next, the present invention will be described in more detail with reference to preferred embodiments.
In the present invention, “part” or “%” is based on mass unless otherwise specified.
First, cationized chitosan constituting the immune adjuvant of the present invention will be described.
本発明で使用するカチオン化キトサンは、天然物からキチンを得て、それを脱アセチル化してキトサンを得、このキトサンをカチオン化したものである。上記キチンは、カニ・エビなど甲殻類の外骨格をはじめとして、イカ、オキアミ及び昆虫並びにキノコなどの菌類などから得られるものであり、その起源は特に限定されることなく、本発明に使用することができる。 The cationized chitosan used in the present invention is obtained by obtaining chitin from a natural product, deacetylating it to obtain chitosan, and cationizing this chitosan. The chitin is obtained from crustacean exoskeletons such as crabs and shrimps, squid, krill and insects, and fungi such as mushrooms, and the origin thereof is not particularly limited and is used in the present invention. be able to.
キチンは、２−アセトアミド−２−デオキシ−Ｄ−グルコース（Ｎ−アセチルグルコサミン）を構成単位とする天然高分子である。一方、キトサンは、キチンの脱アセチル化物であり、２−アミノ−２−デオキシ−Ｄ−グルコース（グルコサミン）を構成単位とする塩基性多糖類である。キトサンは、既に工業的に生産されており、種々のグレードのものを入手することができる。本発明に使用するキトサンは、起源及び製法に特別な制限はなく、従来より工業生産されているキトサンを使用することができる。 Chitin is a natural polymer having 2-acetamido-2-deoxy-D-glucose (N-acetylglucosamine) as a structural unit. On the other hand, chitosan is a deacetylated product of chitin and is a basic polysaccharide having 2-amino-2-deoxy-D-glucose (glucosamine) as a structural unit. Chitosan has already been industrially produced, and various grades are available. The chitosan used in the present invention is not particularly limited in its origin and production method, and chitosan that has been conventionally industrially produced can be used.
本発明のカチオン化キトサンの製造方法には特に制限はないが、
（１）上記キトサンに３級アミノ基又は４級アンモニウム基或いはその両方（以下「カチオン基」という）を持つ化合物を用いて、カチオン基をキトサンに導入する方法；又は、
（２）アルキル化剤を用いてキトサンのアミノ基をアルキル化し、該アミノ基をカチオン化する方法；を好適に使用することができる。
There is no particular limitation on the method for producing the cationized chitosan of the present invention,
(1) A method of introducing a cationic group into chitosan using a compound having a tertiary amino group or a quaternary ammonium group or both (hereinafter referred to as “cationic group”) in the chitosan; or
(2) A method of alkylating an amino group of chitosan using an alkylating agent and cationizing the amino group can be suitably used.
上記（１）の方法に使用できる、カチオン基を持つ化合物としては、例えば、２−クロロエチルジエチルアミン又はその塩酸塩、３−クロロ−２−ヒドロキシプロピルジエチルアミン、２，３−エポキシプロピルジメチルアミン、トリメチル−３−クロロ−２−ヒドロキシプロピルトリメチルアンモニウムクロライド、２，３−エポキシプロピルトリメチルアンモニウムクロライドなどが挙げられる。一方、（２）の方法に使用するアルキル化剤としては、例えば、ヨウ化メチル及びヨウ化エチルなどを挙げることができる。 Examples of the compound having a cationic group that can be used in the method (1) include 2-chloroethyldiethylamine or its hydrochloride, 3-chloro-2-hydroxypropyldiethylamine, 2,3-epoxypropyldimethylamine, and trimethyl. Examples include -3-chloro-2-hydroxypropyltrimethylammonium chloride and 2,3-epoxypropyltrimethylammonium chloride. On the other hand, examples of the alkylating agent used in the method (2) include methyl iodide and ethyl iodide.
本発明におけるカチオン化キトサンのカチオン化度は、以下の式１により定義される。
（式１）カチオン化度＝キトサンに導入されたカチオン基の数÷原料キトサンを構成するグルコサミン残基の数
上記カチオン化度はキトサン及びカチオン化キトサンのコロイド当量値から、以下の式２により求めることができる。
（式２）カチオン化度＝ｐＨ９でのカチオン化キトサンのコロイド当量値÷（ｐＨ３でのカチオン化キトサンのコロイド当量値−ｐＨ９でのカチオン化キトサンのコロイド当量値）
なお、ｐＨ３では全アミノ基及び４級アンモニウム基のコロイド当量値を求めることができ、ｐＨ９では４級アンモニウム基のコロイド当量値を求めることができる。
The degree of cationization of the cationized chitosan in the present invention is defined by the following formula 1.
(Formula 1) Degree of cation = number of cationic groups introduced into chitosan / number of glucosamine residues constituting raw material chitosan The degree of cationization is obtained from the colloid equivalent value of chitosan and cationized chitosan according to the following
(Formula 2) Degree of cationization = colloidal equivalent value of cationized chitosan at pH 9 / (colloidal equivalent value of cationized chitosan at
At
キトサンは水に不溶であり、ほとんどの希酸には可溶であることが知られている。原料キトサンをカチオン化することにより、親水性を高め、その水溶性を向上させることができる。カチオン化キトサンのカチオン化度が０．１未満では、カチオン性、親水性共にキトサン本来の性質と同程度であり、免疫アジュバントとして使用したときに十分な効果が得られない。したがって、本発明の免疫アジュバントに用いるカチオン化キトサンのカチオン化度は、０．１以上であることが好ましい。カチオン化度が約１以上となると、酸を用いずに水に溶解することができ、本発明の免疫アジュバントとしてより好ましい形態の一つである。なお、カチオン化度が３を超えても、免疫アジュバントとしての効果は向上しないため、製造工程上のコスト面で非効率である。 Chitosan is known to be insoluble in water and soluble in most dilute acids. By cationizing raw material chitosan, hydrophilicity can be improved and its water solubility can be improved. When the degree of cationization of the cationized chitosan is less than 0.1, both the cationic property and the hydrophilic property are the same as those of chitosan, and sufficient effects cannot be obtained when used as an immune adjuvant. Therefore, the cationization degree of the cationized chitosan used for the immunoadjuvant of the present invention is preferably 0.1 or more. When the degree of cationization is about 1 or more, it can be dissolved in water without using an acid, which is one of the more preferable forms of the immunoadjuvant of the present invention. In addition, even if the degree of cationization exceeds 3, the effect as an immune adjuvant is not improved, so that it is inefficient in terms of cost in the production process.
前記（１）のカチオン基の導入方法の例として、キトサン８部を７０％含水イソプロピルアルコール１４０部中に分散させ、次いでこの分散液に、２，３−エポキシプロピルトリメチルアンモニウムクロライド１３部を加えて、６５〜７０℃で２４時間撹拌しながら反応させる方法を挙げることができ、この反応後にカチオン化キトサンを得ることができる。 As an example of the method for introducing a cationic group of (1), 8 parts of chitosan is dispersed in 140 parts of 70% hydrous isopropyl alcohol, and then 13 parts of 2,3-epoxypropyltrimethylammonium chloride is added to this dispersion. And a method of reacting with stirring at 65 to 70 ° C. for 24 hours, and cationized chitosan can be obtained after this reaction.
この例において、原料キトサンの脱アセチル化度、分子量、イソプロピルアルコールなどの反応溶媒の含水率、キトサンとカチオン化基を有する化合物（例：２，３−エポキシプロピルトリメチルアンモニウムクロライド）との当量比、反応時間、反応温度などを変えることにより、カチオン化度を調整することができる。 In this example, the degree of deacetylation of the raw material chitosan, the molecular weight, the water content of the reaction solvent such as isopropyl alcohol, the equivalent ratio of chitosan and the compound having a cationized group (eg, 2,3-epoxypropyltrimethylammonium chloride), The degree of cationization can be adjusted by changing the reaction time, reaction temperature, and the like.
前記（２）の方法により、本発明の免疫アジュバントに適するカチオン化度が０．１〜３であるカチオン化キトサンを得るためには、原料キトサンを構成するピラノース環１個（１モル）当たり０．３モル以上１０モル以下のアルキル化剤を加えて反応するとよい。 In order to obtain a cationized chitosan having a degree of cationization of 0.1 to 3 suitable for the immunoadjuvant of the present invention by the method (2), 0 per 1 pyranose ring (1 mole) constituting the raw material chitosan. The reaction may be carried out by adding 3 to 10 moles of an alkylating agent.
