JPH0329854A - Enzyme immunoassay - Google Patents
Enzyme immunoassayInfo
- Publication number
- JPH0329854A JPH0329854A JP16644989A JP16644989A JPH0329854A JP H0329854 A JPH0329854 A JP H0329854A JP 16644989 A JP16644989 A JP 16644989A JP 16644989 A JP16644989 A JP 16644989A JP H0329854 A JPH0329854 A JP H0329854A
- Authority
- JP
- Japan
- Prior art keywords
- hydrogen peroxide
- biological fluid
- measured
- superoxide
- catalase
- 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
Links
- 102000004190 Enzymes Human genes 0.000 title claims description 18
- 108090000790 Enzymes Proteins 0.000 title claims description 18
- 238000003018 immunoassay Methods 0.000 title claims description 12
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 124
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 22
- 102000016938 Catalase Human genes 0.000 claims abstract description 17
- 108010053835 Catalase Proteins 0.000 claims abstract description 17
- 102000004316 Oxidoreductases Human genes 0.000 claims abstract description 8
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 8
- 239000003112 inhibitor Substances 0.000 claims abstract description 7
- 229940123748 Catalase inhibitor Drugs 0.000 claims abstract description 4
- 239000013060 biological fluid Substances 0.000 claims description 39
- 230000001590 oxidative effect Effects 0.000 claims description 9
- 238000000354 decomposition reaction Methods 0.000 claims description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 210000001124 body fluid Anatomy 0.000 abstract 7
- 239000010839 body fluid Substances 0.000 abstract 7
- 102000019197 Superoxide Dismutase Human genes 0.000 abstract 4
- 108010012715 Superoxide dismutase Proteins 0.000 abstract 4
- 238000005259 measurement Methods 0.000 description 22
- 229940088598 enzyme Drugs 0.000 description 15
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 11
- 229940116269 uric acid Drugs 0.000 description 11
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 10
- 239000008363 phosphate buffer Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 108010058683 Immobilized Proteins Proteins 0.000 description 4
- -1 Potassium ferricyanide Chemical compound 0.000 description 4
- 150000001299 aldehydes Chemical class 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 3
- 239000002262 Schiff base Substances 0.000 description 3
- 150000004753 Schiff bases Chemical class 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000012460 protein solution Substances 0.000 description 3
- 238000003127 radioimmunoassay Methods 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 108010092464 Urate Oxidase Proteins 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229940005267 urate oxidase Drugs 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000005373 porous glass Substances 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
Ａ．産業上の利用分野
本発明は、生体液中の成分であって、酸化酵素の作用に
より過酸化水素を生成する被測定成分を測定する方法に
係わり、特に高感度の測定を行えるものに関する。[Detailed Description of the Invention] A. INDUSTRIAL APPLICATION FIELD The present invention relates to a method for measuring a component in a biological fluid that generates hydrogen peroxide through the action of an oxidizing enzyme, and particularly to a method that enables highly sensitive measurement.
Ｂ．発明の概要
本発明は、酵素免疫測定方法において、酸化酵素を生体
液に作用させる工程の前に、生体液中の過酸化水素を除
去する前処理を行うこととし、この前処理として、スー
パーオキサイドを分解する工程と、過酸化水素を分解す
る工程とを行うことにより、測定ブランクを低減し、測
定感度を高めることを可能とする。B. Summary of the Invention The present invention provides an enzyme immunoassay method in which pretreatment for removing hydrogen peroxide from the biological fluid is performed before the step of causing an oxidizing enzyme to act on the biological fluid, and as this pretreatment, superoxide By performing the step of decomposing hydrogen peroxide and the step of decomposing hydrogen peroxide, it is possible to reduce measurement blanks and increase measurement sensitivity.
Ｃ．従来の技術
一般に、臨床検査分野においては、さまざまな微量戊分
を測定するものとして、免疫測定法が用いられている。C. BACKGROUND OF THE INVENTION Generally, in the field of clinical testing, immunoassay methods are used to measure various minute amounts of blood.
