JP2014218496A - Highly-preservable antibody solution - Google Patents

Highly-preservable antibody solution Download PDF

Info

Publication number
JP2014218496A
JP2014218496A JP2014079671A JP2014079671A JP2014218496A JP 2014218496 A JP2014218496 A JP 2014218496A JP 2014079671 A JP2014079671 A JP 2014079671A JP 2014079671 A JP2014079671 A JP 2014079671A JP 2014218496 A JP2014218496 A JP 2014218496A
Authority
JP
Japan
Prior art keywords
antibody
antibody solution
mass
egg yolk
ethanol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
JP2014079671A
Other languages
Japanese (ja)
Inventor
村上 洋一
Yoichi Murakami
洋一 村上
翔一郎 平野
Shoichiro Hirano
翔一郎 平野
バン サー ヌグエン
Sa Van Nguyen
バン サー ヌグエン
浩二 梅田
Koji Umeda
浩二 梅田
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
EADABLE NUTRITION JAPAN KK
Wako Filter Technology Co Ltd
Original Assignee
EADABLE NUTRITION JAPAN KK
Wako Filter Technology Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by EADABLE NUTRITION JAPAN KK, Wako Filter Technology Co Ltd filed Critical EADABLE NUTRITION JAPAN KK
Priority to JP2014079671A priority Critical patent/JP2014218496A/en
Publication of JP2014218496A publication Critical patent/JP2014218496A/en
Pending legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/02Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

PROBLEM TO BE SOLVED: To provide a highly-preservable antibody solution which, without a preservative or a stabilizer, can be preserved at room temperature for the long term while keeping the activity of a yolk antibody, a production method thereof and an antibody preserving method.SOLUTION: A yolk antibody, water, and 1-25 mass% of ethanol are mixed and then subjected to sterilization treatment.

Description

この発明は、防腐剤や安定化剤等を用いなくても、卵黄抗体の活性を維持しつつ長期にわたり常温で保存できる高保存性抗体溶液、その製造方法、及び、抗体の保存方法に関するものである。   The present invention relates to a highly-storable antibody solution that can be stored at room temperature for a long period of time while maintaining the activity of egg yolk antibodies without using preservatives, stabilizers, etc., a method for producing the same, and a method for storing antibodies. is there.

従来、マスクに抗菌性や抗ウイルス性等を付与するために、細菌やウイルスに由来する抗原に対する抗体が担持されたマスクが知られているが(特許文献1)、より手軽にマスクに抗菌性や抗ウイルス性等を付与できるように、マスクに抗体溶液を吹き付けるためのスプレーも知られている。このようなスプレーでは、使用時に粉末状の抗体を水道水等に溶解して抗体溶液を調製し、当該抗体溶液をマスクに向けて噴霧するように構成されている。しかし、粉末状の抗体を用いると、使用の度に水道水等に溶解する必要があるため作業が煩雑となり、使いやすさの点から、予め調製された抗体溶液が充填されているスプレーが求められている。   Conventionally, in order to impart antibacterial and antiviral properties to a mask, a mask in which an antibody against an antigen derived from a bacterium or virus is supported is known (Patent Document 1). A spray for spraying an antibody solution onto a mask is also known so that it can be imparted with antiviral properties and antiviral properties. In such a spray, a powdered antibody is dissolved in tap water or the like at the time of use to prepare an antibody solution, and the antibody solution is sprayed toward a mask. However, when powdered antibodies are used, it is necessary to dissolve them in tap water each time they are used, and the work becomes complicated. From the viewpoint of ease of use, a spray filled with a preliminarily prepared antibody solution is required. It has been.

ところで、抗原抗体反応には、抗体の可変領域等の立体構造が重要であるため、抗体を保存するに際しては、このような立体構造が壊れないように注意を要する。しかし、抗体の立体構造は、温度、化学物質等の影響を受けやすく、とりわけ液体中において活性を維持した状態で抗体を長期間保存することは困難である。このため、液体中の抗体を安定化する様々な方法が検討されている。このような安定化方法としては、例えば、特許文献2には、IgM試薬溶液にウシ血清アルブミン(BSA)を添加する方法が提案されており、また、抗体溶液にポリエチレングリコールを添加する方法も知られている。更に、抗体溶液の腐敗を防止するために、通常、抗体溶液にはアジ化ナトリウム等の防腐剤も添加されている。このような抗体溶液は、通常4℃程度の低温で保存される。   By the way, since the three-dimensional structure such as the variable region of the antibody is important for the antigen-antibody reaction, care must be taken when storing the antibody so that the three-dimensional structure is not broken. However, the three-dimensional structure of an antibody is easily affected by temperature, chemical substances, etc., and it is difficult to store the antibody for a long period of time, particularly while maintaining the activity in a liquid. For this reason, various methods for stabilizing antibodies in a liquid have been studied. As such a stabilization method, for example, Patent Document 2 proposes a method of adding bovine serum albumin (BSA) to an IgM reagent solution, and also known a method of adding polyethylene glycol to an antibody solution. It has been. Furthermore, in order to prevent the antibody solution from decaying, an antiseptic such as sodium azide is usually added to the antibody solution. Such an antibody solution is usually stored at a low temperature of about 4 ° C.