本発明の免疫アジュバントに使用するカチオン化キトサンの脱アセチル化度に特別な制限はなく、用途に応じて最適な条件を適宜選択することができるが、脱アセチル化度が３０％未満では、得られるカチオン化キトサンのカチオン性及び親水性が不十分であり、通常、脱アセチル化度３０％以上が好ましい。カチオン化キトサンのカチオン性及び親水性の点から、脱アセチル化度は、６０％〜１００％であることがより好ましく、さらに好ましくは８０％〜１００％である。 There is no particular limitation on the degree of deacetylation of the cationized chitosan used in the immunoadjuvant of the present invention, and the optimum conditions can be appropriately selected according to the application. However, when the degree of deacetylation is less than 30%, The cationicity and hydrophilicity of the resulting cationized chitosan are insufficient, and usually a degree of deacetylation of 30% or more is preferred. From the viewpoint of cationicity and hydrophilicity of the cationized chitosan, the degree of deacetylation is more preferably 60% to 100%, and still more preferably 80% to 100%.
（脱アセチル化度測定方法）
上記キトサンの脱アセチル化度は、コロイド滴定を行い、その滴定量から算出することができる。具体的には、指示薬にトルイジンブルー溶液を用い、ポリビニル硫酸カリウム水溶液でコロイド滴定することにより、キトサン分子中の遊離アミノ基を定量し、キトサンの脱アセチル化度を求める。以下に脱アセチル化度測定方法の一例を示す。
(Deacetylation degree measuring method)
The degree of deacetylation of chitosan can be calculated from the titration amount after colloid titration. Specifically, a toluidine blue solution is used as an indicator and colloidal titration with an aqueous polyvinyl potassium sulfate solution to quantify the free amino groups in the chitosan molecule and determine the degree of deacetylation of chitosan. An example of a method for measuring the degree of deacetylation is shown below.
１．滴定試験
０．５％酢酸水溶液にキトサン純分濃度が０．５％となるようにキトサンを添加し、キトサンを攪拌、溶解して１００ｇの０．５％キトサン／０．５％酢酸水溶液を調製する。次にこの溶液１０ｇとイオン交換水９０ｇを攪拌混合して、０．０５％のキトサン溶液を調製する。さらにこの０．０５％キトサン溶液１０ｇにイオン交換水５０ｍｌ、トルイジンブルー溶液約０．２ｍｌを添加して試料溶液を調製し、ポリビニル硫酸カリウム溶液（Ｎ／４００ＰＶＳＫ）にて滴定する。滴定速度は２ｍｌ／分〜５ｍｌ／分とし、試料溶液が青から赤紫色に変色後、３０秒間以上保持する点を終点の滴定量とする。
なお、キトサン純分とは、原料キトサン試料中のキトサンの質量を意味し、具体的には、キトサン試料を１０５℃、２時間乾燥して求められる固形分質量である。
1. Titration test Add chitosan to 0.5% acetic acid aqueous solution so that the concentration of pure chitosan is 0.5%, stir and dissolve chitosan to prepare 100 g of 0.5% chitosan / 0.5% acetic acid aqueous solution. To do. Next, 10 g of this solution and 90 g of ion-exchanged water are mixed with stirring to prepare a 0.05% chitosan solution. Further, 50 ml of ion exchange water and about 0.2 ml of toluidine blue solution are added to 10 g of this 0.05% chitosan solution to prepare a sample solution, and titrated with a polyvinyl potassium sulfate solution (N / 400 PVSK). The titration rate is 2 ml / min to 5 ml / min, and the point at which the sample solution is held from 30 to 30 seconds after the color changes from blue to reddish purple is used as the end point titration.
The pure chitosan content means the mass of chitosan in the raw material chitosan sample. Specifically, it is the solid content mass obtained by drying the chitosan sample at 105 ° C. for 2 hours.
２．空試験
上記滴定試験に使用した０．５％キトサン／０．５％酢酸水溶液の代わりに、イオン交換水を使用し、同様の滴定試験を行う。
2. Blank test A similar titration test is performed using ion-exchanged water instead of the 0.5% chitosan / 0.5% acetic acid aqueous solution used in the above titration test.
３．アセチル化度の計算
Ｘ＝１／４００×１６１×ｆ×（Ｖ−Ｂ）／１０００
＝０．４０２５×ｆ×（Ｖ−Ｂ）／１０００
Ｙ＝０．５／１００−Ｘ
Ｘ：キトサン中の遊離アミノ基質量
（グルコサミン残基質量に相当）
Ｙ：キトサン中の結合アミノ基質量
（Ｎ−アセチルグルコサミン残基質量に相当）
ｆ：Ｎ／４００ＰＶＳＫの力価
Ｖ：試料溶液の滴定量（ｍｌ）
Ｂ：空試験滴定量（ｍｌ）
脱アセチル化度（％）
＝（遊離アミノ基）／｛（遊離アミノ基）＋（結合アミノ基）｝×１００
＝（Ｘ／１６１）／（Ｘ／１６１＋Ｙ／２０３）×１００
なお、１６１はグルコサミン残基の分子量、２０３はＮ−アセチルグルコサミン残基の分子量である。
3. Calculation of degree of acetylation X = 1/400 × 161 × f × (V−B) / 1000
= 0.4025 * f * (V-B) / 1000
Y = 0.5 / 100-X
X: Mass of free amino group in chitosan (equivalent to glucosamine residue mass)
Y: Mass of bound amino group in chitosan (corresponding to mass of N-acetylglucosamine residue)
f: titer of N / 400 PVSK V: titration of sample solution (ml)
B: Blank test titration (ml)
Deacetylation degree (%)
= (Free amino group) / {(free amino group) + (bonded amino group)} × 100
= (X / 161) / (X / 161 + Y / 203) × 100
161 is the molecular weight of the glucosamine residue, and 203 is the molecular weight of the N-acetylglucosamine residue.
本発明の免疫アジュバントに含まれるカチオン化キトサンの原料キトサンは、１％酢酸水溶液に、当該原料キトサンを１％となるように溶解させたときに、当該水溶液の粘度が１ｍＰａ・ｓ〜１０，０００ｍＰａ・ｓとなるものが好ましい。上記粘度が１ｍＰａ・ｓ未満となる原料キトサンは、低分子化のためのコストがかさみ、製造コスト上不利である。一方、粘度が１０，０００ｍＰａ・ｓを超えるとカチオン化キトサン水溶液としたときに粘度が高くなりすぎ、水溶液中のカチオン化キトサンの濃度を低くしなければならず、また、接種方法も限定されるため、好ましくない。 The raw material chitosan of cationized chitosan contained in the immunoadjuvant of the present invention has a viscosity of 1 mPa · s to 10,000 mPa when the raw material chitosan is dissolved in 1% acetic acid aqueous solution so as to be 1%. -What becomes s is preferable. The raw material chitosan having a viscosity of less than 1 mPa · s is disadvantageous in terms of production cost due to the high cost for reducing the molecular weight. On the other hand, when the viscosity exceeds 10,000 mPa · s, the viscosity becomes too high when the aqueous cationized chitosan solution is used, the concentration of the cationized chitosan in the aqueous solution must be lowered, and the inoculation method is also limited. Therefore, it is not preferable.
なお、上記キトサン水溶液の粘度は、例えば、１％酢酸水溶液にキトサン純分濃度が１％となるようにキトサンを溶解させ、この溶液を、恒温槽にて２０℃に保ちながらＢ型回転粘度計を用いて粘度測定を行うことにより、決定することができる。 The viscosity of the chitosan aqueous solution is, for example, a B-type rotational viscometer in which chitosan is dissolved in a 1% acetic acid aqueous solution so that the concentration of pure chitosan is 1% and this solution is kept at 20 ° C. in a thermostatic bath Can be determined by measuring the viscosity using
本発明の免疫アジュバントは、前記したように水に対する溶解度が高いので、水に溶かして免疫アジュバント水溶液として使用することができる。
当該免疫アジュバント水溶液におけるカチオン化キトサンの含有量は、全質量中０．１〜２０％を占める量であることが好ましく、１〜５％含有することがより好ましい。カチオン化キトサンの量が０．１％未満では、免疫応答を促進するという免疫アジュバントとしての効果が十分には得られず、一方、添加量が２０％を超えるとカチオン化キトサン水溶液が流動性を失い、接種に不便であり好ましくない。
Since the immunoadjuvant of the present invention has high solubility in water as described above, it can be dissolved in water and used as an immune adjuvant aqueous solution.