免疫測定法の代表的なものには、ラジオ・イムノ・アッ
セイ法（ＲＩＡ）があるが、このＲＩＡは標識として放
射性同位元素を使用するため、環境や健康の保安の観点
から、他の測定法に対する要請が高まっている。Radio immunoassay (RIA) is a typical immunoassay method, but since RIA uses a radioactive isotope as a label, it is not suitable for other measurement methods from the viewpoint of environmental and health safety. There is an increasing demand for
３
他の免疫測定法としては、エンザム・イムノ・アッセイ
法（Ｅ Ｉ Ａ ；酵素免疫測定法）やルミネッセンス
・イムノ・アッセイ法（ＬＩＡ；発光免疫測定法）など
が挙げられる。3 Other immunoassay methods include enzyme immunoassay (EIA) and luminescence immunoassay (LIA).
これらの測定法では、微量戊分の量を過酸化水素などの
量に置き換えて測定を行っている。In these measurement methods, measurements are performed by replacing the amount of trace amounts of bokubun with amounts of hydrogen peroxide, etc.
Ｄ．発明が解決しようとする課題
しかしながら従来、ＥＩＡでは、測定ブランクがあるた
めに、高い測定感度を得ることが困ガ［であるという問
題があった。D. Problems to be Solved by the Invention However, conventional EIA has had a problem in that it is difficult to obtain high measurement sensitivity due to the presence of measurement blanks.
すなわち生体液中には微量の過酸化水素が存在しており
、この過酸化水素の濃度が測定ブランクとなる。被測定
戊分が極めて低濃度である場合、過酸化水素の測定値と
測定ブランクの差異が極めて小さくなってしまい、過酸
化水素の測定値に基４
づいて被測定戊分を正確に定量することが困難となる。That is, a trace amount of hydrogen peroxide exists in the biological fluid, and the concentration of this hydrogen peroxide serves as a measurement blank. If the concentration of the ester to be measured is extremely low, the difference between the measured value of hydrogen peroxide and the measurement blank will be extremely small, making it difficult to accurately quantify the ester to be measured based on the measured value of hydrogen peroxide. This becomes difficult.
本発明は、この問題点に鑑みてなされたものであり、そ
の目的とするところは、過酸化水素の測定ブランクを抑
え、ＥＩＡにおける測定感度を高める点にある。The present invention has been made in view of this problem, and its purpose is to suppress hydrogen peroxide measurement blanks and increase measurement sensitivity in EIA.
Ｅ．課題を解決するための手段
請求項ＩＣこ係る発明は、生体液中の成分であって、酸
化酵素の作用により過酸化水素を生成する被測定成分を
測定する酵素免疫測定方法において、次の工程を順次行
うものである。E. Means for Solving the Problems Claim IC This invention provides an enzyme immunoassay method for measuring a component in a biological fluid that produces hydrogen peroxide by the action of an oxidizing enzyme, which includes the following steps: This is done sequentially.
■ スーパーオキサイド・ジスムダーゼ（ＳＯＤ）とカ
タラーゼとを生体液に混合し、生体液中に含まれるスー
パーオキサイドと過酸化水素とを分解する工程。■ A step in which superoxide dismudase (SOD) and catalase are mixed into a biological fluid to decompose superoxide and hydrogen peroxide contained in the biological fluid.
■ ＳＯＤのスーパーオキサイド分解機能を抑制するＳ
ＯＤ阻害剤と、カタラーゼの過酸化水素分解機能を抑制
するカタラーゼ阻害剤とを生体液に添加する工程。■ S suppresses the superoxide decomposition function of SOD
A step of adding an OD inhibitor and a catalase inhibitor that suppresses the hydrogen peroxide decomposition function of catalase to a biological fluid.
■ 被測定成分と選択的に反応して過酸化水素を生戊す
る酸化酵素を生体液に作用させる工程。■ A process in which an oxidizing enzyme that selectively reacts with the component to be measured to produce hydrogen peroxide acts on the biological fluid.
■ 工程■において生成した過酸化水素の濃度を測定す
る工程。■ A step of measuring the concentration of hydrogen peroxide produced in step ■.
■ 工程■において測定された過酸化水素の濃度から被
測定成分の濃度を決定する工程。■ A step of determining the concentration of the component to be measured from the concentration of hydrogen peroxide measured in step ■.