また、マスクに抗菌性や抗ウイルス性を付与するための抗体としては、卵黄抗体(IgY:immunoglobulin yolk)が用いられることが多い。卵黄抗体は、鳥類の血液中のIgGが卵黄中に移行したものであり、特定の抗原でニワトリ等の鳥類を免疫すると、卵黄からその抗原に特異的な抗体を得ることができる。このように鳥類の卵から抗体を調製する方法には、抗体を大量に調製できること、採血が不要なこと、大量飼育が容易であること、卵黄からの精製は比較的簡単であること等の利点がある。しかし、卵黄抗体は哺乳類のIgGとは物理的化学的な性状が異なることが知られている。   Further, as an antibody for imparting antibacterial and antiviral properties to the mask, an egg yolk antibody (IgY: immunooglobulin yolk) is often used. The egg yolk antibody is obtained by transferring IgG in the blood of birds into the egg yolk. When a bird such as a chicken is immunized with a specific antigen, an antibody specific to the antigen can be obtained from the egg yolk. Thus, the method for preparing antibodies from avian eggs has advantages such as the ability to prepare antibodies in large quantities, the need for blood collection, ease of mass breeding, and the relatively simple purification from egg yolk. There is. However, egg yolk antibodies are known to have different physical and chemical properties from mammalian IgG.

特開2005−169105号公報JP 2005-169105 A 特開平9−127114号公報JP 9-127114 A

そこで本発明は、防腐剤や安定化剤等を用いなくても、卵黄抗体の活性を維持しつつ長期にわたり常温で保存できる高保存性抗体溶液、その製造方法、及び、抗体の保存方法を提供すべく図ったものである。   Therefore, the present invention provides a highly-storable antibody solution that can be stored at room temperature for a long period of time while maintaining the activity of egg yolk antibody without using preservatives, stabilizers, etc., a method for producing the same, and a method for storing the antibody It was something that was planned.

本発明者らは、所定濃度になるようにエタノールを添加し、かつ、滅菌処理を施すことにより、安定化剤や防腐剤等を用いなくとも、卵黄抗体を水溶液中において安定な状態で長期にわたり保存できることを見出した。本発明は、この知見に基づき完成するに至ったものである。   By adding ethanol to a predetermined concentration and applying sterilization, the present inventors can maintain the egg yolk antibody in an aqueous solution in a stable state over a long period of time without using a stabilizer or preservative. I found that it can be saved. The present invention has been completed based on this finding.

すなわち本発明に係る高保存性抗体溶液は、卵黄抗体と、水と、1〜25質量%のエタノールとを含有し、滅菌処理が施されていることを特徴とする。   That is, the highly conserved antibody solution according to the present invention contains egg yolk antibody, water, and 1 to 25% by mass of ethanol, and is sterilized.

このようなものであれば、滅菌処理が施されているので、調製直後は細菌等の混入は抑えられており、また、調製してから時間が経過して若干の細菌等が混入しても、エタノールにより細菌等の繁殖を抑えることができる。   In such a case, since sterilization is performed, contamination of bacteria and the like is suppressed immediately after the preparation, and even if some bacteria or the like are mixed in after a lapse of time after preparation. The growth of bacteria and the like can be suppressed by ethanol.

前記滅菌処理としては、例えば、フィルター滅菌が好適に用いられる。   As the sterilization treatment, for example, filter sterilization is preferably used.

本発明に係る高保存性抗体溶液は、香料や緩衝剤等を含有していてもよい。   The highly conserved antibody solution according to the present invention may contain a fragrance, a buffering agent and the like.

本発明に係る高保存性抗体溶液は、マスク等に抗菌性や抗ウイルス性を付与するためのスプレー用充填液として好適に用いられる。すなわち本発明に係る高保存性抗体溶液が充填されてなるスプレーもまた、本発明の一つである。   The highly conserved antibody solution according to the present invention is suitably used as a spray filling liquid for imparting antibacterial and antiviral properties to a mask or the like. That is, a spray filled with the highly conserved antibody solution according to the present invention is also one aspect of the present invention.

また、本発明に係る高保存性抗体溶液の製造方法もまた、本発明の一つである。すなわち本発明に係る高保存性抗体溶液の製造方法は、卵黄抗体を水性溶媒に溶解して抗体溶液を調製する工程と、前記抗体溶液をフィルター滅菌する工程と、フィルター滅菌が施される前又は後の抗体溶液に、1〜25質量%の濃度となるようにエタノールを加える工程と、を有することを特徴とする。   The method for producing a highly conserved antibody solution according to the present invention is also one aspect of the present invention. That is, the method for producing a highly-storable antibody solution according to the present invention includes a step of dissolving an egg yolk antibody in an aqueous solvent to prepare an antibody solution, a step of filter sterilizing the antibody solution, and before the filter sterilization is performed. Adding ethanol to the subsequent antibody solution to a concentration of 1 to 25% by mass.

