JP2011242408A - Method of measuring anti-gelatin antibody - Google Patents
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本発明は、
イ、血液中の抗ゼラチン抗体、そのアレルゲンとなるゼラチン動物種及びその分画を測定する方法と試薬
ロ、特定動物のみから得られるゼラチンと、これを添加した食品・薬剤に関する物である。The present invention
(A) Anti-gelatin antibodies in blood, gelatin animal species as allergens thereof, methods and reagents for measuring fractions thereof, gelatin obtained from specific animals only, and foods and drugs to which these are added.
従来、ゼラチンアレルギー者を発見するには、反応を起こした食品や薬剤の共通成分を推定いくのが一般的で、時間を要した。又、専門医にゼラチンアレルギー者と診断されても、ゼラチンは、様々な食品や薬剤に微量ずつ含まれているのでどの食品を避けるべきか不明で、又ゼラチンアレルギー者用の食品もない。
更にゼラチンアレルギー者は、一般に用いられる薬剤、例えば疾病予防の為に当然投与されるべき必要なワクチンも、ゼラチンを含む為、時には避けなくてはならなかった。Conventionally, it has been common to estimate the common components of foods and drugs that have caused reactions in order to find gelatin allergic individuals. Even if a specialist diagnoses a person who is allergic to gelatin, gelatin is contained in various foods and medicines in minute amounts, so it is unclear which food should be avoided, and there is no food for people with gelatin allergies.
In addition, gelatin allergies sometimes had to be avoided because commonly used drugs, such as vaccines that should be naturally administered for disease prevention, also contain gelatin.
本発明者は、長い間、ゼラチンアレルギー者の簡便な発見方法、食品・薬剤中の微量ゼラチンの検出方法、及びゼラチンアレルギー者用の食品・薬剤の研究に努めた。For a long time, the present inventor has sought to study a simple method for finding a gelatin allergic person, a method for detecting a minute amount of gelatin in a food or drug, and a food or drug for a gelatin allergic person.
その結果、本発明者は、ゼラチンアレルギー者の簡便な発見方法として、下記１、２、３、４を確立した。As a result, the present inventors established the following 1, 2, 3, and 4 as a simple method for finding gelatin allergic subjects.
１ 血液中の抗ゼラチン抗体とその由来動物種を免疫反応を用いて測定する方法とその試薬。
ヒト血清中の抗ゼラチン抗体を、酵素免疫反応（ＥＬＩＳＡ）で測定する時、どの種のゼラチンと交叉するか知る為、あらかじめ動物種が明確なゼラチンを、種別にプレートにコートする事、及び抗ゼラチン抗体として、ＩｇＥを捕捉する為、第二抗体として、抗ヒトＩｇＥ抗体を用いる測定法及びその試薬。
つまり、試薬として、１）各種動物由来のゼラチンをコートしたプレート、２）酵素標識抗ヒトＩｇＥ抗体、３）発色基質（ＴＭＢ，過酸化水素）、４）反応停止液（硫酸）を用い、一般の酵素免疫反応によりヒト血清を検体として測定する。1. A method and reagent for measuring an anti-gelatin antibody in blood and the animal species derived from it using an immune reaction.
When measuring anti-gelatin antibody in human serum by enzyme immunoreaction (ELISA), in order to know which kind of gelatin crossover, it is necessary to coat the plate with a specific type of gelatin in advance, A measurement method using an anti-human IgE antibody as a second antibody and a reagent for capturing IgE as a gelatin antibody.
That is, as reagents, 1) plates coated with gelatin derived from various animals, 2) enzyme-labeled anti-human IgE antibody, 3) chromogenic substrate (TMB, hydrogen peroxide), 4) reaction stop solution (sulfuric acid), Human serum is measured as a specimen by the enzyme immunoreaction.
