JP2794426B2 - Peroxidase-containing reagent composition and method for preventing peroxidase enzyme activity inhibition - Google Patents
Peroxidase-containing reagent composition and method for preventing peroxidase enzyme activity inhibitionInfo
- Publication number
- JP2794426B2 JP2794426B2 JP63290077A JP29007788A JP2794426B2 JP 2794426 B2 JP2794426 B2 JP 2794426B2 JP 63290077 A JP63290077 A JP 63290077A JP 29007788 A JP29007788 A JP 29007788A JP 2794426 B2 JP2794426 B2 JP 2794426B2
- Authority
- JP
- Japan
- Prior art keywords
- peroxidase
- enzyme activity
- reagent composition
- preventing
- polyalkylene glycol
- 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 - Lifetime
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明はアジ化ナトリウムの共存下においてもペルオ
キシダーゼの酵素活性が阻害されることがないペルオキ
シダーゼ含有試薬組成物及びその酵素活性阻害防止方法
に関する。Description: TECHNICAL FIELD The present invention relates to a peroxidase-containing reagent composition in which the enzyme activity of peroxidase is not inhibited even in the presence of sodium azide, and a method for preventing the enzyme activity from being inhibited.
ペルオキシダーゼは過酸化水素又は有機過酸化物によ
る種々の有機物の酸化を接触する酵素であるが、近年エ
ンザイムイムノアッセイ（EIA）や組織染色用試薬の標
識物質として利用されることが多くなってきた。Peroxidase is an enzyme that comes into contact with the oxidation of various organic substances by hydrogen peroxide or an organic peroxide. In recent years, peroxidase has been increasingly used as a labeling substance for enzyme immunoassay (EIA) and tissue staining reagents.
このペルオキシダーゼを含む試薬には通常、貯蔵安定
性を保つため防腐剤が共存せしめられている。A preservative is usually added to the reagent containing peroxidase in order to maintain storage stability.
一般に防腐剤としてはアジ化ナトリウムが用いられる
が、このアジ化ナトリウムはペルオキシダーゼの酵素活
性を阻害する作用があることが知られており、その代替
物が求められていた。而して、従来その代替物としてチ
メロサールが開発され用いられているのであるが、この
物質は有機水銀系の化合物であるため公害上の問題が心
配され、改善が望まれていた。In general, sodium azide is used as a preservative. It is known that this sodium azide has an effect of inhibiting the enzyme activity of peroxidase, and a substitute has been required. Thus, thimerosal has been conventionally developed and used as a substitute, but since this substance is an organic mercury-based compound, there is a concern about pollution, and improvement has been desired.
本発明者らは上記の問題点を解決するため研究を続け
た結果ペルオキシダーゼとアジ化ナトリウムが共存する
系においてもポリアルキレングリコールを存在せしめる
とペルオキシダーゼの酵素活性阻害が防止できることを
見出し本発明を完成した。The present inventors have continued research to solve the above problems, and found that the presence of polyalkylene glycol can prevent the inhibition of peroxidase enzyme activity even in a system in which peroxidase and sodium azide coexist, and completed the present invention. did.
すなわち本発明は、ペルオキシダーゼとアジ化ナトリ
ウムとポリアルキレングリコールを含有する水溶液から
なる試薬組成物、および、水溶液中においてペルオキシ
ダーゼとアジ化ナトリウムの共存下、ポリアルキレング
リコールを存在せしめることを特徴とするペルオキシダ
ーゼの酵素活性阻害防止方法を提供するものである。That is, the present invention provides a reagent composition comprising an aqueous solution containing peroxidase, sodium azide and polyalkylene glycol, and a peroxidase characterized by allowing the polyalkylene glycol to be present in the aqueous solution in the presence of peroxidase and sodium azide. And a method for preventing enzyme activity inhibition.
本発明に用いるポリアルキレングリコールは一般式
（Ｉ）で示される化合物である。The polyalkylene glycol used in the present invention is a compound represented by the general formula (I).
式中のR1,R2,X,Yは水素又は低級アルキル基を表わ
し、ｎは２以上の整数を表わす。（Ｉ）式で示される化
合物の中で特にポリエチレングリコール、ポリポリプロ
ピレングリコールが好ましい。 In the formula, R 1 , R 2 , X, and Y represent hydrogen or a lower alkyl group, and n represents an integer of 2 or more. Among the compounds represented by the formula (I), polyethylene glycol and polypropylene glycol are particularly preferred.
