JPS5822200B2 - How to measure serum components - Google Patents
How to measure serum componentsInfo
- Publication number
- JPS5822200B2 JPS5822200B2 JP14630980A JP14630980A JPS5822200B2 JP S5822200 B2 JPS5822200 B2 JP S5822200B2 JP 14630980 A JP14630980 A JP 14630980A JP 14630980 A JP14630980 A JP 14630980A JP S5822200 B2 JPS5822200 B2 JP S5822200B2
- Authority
- JP
- Japan
- Prior art keywords
- bilirubin
- edta
- iron
- reagent
- serum components
- 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
Links
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- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】
本発明は血清成分の測定に酵素反応系を用い生成する過
酸化水素をペルオキシダーゼの存在下比色定量する場合
、血清に共存するビリルビンの干渉をEDTA−鉄錯体
を予め加えて置くことによって回避捷たは軽減する方法
に関する。DETAILED DESCRIPTION OF THE INVENTION When the present invention uses an enzymatic reaction system to measure serum components and colorimetrically quantitates generated hydrogen peroxide in the presence of peroxidase, the interference of bilirubin coexisting in serum can be eliminated by pre-containing EDTA-iron complex. Concerning how to avoid or reduce by adding additional information.
血清中には常にビリルビンが存在し、正常人では総ビリ
ルビンとして約0.5Tvdi以下が普通であるが病的
には著しく増加し20Tn9/dlに達することもある
。Bilirubin is always present in serum, and in a normal person, total bilirubin is normally less than about 0.5 Tvdi, but in pathological conditions it increases markedly and can reach 20 Tn9/dl.
これらのビリルビンは血清成分の比色法による測定に種
々の干渉を行ない測定値に誤差を与える原因となる。These bilirubins cause various interferences in the colorimetric measurement of serum components, causing errors in the measured values.
特に最近では酵素を用いる生体成分の渭淀か繁用されて
いるが、その中でも過酸化水素−ペルオキシダーゼ−水
素供与体反応系(過酸化水素系)を用いる方法か数多く
使用されている。Particularly recently, biological components using enzymes have been frequently used, and among these methods, many methods using hydrogen peroxide-peroxidase-hydrogen donor reaction system (hydrogen peroxide system) have been used.
この系においてビリルビンは水素供与体と競合し測定値
に負の影響を与える(臨床化学第8巻、第1号63〜7
2(1979))。In this system, bilirubin competes with the hydrogen donor and has a negative effect on the measured values (Clinical Chemistry Vol. 8, No. 1, 63-7
2 (1979)).
このようなビリルビンの干渉は測定する成分が血清中に
少量しか存在しない場合特に問題となる。Such bilirubin interference is particularly problematic when the component to be measured is present in only a small amount in serum.
たとえば尿酸、クレアチニン、遊離脂肪酸、スフィンゴ
ミエリン、リゾレシチンの場合がそわであり、さらに微
量成分の場合は問題は深刻で過酸化水素系測定法の適用
が困難という隘路があった。For example, it is difficult to measure uric acid, creatinine, free fatty acids, sphingomyelin, and lysolecithin, and the problem is even more serious when it comes to trace components, making it difficult to apply hydrogen peroxide-based measurement methods.
従って過酸化水素系においてビリルビンの一干渉を受け
ない方法の開発が強く望まれていた。Therefore, there has been a strong desire to develop a method that does not suffer from the interference of bilirubin in hydrogen peroxide systems.
これらの問題に関する先行技術としてはすでにフェロシ
アン化合物を用いる方法が示されている。As a prior art related to these problems, a method using a ferrocyan compound has already been shown.
たとえば特許公報昭55−25840号あるいはP。For example, Patent Publication No. 55-25840 or P.
Fossati他、クリニカルケミストリー26/2.
227−231.(1980)の中でフェロシアン化合
物によるビリルビンの干渉回避を次のように説明してい
る。Fossati et al., Clinical Chemistry 26/2.
227-231. (1980) explains the avoidance of bilirubin interference by ferrocyanide compounds as follows.