The content of the cationized chitosan in the immune adjuvant aqueous solution is preferably an amount that occupies 0.1 to 20% of the total mass, and more preferably 1 to 5%. If the amount of the cationized chitosan is less than 0.1%, the effect as an immune adjuvant for promoting the immune response is not sufficiently obtained. On the other hand, if the amount added exceeds 20%, the aqueous cationized chitosan solution becomes fluid. Lost, inconvenient to inoculate, not preferred.
本発明の免疫アジュバント水溶液は、そのｐＨが４．５〜９．０であることが好ましい。ｐＨが４．５より低いと生物に悪影響を与えるという点で問題があり、９．０を超えるとカチオン化キトサンが析出するという問題があり好ましくない。 The aqueous immune adjuvant solution of the present invention preferably has a pH of 4.5 to 9.0. If the pH is lower than 4.5, there is a problem in that it adversely affects the organism, and if it exceeds 9.0, there is a problem that cationized chitosan precipitates, which is not preferable.
ｐＨの調整に使用する物質は、特に制限されず、当業者に周知の物質を使用することができる。ｐＨ調製に使用するアルカリ物質としては、アルカリ金属の水酸化物又は炭酸塩、アンモニア及びアミン類があり、具体的には、水酸化リチウム、水酸化ナトリウム、水酸化カリウム、炭酸ナトリウム、炭酸水素ナトリウム、炭酸カリウム、アンモニア、モルホリン、Ｎ−メチルモルホリン、トリメチルアミン、トリエチルアミン、エタノールアミン、ジエタノールアミン、トリエタノールアミン、プロパノールアミン、ジプロパノールアミン、トリプロパノールアミン、Ｎ−メチルエタノールアミン、Ｎ，Ｎ−ジメチルエタノールアミン、Ｎ−エチルエタノールアミン、Ｎ，Ｎ−ジエチルエタノールアミン、Ｎ−メチルジエタノールアミン、Ｎ−エチルジエタノールアミン、Ｎ−メチルプロパノールアミン、Ｎ，Ｎ−ジメチルプロパノールアミン及びアミノメチルプロパノールなどを挙げることができる。特に好ましいものは、水酸化ナトリウム及びアンモニアである。 The substance used for adjusting the pH is not particularly limited, and substances well known to those skilled in the art can be used. Alkali substances used for pH adjustment include alkali metal hydroxides or carbonates, ammonia and amines, specifically lithium hydroxide, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate. , Potassium carbonate, ammonia, morpholine, N-methylmorpholine, trimethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine, propanolamine, dipropanolamine, tripropanolamine, N-methylethanolamine, N, N-dimethylethanolamine N-ethylethanolamine, N, N-diethylethanolamine, N-methyldiethanolamine, N-ethyldiethanolamine, N-methylpropanolamine, N, N-dimethylpropanol , And the like Min and aminomethyl propanol. Particularly preferred are sodium hydroxide and ammonia.
また、本発明の免疫アジュバント水溶液は、水溶性の有機酸をさらに含んでもよい。本発明の免疫アジュバント水溶液が水溶性の有機酸をさらに含むことにより、カチオン化キトサンの不溶解分が減少し、カチオン化キトサンの使用量を減らすことができるという利点がある。当該有機酸は水溶性であれば特に制限されず、例えば、ギ酸、酢酸、プロピオン酸、酪酸、タウリン、ピロリドンカルボン酸、クエン酸、リンゴ酸、乳酸、ヒドロキシマロン酸、マロン酸、コハク酸、アジピン酸、安息香酸、サリチル酸、アミノ安息香酸、フタル酸、けい皮酸及びビタミンＣなどを挙げることができる。
本発明の免疫アジュバント水溶液が有機酸を含む場合、カチオン化キトサン１００部に対し有機酸は３００部以下であることが好ましい。
Moreover, the immune adjuvant aqueous solution of the present invention may further contain a water-soluble organic acid. When the immunoadjuvant aqueous solution of the present invention further contains a water-soluble organic acid, there is an advantage that the insoluble content of the cationized chitosan is reduced and the amount of the cationized chitosan used can be reduced. The organic acid is not particularly limited as long as it is water-soluble. Examples thereof include acid, benzoic acid, salicylic acid, aminobenzoic acid, phthalic acid, cinnamic acid, and vitamin C.
When the aqueous immune adjuvant solution of the present invention contains an organic acid, the organic acid is preferably 300 parts or less with respect to 100 parts of cationized chitosan.
本発明の免疫アジュバント水溶液は、水溶性の無機酸を含んでもよく、このような無機酸の例として、塩酸、リン酸、硝酸及び硫酸などを挙げることができる。 The aqueous immune adjuvant solution of the present invention may contain a water-soluble inorganic acid, and examples of such inorganic acid include hydrochloric acid, phosphoric acid, nitric acid and sulfuric acid.
また、本発明の免疫アジュバント水溶液は、水に不溶のキトサンをさらに含む分散液であってもよい。キトサンを含むことにより、その接種部位によっては、アジュバント残存量が増加し、免疫促進効果が持続するという効果を生ずるため好ましい。 Moreover, the immunoadjuvant aqueous solution of the present invention may be a dispersion further containing chitosan insoluble in water. By including chitosan, depending on the inoculation site, the amount of residual adjuvant is increased, and the effect of sustaining the immune promoting effect is preferable.
本発明の免疫アジュバント水溶液は、当業者に周知の種々の接種方法により接種することができるが、当該水溶液を霧状として粘膜に噴霧することにより、より適切なアジュバント効果を発揮することができる。接種部位にも特に制限はないが、鼻腔内、口腔内、肺内、膣内及び直腸内に接種することが好ましく、肺内に接種することが最も好ましい。本発明の免疫アジュバント水溶液を、経鼻接種及び経口接種する場合、カチオン化キトサンの濃度は０．５〜５％が好ましい。また、経肺接種の場合、０．１〜３％が好ましい。 The immunoadjuvant aqueous solution of the present invention can be inoculated by various inoculation methods well known to those skilled in the art, but a more appropriate adjuvant effect can be exhibited by spraying the aqueous solution as a mist on the mucous membrane. The inoculation site is not particularly limited, but it is preferably inoculated intranasally, in the oral cavity, in the lung, in the vagina and in the rectum, and most preferably inoculated into the lung. When the immunoadjuvant aqueous solution of the present invention is nasally or orally inoculated, the concentration of cationized chitosan is preferably 0.5 to 5%. In the case of transpulmonary inoculation, 0.1 to 3% is preferable.
本発明の免疫アジュバント、その水溶液及び分散液を接種できる動物は、特に制限されず、例として、ニワトリ、ブタ、ウマ、ヤギ、ヒツジなどの家畜類、ネコ、イヌ、ハムスター、ウサギ、鳥などの愛玩動物、ペット類、魚類、海洋生物並びにマウス、サル及びヒトを挙げることができる。 Animals that can be inoculated with the immune adjuvant of the present invention, aqueous solutions and dispersions thereof are not particularly limited, and examples thereof include domestic animals such as chickens, pigs, horses, goats, sheep, cats, dogs, hamsters, rabbits, birds, etc. Mention may be made of pets, pets, fish, marine life and mice, monkeys and humans.
抗体産生能の高い免疫アジュバントの条件としては、まず目的の抗原物質と結合しやすいことが必要である。また、細胞内に取り込まれやすいこと、および、細胞内で長期間にわたって抗原物質を徐放することが必要である。本発明の免疫アジュバントの作用機序は不明であるが、このような条件を満たしているために、少ないワクチン接種量で長期間免疫効果を発揮することができるものと考えられる。 As conditions for an immunoadjuvant with high antibody-producing ability, it is first necessary to easily bind to a target antigenic substance. In addition, it is necessary to be easily taken up into cells and to release the antigen substance over a long period of time in the cells. Although the mechanism of action of the immunoadjuvant of the present invention is unknown, it is considered that the immune effect can be exerted for a long period of time with a small amount of vaccination because such conditions are satisfied.