更に、本発明に係る抗体の保存方法もまた、本発明の一つである。すなわち本発明に係る抗体の保存方法は、卵黄抗体を水性溶媒に溶解して抗体溶液を調製する工程と、前記抗体溶液をフィルター滅菌する工程と、フィルター滅菌が施される前又は後の抗体溶液に、1〜25質量%の濃度となるようにエタノールを加える工程と、前記抗体溶液を滅菌済みの密閉容器内に充填する工程と、を有することを特徴とする。   Furthermore, the method for preserving an antibody according to the present invention is also one aspect of the present invention. That is, the method for preserving an antibody according to the present invention includes a step of preparing an antibody solution by dissolving egg yolk antibody in an aqueous solvent, a step of filter sterilizing the antibody solution, and an antibody solution before or after filter sterilization is performed. And a step of adding ethanol to a concentration of 1 to 25% by mass and a step of filling the antibody solution in a sterilized sealed container.

このような構成を有する本発明によれば、防腐剤や安定化剤等を用いなくても、卵黄抗体を液体中において活性を維持しつつ長期にわたり常温で保存することが可能となる。   According to the present invention having such a configuration, it is possible to store an egg yolk antibody at room temperature for a long period of time while maintaining activity in a liquid without using a preservative or a stabilizer.

エタノール濃度を変えた抗コナヒョウヒダニ虫体由来粗精製アレルゲンIgYの各抗体溶液の抗体価維持率の経時変化を示すグラフである。It is a graph which shows a time-dependent change of the antibody titer rate of each antibody solution of the anti-Pleurotus mite parasite purified crude allergen IgY which changed ethanol concentration. エタノール濃度を変えた抗ヤケヒョウヒダニ虫体由来粗精製アレルゲンIgYの各抗体溶液の抗体価維持率の経時変化を示すグラフである。It is a graph which shows a time-dependent change of the antibody titer ratio of each antibody solution of the anti-Yake-Leopard mite body origin crude refined allergen IgY which changed ethanol concentration. エタノール濃度を変えた抗コナヒョウヒダニ虫体由来粗精製アレルゲンIgYの各抗体溶液の1週間保存後の様子を示す写真である。It is a photograph which shows the mode after 1 week preservation | save of each antibody solution of the crude purified allergen IgY derived from the anti-carp leopard mite body which changed ethanol concentration.

以下に本発明を詳述する。   The present invention is described in detail below.

本発明に係る高保存性抗体溶液は、卵黄抗体と、水と、1〜25質量%のエタノールとを含有し、滅菌処理が施されているものである。   The highly conserved antibody solution according to the present invention contains egg yolk antibody, water, and 1 to 25% by mass of ethanol, and has been sterilized.

前記卵黄抗体としては特に限定されないが、例えば、アレルゲン、ウイルス、細菌、カビ等に由来する抗原に対して結合能を有するものである。前記アレルゲンとしては、例えば、ダニアレルゲン、花粉、ネコアレルゲン等が挙げられる。前記ウイルスとしては、例えば、インフルエンザウイルス、コロナウイルス(SARSウイルス)、アデノウイルス、ライノウイルス等が挙げられる。前記細菌としては、例えば、黄色ブドウ球菌や表皮ブドウ球菌等のブドウ球菌属、ミクロコッカス属、炭疽菌、セレウス菌、枯草菌、アクネ菌等のグラム陽性菌;緑膿菌、セラチア菌、セパシア菌、肺炎球菌、レジオネラ菌、結核菌等のグラム陰性菌等が挙げられる。前記カビとしては、例えば、アスペルギルス、ペニシリウス、クラドスポリウム等が挙げられる。   Although it does not specifically limit as said egg yolk antibody, For example, it has a binding ability with respect to the antigen derived from allergen, a virus, bacteria, mold, etc. Examples of the allergen include mite allergen, pollen, cat allergen and the like. Examples of the virus include influenza virus, coronavirus (SARS virus), adenovirus, rhinovirus and the like. Examples of the bacteria include Gram-positive bacteria such as Staphylococcus aureus and Staphylococcus epidermidis, Micrococcus, Anthrax, Bacillus cereus, Bacillus subtilis and Acne; Pseudomonas aeruginosa, Serratia and Sephacia And Gram-negative bacteria such as Streptococcus pneumoniae, Legionella, and Mycobacterium tuberculosis. Examples of the mold include Aspergillus, Penicillius, and Cladosporium.

前記卵黄抗体を産生する鳥類としては特に限定されず、例えば、ニワトリ、ウズラ、アヒル、ダチョウ、ガチョウ、エミュー、七面鳥等が挙げられる。なかでも大量飼育が容易であり、安定かつ安価に卵黄抗体を産生できること等からニワトリが好適に用いられる。   The birds that produce the egg yolk antibody are not particularly limited, and examples thereof include chickens, quails, ducks, ostriches, geese, emu, and turkeys. Of these, chickens are preferably used because they are easily bred in large quantities and can produce egg yolk antibodies stably and inexpensively.