もちろん、免疫反応として、酵素免疫反応に限定されず、ＲＩＡ，免疫発光法、凝集反応他を含み、酵素標識の抗体としては、ポリクローナル又はモノクローナル抗体を問わず、又それを放射性物質、発光物質で標識した物、無標識物でも良い。
プレートをゼラチンでコートする時、プレート毎に動物種を変えても良いが、同一プレートの穴毎にかえて、パネルにする方が望ましい。同一検体と複数のゼラチンとの反応を、一目で観察できるからである。又、動物種毎にプレートや穴に、マークや色をつければ識別しやすい。
用いるゼラチンの動物種としては、ウシ、ブタ、ニワトリ、羊、鮭、タコ等、又これに限定されない。
単に、ゼラチン抗体を検出するなら、プレートにコートするゼラチンは、複数種を混合したもので良いが、それでは、避けるべきゼラチンの動物種までは明確に成らない。更に第二抗体は、抗ヒトＩｇＥ抗体に限定されず、抗ヒトイムノグロブリン抗体でも良いが、抗ヒトＩｇＥ抗体の方が望ましい。Of course, the immune reaction is not limited to the enzyme immune reaction, but includes RIA, immunoluminescence method, agglutination reaction, etc. The enzyme-labeled antibody may be a polyclonal or monoclonal antibody, and may be a radioactive substance or a luminescent substance. It may be labeled or unlabeled.
When the plate is coated with gelatin, the animal species may be changed from plate to plate, but it is preferable to change the plate to a panel instead of every hole in the same plate. This is because the reaction between the same specimen and a plurality of gelatins can be observed at a glance. In addition, it is easy to identify each animal species by adding marks and colors to the plates and holes.
The animal species of gelatin to be used is not limited to cattle, pigs, chickens, sheep, rabbits, octopuses and the like.
If the gelatin antibody is simply detected, the gelatin coated on the plate may be a mixture of a plurality of species, but this does not clearly define the animal species of gelatin to be avoided. Further, the second antibody is not limited to the anti-human IgE antibody, and may be an anti-human immunoglobulin antibody, but the anti-human IgE antibody is more preferable.
２ 血液中の抗特定分画ゼラチン抗体を免疫反応を用いて測定する方法と試薬。
各動物種のゼラチンを電気泳動で分画した後、この各分画を採取し、前述「１」と同様に、プレートにコートする。他は前述「１」と同様の試薬と測定法である。これにより、抗ゼラチン抗体と反応する動物種のみならず、反応するゼラチンの分画まで確定できる。2 Method and reagent for measuring anti-specific fraction gelatin antibody in blood using immune reaction.
After gelatin of each animal species is fractionated by electrophoresis, each fraction is collected and coated on a plate in the same manner as in “1” above. Others are the same reagents and measurement methods as in the above-mentioned “1”. Thereby, not only the animal species that react with the anti-gelatin antibody but also the fraction of the gelatin that reacts can be determined.
３ ゼラチンを用いた皮内注射法とその注射剤各動物種由来の微量のゼラチンを、例えばウシ、ニワトリ、サケの、１μｇ〜０．０１μｇ／ｍｌを各別個のリン酸バッファーに溶解し、その０．１ｍｌ〜０．０１ｍｌを皮内注射し、その部位に発赤を生じる時にどの動物種由来のゼラチンに陽性かを判定できる。
この時、ゼラチンは、前述「２」の電気泳動分画を用いれば、より適格にアレルゲンを推定できる。
ゼラチンの注射量は、濃度０．０１μｇ／ｍｌの液を、０．０１ｍｌ投与で、充分であるが、これに限定されない。溶解液は、リン酸バッファーが望ましいが、それ自身が反応に影響を与えないものであれば、代用できる。3 Intradermal injection method using gelatin and its injection A small amount of gelatin derived from each animal species, for example, 1 μg to 0.01 μg / ml of bovine, chicken and salmon is dissolved in each separate phosphate buffer, When 0.1 ml to 0.01 ml is injected intradermally and redness occurs at the site, it can be determined which animal species-derived gelatin is positive.
At this time, gelatin can estimate the allergen more appropriately by using the electrophoresis fraction of “2”.
As for the injection amount of gelatin, 0.01 ml of a solution having a concentration of 0.01 μg / ml is sufficient, but is not limited thereto. The lysis solution is preferably a phosphate buffer, but can be substituted if it does not affect the reaction itself.