以下、実施例及び比較例を挙げて本発明を具体的に説明するが、本発明はこれらに限定されるものではない。 EXAMPLES Hereinafter, although an Example and a comparative example are given and this invention is demonstrated concretely, this invention is not limited to these.
［実施例１］
（マウスへのＯＶＡ抗原の免疫）
カニ殻より調製した脱アセチル化度８７％のキトサンフレーク１部に２，３−エポキシプロピルトリメチルアンモニウムクロライド１部及び精製水１部を混合後、６５℃にて２４時間加熱し、カチオン化度１．１のカチオン化キトサンを得た。このカチオン化キトサンを含水イソプロピルアルコールで洗浄後、乾燥して固形カチオン化キトサンを得、これを以下の実験に使用した。なお、カチオン化度はキトサン及びカチオン化キトサンのコロイド当量値より算出した。
[Example 1]
(Immune immunization of mice with OVA antigen)
After mixing 1 part of 2,3-epoxypropyltrimethylammonium chloride and 1 part of purified water with 1 part of chitosan flakes with 87% deacetylation prepared from crab shell, the mixture was heated at 65 ° C. for 24 hours, and the degree of cationization was 1 .1 cationized chitosan was obtained. This cationized chitosan was washed with hydrous isopropyl alcohol and then dried to obtain solid cationized chitosan, which was used in the following experiments. The degree of cationization was calculated from the colloid equivalent value of chitosan and cationized chitosan.
固形カチオン化キトサンを４％クエン酸溶液に溶解し、不溶解分を濾過して２％カチオン化キトサン水溶液を得た。次いで、当該２％カチオン化キトサン水溶液５部に対して０．２５％ＯＶＡ（卵白由来アルブミン）／１×ＰＢＳ溶液２部を加え、塩酸及び水酸化ナトリウムを用いてｐＨを８．０に調製し、カチオン化キトサン−ＯＶＡ混合液を得た。 Solid cationized chitosan was dissolved in 4% citric acid solution, and the insoluble matter was filtered to obtain a 2% cationized chitosan aqueous solution. Next, 2 parts of 0.25% OVA (egg white albumin) / 1 × PBS solution is added to 5 parts of the 2% cationized chitosan aqueous solution, and the pH is adjusted to 8.0 using hydrochloric acid and sodium hydroxide. A cationized chitosan-OVA mixed solution was obtained.
当該混合液７μＬを、ジエチルエーテルで麻酔した６週齢、雌のＢＡＬＢ／ｃマウス５匹に経鼻接種した。この経鼻接種の日から１週間ごとに計３回、上記混合液７μＬを経鼻接種した。この３回目の経鼻接種の日を第０週としてその後、週に一度、以下の要領で血液、便及び膣洗浄液を得た。また、ブースター効果を確認するために、上記３回目の経鼻接種の日から２〜３ヶ月の後、同様に４回目の経鼻接種を行った。
7 μL of the mixed solution was intranasally inoculated into five 6-week-old female BALB / c mice anesthetized with diethyl ether. From the day of this nasal inoculation, 7 μL of the above mixed solution was inoculated nasally three times every week. The day of this third nasal inoculation was set as
血液：ジエチルエーテルで麻酔後、尾静脈から採血した。
便：各個体を樹脂製ケージ中に数分間放置し、便をエッペンドルフチューブに回収した。
膣洗浄液：１％ＢＳＡ（ウシ血清アルブミン）５０μＬを膣中でピペッティングした。この作業を２回行い、膣洗浄液を得た。
Blood: Blood was collected from the tail vein after anesthesia with diethyl ether.
Stool: Each individual was left in a resin cage for several minutes, and the stool was collected in an Eppendorf tube.
Vaginal lavage fluid: 50 μL of 1% BSA (bovine serum albumin) was pipetted into the vagina. This operation was performed twice to obtain a vaginal wash.
（サンプルの調製）
上記手順により得た血液、便及び膣洗浄液を以下のように処理し、抗体価測定のためのサンプルを調製した。
血清サンプル：上記血液を室温にて、１５，０００ｒ．ｐ．ｍ．で１５分間遠心分離し、その上清を再度同じ条件で遠心分離して得た上清を血清サンプルとした。当該サンプルは、使用するまで−３０℃で冷凍保存した。
便サンプル：上記便を４℃にて１％ＢＳＡ中に分散し、この分散液を４℃にて、１５，０００ｒ．ｐ．ｍ．で１５分間遠心分離して、その上清を便サンプルとした。当該サンプルは、使用するまで−３０℃で冷凍保存した。
膣洗浄液サンプル：上記膣洗浄液を室温にて、１５，０００ｒ．ｐ．ｍ．で１５分間遠心分離し、その上清を膣洗浄液サンプルとした。当該サンプルは使用するまで−３０℃で冷凍保存した。
(Sample preparation)
The blood, stool and vaginal lavage fluid obtained by the above procedure were processed as follows to prepare samples for antibody titer measurement.
Serum sample: The above blood was collected at room temperature at 15,000 r. p. m. Was centrifuged for 15 minutes, and the supernatant was centrifuged again under the same conditions to obtain a serum sample. The sample was stored frozen at −30 ° C. until use.
Stool sample: The above stool was dispersed in 1% BSA at 4 ° C., and this dispersion was dispersed at 15,000 r. p. m. The supernatant was used as a stool sample. The sample was stored frozen at −30 ° C. until use.
Vaginal washing liquid sample: The above-mentioned vaginal washing liquid was mixed at room temperature at 15,000 r. p. m. For 15 minutes, and the supernatant was used as a vaginal wash sample. The sample was stored frozen at −30 ° C. until use.
ＥＬＩＳＡ法を用いて、各サンプルについてＩｇＧ抗体価及びＩｇＡ抗体価を測定した。当該測定は以下の手順で行った。
（ＩｇＧ抗体価の測定）
ＯＶＡ抗原（生化学工業株式会社製）をコーティングバッファー（精製水１ＬにＮａ2ＣＯ3：１．６ｇ、ＮａＨＣＯ3：２．８２ｇ、ＮａＮ3：０．１ｇを含む；ｐＨ９．０）で５μｇ／ｍＬとなるように調製し、この溶液を９６穴ＥＬＩＳＡプレート（Costar3690 A/2、コーニング社）に１穴あたり５０μＬずつ分注し、４℃で一晩静置して、抗原を吸着（コーティング）させた。この際、プレート表面にプレートシールを貼って溶液の乾燥を防いだ。
Using the ELISA method, IgG antibody titer and IgA antibody titer were measured for each sample. The measurement was performed according to the following procedure.
(Measurement of IgG antibody titer)
OVA antigen (manufactured by Seikagaku Corporation) with a coating buffer (containing 1 g of purified water containing Na 2 CO 3 : 1.6 g, NaHCO 3 : 2.82 g, NaN 3 : 0.1 g; pH 9.0) at 5 μg / Prepare 50 mL of the solution in a 96-well ELISA plate (Costar3690 A / 2, Corning) and leave it at 4 ° C. overnight to adsorb the antigen (coating). I let you. At this time, a plate seal was stuck on the plate surface to prevent the solution from drying.
上記コーティング終了後、コーティングバッファーを廃棄し、次いでＥＬＩＳＡプレートにブロッキングバッファー（精製水１Ｌに１０×ダルベッコＰＢＳ：１００ｍＬ、アルブミン（Ｆｒ．Ｖ）：１０ｇ、ＮａＮ3：１ｇを含む）を１穴あたり１００μＬずつ分注し、１時間放置した。
ＥＬＩＳＡプレートとは別にＵ底プレートを用意し、上記各サンプルを２6〜２15倍に希釈した１０段階の２倍希釈系列を調製した。希釈にはブロッキングバッファーを用い、最終希釈測定法（エンド・ポイント・ダイリューション法）を行った。
After the above coating is completed, the coating buffer is discarded, and then blocking buffer (containing 10 × Dulbecco's PBS: 100 mL, albumin (Fr.V): 10 g, NaN 3 : 1 g in 1 L of purified water) is added to the ELISA plate at 100 μL per well. Aliquoted and left for 1 hour.