また請求項２に係る発明は、同じく酵素免疫測定方法に
おいて、次の工程を順次行うものである。Furthermore, the invention according to claim 2 is also an enzyme immunoassay method in which the following steps are sequentially performed.
■ 生体液を固定化ＳＯＤに接触させて、生体液中に含
まれるスーパーオキサイドを分解する工程。■ A step in which the biological fluid is brought into contact with immobilized SOD to decompose superoxide contained in the biological fluid.
■ 生体液を固定化力タラーゼに接触さぜて、生体液中
に含まれる過酸化水素を分解する工程。■ A process in which the biological fluid is brought into contact with immobilized talase to decompose hydrogen peroxide contained in the biological fluid.
■ 被測定成分と選択的に反応して過酸化水素を生成す
る酸化酵素を生体液に作用させる工程。■ A process in which an oxidizing enzyme that selectively reacts with the component to be measured to produce hydrogen peroxide acts on the biological fluid.
■ 工程■において生成した過酸化水素の濃度を測定す
る工程。■ A step of measuring the concentration of hydrogen peroxide produced in step ■.
■ 工程■において測定された過酸化水素の濃度から被
測定成分の濃度を決定する工程。■ A step of determining the concentration of the component to be measured from the concentration of hydrogen peroxide measured in step ■.
Ｆ．作用
酵素免疫測定方法における測定ブランクは、生体液中に
存在する過酸化水素Ｃ８：より生じる。F. The measurement blank in the action enzyme immunoassay method is generated from hydrogen peroxide C8: present in biological fluids.
そこで本発明では、酸化酵素を生体液に作用させる前に
、カタラーゼを生体液に混合する操作を行うことにより
、生体液中の過酸化水素を分解する。この反応を式（１
）に示す。Therefore, in the present invention, hydrogen peroxide in the biological fluid is decomposed by mixing catalase with the biological fluid before allowing the oxidase to act on the biological fluid. This reaction is expressed by the formula (1
).
７
２１−Ｉ，Ｏｆ→ Ｏ，＋２Ｈ２０ ・
・・（１）しかしこの操作だけでは、この操作の後にス
ーパーオキサイドＯｔｈ−から新たに発生する過酸化水
素を除去することはできない。一般にスーパーオキサイ
ドＯ，−は、菌体等の生物体において各種の酸化反応が
起こるにつれて発生しており、寿命が短かく、式（２）
に示すように、過酸化水素Ｈ，Ｏ，に変化すると考えら
れる。7 21-I, Of→ O, +2H20 ・
(1) However, this operation alone cannot remove newly generated hydrogen peroxide from superoxide Oth- after this operation. In general, superoxide O,- is generated as various oxidation reactions occur in living organisms such as bacterial cells, and has a short lifespan, and is expressed by the formula (2).
As shown in the figure, it is thought that hydrogen peroxide changes to H, O, and so on.
Ｏ ｔ−＋ Ｏ ，−＋２ Ｈ ’→ Ｏ ，十Ｈ ２
０’２ ・・・（２）そこで上記の操作を行う前か
、上記の操作と共に、生体液にＳＯＤを混合する操作を
行う。ＳＯＤは、広く好気性生物に存在する酵素であり
、式（２）の反応を促進するものである。この操作によ
り、スーパーオキサイドＯ，一を除去することができる
。O t-+ O ,-+2 H'→ O , 10H 2
0'2...(2) Then, before or together with the above operation, an operation of mixing SOD into the biological fluid is performed. SOD is an enzyme that widely exists in aerobic organisms, and promotes the reaction of formula (2). By this operation, superoxide O,1 can be removed.
８
この後、ＳＯＤ阻害剤とカタラーゼ阻害剤とを生体液に
添加し、ＳＯＤと過酸化水素の分解を抑制したうえで、
酸化酵素を生体液に作用させて過酸化水素を生成する。8 After this, an SOD inhibitor and a catalase inhibitor are added to the biological fluid to suppress the decomposition of SOD and hydrogen peroxide, and then
Hydrogen peroxide is produced by oxidizing enzymes acting on biological fluids.