前記鳥類への免疫は、常法に従い行えばよく、目的の抗原を、必要に応じて適宜アジュバントと共に、鳥類に筋肉注射や皮下注射等の方法により接種する。前記アジュバントとしては、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等を用いることができる。抗原やアジュバントの接種量、接種時期、接種回数は、目的の抗体が鳥類の体内で産生され、卵黄中に移行し所望の量が蓄積されるように適宜決定すればよい。また、卵黄中の抗体価を高めるために、追加免疫してもよい。   The bird may be immunized according to a conventional method, and the target antigen is inoculated to the bird together with an appropriate adjuvant as necessary by intramuscular injection, subcutaneous injection, or the like. As the adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, or the like can be used. The inoculation amount, the inoculation time, and the inoculation number of the antigen and adjuvant may be appropriately determined so that the target antibody is produced in the body of the bird and moves into the yolk to accumulate a desired amount. In order to increase the antibody titer in egg yolk, booster immunization may be performed.

卵黄から抗体を精製する方法としては、公知の方法を用いることができ、例えば、超遠心分離法、有機溶剤脱脂による分離法、リポタンパク質凝集剤(ポリエチレングリコール、デキストラン硫酸ナトリウム、ポリアクリル酸樹脂、食品増粘安定剤等)を用いる方法、硫安沈澱等の塩析、限外濾過等の濾過、エタノールと超臨界炭酸ガス抽出を併用する脱脂法等が挙げられる。   As a method for purifying an antibody from egg yolk, a known method can be used. For example, ultracentrifugation, separation by organic solvent degreasing, lipoprotein flocculant (polyethylene glycol, sodium dextran sulfate, polyacrylic acid resin, Food thickening stabilizers), salting out such as ammonium sulfate precipitation, filtration such as ultrafiltration, degreasing method using ethanol and supercritical carbon dioxide extraction in combination.

本発明で用いられる卵黄抗体は、目的の抗原に対する結合能を有していれば特に限定されず、完全な状態の抗体以外に、抗体の分解物であるF(ab′)、Fab′、Fab等であってもよい。これらは単独で用いられてもよく、2種以上が併用されてもよい。また、複数種類の抗原に対する抗体を併用してもよい。 The egg yolk antibody used in the present invention is not particularly limited as long as it has the ability to bind to the target antigen. In addition to the intact antibody, F (ab ′) 2 , Fab ′, It may be Fab or the like. These may be used independently and 2 or more types may be used together. Moreover, you may use together the antibody with respect to multiple types of antigen.

本発明に係る高保存性抗体溶液における卵黄抗体の濃度は、卵黄抗体として凍結乾燥品を用いた場合、1μg/mL〜20mg/mLであることが好ましく、より好ましくは8μg/mL〜10mg/mLである。1μg/mL未満であると、充分な量の卵黄抗体をマスク等へ吹き付けることが難しく、20mg/mLを超えると、卵黄抗体の凝集等が起こることがある。   The concentration of the yolk antibody in the highly conserved antibody solution according to the present invention is preferably 1 μg / mL to 20 mg / mL, more preferably 8 μg / mL to 10 mg / mL when a freeze-dried product is used as the yolk antibody. It is. When it is less than 1 μg / mL, it is difficult to spray a sufficient amount of egg yolk antibody onto a mask or the like, and when it exceeds 20 mg / mL, aggregation of egg yolk antibody may occur.

本発明で用いられる水としては特に限定されず、水道水であってもよいが、脱イオン水や超純水等が好適に用いられる。   It does not specifically limit as water used by this invention, Although tap water may be sufficient, deionized water, ultrapure water, etc. are used suitably.

本発明に係る高保存性抗体溶液は、1〜25質量%のエタノールを含有している。エタノール濃度が1質量%未満であると、充分な保存性を得るためには精度の高い滅菌処理が必要となり、エタノール濃度が25質量%を超えると、保存中に抗体が変性し活性が低下してしまう。当該エタノール濃度の、好ましい下限は5質量%であり、より好ましい下限は10質量%である。また、好ましい上限は20質量%であり、より好ましい上限は15質量%である。   The highly conserved antibody solution according to the present invention contains 1 to 25% by mass of ethanol. If the ethanol concentration is less than 1% by mass, high-precision sterilization is necessary to obtain sufficient storage stability. If the ethanol concentration exceeds 25% by mass, the antibody is denatured during storage and the activity decreases. End up. A preferable lower limit of the ethanol concentration is 5% by mass, and a more preferable lower limit is 10% by mass. Moreover, a preferable upper limit is 20 mass% and a more preferable upper limit is 15 mass%.