A U-bottom plate was prepared separately from the ELISA plate, and a 10-fold two-fold dilution series was prepared by diluting the above samples by 2 6 to 2 15 times. For the dilution, a blocking buffer was used, and the final dilution measurement method (end point dilution method) was performed.
上記ブロッキング終了後、ＥＬＩＳＡプレートからブロッキングバッファーを廃棄し、Ｕ底プレートで調製したサンプルを１穴あたり５０μＬずつ分注して３７℃で１時間インキュベートした。この際、プレート表面にプレートシールを貼って溶液の乾燥を防いだ。
なお、陽性コントロールとしては、単クローン抗体抗鶏卵アルブミン（MONOCLONAL ANTI-CHICKEN EGG ALBUMIN CLONE OVA）（ＳＩＧＭＡ社）を１万〜５１２万倍に希釈した１０段階の２倍希釈系列を用いた。また、陰性コントロールとしては、ＯＶＡ抗原を免疫していないマウスから、上記と同様にして調製した血清サンプルをブロッキングバッファーで希釈して調製した２6〜２15の１０段階の２倍希釈系列を用いた。
After the blocking, the blocking buffer was discarded from the ELISA plate, and the sample prepared on the U-bottom plate was dispensed at 50 μL per well and incubated at 37 ° C. for 1 hour. At this time, a plate seal was stuck on the plate surface to prevent the solution from drying.
As a positive control, a 10-fold 2-fold dilution series in which a monoclonal antibody anti-hen egg albumin (MONOCLONAL ANTI-CHICKEN EGG ALBUMIN CLONE OVA) (SIGMA) was diluted 10,000 to 512 thousand times was used. Use As the negative control, the mice not immunized with OVA antigen, a 2-fold dilution series of 10 stages of 2 6-2 15 prepared by diluting the serum sample prepared in the same manner as above in blocking buffer It was.
インキュベート終了後サンプル液を廃棄し、ＥＬＩＳＡプレート洗浄機（バイオテック社製、MODEL MW-96F）を用いて、洗浄液（１×ＰＢＳ、０．２％Tween２０）を１穴あたり２００μＬ分注後、廃棄することを４回繰り返してプレートを洗浄した。 After completion of the incubation, the sample solution is discarded, and 200 μL of the washing solution (1 × PBS, 0.2% Tween 20) is dispensed per well using an ELISA plate washer (Biotech, MODEL MW-96F) and discarded. This was repeated 4 times to wash the plate.
２次抗体として、ビオチン標識ヤギ抗マウスＩｇＧ（Jackson ImmunoResearch社）を希釈液（１×ＰＢＳ、０．２％Tween２０）で１，０００倍希釈し、これを１穴あたり５０μＬずつ分注して３７℃で１時間インキュベートした。この際、プレート表面にプレートシールを貼って溶液の乾燥を防いだ。インキュベート終了後溶液を廃棄し、洗浄液（１×ＰＢＳ、０．２％Tween２０）を１穴あたり２００μＬ分注後、廃棄することを４回繰り返してプレートを洗浄した。 As a secondary antibody, biotin-labeled goat anti-mouse IgG (Jackson ImmunoResearch) was diluted 1,000 times with a diluent (1 × PBS, 0.2% Tween 20), and 50 μL was dispensed per well. Incubated for 1 hour at ° C. At this time, a plate seal was stuck on the plate surface to prevent the solution from drying. After the incubation was completed, the solution was discarded, and 200 μL of a washing solution (1 × PBS, 0.2% Tween 20) was dispensed per well, and then the plate was washed by repeating discarding 4 times.
３次抗体として、アルカリフォスファターゼ標識ストレプトアビジン（インビトロジェン社）を希釈液（１×ＰＢＳ、０．２％Tween２０）で２，０００倍に希釈し、これを１穴あたり５０μＬずつ分注して３７℃で１時間インキュベートした。この際、プレート表面にプレートシールを貼って溶液の乾燥を防いだ。インキュベート終了後溶液を廃棄し、洗浄液（１×ＰＢＳ、０．２％Tween２０）を１穴あたり２００μＬ分注後、廃棄することを４回繰り返してプレートを洗浄した。 As a tertiary antibody, alkaline phosphatase-labeled streptavidin (Invitrogen) was diluted 2,000 times with a diluent (1 × PBS, 0.2% Tween 20), and this was dispensed at 50 μL per well at 37 ° C. And incubated for 1 hour. At this time, a plate seal was stuck on the plate surface to prevent the solution from drying. After the incubation was completed, the solution was discarded, and 200 μL of a washing solution (1 × PBS, 0.2% Tween 20) was dispensed per well, and then the plate was washed by repeating discarding 4 times.
フォスファターゼ用発色基質ｐ−ニトロフェニルリン酸（ＫＰＬ社）の１粒を発色基質用緩衝液（精製水１Ｌにジエタノールアミン：０．９７ｍＬ、ＭｇＣｌ2・６Ｈ2Ｏ：０．１ｇ、ＮａＮ3：０．１ｇを含む；ｐＨ９．８）５ｍＬに溶解して発色液を調製し、この発色液を１穴あたり１００μＬずつ分注して３７℃で１時間インキュベートした。この際、プレート表面にプレートシールを貼って溶液の乾燥を防いだ。インキュベート終了後反応停止液として１Ｎ ＮａＯＨを１穴あたり５０μＬずつ分注して反応を止め、波長４０５ｎｍ及び６３０ｎｍ（主波長４０５ｎｍ、副波長６３０ｎｍ）で各穴の吸光度を測定した。なお、吸光度の測定には、ＥＬＩＳＡ用プレートリーダー Multiskan EX（Thermo Labsystems社）を使用した。 One phosphatase chromogenic substrate p-nitrophenyl phosphate (KPL) was added to a chromogenic substrate buffer solution (diethanolamine: 0.97 mL in 1 L of purified water, MgCl 2 .6H 2 O: 0.1 g, NaN 3 : 0. 1 g containing; pH 9.8) Dissolving in 5 mL to prepare a coloring solution, dispensing 100 μL of this coloring solution per well and incubating at 37 ° C. for 1 hour. At this time, a plate seal was stuck on the plate surface to prevent the solution from drying. After completion of the incubation, 50 μL of 1N NaOH was dispensed per well as a reaction stop solution to stop the reaction, and the absorbance of each well was measured at wavelengths of 405 nm and 630 nm (main wavelength 405 nm, subwavelength 630 nm). For the measurement of absorbance, ELISA plate reader Multiskan EX (Thermo Labsystems) was used.
吸光度の測定値から、陰性コントロールの吸光度平均値とその標準偏差（ＳＤ）を求めた。次いで、サンプルの吸光度を希釈率に対してプロットし標準曲線を作成し、陰性コントロールの吸光度平均値＋２ＳＤの標準曲線との交点を求め、この交点の２の乗数をＩｇＧ抗体価として得た。３回目の経鼻接種の日を第０週として、経鼻接種の日からの時間経過に対してプロットした血清サンプルのＩｇＧ抗体価を図１中に◆で示す。
From the measured absorbance, the average absorbance of the negative control and its standard deviation (SD) were determined. Next, the absorbance of the sample was plotted against the dilution rate to prepare a standard curve, the intersection of the negative control absorbance average value + 2SD standard curve was determined, and the multiplier of 2 at this intersection was obtained as the IgG antibody titer. The IgG antibody titer of the serum sample plotted against the time course from the day of nasal inoculation, with the third day of nasal inoculation as
（ＩｇＡ抗体価の測定）
ＯＶＡ抗原（生化学工業株式会社製）をコーティングバッファー（精製水１ＬにＮａ2ＣＯ3：１．６ｇ、ＮａＨＣＯ3：２．８２ｇ、ＮａＮ3：０．１ｇを含む；ｐＨ９．０）で５μｇ／ｍＬに調製し、この溶液を９６穴ＥＬＩＳＡプレート（SUMILON ELISAプレートＳ MS-8496F、住友ベークライト株式会社）に１穴あたり１００μＬずつ分注し、４℃で一晩静置して、抗原を吸着させた。この際、プレート表面にプレートシールを貼って溶液の乾燥を防いだ。
(Measurement of IgA antibody titer)
OVA antigen (manufactured by Seikagaku Corporation) with a coating buffer (containing 1 g of purified water containing Na 2 CO 3 : 1.6 g, NaHCO 3 : 2.82 g, NaN 3 : 0.1 g; pH 9.0) at 5 μg / Prepare 100 mL of the solution in a 96-well ELISA plate (SUMILON ELISA plate S MS-8496F, Sumitomo Bakelite Co., Ltd.), and allow to stand overnight at 4 ° C. to adsorb the antigen. It was. At this time, a plate seal was stuck on the plate surface to prevent the solution from drying.