生１戊した過酸化水素の量は、被測定成分の量を置き換
えたものとなる。酸化酵素を作用させる前に、スーパー
オキサイド０２−と過酸化水素を除去することにより、
測定ブランクを極めて小さく抑えているので、過酸化水
素の濃度を測定することにより、被測定戊分の濃度を高
精度に決定することが可能となる。The amount of raw hydrogen peroxide replaces the amount of the component to be measured. By removing superoxide 02- and hydrogen peroxide before allowing the oxidizing enzyme to act,
Since the measurement blank is kept extremely small, by measuring the concentration of hydrogen peroxide, it is possible to determine the concentration of the hydrogen peroxide with high precision.
また生体液中のスーパーオキサイドＯｆｆｉ−と過酸化
水素を除去する前処理において、固定化ＳＯＤと固定化
カタラーゼを使用することとすれば、ＳＯＤ阻害剤とカ
タラーゼ阻害剤を生体液に添加する必要がなくなり、生
体液中の成分の測定かより容易になる。Furthermore, if immobilized SOD and immobilized catalase are used in pretreatment to remove superoxide Offi- and hydrogen peroxide from biological fluids, it is necessary to add SOD inhibitors and catalase inhibitors to biological fluids. This makes it easier to measure components in biological fluids.
Ｇ．実施例１
ここで実胤例１を説明する。実施例ｌは、尿酸の測定法
へ本発明を適用した例である。G. Example 1 Here, actual seed example 1 will be explained. Example 1 is an example in which the present invention is applied to a method for measuring uric acid.
まず、実施例ｌに使用する試薬等の調製を示す。First, the preparation of reagents and the like used in Example 1 will be described.
■ Ｏ．ｌｍｏｌ／（！ リン酸緩衝液 ｐＨ．７、０
■ ＳＯＤ （フナコシ製）
１０，ＯＯＯ’Ｕ／Ｉｎ１２ ０．０５’ｍｏｌ／（！
リン酸緩衝液 ｐＨ７．８
■ カタラーゼ（シグマ製）
１０，Ｏ’００Ｕ／ｍＱ Ｏ．’０ １ｍｏｌ／１２
リン酸緩衝液 ｐＨ７．０
■ ＫＣＮ Ｏ’． ｌｍｏ＋／１２（０． １
ｍｏｌｚ’２ トリス−塩酸緩衝液ｐ Ｈ ８．０溶液
）
■ 尿酸オキシダーゼ（ベーリンガー・マンハイム社製
）
４．’５Ｕ／ｍｇ Ｏ．’ＩＭ炭酸ナトリウム溶液■
ルミノール
２Ｘ１０−’ｍｏｌ／１２ （０．２ｍｏｌ／（！炭
酸緩衝液ｐＨ９．８）溶液
■ フェリシアン化カリウム６ Ｘ １ ０ −”ｍｏ
ｌ７ Ｑ水溶液
■ 標準液：尿酸水溶液（ ５ ｍｇ／ｄｌｌ！，
１ ０ ｍｇ／ｄＱ． １ ４ ｍｇ／ ｄｌ２）
次に測定手順を説明する。■ O. lmol/(! Phosphate buffer pH.7,0
■ SOD (manufactured by Funakoshi) 10, OOO'U/In12 0.05'mol/(!
Phosphate buffer pH 7.8 ■ Catalase (manufactured by Sigma) 10, O'00U/mQ O. '0 1mol/12
Phosphate buffer pH 7.0 ■ KCN O'. lmo+/12(0.1
molz'2 Tris-HCl buffer pH 8.0 solution) ■ Urate oxidase (manufactured by Boehringer Mannheim) 4. '5U/mg O. 'IM sodium carbonate solution■
Luminol 2X10-'mol/12 (0.2mol/(!carbonate buffer pH 9.8) solution ■ Potassium ferricyanide 6X 10-'mo
l7 Q aqueous solution■ Standard solution: uric acid aqueous solution (5 mg/dll!,
10 mg/dQ. 14 mg/dl2) Next, the measurement procedure will be explained.