すなわち■フェロシアン化合物が過酸化水素−ペルオキ
シダーゼにより酸化されてフェリシアン化合物となる。That is, (1) a ferrocyan compound is oxidized by hydrogen peroxide-peroxidase to become a ferricyan compound;
■フェリシアン化合物はビリルビンよりも発色性水素供
与体を選択的に酸化し発色させ自身はフェロシアン化合
物に戻る。■Ferrocyanide compounds selectively oxidize color-forming hydrogen donors than bilirubin, develop color, and return to ferrocyanide compounds.
このほかフェロシアン化合物による効果についてはP、
Fossati等により詳しく述べられているが、欠点
としてフェロシアン化合物あるいはフェリシアン化合物
はそれ自体毒性はないとされているか光または酸処理で
分解しシアン化合物を遊離するとされているので排水の
水質保全上問題がある。In addition, regarding the effects of ferrocyanide compounds, see P.
As described in detail by Fossati et al., the disadvantage is that ferrocyanic compounds or ferricyanide compounds are said to be either not toxic in themselves or to decompose with light or acid treatment to liberate cyanide compounds, so they cannot be used to preserve the water quality of wastewater. There's a problem.
本発明者等はフェロシアン化合物に替る毒性の全く心配
のない多数の化合物を検索した結果EDTA−鉄(I)
ま、たはEDTA−鉄(I)の錯体に著しい効果のある
ことを見出し本発明を完成した。The present inventors searched for a large number of compounds that are completely free of toxicity as an alternative to ferrocyanide compounds, and found that EDTA-iron(I)
Furthermore, the present invention was completed by discovering that a complex of EDTA-iron(I) has a remarkable effect.
本発明によれば血清成分の測定に酵素反応系を適用し生
成する過酸化水素によりペルオキシダーゼの存在下発色
性水素供与体を酸化することによシ当該成分を比色定量
する方法においてEDTA−鉄錯体を添加することによ
り血清に共存するビリルビンの干渉を回避または軽減す
ることを特徴とする血清成分の測定方法が提供される。According to the present invention, an enzymatic reaction system is applied to the measurement of serum components, and a chromogenic hydrogen donor is oxidized by the generated hydrogen peroxide in the presence of peroxidase. A method for measuring serum components is provided, which is characterized by adding a complex to avoid or reduce interference of bilirubin coexisting in serum.
このことはフェロシアン化合物の知見からは全く予期で
きない効果である。This is a completely unexpected effect based on the knowledge of ferrocyan compounds.
なぜならばEDTA−鉄@)錯体はフェリシアン化合物
のように発色剤として用いる水素供与体を酸化すること
はない。This is because the EDTA-iron@) complex does not oxidize the hydrogen donor used as a coloring agent, unlike the ferricyanide compound.
またEDTA−鉄(II)あるいはEDTA−鉄@)の
錯体の添加がビリルビンの干渉を回避しているがこの二
つの錯体の間の酸化還元反応を拌ったものとは考えられ
ず。Furthermore, although the addition of the EDTA-iron(II) or EDTA-iron@) complex avoided the interference of bilirubin, it cannot be considered that this stirred the redox reaction between these two complexes.
さらに塩化第二鉄塩または塩化第一鉄塩にこのような効
果がないことから単に鉄塩であればビリルビンの干渉を
回避できるものでもない。Furthermore, since ferric chloride salts and ferrous chloride salts do not have such effects, interference with bilirubin cannot be avoided simply by using iron salts.
現状ではEDTA−鉄錯体の効果の機構は明らかでない
。At present, the mechanism of the effect of the EDTA-iron complex is not clear.
次に参考例および実施例によシさらに詳しく本発明を説
明する。Next, the present invention will be explained in more detail with reference to reference examples and examples.
参考例
過酸化水素系におけるビリルビンの干渉はpHが高い程
大きいとされている。Reference Example It is said that the higher the pH, the greater the interference of bilirubin in hydrogen peroxide systems.
pH8における過酸化水素そのもの\発色とビリルビン
の存在する場合のEDTA−鉄錯体の効果を示すため次
の試験を行なった。The following test was conducted to demonstrate the effect of hydrogen peroxide itself\color development at pH 8 and the EDTA-iron complex in the presence of bilirubin.