上記コーティング終了後、コーティングバッファーを廃棄し、次いで当該ＥＬＩＳＡプレートにブロッキングバッファー（精製水１Ｌに１０×ダルベッコＰＢＳ：１００ｍＬ、アルブミン（Ｆｒ．Ｖ）：１０ｇ、ＮａＮ3：１ｇを含む）を１穴あたり２００μＬずつ分注し、１時間放置した。 After the above coating is completed, the coating buffer is discarded, and then the blocking buffer (containing 10 × Dulbecco's PBS: 100 mL, albumin (Fr.V): 10 g, NaN 3 : 1 g in 1 L of purified water) per hole is applied to the ELISA plate. 200 μL was dispensed and allowed to stand for 1 hour.
ＥＬＩＳＡプレートとは別にＵ底プレートを用意し、各サンプルについて５倍及び１０倍の希釈液を調製した。希釈にはブロッキングバッファーを使用した。 A U-bottom plate was prepared separately from the ELISA plate, and 5-fold and 10-fold dilutions were prepared for each sample. Blocking buffer was used for dilution.
インキュベーション終了後、ＥＬＩＳＡプレートからブロッキングバッファーを廃棄し、Ｕ底プレートで調製した希釈サンプルを１穴あたり１００μＬずつ分注して３７℃で１時間インキュベートした。インキュベートの際、プレート表面にプレートシールを貼って溶液の乾燥を防いだ。
なお、陽性コントロールとしては、コレラ毒素（以下、ＣＴと略す）−ＯＶＡ混合液（１×ＰＢＳ緩衝液で２ｍｇ／ｍＬに調製したＣＴ溶液１部に対して０．２５％ＯＶＡ水溶液２部を加え、ピペッティングにより十分に混合して調製したもの）を多量に接種してＩｇＡ抗体を産生させたマウスから、上記と同様にして調製した血清サンプルをブロッキングバッファーで５倍及び１０倍希釈したものを用いた。また陰性コントロールとしては、ＯＶＡ抗原を免疫していないマウスから、上記と同様にして調製した血清サンプルをブロッキングバッファーで５倍及び１０倍希釈したものを用いた。
After the incubation was completed, the blocking buffer was discarded from the ELISA plate, and the diluted sample prepared on the U-bottom plate was dispensed at 100 μL per well and incubated at 37 ° C. for 1 hour. During incubation, a plate seal was applied to the plate surface to prevent the solution from drying.
As a positive control, cholera toxin (hereinafter abbreviated as CT) -OVA mixed solution (2 parts of 0.25% OVA aqueous solution was added to 1 part of CT solution prepared to 2 mg / mL with 1 × PBS buffer). , Prepared by mixing well by pipetting), and a serum sample prepared in the same manner as described above from a mouse inoculated with a large amount of IgA antibody and diluted 5 times and 10 times with a blocking buffer. Using. As a negative control, a serum sample prepared in the same manner as described above from a mouse not immunized with the OVA antigen was diluted 5 times and 10 times with a blocking buffer.
インキュベーション終了後サンプル液を廃棄し、ＥＬＩＳＡプレート洗浄機（バイオテック社、ＭＷ−９６Ｆ）を用いて、洗浄液（１×ＰＢＳ、０．２％Tween２０）を１穴あたり４００μＬ分注後、廃棄することを４回繰り返してプレートを洗浄した。 After the incubation, discard the sample solution, and use an ELISA plate washer (Biotech, MW-96F) to dispense 400 μL of the washing solution (1 × PBS, 0.2% Tween20) per well, and then discard it. Was repeated 4 times to wash the plate.
２次抗体として、ビオチン標識ヤギ抗マウスＩｇＡ（ＫＰＬ社）を希釈液（１×ＰＢＳ、０．２％Tween２０）で１，０００倍希釈し、これを１穴あたり１００μＬずつ分注して３７℃で１時間インキュベートした。この際、プレート表面にプレートシールを貼って溶液の乾燥を防いだ。インキュベーション終了後溶液を廃棄し、洗浄液（１×ＰＢＳ、０．２％Tween２０）を１穴あたり４００μＬずつ分注後、廃棄することを４回繰り返してプレートを洗浄した。
As a secondary antibody, biotin-labeled goat anti-mouse IgA (KPL) was diluted 1,000 times with a diluent (1 × PBS, 0.2% Tween 20), and dispensed at 100 μL per well at 37 ° C. And incubated for 1 hour. At this time, a plate seal was stuck on the plate surface to prevent the solution from drying. After the incubation was completed, the solution was discarded, and the plate was washed by repeating the discarding
３次抗体として、アルカリフォスファターゼ標識ストレプトアビジン（インビトロジェン社）を希釈液（１×ＰＢＳ、０．２％Tween２０）で２，０００倍に希釈し、これを１穴あたり１００μＬずつ分注して３７℃で１時間インキュベートした。この際、プレート表面にプレートシールを貼って溶液の乾燥を防いだ。インキュベート終了後、ＥＬＩＳＡプレートから溶液を廃棄し、次いで洗浄液（１×ＰＢＳ、０．２％Tween２０）を１穴あたり４００μＬ分注後、廃棄することを４回繰り返してプレートを洗浄した。
As a tertiary antibody, alkaline phosphatase-labeled streptavidin (Invitrogen) was diluted 2,000 times with a diluent (1 × PBS, 0.2% Tween 20), and this was dispensed at 100 μL per well at 37 ° C. And incubated for 1 hour. At this time, a plate seal was stuck on the plate surface to prevent the solution from drying. After completion of the incubation, the solution was discarded from the ELISA plate, and then the plate was washed by repeating the discarding
フォスファターゼ用発色基質ｐ−ニトロフェニルリン酸（ＫＰＬ社）の１粒を発色基質用緩衝液（精製水１Ｌにジエタノールアミン：０．９７ｍＬ、ＭｇＣｌ2・６Ｈ2Ｏ：０．１ｇ、ＮａＮ3：０．１ｇを含む；ｐＨ９．８）５ｍＬに溶解して発色液を調製し、この発色液を１穴あたり１００μＬずつ分注して３７℃で１時間インキュベートした。この際、プレート表面にプレートシールを貼って溶液の乾燥を防いだ。インキュベート終了後、反応停止液として１Ｎ ＮａＯＨを１穴あたり５０μＬずつ分注して反応を止め、波長４０５ｎｍ及び６３０ｎｍで各穴の吸光度を測定した。なお、吸光度の測定には、ＥＬＩＳＡ用プレートリーダー Multiskan EX（Thermo Labsystems社）を使用した。
５倍及び１０倍希釈したサンプルの吸光度の各平均値をＩｇＡ抗体価として得た。３回目の経鼻接種の日を第０週として、経鼻接種の日からの時間経過に対してプロットした血清サンプルのＩｇＡ抗体価を図２中に◆で示す。
One phosphatase chromogenic substrate p-nitrophenyl phosphate (KPL) was added to a chromogenic substrate buffer solution (diethanolamine: 0.97 mL in 1 L of purified water, MgCl 2 .6H 2 O: 0.1 g, NaN 3 : 0. 1 g containing; pH 9.8) Dissolving in 5 mL to prepare a coloring solution, dispensing 100 μL of this coloring solution per well and incubating at 37 ° C. for 1 hour. At this time, a plate seal was stuck on the plate surface to prevent the solution from drying. After completion of the incubation, 50 μL of 1N NaOH was dispensed per well as a reaction stop solution to stop the reaction, and the absorbance of each well was measured at wavelengths of 405 nm and 630 nm. For the measurement of absorbance, ELISA plate reader Multiskan EX (Thermo Labsystems) was used.