４ ゼラチンを用いたパッチ反応法とその試薬。
各種動物由来の、例えばウシ、ニワトリ、鮭由来の微量のゼラチン、例えば１ｍｇ〜０．５μｇ／平方ｃｍを各別個のパッチに付着させ、これを、通常のパッチ試験と同様、背部に貼り、その部位に発赤を生じる時、どの動物由来のゼラチンに陽性か判定できる。
この時、ゼラチンは、前述「２」の電気泳動分画を用いれば、より適格にアレルゲンを推定できる。4 Patch reaction method using gelatin and its reagents.
A small amount of gelatin derived from various animals, such as bovine, chicken, and rabbit, for example, 1 mg to 0.5 μg / cm 2 is attached to each individual patch, and this is applied to the back as in a normal patch test. When redness occurs in a region, it can be determined which animal-derived gelatin is positive.
At this time, gelatin can estimate the allergen more appropriately by using the electrophoresis fraction of “2”.
従来ゼラチンアレルギーと称されていた人が、全ての動物由来のゼラチンに反応するものではなく、ウシ由来のみに反応することを発見した。又、特定分画としてウシ由来α２のみと反応した。他方各種市販ゼラチンを調べたところその由来は全てウシであった。ウシがゼラチンの原料として繁用されるから、いわゆるゼラチンアレルギー者は、ウシ由来のゼラチンのみに反応するのである。
ゼラチンとしてブタが汎用されれば、ゼラチンアレルギー者のアレルゲンは、ブタゼラチンに特定される可能性を示す。
これは、「ゼラチンはコラーゲンの変性したものであり、コラーゲンは構造上動物種差がないから、どの動物由来のゼラチンも一緒だろう。だから、ゼラチンアレルギー者は、全ての動物由来のゼラチンに反応するはずだ。」と言う一般の思い込みを否定するものであった。
つまり、ゼラチンは、構造上、機能上、由来動物の種差はないが、アレルギー反応のような免疫上の違いを示すと言う事である。
本発明者は、以上の発明と発見から、更に下記５・６の発明を完成した。It has been discovered that a person previously called gelatin allergy does not react to all animal-derived gelatin, but only cattle-derived. Moreover, it reacted only with bovine-derived α2 as a specific fraction. On the other hand, when various commercially available gelatins were examined, their origin was all bovine. Since cattle are frequently used as a raw material for gelatin, so-called gelatin allergic individuals react only to bovine-derived gelatin.
If pigs are widely used as gelatin, allergens of people with gelatin allergies may be identified as porcine gelatin.
This is because “gelatin is a modified form of collagen and there is no animal species difference in the structure of collagen, so any animal-derived gelatin will be together. So, allergic to gelatin will react to gelatin from all animals. It was a denial of the general belief.
In other words, gelatin is structurally and functionally different from species of origin, but it shows immune differences such as allergic reactions.
The present inventor completed the following inventions 5 and 6 based on the above inventions and discoveries.
５ 食品・薬剤中のゼラチンの存在と、その由来動物種を免疫反応を用いて測定する方法と試薬。
例えば、食品や薬剤、具体的には、ケーキやワクチン中のゼラチンの存在と、その由来動物種を知る為、サンドイッチ法による酵素免疫反応（ＥＬＩＳＡ）で測定する時、サンドイッチ法の一方又は両方を、特定動物種のゼラチンにのみ反応する抗ゼラチン抗体を用いることを特長とする測定法及びその試薬である。
試薬としては、１）特定動物種のゼラチンに反応する抗ゼラチン抗体をコートしたプレート、２）特定動物種のゼラチンに反応する酵素標識抗体、３）発色基質（ＴＭＢ、過酸化水素）、４）反応停止液（硫酸）を用い一般の酵素免疫反応により、食品又は薬剤を検体として測定する。
食品や薬剤が直接検体として用い難い形状の時は、これを強酸（又はアルカリ）で処理すれば、ゼラチンが抽出されるので、強酸（又はアルカリ）を除き、適当な緩衝液、例えばリン酸バッファーに溶解させて検体とする。
免疫反応としては、酵素免疫反応のみでなく、ＲＩＡ、免疫発光法、凝集反応他を含み、サンドイッチ法で使用する抗体はポリクローナル又はモノクローナル抗体を問わず、又、それを、酵素、放射性物質、発光物質で標識したものでも良い。特定動物種としては、ウシ、ブタ、ニワトリ、鮭に限定されない。5. A method and reagent for measuring the presence of gelatin in foods and drugs and the animal species derived from it using an immune reaction.