第１図は、実施例１における測定手順を示すフローチャ
ートである。FIG. 1 is a flowchart showing the measurement procedure in Example 1.
１１
まず試験管に試料（ ５ ｍｇ／ ｄＱ． １ ０
ｍｇ／ ｄＱ． １４ ｍｇ／ ｄＱ尿酸水溶液）０
．５ｍＱを分取し、０．１ｍｏｌ／１２リン酸緩衝液を
０．５ｍｌ２入れｐＨ７．０に調製した（ステップの）
。11 First, put the sample (5 mg/dQ. 10) into a test tube.
mg/dQ. 14 mg/dQ uric acid aqueous solution) 0
．． 5 mQ was taken out and 0.5 ml of 0.1 mol/12 phosphate buffer was added to adjust the pH to 7.0 (in step).
.
次にＳＯＤとカタラーゼをそれぞれ１０μＱ添加し（ス
テップ■■）、３０℃で３０分反応させ、試料中のスー
パーオキサイドと過酸化水素を除去した。Next, 10 μQ each of SOD and catalase were added (step ■■) and reacted at 30° C. for 30 minutes to remove superoxide and hydrogen peroxide in the sample.
次にＳＯＤとカタラーゼの阻害剤としてＫＣＮを１０μ
ｇ添加したくステップ■）。Next, add 10μ of KCN as an inhibitor of SOD and catalase.
g Addition step ■).
次にこの試料を１００倍に希釈したうえで１ｍｌ２分取
し、尿酸オキシダーゼを１０μｅ添加した。Next, this sample was diluted 100 times, two 1 ml portions were taken, and 10 μe of uric acid oxidase was added.
そして３０℃で３０分反応させ、過酸化水素を発生させ
た（ステップ■）。A reaction was then carried out at 30° C. for 30 minutes to generate hydrogen peroxide (step ①).
次にこの試料を１００μｅ分取し、ルミノメー１２
夕（ＵＰＤ−８０００ ：器明電舎製）を使用し、ルミ
ノール、フェリシアン化カリウムをそれぞれ５００μｅ
自動分注し、発光反応を起こさせて過酸化水素の量を測
定した（ステップ■）。Next, 100 μe of this sample was collected, and 500 μe each of luminol and potassium ferricyanide were collected using Luminome 12 (UPD-8000, manufactured by Kimeidensha).
The amount of hydrogen peroxide was measured by automatic dispensing and a luminescence reaction (Step ■).
以上の手順により測定した過酸化水素の量に基づいて、
標準液から検量線を求め、尿酸の量を定量分析した。こ
の結果を第２図に示す。Based on the amount of hydrogen peroxide measured by the above procedure,
A calibration curve was obtained from the standard solution, and the amount of uric acid was quantitatively analyzed. The results are shown in FIG.
Ｈ．実施例２
次に、実施例２を説明する。本実施例も実施例ｌと同様
に尿酸の測定法への適用例である。H. Example 2 Next, Example 2 will be described. Like Example 1, this example is also an example of application to a method for measuring uric acid.
実施例２では、固定化ＳＯＤと固定化カタラーゼを使用
して、生体液中のスーパーオキシドと過酸化水素を除去
する手法をとっている。Example 2 employs a method of removing superoxide and hydrogen peroxide from biological fluids using immobilized SOD and immobilized catalase.
まず、固定化ＳＯＤおよび固定化カタラーゼの調製を説
明する。水不溶性媒体として、式（３）に示すアミノブ
ロビルＣＰＧ（多孔性ガラスの一種であり、修飾型を使
用（フナコシ製））を使用する。また官能性試薬として
２．５％グルタルアルデヒド溶液（０．ＯＩＭリン酸緩
衝液（ｐＨ７．０））を使用した。１ｇのアミノブロピ
ルＣ Ｉ３ Ｇを２５ｍ（２のグルタルアルデヒド溶液
に接触させ、２時間の室温撹拌（スターラは×）を行い
、式（３）に示すように反応させる。この後、蒸留水に
より洗浄してアルデヒドＣＰＧを得る。First, the preparation of immobilized SOD and immobilized catalase will be explained. As the water-insoluble medium, aminobrovir CPG (a type of porous glass, a modified type (manufactured by Funakoshi)) shown in formula (3) is used. In addition, a 2.5% glutaraldehyde solution (0.0M phosphate buffer (pH 7.0)) was used as a functional reagent. 1 g of aminopropyl C I3 G was brought into contact with the glutaraldehyde solution of 25 m (2) and stirred at room temperature for 2 hours (with the stirrer x) to react as shown in formula (3). After this, it was washed with distilled water and to obtain aldehyde CPG.