ポリアルキレングリコールの反応液中の濃度は0.5〜1
0％が好ましい。10％を越えると反応液の粘度が上昇し
たり、液中の成分の不溶化等が起り好ましくない。The concentration of the polyalkylene glycol in the reaction solution is 0.5 to 1
0% is preferred. If it exceeds 10%, the viscosity of the reaction solution increases and the components in the solution are insolubilized.
なお、ポリアルキレングリコールは試薬組成物中に含
有させておく他に、反応液中に添加することにより共存
させてもよい。In addition to the polyalkylene glycol being contained in the reagent composition, the polyalkylene glycol may be made to coexist by being added to the reaction solution.
以下実施例で本発明を具体的に説明する。 Hereinafter, the present invention will be described specifically with reference to Examples.
実施例1:〔ペルオキシダーゼ活性阻害防止効果〕 （１）市販ペルオキシダーゼの調製 市販ホースラデッシュ．パーオキシダーゼ（東洋紡績
（株）製）を水で溶解し、500ng/mlにする。Example 1: [Effect of inhibiting peroxidase activity inhibition] (1) Preparation of commercially available peroxidase Commercially available horseradish. Peroxidase (manufactured by Toyobo Co., Ltd.) is dissolved in water to make 500 ng / ml.
（２）緩衝液の調製 0.05％アジ化ソーダ及び0.9％塩化ナトリウムを含
む10mMリン酸緩衝液（pH7.4），即ち無添加緩衝液を調
製する。(2) Preparation of buffer solution Prepare a 10 mM phosphate buffer solution (pH 7.4) containing 0.05% sodium azide and 0.9% sodium chloride, that is, a buffer solution without addition.
0.05％アジ化ソーダ,0.9％塩化ナトリウム及び２％
ポリエチレングリコール6000を含む10mMリン酸緩衝液
（pH7.4），即ち添加緩衝液を調製する。0.05% sodium azide, 0.9% sodium chloride and 2%
Prepare a 10 mM phosphate buffer (pH 7.4) containing polyethylene glycol 6000, ie, an addition buffer.
（３）酸素活性の測定 500ng/mlに調製されたペルオキシダーゼを精製水と前
記無添加緩衝液および添加緩衝液で２倍,4倍,8倍,16倍,
32倍並びに64倍に希釈し、１時間室温に放置する。(3) Measurement of oxygen activity Peroxidase adjusted to 500 ng / ml was purified with purified water and the above-mentioned non-supplemented buffer solution and the added buffer solution two-fold, four-fold, eight-fold, 16-fold,
Dilute 32 and 64 times and leave at room temperature for 1 hour.
次に各サンプルを100μ取り、0.5mlの基質液中（オ
ルトフェニレンジアミンと過酸化水素を含む反応液）に
加え、室温で５分間反応させた後、2N−H2SO4で反応を
停止させる。得られた反応液の492nmにおける吸光度を
測定する。Next, 100 μl of each sample was taken, added to 0.5 ml of a substrate solution (a reaction solution containing orthophenylenediamine and hydrogen peroxide), allowed to react at room temperature for 5 minutes, and then stopped with 2N—H 2 SO 4 . . The absorbance at 492 nm of the obtained reaction solution is measured.
結果を図１に示す。 The results are shown in FIG.
図１にみるとうり、0.05％アジ化ソーダ（NaN3）によ
って阻害を受け完全に失活してしまうパーオキシダーゼ
（POD）が、２％ポリエチレングリコール6000（PEG）を
緩衝液中に含むことにより全く阻害を受けず活性が維持
されている。As shown in FIG. 1, peroxidase (POD), which is inhibited by 0.05% sodium azide (NaN 3 ) and completely inactivated, contains 2% polyethylene glycol 6000 (PEG) in the buffer. Activity is maintained without any inhibition.