For example, in order to know the presence of gelatin in foods and drugs, specifically cakes and vaccines, and the animal species derived from it, when measuring by enzyme-linked immunosorbent assay (ELISA) using the sandwich method, one or both of the sandwich methods are used. And a reagent and its measuring method characterized by using an anti-gelatin antibody that reacts only with gelatin of a specific animal species.
As reagents, 1) a plate coated with an anti-gelatin antibody that reacts with gelatin of a specific animal species, 2) an enzyme-labeled antibody that reacts with gelatin of a specific animal species, 3) a chromogenic substrate (TMB, hydrogen peroxide), 4) Using a reaction stop solution (sulfuric acid), a food or drug is measured by a general enzyme immunoreaction.
When it is difficult to use food or drug directly as a specimen, if it is treated with a strong acid (or alkali), gelatin is extracted. Therefore, an appropriate buffer solution such as a phosphate buffer can be used except for the strong acid (or alkali). Dissolve the sample to make a sample.
The immune reaction includes not only enzyme immune reaction but also RIA, immunoluminescence method, agglutination reaction, etc. The antibody used in the sandwich method may be polyclonal or monoclonal antibody, and it can be expressed as enzyme, radioactive substance, luminescence. It may be labeled with a substance. Specific animal species are not limited to cattle, pigs, chickens, and rabbits.
６ 特定動物由来のゼラチンとこれを含む食品、薬剤。
特定動物由来のゼラチンを得るには、ゼラチン製造時には、単一動物種のみを扱い、得られたゼラチンを他種のものと混合しない。用いるゼラチンの動物種としては、ウシ、ブタ、ニワトリ、羊、鮭、タコなど、これに限定されない。
ウシゼラチンは、その骨を原料とし、強酸（強アルカリ）、脱灰の処理で製するが、他の動物でも、例えば、ブタ、羊、ニワトリでも同様に可能で有る。
動物でも、魚類は、例えば鮭は、皮を用いる。軟体類は、例えばタコ、イカは、そのまま用いる。又、ウシ、ブタ、羊、ニワトリは、皮も利用できる。
皮を原料とする時は、真皮のみを用いる事が望ましい。軟体類や皮からの場合、強酸（強アルカリ）に漬け、加熱することで抽出される。
ゼラチンの製法は、本法に限定されず、例えば、コラーゲンを製し、これを、加熱変性させても良い。6 Gelatin derived from a specific animal and foods and drugs containing it.
In order to obtain gelatin derived from a specific animal, only a single animal species is handled during gelatin production, and the obtained gelatin is not mixed with other species. Examples of the animal species of gelatin to be used include, but are not limited to, cows, pigs, chickens, sheep, rabbits and octopus.
Bovine gelatin is produced from its bone as a raw material by a strong acid (strong alkali) and decalcification treatment, but it can be similarly applied to other animals, for example, pigs, sheep and chickens.
Even in animals, fish use skins, for example, salmon. For example, octopus and squid are used as they are. Cows, pigs, sheep and chickens can also be used with skin.
When using leather as the raw material, it is desirable to use only the dermis. In the case of mollusks and skin, it is extracted by soaking in a strong acid (strong alkali) and heating.
The manufacturing method of gelatin is not limited to this method, For example, collagen may be manufactured and this may be heat-denatured.