本発明に係る高保存性抗体溶液は、必要に応じて香料や緩衝剤等を含有してもよい。前記香料としては、例えば、メントール、D−リモネン、デシルアルデヒド、メントン、プレゴン、オイゲノール、シンナムアルデヒド、ベンズアルデヒド、ペパーミント油、レモン油、オレンジ油等が挙げられる。前記緩衝剤としては一般的にタンパク質に用いられるものであれば特に限定されず、例えば、リン酸緩衝液、酢酸緩衝液、ホウ酸緩衝液、グリシン緩衝液、トリス緩衝液、グッドの緩衝液等の緩衝液を調製するためのものが挙げられる。   The highly conserved antibody solution according to the present invention may contain a fragrance, a buffering agent or the like as necessary. Examples of the fragrances include menthol, D-limonene, decylaldehyde, menthone, pulegone, eugenol, cinnamaldehyde, benzaldehyde, peppermint oil, lemon oil, orange oil and the like. The buffer is not particularly limited as long as it is generally used for proteins. For example, phosphate buffer, acetate buffer, borate buffer, glycine buffer, Tris buffer, Good's buffer, etc. For preparing a buffer solution of

本発明に係る高保存性抗体溶液のpHとしては特に限定されないが、中性付近(例えば、pH6〜8)であればよい。   Although it does not specifically limit as pH of the highly-storable antibody solution based on this invention, What is necessary is just near neutrality (for example, pH 6-8).

本発明に係る高保存性抗体溶液には、滅菌処理を施すことが必要である。当該滅菌処理としては抗体活性を低下させない方法であれば特に限定されないが、フィルター滅菌が好適に用いられる。フィルター滅菌を用いれば、粗精製品である卵黄抗体を用いた場合、滅菌とともに卵黄抗体の精製を行うことも可能となる。   The highly conserved antibody solution according to the present invention needs to be sterilized. The sterilization treatment is not particularly limited as long as it does not reduce antibody activity, but filter sterilization is preferably used. If filter sterilization is used, when an egg yolk antibody, which is a crude product, is used, it becomes possible to purify the egg yolk antibody together with sterilization.

前記フィルター滅菌に用いるフィルターとしては、孔径が0.50μm以下のものが好ましく、より好ましくは孔径が0.25μm以下のものである。孔径が0.50〜0.25μmであると、一部の細菌は通過可能であるが、本発明に係る高保存性抗体溶液はエタノールを含有しているので、腐敗は起こらない。フィルターの孔径が0.25μm以下であれば、略完全に細菌を除去することができる。   The filter used for filter sterilization preferably has a pore size of 0.50 μm or less, and more preferably has a pore size of 0.25 μm or less. When the pore size is 0.50 to 0.25 μm, some bacteria can pass through, but since the highly conserved antibody solution according to the present invention contains ethanol, no spoilage occurs. If the pore size of the filter is 0.25 μm or less, bacteria can be removed almost completely.

本発明に係る高保存性抗体溶液を製造するには、まず、卵黄抗体を水(脱イオン水、超純水等)や緩衝液等の水性溶媒に溶解して抗体溶液を調製し、次いで、抗体溶液をフィルター滅菌する。抗体溶液へのエタノールの添加はフィルター滅菌が施される前であってもよく、後であってもよい。   To produce a highly conserved antibody solution according to the present invention, first, an antibody solution is prepared by dissolving an egg yolk antibody in an aqueous solvent such as water (deionized water, ultrapure water, etc.) or a buffer solution, Filter sterilize the antibody solution. The ethanol may be added to the antibody solution before or after filter sterilization.

得られた高保存性抗体溶液を滅菌済みの密閉容器内に収容すれば、長期間安定して保存することができる。また、前記密閉容器としてスプレー容器を用いれば、マスク等に抗体溶液を吹き付けて抗菌性や抗ウイルス性等を付与するためのスプレーとして用いることができる。   If the obtained highly-storable antibody solution is accommodated in a sterilized sealed container, it can be stably stored for a long period of time. In addition, if a spray container is used as the closed container, it can be used as a spray for imparting antibacterial and antiviral properties by spraying an antibody solution onto a mask or the like.

また、得られた高保存性抗体溶液を用いてエアゾール製品を構成してもよい。この場合、滅菌済みの密閉容器内に高保存性抗体溶液とともに充填する噴射剤としては、液体中の抗体活性を低下させないために、窒素ガスを用いることが望ましい。また、前記密閉容器内にメチルパラベン等の防腐剤を入れても良い。防腐剤としてメチルパラベンを用いることで、エアゾール化に伴う爆発の心配がない。   Moreover, you may comprise an aerosol product using the obtained highly conserved antibody solution. In this case, it is desirable to use nitrogen gas as a propellant filled together with a highly-storable antibody solution in a sterilized sealed container so as not to reduce antibody activity in the liquid. Moreover, you may put antiseptic | preservatives, such as methyl paraben, in the said airtight container. By using methylparaben as a preservative, there is no worry of explosion associated with aerosolization.

以下に実施例を掲げて本発明を更に詳細に説明するが、本発明はこれら実施例のみに限定されるものではない。   The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to these examples.

以下のようにして調製した抗体溶液を用いて、その抗体価の経時変化を観察した。   Using an antibody solution prepared as follows, the antibody titer was observed over time.