ＧＩ ａｓｓ−−−ＮＨ，＋ ＣＨＯ（ＣＨ．）．ＣＨ
ＯアミノプロピルＣＰＧ グルタルアルデヒド→
Ｇ Ｉ ａ ｓ ｓ ・・・Ｎ＝ＣＨ（ＣＨ，）３Ｃ
ＨＯ − （３）アルデヒドＣｌ
）Ｇ
このアルデヒドＣＰＧをプロテイン溶液と接触させ、３
時間の室温撹拌（スターラは×）を行い、式（４）に示
すように反応させる。GI ass---NH, + CHO (CH.). CH
O aminopropyl CPG glutaraldehyde→
G I a s s...N=CH(CH,)3C
HO - (3) Aldehyde Cl
)G This aldehyde CPG is contacted with a protein solution, 3
Stir at room temperature for hours (stirrer: x) to react as shown in formula (4).
ＧＩ ａｓｓ ・・・Ｎ＝ＣＨ（ＣＨＪ３ＣＨＯ ＋
ＮＨｔＲアルデヒドＣＰＧ
→Ｇ Ｉ ａ ｓ ｓ −・Ｎ’Ｃ Ｈ （Ｃ Ｈ ２
）３Ｃ Ｈ ＝Ｎ−Ｒ固定化プロテイン（シッフ塩基生
成物）・・・（４）
この後、式（５）に示すように、このシッフ塩基生成物
を還元剤ＮａＢＨ．により還元して、固定化プロテイン
を得る。GI ass...N=CH(CHJ3CHO+
NHtR aldehyde CPG → G I a s s −・N'C H (C H 2
)3C H =N-R immobilized protein (Schiff base product) (4) After this, as shown in formula (5), this Schiff base product is treated with the reducing agent NaBH. to obtain the immobilized protein.
Ｇ Ｉ ａ ｓｓ−Ｎ＝ＣＨ（ＣＨｔ）ａｃＨ＝Ｎ−Ｒ
固定化プロテイン（シッフ塩基生成物）→ ＧＩ ａＳ
Ｓ・＝ＮＨＣＨ，（ＣＨ．）３ＣＨ．ＮＨ−Ｒ ・
（５）ＮａＢＨ４ 固定化プロテイン
以上の手順において、プロテイン溶液としてＳＯＤ溶液
（１０，ＯＯＯＵ／ｍＩ２、０．０５ｍｏ＋／ｅリン酸
緩衝液（ｐＨ７．８））を使用することにより、固定化
ＳＯＤを得る。またプロテイン溶液としてカタラーゼ溶
液（１００，ＯＯＯＵ／ｍ（１、Ｏ ． Ｏ ｌ ｍ
ｏｌ／ Ｑリン酸緩衝液（ｐＨ７．０））を使用するこ
とにより、固定化カタラーゼを得る。G I a ss-N=CH(CHt)acH=N-R
Immobilized protein (Schiff base product) → GI aS
S.=NHCH, (CH.)3CH. NH-R・
(5) NaBH4 Immobilized protein In the above procedure, immobilized SOD is obtained by using SOD solution (10,OOOU/mI2, 0.05 mo+/e phosphate buffer (pH 7.8)) as the protein solution. . In addition, as a protein solution, a catalase solution (100, OOOU/m (1, O.Olm
Obtain immobilized catalase by using ol/Q phosphate buffer (pH 7.0).
１５ １６ 次に測定手順を説明する。15 16 Next, the measurement procedure will be explained.
第３図は本実施例の測定手順を示すフローチャートであ
る。FIG. 3 is a flowchart showing the measurement procedure of this embodiment.