７ 特定動物由来のゼラチンを含む食品及び又は薬剤。
前述の各種由来のゼラチンを、従来の原料不明のゼラチンに代えて、食品や薬剤に加えれば、特定動物由来のゼラチンを含む食品・薬剤が得られる。特定動物由来のゼラチンを、できるだけ多くそろえることは、将来、ウシ以外の動物ゼラチンにアレルギーが現れた時に、有用である。
食品に加えるゼラチンは、その食味の点から他の材料に代え難い。例えば、ゼラチンゼリーや、煮凝である。
薬剤に加えるゼラチンは、薬剤の安定化の為に代え難い。例えば、ワクチンやカプセルである。ワクチンに、代用物質としてウシ血清アルブミンやヒト血清アルブミンを加える事は、抗原性、安全性で問題がある。
又、ワクチンは、時に、ゼラチンを培地として用い、ふけい剤として用いる。
これらの時、一般に用いられるゼラチンに代えて、特定動物種、例えば、ニワトリ由来のゼラチンを使用すれば、従来製していたものと同じ食品や薬剤が、同じ製法で得られ、同じ効果を得られ、ウシゼラチンアレルギー者は安心して利用できる。7. Foods and / or drugs containing gelatin derived from specific animals.
If the above-mentioned gelatins of various origins are added to foods and drugs in place of conventional gelatins whose raw materials are unknown, foods / drugs containing gelatin derived from specific animals can be obtained. Having as much gelatin as possible from a specific animal is useful when allergies appear in animal gelatin other than bovine in the future.
Gelatin added to food is difficult to replace with other ingredients because of its taste. For example, gelatin jelly or boiled.
Gelatin added to the drug is difficult to replace for drug stabilization. For example, vaccines and capsules. Adding bovine serum albumin or human serum albumin as a substitute substance to a vaccine has problems in antigenicity and safety.
Also, vaccines sometimes use gelatin as a medium and as a dandruff agent.
In these cases, if gelatin derived from a specific animal species, such as chicken, is used instead of gelatin generally used, the same foods and drugs as those conventionally produced can be obtained by the same manufacturing method, and the same effect can be obtained. It can be safely used by those who are allergic to bovine gelatin.
ゼラチンアレルギー患者血清中の抗ゼラチン抗体とその由来動物種を測定した。
健常者血清を対照とし、次の試薬で酵素免疫反応を行った。
試薬１、ウシ（タイプＩコラーゲン型）ゼラチン、ニワトリ（タイプＩコラーゲン型）ゼラチン、鮭由来ゼラチンを各０．５μｇ／穴をコートしたマイクロプレート
試薬２、ＨＲＰ標識抗ヒトＩｇＥ抗体（ラット由来ポリクローナル）
試薬３、ＴＭＢ試薬（ＴＭＢ０．１％，過酸化水素０．０２％、０．１Ｍクエン酸緩衝液）
試薬４、反応停止液（０．５Ｍ硫酸）
検体は、リン酸緩衝液で１００倍に希釈し、１００μｌ／穴を用いた。
測定方法：試薬１に検体１００μｌ／穴→２時間インキュベーション及び洗浄→試薬２、１００μｌ／穴→１時間インキュベーションおよび洗浄→試薬３、１００μｌ→３０分インキュベーション→試薬４、５０μｌ→測定（吸光度４５０ｎｍ）
結果：検体中には、抗ウシゼラチン抗体が認められたが、抗ニワトリゼラチン抗体、抗鮭ゼラチン抗体は認められなかった。よって、このゼラチンアレルギー患者は、ウシゼラチンアレルギーで、ウシゼラチンのみ避ければ良い。又、本法は、血清中の抗ゼラチン抗体の存在とその由来動物種を測定する方法となり得る。Anti-gelatin antibodies and their derived animal species in the serum of gelatin allergy patients were measured.
Serum of healthy subjects was used as a control, and an enzyme immunoreaction was performed with the following reagents.
Reagent 1, microplate reagent 2 coated with 0.5 μg / well each of bovine (type I collagen type) gelatin, chicken (type I collagen type) gelatin, and rabbit-derived gelatin, HRP-labeled anti-human IgE antibody (rat-derived polyclonal)
Reagent 3, TMB reagent (TMB 0.1%, hydrogen peroxide 0.02%, 0.1M citrate buffer)
Reagent 4, reaction stop solution (0.5M sulfuric acid)
The specimen was diluted 100 times with a phosphate buffer, and 100 μl / well was used.