Each average value of the absorbance of the 5-fold and 10-fold diluted samples was obtained as an IgA antibody titer. The IgA antibody titer of the serum sample plotted against the time course from the day of nasal inoculation, with the third day of nasal inoculation as
［比較例１］
実施例１で用いたカチオン化キトサン水溶液に代えて、粘膜免疫アジュバントとして定評のあるコレラ毒素（ＣＴ）を用いて実施例１と同様の実験を行った。すなわち、ＣＴ−ＯＶＡ混合液を実施例１と同様にして調製し、当該混合液３μＬを、実施例１と同様にジエチルエーテルで麻酔した６週齢、雌のＢＡＬＢ／ｃマウス５匹に経鼻接種し、血液、便及び膣洗浄液を得てサンプルを調製し、ＥＬＩＳＡ法を用いてＩｇＧ及びＩｇＡの抗体価を求めた。
[Comparative Example 1]
In place of the cationized chitosan aqueous solution used in Example 1, cholera toxin (CT), which has a well-established mucosal immune adjuvant, was used to conduct the same experiment as in Example 1. That is, a CT-OVA mixed solution was prepared in the same manner as in Example 1, and 3 μL of the mixed solution was nasally applied to five 6-week-old, female BALB / c mice anesthetized with diethyl ether as in Example 1. Inoculated, blood, stool and vaginal lavage fluid were obtained, samples were prepared, and antibody titers of IgG and IgA were determined using ELISA.
実施例１と同様にして得た血清サンプルのＩｇＧ抗体価及びＩｇＡ抗体価を、それぞれ図１及び図２中に△で示す。 The IgG antibody titer and IgA antibody titer of a serum sample obtained in the same manner as in Example 1 are indicated by Δ in FIGS. 1 and 2, respectively.
図１及び図２から、ＣＴ−ＯＶＡ混合液を用いて免疫したマウスよりも、カチオン化キトサン−ＯＶＡ混合液を用いて免疫したマウスの方が、ＯＶＡに対する抗体価が有意に高いことが明らかとなった。この血清サンプルについての結果と同様に、便サンプル、膣洗浄液サンプルについても、キトサン−ＯＶＡ混合液を用いて免疫したマウスの方が、ＣＴ−ＯＶＡ混合液を用いて免疫したマウスよりも、ＯＶＡに対する抗体価が有意に高いという結果を示した。 FIG. 1 and FIG. 2 clearly show that the antibody titer against OVA is significantly higher in the mouse immunized with the cationized chitosan-OVA mixture than in the mouse immunized with the CT-OVA mixture. became. Similar to the results for this serum sample, for stool samples and vaginal lavage samples, mice immunized with the chitosan-OVA mixture were more effective against OVA than mice immunized with the CT-OVA mixture. The result showed that the antibody titer was significantly high.
［実施例２］
実施例１と同様にして調製したカチオン化キトサン−ＯＶＡ混合液７μＬを、ジエチルエーテルで麻酔した６週齢、雌の多量体免疫グロブリンレセプターノックアウトマウス(Polymeric immunoglobulin receptor knockout mice)５匹に経鼻接種し、実施例１と同様の手順及び方法で血液を得て血清サンプルを調製した。ＩｇＡ抗体価の測定において血清サンプルを１００倍及び２００倍に希釈して用いたこと以外は、実施例１と同様の手順及び方法でＥＬＩＳＡ法を用いてＩｇＧ及びＩｇＡの抗体価を求めた。ＩｇＧ及びＩｇＡの抗体価をそれぞれ図３及び図４中に◆で示す。
[Example 2]
7 μL of the cationized chitosan-OVA mixed solution prepared in the same manner as in Example 1 was intranasally inoculated into 6 female 6-week-old female polymeric immunoglobulin receptor knockout mice anesthetized with diethyl ether. Then, blood was obtained by the same procedure and method as in Example 1 to prepare a serum sample. The IgG and IgA antibody titers were determined using the ELISA method in the same procedure and manner as in Example 1, except that the serum samples were diluted 100-fold and 200-fold for measurement of IgA antibody titers. The antibody titers of IgG and IgA are indicated by ♦ in FIGS. 3 and 4, respectively.
［比較例２］
実施例２で用いたカチオン化キトサン水溶液に代えて、コレラ毒素（ＣＴ）を用いて実施例２と同様の実験を行った。すなわち、比較例１と同様にして調製したＣＴ−ＯＶＡ混合液３μＬを、実施例２と同様にジエチルエーテルで麻酔した６週齢、雌の多量体免疫グロブリンレセプターノックアウトマウス５匹に経鼻接種し、実施例２と同様の手順及び方法で血液を得て血清サンプルを調製し、ＥＬＩＳＡ法を用いてＩｇＧ及びＩｇＡの抗体価を求めた。ＩｇＧ及びＩｇＡの抗体価をそれぞれ図３及び図４中に△で示す。
[Comparative Example 2]
An experiment similar to that of Example 2 was performed using cholera toxin (CT) instead of the cationized chitosan aqueous solution used in Example 2. That is, 3 μL of a CT-OVA mixed solution prepared in the same manner as in Comparative Example 1 was intranasally inoculated into 5 female multimeric immunoglobulin receptor knockout mice anesthetized with diethyl ether as in Example 2. Blood was obtained by the same procedure and method as in Example 2, serum samples were prepared, and antibody titers of IgG and IgA were determined using ELISA. Antibody titers of IgG and IgA are indicated by Δ in FIGS. 3 and 4, respectively.
図３及び図４から、ＣＴ−ＯＶＡ混合液を用いて免疫したマウスよりも、カチオン化キトサン−ＯＶＡ混合液を用いて免疫したマウスの方が、ＯＶＡに対する抗体価が有意に高いことが明らかとなった。 FIG. 3 and FIG. 4 clearly show that the antibody titer against OVA is significantly higher in the mouse immunized with the cationized chitosan-OVA mixture than in the mouse immunized with the CT-OVA mixture. became.
［実施例３］
実施例１と同様にして調製した２％カチオン化キトサン水溶液５部に対して、０．２５％ＯＶＡ水溶液２部及び１×ＰＢＳ溶液を１８部加え、ピペッティングにより十分に混合し、カチオン化キトサン−ＯＶＡ混合液を得た。当該混合液２５μＬを、５ｍｇ／ｍＬネンブタール（マウス体重１ｇ当たり、約０．０１ｍＬ使用）腹腔内注射で麻酔した６週齢、雌のＢＡＬＢ／ｃマウス５匹に経肺接種した。なお、肺への接種の噴霧器としてはPENNCENTURY社製、モデル１Ａ−１Ｃを使用した。
[Example 3]
To 5 parts of 2% cationized chitosan aqueous solution prepared in the same manner as in Example 1, 2 parts of 0.25% OVA aqueous solution and 18 parts of 1 × PBS solution were added, and mixed well by pipetting. A -OVA mixture was obtained. 25 μL of the mixed solution was inoculated into 5 6-week-old, female BALB / c mice anesthetized by intraperitoneal injection with 5 mg / mL Nembutal (use of about 0.01 mL per 1 g of mouse body weight). In addition, as a nebulizer for inoculating the lung, model 1A-1C manufactured by PENNCENTURY was used.