まず試験管に試料（標準液： ５ｍｇ／ｄｌ２， １
０ｍｇ／ｄｌ２， １ ４ｍｇ／ｄＱ尿酸水溶液）
０．５ｍｌ２を分取し、０． １ｎｏ１／１２リン酸
緩衝液を０．５ｍｌ２入れｐ Ｈ ７ ．０に調製した
（ステップ■）。First, add the sample (standard solution: 5mg/dl2, 1
0mg/dl2, 14mg/dQ uric acid aqueous solution)
Aliquot 0.5ml2 and add 0.5ml. Add 0.5ml2 of 1no1/12 phosphate buffer to pH 7. 0 (step ■).
次に固定化ＳＯＤを充填したカラムに試料を通し、その
濾液を収集する（ステップ■）。Next, the sample is passed through a column packed with immobilized SOD, and the filtrate is collected (step ①).
さらにこの溶液を、固定化力タラーゼを充填したカラム
に通し、その濾液を収集する（ステップ■）。Further, this solution is passed through a column packed with immobilized talase, and the filtrate is collected (step ①).
次にこの試料を１００倍に希釈したうえで１ｍｌ２分取
し、尿酸オキシダーゼを１０μσ添加し、実施例１と同
様に過酸化水素を発生させ（ステップ■）、その過酸化
水素の発生量を測定することにより、尿酸の量を定量分
析したくステップ■）。Next, this sample was diluted 100 times, 1 ml was taken out, 10 μσ of urate oxidase was added, hydrogen peroxide was generated in the same manner as in Example 1 (step ■), and the amount of hydrogen peroxide generated was measured. Step ■) to quantitatively analyze the amount of uric acid.
この結果、実施例１と同等の効果を得た。As a result, the same effect as in Example 1 was obtained.
■．発明の効果
以上説明したように、本発明によれば、酵素免疫測定方
法において、酸化酵素を生体液に作用させる工程の前に
、生体液中の過酸化水素を除去する前処理を行うことと
し、この前処理として、スーパーオキサイドを分解する
工程と、過酸化水素を分解する工程とを行うこととして
いる。■． Effects of the Invention As explained above, according to the present invention, in the enzyme immunoassay method, a pretreatment for removing hydrogen peroxide from the biological fluid is performed before the step of allowing the oxidizing enzyme to act on the biological fluid. As this pretreatment, a step of decomposing superoxide and a step of decomposing hydrogen peroxide are performed.
これにより生体液中に存在する過酸化水素を除去し、し
かもスーパーオキシターゼより新たに過酸化水素が発生
することを防止することができ、過酸化水素の測定ブラ
ンクを大幅に低減できる。This makes it possible to remove hydrogen peroxide present in the biological fluid and prevent new hydrogen peroxide from being generated by superoxidase, thereby significantly reducing hydrogen peroxide measurement blanks.
したがって測定感度が向上し、被測定戊分が極めて低濃
度である場合でも、正確な測定が可能となる。Therefore, measurement sensitivity is improved, and even when the concentration of the substance to be measured is extremely low, accurate measurement is possible.
４．図面の簡単な説明
第ｌ図は実施例１における測定手順を示すフローチャー
ト、第２図は実施例１による尿酸の測定結果を示すグラ
フ、第３図は実施例２における測定手順を示すフローチ
ャートである。4. Brief Description of the Drawings Figure 1 is a flowchart showing the measurement procedure in Example 1, Figure 2 is a graph showing the measurement results of uric acid in Example 1, and Figure 3 is a flowchart showing the measurement procedure in Example 2. .