Measurement method: 100 μl / well of specimen in reagent 1 → 2 hours incubation and washing → reagent 2, 100 μl / well → 1 hour incubation and washing → reagent 3, 100 μl → 30 minutes incubation → reagent 4, 50 μl → measurement (absorbance 450 nm)
Result: Anti-bovine gelatin antibody was observed in the specimen, but anti-chicken gelatin antibody and anti-wrinkle gelatin antibody were not observed. Therefore, this gelatin allergic patient is allergic to bovine gelatin and only bovine gelatin should be avoided. In addition, this method can be a method for measuring the presence of anti-gelatin antibodies in serum and the animal species derived from them.
ウシ由来ゼラチンを電気泳動により分画した。この分画の中から、α１とα２を、「実施例２」の「試薬１」を置き換え「実施例２」と同様の実験を行った。
結果：検体中の抗ウシ由来ゼラチン抗体はα２分画にのみ反応し、α１分画とは反応しなかった。よって、このゼラチンアレルギー患者は、ウシα２分画のみがアレルゲンで、ウシ由来ゼラチンを食品や薬剤に用いる時、α２分画を除いて用いれば良い。Bovine gelatin was fractionated by electrophoresis. From this fraction, α1 and α2 were replaced with “Reagent 1” in “Example 2”, and the same experiment as in “Example 2” was performed.
Results: The anti-bovine-derived gelatin antibody in the sample reacted only with the α2 fraction and did not react with the α1 fraction. Therefore, in this gelatin allergic patient, only the bovine α2 fraction is the allergen, and when the bovine-derived gelatin is used for foods and drugs, the α2 fraction may be used.
ゼラチンアレルギー者にウシ由来ゼラチン、ニワトリ由来ゼラチン、サケ由来ゼラチンの各０．００１μｇ／１０μｌリン酸バッファーを皮内注射した。
又、日をあらため、これらの注射薬を、同量、同一人に皮下注射した。
結果：皮内注射後、ウシ由来ゼラチンのみ、翌日に発赤を示し、ニワトリ及びサケ由来ゼラチンは発赤を観察されなかった。皮下注射についても、異常がなかった。
よって、ゼラチンを用いた皮内注射反応法は、ゼラチンアレルギー患者の発見と、そのゼラチン由来種を明確にする。又、非ウシ由来ゼラチンを含む注射薬剤はウシゼラチンアレルギー者に安全に投与し得る。Those who were allergic to gelatin were injected intradermally with 0.001 μg / 10 μl phosphate buffer of bovine-derived gelatin, chicken-derived gelatin, and salmon-derived gelatin.
In addition, these injections were subcutaneously injected to the same person in the same amount for another day.
Result: After intradermal injection, only bovine-derived gelatin showed redness the following day, and chicken and salmon-derived gelatin did not show redness. Even with the subcutaneous injection, there was no abnormality.
Therefore, the intradermal injection reaction method using gelatin clarifies the discovery of gelatin allergy patients and the gelatin-derived species. Injectable drugs containing non-bovine derived gelatin can be safely administered to those who are allergic to bovine gelatin.
ゼラチンアレルギー患者の背部に、ウシ由来ゼラチン、ニワトリ由来ゼラチン、サケ由来ゼラチンの１００μｇ／９平方ミリメートルのガーゼをしっかりと貼り付けた。
結果：付着後、ウシ由来ゼラチンのみが、２日後赤色斑を示し、ニワトリ及びワケ由来ゼラチンには赤色斑が観察されなかった。よって、ゼラチンを用いたパッチ反応法は、ゼラチンアレルギー患者の発見と、そのアレルギー由来種を明確にする。100 μg / 9 square millimeter gauze of bovine-derived gelatin, chicken-derived gelatin, and salmon-derived gelatin was firmly attached to the back of a gelatin allergy patient.
Results: After attachment, only bovine-derived gelatin showed red spots after 2 days, and no red spots were observed on chicken and salmon-derived gelatin. Therefore, the patch reaction method using gelatin clarifies the discovery of gelatin allergy patients and the allergen-derived species.