この経肺接種の日から１週間ごと計３回、同様に上記混合液の経肺接種を行った。この３回目の経肺接種の日を第０週として、その後、実施例１と同様の手順及び方法により、週に一度、血液、便及び膣洗浄液を得てサンプルを調製し、ＥＬＩＳＡ法を用いてＩｇＧ及びＩｇＡの抗体価を求めた。また、ブースター効果を確認するために、上記３回目の経肺接種の日から２〜３ヶ月の後、同様に４回目の経肺接種を行った。血清サンプルについて求めたＩｇＧ及びＩｇＡの抗体価をそれぞれ図５及び図６中に◆で示す。
From the day of this pulmonary inoculation, pulmonary inoculation of the above mixed solution was similarly performed three times a week. The day of this third transpulmonary inoculation was set as
［比較例３］
実施例３で用いたカチオン化キトサン水溶液に代えて、コレラ毒素（ＣＴ）を用いて実施例３と同様の実験を行った。すなわち、１×ＰＢＳで２ｍｇ／ｍＬに調製したＣＴ溶液１部に０．２５％ＯＶＡ溶液２部を加え、さらに１×ＰＢＳを２２部加えて希釈し、ピペッティングにより十分に混合した。このＣＴ−ＯＶＡ混合液２５μＬを実施例３と同様に６週齢、雌のＢＡＬＢ／ｃマウス５匹に経肺接種した。その後、実施例３と同様の手順及び方法で経肺接種、サンプル調製をし、ＥＬＩＳＡ法を用いてＩｇＧ及びＩｇＡの抗体価を求めた。血清サンプルについて求めたＩｇＧ及びＩｇＡの抗体価をそれぞれ図５及び図６に△で示す。
[Comparative Example 3]
The same experiment as in Example 3 was performed using cholera toxin (CT) instead of the cationized chitosan aqueous solution used in Example 3. That is, 2 parts of 0.25% OVA solution was added to 1 part of CT solution prepared to 2 mg / mL with 1 × PBS, and further diluted with 22 parts of 1 × PBS, and mixed well by pipetting. In the same manner as in Example 3, 25 μL of this CT-OVA mixed solution was inoculated into five 6-week-old female BALB / c mice via lung. Thereafter, transpulmonary inoculation and sample preparation were carried out by the same procedure and method as in Example 3, and the antibody titers of IgG and IgA were determined using the ELISA method. The antibody titers of IgG and IgA determined for serum samples are indicated by Δ in FIGS. 5 and 6, respectively.
図５及び図６から、ＣＴ−ＯＶＡ混合液を用いて免疫したマウスよりも、カチオン化キトサン−ＯＶＡ混合液を用いて免疫したマウスの方が、ＯＶＡに対する抗体価が有意に高いことが明らかとなった。この血清サンプルについての結果と同様に、便サンプル及び膣洗浄液サンプルについても、カチオン化キトサン−ＯＶＡ混合液を用いて免疫したマウスの方が、ＣＴ−ＯＶＡ混合液を用いて免疫したマウスよりも、ＯＶＡに対する抗体価が有意に高いという結果を示した。 FIG. 5 and FIG. 6 clearly show that the antibody titer against OVA is significantly higher in the mouse immunized with the cationized chitosan-OVA mixture than in the mouse immunized with the CT-OVA mixture. became. Similar to the results for this serum sample, for stool samples and vaginal lavage samples, mice immunized with the cationized chitosan-OVA mixture were more responsive than those immunized with the CT-OVA mixture. The result showed that the antibody titer against OVA was significantly high.
［実施例４］
実施例３と同様の手順及び方法でカチオン化キトサン−ＯＶＡ混合液を調製した。当該混合液２５μＬを、５ｍｇ／ｍＬネンブタール（マウス体重１ｇ当たり、約０．０１ｍＬ使用）腹腔内注射で麻酔した６週齢、雌の多量体免疫グロブリンレセプターノックアウトマウス５匹に経肺接種した。なお、肺への接種は実施例３と同様に行った。
[Example 4]
A cationized chitosan-OVA mixture was prepared in the same procedure and manner as in Example 3. 25 μL of the mixed solution was inoculated via pulmonary inoculation into five 6-week-old female multimeric immunoglobulin receptor knockout mice anesthetized by intraperitoneal injection of 5 mg / mL Nembutal (use about 0.01 mL per g of mouse body weight). The lung was inoculated in the same manner as in Example 3.
実施例３と同様の手順及び方法により、この経肺接種の日から１週間ごとに計３回、上記混合液の経肺接種を行った。この３回目の経肺接種の日を第０週として、その後、実施例３と同様の手順及び方法により、週に一度血液を得て血清サンプルを調製し、ＥＬＩＳＡ法を用いてＩｇＧ及びＩｇＡの抗体価を求めた。また、ブースター効果を確認するために、３回目の経肺接種の日から２〜３ヶ月の後、４回目の経肺接種を行った。血清サンプルについて求めたＩｇＧ及びＩｇＡの抗体価をそれぞれ図７及び図８中に◆で示す。
According to the same procedure and method as in Example 3, the mixture was pulmonary inoculated three times every week from the day of pulmonary inoculation. The day of this third pulmonary inoculation was set as
［比較例４］
実施例４で用いたカチオン化キトサン水溶液に代えて、コレラ毒素（ＣＴ）を用いて実施例４と同様の実験を行った。すなわち、比較例３と同様にして調製したＣＴ−ＯＶＡ混合液２５μＬを実施例４と同様にネンブタールで麻酔した６週齢、雌の多量体免疫グロブリンレセプターノックアウトマウス５匹に計４回経肺接種した。実施例４と同様の手順及び方法により、週に一度、血清サンプルを調製し、ＥＬＩＳＡ法を用いてＩｇＧ及びＩｇＡの抗体価を求めた。血清サンプルについて求めたＩｇＧ及びＩｇＡの抗体価をそれぞれ図７及び図８中に△で示す。
[Comparative Example 4]
The same experiment as in Example 4 was performed using cholera toxin (CT) instead of the cationized chitosan aqueous solution used in Example 4. That is, 25 μL of CT-OVA mixed solution prepared in the same manner as in Comparative Example 3 was inoculated with Nembutal in the same manner as in Example 4 into 6 week-old female multimeric immunoglobulin receptor knockout mice for a total of 4 inoculations. did. Serum samples were prepared once a week by the same procedure and method as in Example 4, and the antibody titers of IgG and IgA were determined using the ELISA method. The IgG and IgA antibody titers determined for the serum samples are indicated by Δ in FIGS. 7 and 8, respectively.
図７及び図８から、ＣＴ−ＯＶＡ混合液を用いて免疫したマウスよりも、カチオン化キトサン−ＯＶＡ混合液を用いて免疫したマウスの方が、ＯＶＡに対する抗体価が有意に高いことが明らかとなった。 FIG. 7 and FIG. 8 clearly show that the antibody titer against OVA is significantly higher in the mouse immunized with the cationized chitosan-OVA mixture than in the mouse immunized with the CT-OVA mixture. became.
全ての実施例において、ＯＶＡの免疫アジュバントとしてコレラ毒素を使用した比較例よりも、カチオン化キトサンを使用した実施例の方が、ＯＶＡに対する抗体価が有意に高いという結果を示した。また、図中１４週目の抗体価が高まっていることから４回目の免疫を行うことにより、急激に抗体産生能が高まるブースター効果が確認できた。 In all the examples, the antibody titer against OVA was significantly higher in the example using cationized chitosan than in the comparative example using cholera toxin as an immune adjuvant for OVA. Moreover, since the antibody titer in the 14th week in the figure was increased, a booster effect in which the antibody producing ability was rapidly increased was confirmed by performing the fourth immunization.
本発明によれば、生体への安全性が高く、全身系免疫に関わるＩｇＧのみならず、粘膜などで局所的に感染防御に関わり、抗ウイルス作用を有する分泌型ＩｇＡの産生能に優れた免疫アジュバント水溶液、免疫アジュバント分散液及び当該免疫アジュバントの接種方法が提供される。本発明の免疫アジュバント水溶液、免疫アジュバント分散液を本発明の接種方法で粘膜に接種することにより、接種部位に局所的に免疫を誘導することができる。 According to the present invention, immunity that is highly safe to the living body, is involved not only in IgG related to systemic immunity, but also locally in the mucous membrane, etc., and has excellent ability to produce secretory IgA having antiviral activity. An aqueous adjuvant solution, an immune adjuvant dispersion, and a method for inoculating the immune adjuvant are provided. By inoculating the mucosa with the immune adjuvant aqueous solution and immune adjuvant dispersion liquid of the present invention by the inoculation method of the present invention, immunity can be locally induced at the site of inoculation.
Claims (4)
上記カチオン化キトサン水溶液のｐＨを７．０超、９．０以下に調整したことを特徴とする免疫アジュバント水溶液。 Cationized chitosan having a cationic group introduced by 2,3-epoxypropyltrimethylammonium chloride and having a cationization degree of 1 to 3 was dissolved in an organic acid aqueous solution containing a water-soluble organic acid to obtain a cationized chitosan aqueous solution. rear,
An aqueous immune adjuvant solution, characterized in that the pH of the aqueous cationized chitosan solution is adjusted to more than 7.0 and 9.0 or less.
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