<被試験抗体>
被試験抗体として、コナヒョウヒダニ虫体由来粗精製アレルゲン又はヤケヒョウヒダニ虫体由来粗精製アレルゲンにより免疫したニワトリに産生させたIgYを、硫安沈澱により粗精製し、これを凍結乾燥してなる粉末状の抗体を用いた。
<Test antibody>
As an antibody to be tested, a powdered antibody obtained by roughly purifying IgY produced in a chicken immunized with a crudely purified allergen derived from an Aedes acarina mite or a crudely purified allergen derived from an Aedes albopictus by ammonium sulfate precipitation and freeze-dried. Using.

<抗体溶液の調製>
PBS(−)に上記の各IgY粉末を10mg/mL濃度になるよう溶解して、完全に溶解させた後、孔径0.45μmのフィルターを用いて滅菌を行った。並行して、脱イオン水に95%エタノールを10、20、40、60、80質量%の濃度になるように加えて、濃度の異なるエタノール水溶液を調製した。各濃度のエタノール水溶液と滅菌済みIgY水溶液とを1:1で混合した後、得られた抗体溶液を1.0mLずつスクリューキャップチューブに分注し、37℃で遮光して静置保存した。
<Preparation of antibody solution>
The above IgY powders were dissolved in PBS (−) to a concentration of 10 mg / mL and completely dissolved, and then sterilized using a filter having a pore diameter of 0.45 μm. In parallel, 95% ethanol was added to deionized water to a concentration of 10, 20, 40, 60, and 80 mass% to prepare ethanol aqueous solutions having different concentrations. After mixing ethanol aqueous solution of each concentration and sterilized IgY aqueous solution 1: 1, the obtained antibody solution was dispensed into a screw cap tube in an amount of 1.0 mL, and stored at 37 ° C. with light shielding.

<抗体価(力価)の測定>
各抗体溶液を1週間保存後及び34週間保存後(加速試験)にサンプリングして、各抗体溶液中のIgYの抗体価の維持率を、各アレルゲン固相化プレートを用いてELISAにより測定した。なお、0週目(試験開始時)及び1週間保存後の各抗体溶液は、サンプリング後−30℃で凍結保存し、34週間保存後の各抗体溶液とともに抗体価の測定に供した。
<Measurement of antibody titer (titer)>
Each antibody solution was sampled after storage for 1 week and 34 weeks (acceleration test), and the maintenance rate of the antibody titer of IgY in each antibody solution was measured by ELISA using each allergen-immobilized plate. Each antibody solution stored at 0 week (at the start of the test) and after 1 week was stored frozen at −30 ° C. after sampling, and subjected to antibody titer measurement together with each antibody solution stored for 34 weeks.

得られた結果は、抗コナヒョウヒダニ虫体由来粗精製アレルゲンIgYの抗体価を下記表1及び図1のグラフに示し、抗ヤケヒョウヒダニ虫体由来粗精製アレルゲンIgYの抗体価を下記表2及び図2のグラフに示す。また、各エタノール濃度の抗コナヒョウヒダニ虫体由来粗精製アレルゲンIgYの抗体溶液を1週間保存した後の様子を図3の写真に示す。   The obtained results show the antibody titers of the crude purified allergen IgY derived from the anti-carp leopard mite body in the following Table 1 and the graph of FIG. Shown in the graph. Moreover, the mode after preserve | saving the antibody solution of the crude purified allergen IgY derived from the anti-carp leopard mite parasite of each ethanol concentration for one week is shown in the photograph of FIG.

なお、表1及び表2中、「−」と記載されている箇所は、測定不能であったことを示す。   In Tables 1 and 2, “-” indicates that measurement was impossible.

<結果>
抗コナヒョウヒダニ虫体由来粗精製アレルゲンIgYの抗体溶液では、表1及び図1に示すように、エタノール濃度が5質量%、10質量%、20質量%及び25質量%である場合は、1週間保存後でも抗体価が90%前後維持されており、34週間保存後でも抗体価が40%前後も維持されていた。一方、エタノール濃度が30質量%の抗体溶液の抗体価維持率は1週間保存後には約50%にまで低下し、34週間保存後には約20%にまで低下した。更に、エタノール濃度が40質量%の抗体溶液の抗体価維持率は1週間保存後で約20%にまで低下した。また、図3に示すように、1週間保存後において、エタノール濃度が30質量%及び40質量%の抗体溶液は白濁しているのに対して、エタノール濃度が5質量%、10質量%及び20質量%の抗体溶液では白濁は認められなかった。なお、エタノール濃度が40質量%の抗体溶液は、エタノール水溶液の混合直後から白濁した。
<Result>
As shown in Table 1 and FIG. 1, the antibody solution of the anti-Pleurotus mite parasite-derived crude purified allergen IgY is stored for 1 week when the ethanol concentration is 5% by mass, 10% by mass, 20% by mass, and 25% by mass. Later, the antibody titer was maintained at around 90%, and the antibody titer was maintained at around 40% even after storage for 34 weeks. On the other hand, the antibody titer maintenance rate of the antibody solution having an ethanol concentration of 30% by mass decreased to about 50% after storage for 1 week and decreased to about 20% after storage for 34 weeks. Furthermore, the antibody titer maintenance rate of the antibody solution having an ethanol concentration of 40% by mass decreased to about 20% after storage for 1 week. In addition, as shown in FIG. 3, after storage for 1 week, antibody solutions with ethanol concentrations of 30% by mass and 40% by mass are cloudy, whereas ethanol concentrations are 5% by mass, 10% by mass, and 20%. White turbidity was not observed in the mass% antibody solution. The antibody solution having an ethanol concentration of 40% by mass became cloudy immediately after mixing the ethanol aqueous solution.