実施例2:〔アルギニノコハク酸合成酵素（ASS）定量系
における本発明の効果〕 （１）ラット肝の精製 ウイスター系の雄のラットから摘出したラット肝をテ
フロンホモジナイザーを用いて0.25M蔗糖液中でホモジ
ナイズした後、得られたホモジネートを27,000×ｇで30
分遠心分離する。上清液を45〜65％飽和硫酸アンモニウ
ム溶液で分画し、遠心分離する。沈渣を緩衝液で溶解
し、透析する。次に、予め平衡化したDEAEセルロースを
混合し、不純物を吸着したDEAEセルロースを遠心分離し
て除去する。Example 2: [Effect of the present invention in the argininosuccinate synthase (ASS) assay system] (1) Purification of rat liver Rat liver isolated from male Wistar rats was subjected to 0.25 M sucrose solution using a Teflon homogenizer. After homogenization, the obtained homogenate was added at 27,000 × g for 30 minutes.
Centrifuge for minutes. The supernatant is fractionated with a 45-65% saturated ammonium sulfate solution and centrifuged. Dissolve the sediment with buffer and dialyse. Next, the DEAE cellulose pre-equilibrated is mixed, and the DEAE cellulose adsorbing the impurities is removed by centrifugation.
次に上清を６〜12％（W/V）ポリエチレングリコール
で分画し、沈渣を緩衝液で溶解後、65℃で熱処理する。
次に、不溶性タンパクを遠心分離で除去した後、DEAE−
セファデックスＡ−50カラムクロマトグラフに通し、次
いでセファデックスＧ−200カラムクロマトグラフに通
す。２つの活性ピークの中でメインピークを濃縮後、5m
Mアルギニノサクシネートおよび45％飽和硫酸アンモニ
ウムを含む0.05Mトリス塩酸水溶液（pH＝7.5）中で結晶
化して精製ラットASSを得る。Next, the supernatant is fractionated with 6 to 12% (W / V) polyethylene glycol, and the precipitate is dissolved in a buffer and then heat-treated at 65 ° C.
Next, after removing the insoluble protein by centrifugation, DEAE-
Pass through a Sephadex A-50 column chromatograph and then through a Sephadex G-200 column chromatograph. After concentrating the main peak among the two active peaks, 5m
Crystallization in a 0.05 M aqueous solution of Tris-HCl (pH = 7.5) containing M-argininosuccinate and 45% saturated ammonium sulfate gives purified rat ASS.
（２）血中ヒトASSの定量 上記の精製ラットASSをフロイントアジュバント（1:
1）を加えて乳化した乳化液0.5mlを家兎の背部下、皮肉
及び足蹠部に分割注射して免疫した。１週間後に同量の
乳化液を同様にして追加免疫し、さらに初回の免疫より
１ヶ月後に倍量の乳化液を再度追加免疫した。最後の免
疫の１週間後より数回にわたって採血し、血清を分離し
た。この抗血清を、硫安処理、DEAEセルロースカラムに
通してIgGに精製する。次にこのIgGを石川等の方法（J.
Biochem.92:1413〜1424（1982））を用いて、Fab′に調
整後、ペルオキシダーゼを標識したコンジュゲート（標
識抗体）を作る。(2) Quantification of human ASS in blood The above purified rat ASS was purified using Freund's adjuvant (1:
0.5 ml of the emulsion obtained by adding 1) was emulsified by split injection into the lower part of the back, the sarcasm and the footpad of the rabbit. One week later, the same amount of the emulsion was boosted in the same manner, and one month after the first immunization, a double amount of the emulsion was boosted again. Blood was collected several times one week after the last immunization, and the serum was separated. The antiserum, ammonium sulfate process, purified to I g G through DEAE cellulose column. Next, this IgG was obtained by the method of Ishikawa et al.
Biochem. 92: 1413-1424 (1982)), and a conjugate (labeled antibody) labeled with peroxidase is prepared after adjusting to Fab '.
次に表１に示す濃度の被検試料20μと0.4mlの各ア
ルブミン−ホウ酸緩衝液（脚注に緩衝液組成を示す）を
加えた試験管に、常法により精製IgGをコートしたビー
ズを加え室温で２時間反応させる。次に上記のFab′−
ペルオキシダーゼ溶液を0.1ml加え、再び室温で１時間
反応させる。反応液を洗浄した後、pH5.5のクエン酸リ
ン酸緩衝液50ml中にオルト−フェニレンジアミン（OP
D）100mgを溶解させ、市販の過酸化水素水0.015％を添
加して作った溶液500μを基質溶液とし、2N−H2SO4を
停止溶液として492nmの波長で吸光度を測定した。Then the albumin test sample 20μ and 0.4ml of concentration shown in Table 1 - borate buffer to a test tube (buffer shows the composition in the footnote) was added, the beads coated with purified I g G by conventional methods And react for 2 hours at room temperature. Next, the above Fab'-
Add 0.1 ml of peroxidase solution and react again for 1 hour at room temperature. After washing the reaction solution, ortho-phenylenediamine (OP) was added to 50 ml of pH 5.5 citrate phosphate buffer.