ｌ９ 第２図 尿酸分析結果 尿酸含量（ｍｇ／ｄＮ） 第１図 実施例１の手順l9 Figure 2 Uric acid analysis results Uric acid content (mg/dN) Figure 1 Procedure of Example 1
Claims (2)
過酸化水素を生成する被測定成分を測定する方法におい
て、 スーパーオキサイド・ジスムダーゼとカタラーゼとを生
体液に混合し、生体液中に含まれるスーパーオキサイド
と過酸化水素とを分解する工程と、スーパーオキサイド
・ジスムダーゼのスーパーオキサイド分解機能を抑制す
るスーパーオキサイド・ジスムダーゼ阻害剤と、カタラ
ーゼの過酸化水素分解機能を抑制するカタラーゼ阻害剤
とを生体液に添加する工程と、 被測定成分と選択的に反応して過酸化水素を生成する酸
化酵素を生体液に作用させる工程と、この工程において
生成した過酸化水素の濃度を測定する工程と、 この工程において測定された過酸化水素の濃度から被測
定成分の濃度を決定する工程と を順次行うことによる酵素免疫測定方法。(1) In a method for measuring a component in a biological fluid that produces hydrogen peroxide through the action of an oxidase, superoxide dismudase and catalase are mixed with the biological fluid, and the component is mixed into the biological fluid. A step of decomposing the superoxide and hydrogen peroxide contained, a superoxide dismudase inhibitor that suppresses the superoxide decomposition function of superoxide dismudase, and a catalase inhibitor that suppresses the hydrogen peroxide decomposition function of catalase. a step of adding the hydrogen peroxide to the biological fluid; a step of causing an oxidizing enzyme that selectively reacts with the component to be measured to produce hydrogen peroxide to act on the biological fluid; and a step of measuring the concentration of the hydrogen peroxide produced in this step. and determining the concentration of the component to be measured from the concentration of hydrogen peroxide measured in this step.
過酸化水素を生成する被測定成分を測定する方法におい
て、 生体液を固定化スーパーオキサイド・ジスムダーゼに接
触させて、生体液中に含まれるスーパーオキサイドを分
解する工程と、 生体液を固定化カタラーゼに接触させて、生体液中に含
まれる過酸化水素を分解する工程と、被測定成分と選択
的に反応して過酸化水素を生成する酸化酵素を生体液に
作用させる工程と、この工程において生成した過酸化水
素の濃度を測定する工程と、 この工程において測定された過酸化水素の濃度から被測
定成分の濃度を決定する工程と を順次行うことによる酵素免疫測定方法。(2) In a method for measuring a component in a biological fluid that produces hydrogen peroxide through the action of an oxidase, the biological fluid is brought into contact with immobilized superoxide dismudase, and A step of decomposing the superoxide contained in the biological fluid, a step of bringing the biological fluid into contact with immobilized catalase to decompose the hydrogen peroxide contained in the biological fluid, and a step of decomposing the hydrogen peroxide by selectively reacting with the component to be measured. A step of causing the generated oxidase to act on a biological fluid, a step of measuring the concentration of hydrogen peroxide generated in this step, and a step of determining the concentration of the component to be measured from the concentration of hydrogen peroxide measured in this step. An enzyme immunoassay method in which the following steps are performed sequentially.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16644989A JPH0329854A (en) | 1989-06-28 | 1989-06-28 | Enzyme immunoassay |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16644989A JPH0329854A (en) | 1989-06-28 | 1989-06-28 | Enzyme immunoassay |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0329854A true JPH0329854A (en) | 1991-02-07 |
Family
ID=15831615
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP16644989A Pending JPH0329854A (en) | 1989-06-28 | 1989-06-28 | Enzyme immunoassay |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0329854A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0405988A2 (en) * | 1989-06-28 | 1991-01-02 | Sankyo Company Limited | Use of superoxide dismutase in assays involving an oxidase |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5966899A (en) * | 1982-10-07 | 1984-04-16 | Ono Pharmaceut Co Ltd | Determination of hydrogen peroxide |
| JPS63291595A (en) * | 1987-05-22 | 1988-11-29 | Meidensha Electric Mfg Co Ltd | Determination of glucose in biospecimen |
-
1989
- 1989-06-28 JP JP16644989A patent/JPH0329854A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5966899A (en) * | 1982-10-07 | 1984-04-16 | Ono Pharmaceut Co Ltd | Determination of hydrogen peroxide |
| JPS63291595A (en) * | 1987-05-22 | 1988-11-29 | Meidensha Electric Mfg Co Ltd | Determination of glucose in biospecimen |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0405988A2 (en) * | 1989-06-28 | 1991-01-02 | Sankyo Company Limited | Use of superoxide dismutase in assays involving an oxidase |
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