また、抗ヤケヒョウヒダニ虫体由来粗精製アレルゲンIgYの抗体溶液では、表2及び図2に示すように、エタノール濃度が5質量%、10質量%、20質量%及び25質量%である場合は、1週間保存後では抗体価はほとんど低下しておらず、34週間保存後でも抗体価は約40%以上も維持されていた。一方、エタノール濃度が30質量%の抗体溶液の抗体価維持率は1週間保存後には約50%にまで低下し、34週間保存後には約20%にまで低下した。更に、エタノール濃度が40質量%の抗体溶液の抗体価維持率は1週間保存後には約25%にまで低下した。   In addition, in the antibody solution of the anti-Dark leopard mite parasite-derived crude purified allergen IgY, as shown in Table 2 and FIG. 2, when the ethanol concentration is 5% by mass, 10% by mass, 20% by mass, and 25% by mass, 1 The antibody titer hardly decreased after weekly storage, and the antibody titer was maintained at about 40% or more after 34 weeks of storage. On the other hand, the antibody titer maintenance rate of the antibody solution having an ethanol concentration of 30% by mass decreased to about 50% after storage for 1 week and decreased to about 20% after storage for 34 weeks. Furthermore, the antibody titer maintenance rate of the antibody solution having an ethanol concentration of 40% by mass decreased to about 25% after storage for 1 week.

Claims (7)

卵黄抗体と、水と、1〜25質量%のエタノールとを含有し、滅菌処理が施されていることを特徴とする高保存性抗体溶液。   A highly conserved antibody solution comprising an egg yolk antibody, water, and 1 to 25% by mass of ethanol and sterilized. 前記滅菌処理が、フィルター滅菌である請求項1記載の高保存性抗体溶液。   The highly conserved antibody solution according to claim 1, wherein the sterilization treatment is filter sterilization. 香料を含有している請求項1又は2記載の高保存性抗体溶液。   The highly conserved antibody solution according to claim 1 or 2, which contains a fragrance. 緩衝剤を含有している請求項1、2又は3記載の高保存性抗体溶液。   The highly conserved antibody solution according to claim 1, 2 or 3, which contains a buffer. 請求項1、2、3又は4記載の高保存性抗体溶液が充填されてなることを特徴とするスプレー。   A spray comprising the highly conserved antibody solution according to claim 1, 2, 3 or 4. 卵黄抗体を水性溶媒に溶解して抗体溶液を調製する工程と、
前記抗体溶液をフィルター滅菌する工程と、
フィルター滅菌が施される前又は後の抗体溶液に、1〜25質量%の濃度となるようにエタノールを加える工程と、を有することを特徴とする高保存性抗体溶液の製造方法。
Dissolving an egg yolk antibody in an aqueous solvent to prepare an antibody solution;
Filter sterilizing the antibody solution;
And a step of adding ethanol to the antibody solution before or after filter sterilization so as to obtain a concentration of 1 to 25% by mass.
卵黄抗体を水性溶媒に溶解して抗体溶液を調製する工程と、
前記抗体溶液をフィルター滅菌する工程と、
フィルター滅菌が施される前又は後の抗体溶液に、1〜25質量%の濃度となるようにエタノールを加える工程と、
前記抗体溶液を滅菌済みの密閉容器内に収容する工程と、を有することを特徴とする抗体の保存方法。
Dissolving an egg yolk antibody in an aqueous solvent to prepare an antibody solution;
Filter sterilizing the antibody solution;
Adding ethanol to the antibody solution before or after filter sterilization to a concentration of 1 to 25% by mass;
And storing the antibody solution in a sterilized sealed container.
JP2014079671A 2013-04-08 2014-04-08 Highly-preservable antibody solution Pending JP2014218496A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2014079671A JP2014218496A (en) 2013-04-08 2014-04-08 Highly-preservable antibody solution

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2013080257 2013-04-08
JP2013080257 2013-04-08
JP2014079671A JP2014218496A (en) 2013-04-08 2014-04-08 Highly-preservable antibody solution

Publications (1)

Publication Number Publication Date
JP2014218496A true JP2014218496A (en) 2014-11-20

Family

ID=51937305

Family Applications (1)

Application Number Title Priority Date Filing Date
JP2014079671A Pending JP2014218496A (en) 2013-04-08 2014-04-08 Highly-preservable antibody solution

Country Status (1)