食品及び薬剤のゼラチンの存在とその由来動物を測定した。ゼラチンを大量に含む水菓子及びＭＭＲワクチンを検体とし、次の試薬で酵素免疫反応を行った。
対象検体には、ウシゼラチン、ニワトリゼラチン（共にタイプＩコラーゲン型）を使用した。
試薬１、抗ウシ（タイプＩコラーゲン型）ゼラチン抗体、抗ニワトリ（タイプＩコラーゲン型）ゼラチン抗体を各０．５μｇ／穴をコートしたマイクロプレート
試薬２−１、ＨＲＰ標識抗ウシ（タイプＩコラーゲン型）ゼラチン抗体
試薬２−２、ＨＲＰ標識抗ニワトリ（タイプＩコラーゲン型）ゼラチン抗体
試薬３、ＴＭＢ試薬（ＴＭＢ０．１％，過酸化水素０．０２％、０．１Ｍクエン酸緩衝液）
試薬４、反応停止液（０．５Ｍ硫酸）
検体は、リン酸緩衝液に溶解させ、１００μｌ／穴を用いた。
測定方法：試薬１に検体１００μｌ／穴→２時間インキュベーション及び洗浄→試薬２−１、２−２、各１００μｌ／穴→１時間インキュベーションおよび洗浄→試薬３、１００μｌ→３０分インキュベーション→試薬４、５０μｌ→測定（吸光度４５０ｎｍ）し、ＯＤ値の高さで判定。
結果：検体中には、ウシ由来ゼラチンが認められたが、ニワトリ由来ゼラチンは認められなかった。よって、本法は、食品・薬剤中のゼラチンの存在とその由来動物種を測定する方法となり得る。The presence of food and drug gelatin and its derived animals were determined. Enzyme immunoreaction was carried out with the following reagents using water confectionery and MMR vaccine containing a large amount of gelatin as samples.
Bovine gelatin and chicken gelatin (both type I collagen type) were used as the target specimens.
Reagent 1, anti-bovine (type I collagen type) gelatin antibody, anti-chicken (type I collagen type) gelatin antibody 0.5 μg / well coated microplate reagent 2-1, HRP labeled anti-bovine (type I collagen type) ) Gelatin antibody reagent 2-2, HRP-labeled anti-chicken (type I collagen type) gelatin antibody reagent 3, TMB reagent (TMB 0.1%, hydrogen peroxide 0.02%, 0.1M citrate buffer)
Reagent 4, reaction stop solution (0.5M sulfuric acid)
The specimen was dissolved in a phosphate buffer and 100 μl / well was used.
Measurement method: Reagent 1 100 μl / well → 2 hours incubation and washing → Reagents 2-1 and 2-2, each 100 μl / well → 1 hour incubation and washing → Reagent 3, 100 μl → 30 minutes incubation → Reagent 4, 50 μl → Measure (absorbance: 450 nm) and judge by OD value height.
Result: Bovine-derived gelatin was observed in the specimen, but chicken-derived gelatin was not observed. Therefore, this method can be a method for measuring the presence of gelatin in foods and medicines and the animal species derived from it.
ゼラチンアレルギー者に、ウシ由来ゼラチン、ニワトリ由来ゼラチン、サケ由来ゼラチン各０．０３％含有０．０１Ｍクエン酸水１０ｍｌを口くう内に２０秒間含ませ、吐き出させた後、観察した。
結果：ウシ由来ゼラチンの時のみ、口くう内に発赤が見られたが、ニワトリ由来ゼラチン及びサケ由来ゼラチンの時には、口くう内に変化は見られなかった。
よって、非ウシ由来ゼラチンを用いた食品や内服薬剤はウシゼラチンアレルギー者に使用できる。The subjects were allergic to gelatin, and 10 ml of 0.01M citric acid solution containing 0.03% of bovine-derived gelatin, chicken-derived gelatin, and salmon-derived gelatin were each contained in the mouth for 20 seconds.
Results: Redness was seen in the mouth only with bovine-derived gelatin, but no change was seen in the mouth with chicken-derived gelatin and salmon-derived gelatin.
Therefore, foods and internal medicines using non-bovine derived gelatin can be used for those who are allergic to bovine gelatin.