D) dissolved 100mg, solution 500μ made by adding 0.015% commercial hydrogen peroxide solution as a substrate solution, and absorbance was measured at 492nm wavelength of 2N-H 2 SO 4 as stop solution.
得られた結果を表１に示す。 Table 1 shows the obtained results.
表１から明らかなように、アジ化ソーダを用いたEIA
測定系において、ポリエチレングリコールを加えた本発
明の場合には、完全にEIAを遂行させることができた。 As is clear from Table 1, EIA using sodium azide
In the case of the present invention to which polyethylene glycol was added in the measurement system, EIA could be completely performed.
本発明によりペルオキシダーゼを含む試薬の貯蔵安定
性の向上には効果がある反面、該ペルオキシダーゼの酵
素活性を阻害する作用があるため使用が困難であったア
ジ化ナトリウムの使用が可能になった。このためペルオ
キシダーゼを用いたEIA或いは組織染色が無公害で安定
的に実施できるようになったのである。The present invention is effective in improving the storage stability of a reagent containing peroxidase, but allows the use of sodium azide, which has been difficult to use because it has an action of inhibiting the enzyme activity of peroxidase. Thus, EIA or tissue staining using peroxidase can be performed stably without pollution.
図１は実施例１における酵素活性（吸光度）の測定結果
を示すグラフである。FIG. 1 is a graph showing the measurement results of the enzyme activity (absorbance) in Example 1.
Claims (4)
リアルキレングリコールを含有する水溶液からなる試薬
組成物。1. A reagent composition comprising an aqueous solution containing peroxidase, sodium azide and polyalkylene glycol.
（Ｉ）で示される化合物である請求項１記載の試薬組成
物。 （式中、R1,R2,X,Yは水素又は低級アルキル基を表わ
し、ｎは２以上の整数を表わす）2. The reagent composition according to claim 1, wherein the polyalkylene glycol is a compound represented by the following general formula (I). (In the formula, R 1 , R 2 , X, and Y represent hydrogen or a lower alkyl group, and n represents an integer of 2 or more.)
化ナトリウムの共存下、ポリアルキレングリコールを存
在せしめることを特徴とするペルオキシダーゼの酵素活
性阻害防止方法。3. A method for preventing the inhibition of enzyme activity of peroxidase, wherein a polyalkylene glycol is present in an aqueous solution in the presence of peroxidase and sodium azide.
（Ｉ）で示される化合物である請求項３記載のペルオキ
シダーゼの酵素活性阻害防止方法。 （式中のR1,R2,X,Y,nは前記の通り）4. The method of claim 3, wherein the polyalkylene glycol is a compound represented by the following general formula (I). (Wherein R 1 , R 2 , X, Y, and n are as described above)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63290077A JP2794426B2 (en) | 1988-11-18 | 1988-11-18 | Peroxidase-containing reagent composition and method for preventing peroxidase enzyme activity inhibition |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63290077A JP2794426B2 (en) | 1988-11-18 | 1988-11-18 | Peroxidase-containing reagent composition and method for preventing peroxidase enzyme activity inhibition |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02138999A JPH02138999A (en) | 1990-05-28 |
| JP2794426B2 true JP2794426B2 (en) | 1998-09-03 |
Family
ID=17751496
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63290077A Expired - Lifetime JP2794426B2 (en) | 1988-11-18 | 1988-11-18 | Peroxidase-containing reagent composition and method for preventing peroxidase enzyme activity inhibition |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2794426B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPWO2006030866A1 (en) * | 2004-09-16 | 2008-05-15 | デンカ生研株式会社 | Method for quantitative determination of uric acid |
-
1988
- 1988-11-18 JP JP63290077A patent/JP2794426B2/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPWO2006030866A1 (en) * | 2004-09-16 | 2008-05-15 | デンカ生研株式会社 | Method for quantitative determination of uric acid |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH02138999A (en) | 1990-05-28 |
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