Country Link
JP (1) JP2014218496A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019024311A1 (en) * 2017-08-02 2019-02-07 Wwhs Biotech, Inc Solution and method for preserving fluorescent antibody

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0253716A (en) * 1988-08-18 1990-02-22 Lion Corp Composition for oral cavity application
JP2002510601A (en) * 1998-04-06 2002-04-09 イノセント リミテッド Oral antimicrobial and deodorant composition
JP2003159094A (en) * 2001-11-26 2003-06-03 Japan Science & Technology Corp Method for aseptically producing yolk antibody
JP2003212898A (en) * 2002-01-22 2003-07-30 Pharmafoods Kenkyusho:Kk Method for producing yolk antibody
JP2005169105A (en) * 2003-03-28 2005-06-30 Daikin Ind Ltd Mask equipped with noxious substance eliminating material for mask, and storage method for noxious substance eliminating material for mask
JP2009528352A (en) * 2006-03-01 2009-08-06 デンテック インコーポレイテッド Oral hygiene products and their use
JP2010516688A (en) * 2007-01-16 2010-05-20 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Antibacterial peptide

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0253716A (en) * 1988-08-18 1990-02-22 Lion Corp Composition for oral cavity application
JP2002510601A (en) * 1998-04-06 2002-04-09 イノセント リミテッド Oral antimicrobial and deodorant composition
JP2003159094A (en) * 2001-11-26 2003-06-03 Japan Science & Technology Corp Method for aseptically producing yolk antibody
JP2003212898A (en) * 2002-01-22 2003-07-30 Pharmafoods Kenkyusho:Kk Method for producing yolk antibody
JP2005169105A (en) * 2003-03-28 2005-06-30 Daikin Ind Ltd Mask equipped with noxious substance eliminating material for mask, and storage method for noxious substance eliminating material for mask
JP2009528352A (en) * 2006-03-01 2009-08-06 デンテック インコーポレイテッド Oral hygiene products and their use
JP2010516688A (en) * 2007-01-16 2010-05-20 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Antibacterial peptide

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019024311A1 (en) * 2017-08-02 2019-02-07 Wwhs Biotech, Inc Solution and method for preserving fluorescent antibody

Similar Documents

Publication Publication Date Title
RU2728575C1 (en) Stabilized preparations containing antibodies against ngf
TWI445714B (en) A method to produce a highly concentrated immunoglobulin preparation for subcutaneous use
JP2018203784A (en) Stabilization of immunoglobulin through aqueous formulation with histidine at weak acidic to neutral ph
ES2584190T3 (en) Procedure for the manufacture of a pharmaceutical preparation containing glatiramer acetate
EP1958626B1 (en) Method for inactivating viruses by addition of slightly acidic arginine
EA028227B1 (en) Stabilized formulations containing antibodies to human proprotein convertase subtilisin/kexin type 9 (pcsk9)
JP2017222654A5 (en)
MX2010011117A (en) Aqueous pharmaceutical formulation.
AR082257A1 (en) COMPOSITION OF CONCENTRATED HUMAN IMMUNOGLOBULINS, PROCEDURE
US20160251261A1 (en) Method of Handling a Liquid Drug Formation
ZA202102986B (en) Protein solution formulation containing high concentration of an anti-vegf antibody
JP2014218496A (en) Highly-preservable antibody solution
JP2022546275A (en) Stable medicinal products containing diluted povidone-iodine preparations
US8501220B2 (en) Stabilized liquid egg material for extended shelf life
RU2019111152A (en) NEW METHOD FOR STABILIZING BIOPHARMACEUTICAL MEDICINAL PRODUCT IN PRODUCTION
WO2018102145A1 (en) Levothyroxine liquid formulations
PT92163B (en) METHOD FOR PREPARING A LIOFILIZED MDM COMPOSITION
CA2390897A1 (en) Method of improving hatchability of eggs
CN104412970B (en) Natural latex vessel disinfectant and using method thereof
US9408804B2 (en) Method for keeping an immunogenic composition available for administration to an animal
RU2561596C2 (en) Method for producing virus-inactivated immunoglobulin solutions low in residual caprylic acid
RU2521513C1 (en) Application of aethonium as adjuvant for production of sorbed foot-mouth disease vaccine
WO2019226058A1 (en) Medicinal product containing improved stability water solution of methisoprinol
US20160144033A1 (en) Concentrated acetaminophen solution
US20240209066A1 (en) 2019 novel coronavirus antibody-containing pharmaceutical formulations

Legal Events

Date Code Title Description
A621 Written request for application examination

Free format text: JAPANESE INTERMEDIATE CODE: A621

Effective date: 20170307

A977 Report on retrieval

Free format text: JAPANESE INTERMEDIATE CODE: A971007

Effective date: 20180131

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20180220

A601 Written request for extension of time

Free format text: JAPANESE INTERMEDIATE CODE: A601

Effective date: 20180418

A521 Request for written amendment filed

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20180621

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20181101

A02 Decision of refusal

Free format text: JAPANESE INTERMEDIATE CODE: A02

Effective date: 20190423