鮭由来ゼラチン、ニワトリ皮膚由来ゼラチンを、別々に、温湯で希釈した出汁に加え、加熱した後、冷却し、鮭由来ゼラチンとニワトリ由来ゼラチンの煮凝を得た。ウシゼラチンアレルギー者に、各１０ｇを、食してもらったが、まったく変化はなかった。
これにより、単一動物由来のゼラチンを加えた食品が得られる事、非ウシゼラチン添加の食品を、ウシゼラチンアレルギー者は、食することができることが明らかである。Salmon-derived gelatin and chicken skin-derived gelatin were separately added to the stock diluted with warm water, heated and then cooled to obtain a boiled mixture of salmon-derived gelatin and chicken-derived gelatin. A person with bovine gelatin allergies ate 10 g each, but there was no change at all.
As a result, it is clear that foods to which gelatin derived from a single animal is added can be obtained, and those who are allergic to bovine gelatin can eat foods added with non-bovine gelatin.
ニワトリ由来ゼラチンを３％濃度に、０．０１Ｍ酢酸に溶解させた後、乾燥させて、シート状にした。このシートで、乳糖１００ｍｇを包み、ウシゼラチンアレルギー者に、内服してもらったが、まったく変化は見られなかった。
これにより、非ウシ由来のゼラチンは、ウシゼラチンアレルギー者に、カプセルとして使用できる。Chicken-derived gelatin was dissolved in 0.01 M acetic acid at a concentration of 3% and then dried to form a sheet. This sheet wrapped 100 mg of lactose and was taken by a bovine gelatin allergy person, but no change was seen.
As a result, gelatin derived from non-bovine can be used as a capsule for those who are allergic to bovine gelatin.
従来、ゼラチンアレルギーは、卵、牛乳、そば等の食物アレルギーと同様切実な問題であった。卵、牛乳、そばは、その食品を食べなければ済むが、ゼラチンは、そのままの形の事はなく、安定剤として薬剤に、食感剤として食品に、又その他の理由で、多くの食品、薬剤に含まれている。しかし含有を明示されていることは少ない。
それ故、薬剤や食品で、異常反応を示した時、主成分以外のゼラチンが原因であることを特定することは困難であった。又、ゼラチンがアレルゲンと判明しても、避けるべき食品は不明であった。治療上、予防上、必要な薬剤を使用するのに不安であった。
本発明は、このような状況を憂慮し、ゼラチンアレルギー者を見つけだし、アレルゲンとなる動物種のゼラチン及びその分画を特定し、避けるべき食品、薬剤を示すことを可能にし、更に彼等の為に必要な食品と薬剤の供給を可能にした。Traditionally, gelatin allergies have been a serious problem, as are food allergies such as eggs, milk and buckwheat. Eggs, milk, and buckwheat do not have to eat the food, but gelatin is not in its original form, it is a drug as a stabilizer, a food as a sensitizer, and for many other reasons, Included in the drug. However, it is rare that the inclusion is clearly stated.
Therefore, it has been difficult to specify that gelatin other than the main component is the cause when the drug or food shows an abnormal reaction. Even if gelatin was found to be an allergen, the food to be avoided was unknown. I was worried about using the necessary medicine for treatment and prevention.
The present invention worries about such a situation, makes it possible to find gelatin allergic individuals, identify gelatins of animal species that become allergens and their fractions, indicate foods and drugs to be avoided, and further The supply of foods and medicines necessary for the plant was made possible.
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| JPS6434925A (en) * | 1987-04-24 | 1989-02-06 | Univ Osaka | Method for cultivating bordetella pertussis, pertussis toxoid and mixed vaccine thereof |
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| JPS6434925A (en) * | 1987-04-24 | 1989-02-06 | Univ Osaka | Method for cultivating bordetella pertussis, pertussis toxoid and mixed vaccine thereof |
| JPH01128927A (en) * | 1987-09-24 | 1989-05-22 | Merck & Co Inc | Controlled porous permeable pump |
| JPH06321803A (en) * | 1993-05-17 | 1994-11-22 | Kirin Brewery Co Ltd | Gradual releasing preparation of water soluble peptide hormone |
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| JPN6008025506; Ｊ． Ａｌｌｅｒｇｙ Ｃｌｉｎ． Ｉｍｍｕｎｏｌ．， １９９３， ９１（４）， ｐ．８６７-８７２ * |
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