■、試薬
(1)試薬
4−アミノアンチピリy0.5mmol/IN−エチル
−N−スルホブ
ロピル−m−)ルビ2フ0.2mmol/lペルオキシ
ダーゼ200則旬
以上を0.1mol/l)’)ス緩衝液に溶解しpHを
8,0とする。■, Reagent (1) Reagent 4-aminoantipyryy0.5mmol/IN-ethyl-N-sulfopropyl-m-)ruby2F0.2mmol/l peroxidase 200 or more in 0.1mol/l)') Dissolve in buffer solution and adjust pH to 8.0.
(2)EDTA−鉄錯体添加物 0.5mmo、g/7EDTA− 鉄(II)まだはEDTA− 鉄(IIJを上記試薬に溶解 する。(2) EDTA-iron complex additive 0.5mmo, g/7EDTA- Iron (II) still EDTA- Dissolve iron (IIJ in the above reagent) do.
塩化鉄添加物0.5mmol/1Fect3またばFe
c13を上記
試薬に溶解する。Iron chloride additive 0.5 mmol/1Fect3 or Fe
Dissolve c13 in the above reagent.
対照上記試薬のみで添加物 なし。Control above reagents only with additives none.
(3)ビリルビン
ビリルビンコントロール
(ディト社製品)20m97dl
(4)過酸化水素
1mmob/を過酸化水素溶液
2、操作
試薬3r/11にビリルビン50μtまたは水50μt
を加えさらに過酸化水素50μ2−または水50μlを
加え37℃5分間加温する。(3) Bilirubin Bilirubin Control (Dito product) 20 m 97 dl (4) 1 mmob of hydrogen peroxide/2 hydrogen peroxide solution, 3 r/11 operating reagents and 50 μt of bilirubin or 50 μt of water
Add 50 μl of hydrogen peroxide or 50 μl of water, and heat at 37° C. for 5 minutes.
同様のことを試薬の替りにEDTA−鉄錯体添加物、塩
化鉄添加物及び対照について行ない、全て波長550n
mで吸光度を測定した。The same thing was done with EDTA-iron complex additive, iron chloride additive and control instead of reagent, all at wavelength 550n.
Absorbance was measured at m.
その結果を表1に示す。表1からEDTA−鉄錯体の添
加(本発明方法)により過酸化水素の発色へのビリルビ
ンの干渉は殆ど無視できる程回避できることまたFec
13.Fec12の添加は若干の効果は認められるが充
分でないことか明らかである。The results are shown in Table 1. Table 1 shows that by adding the EDTA-iron complex (method of the present invention), the interference of bilirubin with the color development of hydrogen peroxide can be avoided to an almost negligible extent.
13. Although the addition of Fec12 has some effect, it is clear that it is not sufficient.
実施例1
尿酸測定
(1)試薬
N−エチル−N−スルホプロ
ピルーm−トルイジ70.5mmol/14−アミノア
ンチピリン0.2mmol、/1ウリカーゼ0.IU/
7!
ペルオキシダーゼ2000U/rnl
EDTA−鉄(I)0.5mmolL/1以上を0.1
mol/l!Jン酸ホウ砂緩衝液に溶解しpH7,0と
する。Example 1 Uric acid measurement (1) Reagent N-ethyl-N-sulfopropyl-m-toluidi 70.5 mmol/14-aminoantipyrine 0.2 mmol/1 uricase 0. IU/
7! Peroxidase 2000U/rnl EDTA-Iron(I) 0.5mmolL/1 or more at 0.1
mol/l! Dissolve in J-phosphate borax buffer and adjust to pH 7.0.
たソし後述の対照の場合りはEDTA−鉄@)を含まな
いものを用いる。For the control described below, use one that does not contain EDTA-iron.
(2)操作
検体血清50μtをとり試薬3mを加え370G、10
分1間加温後、波長550nmで吸光度を測定し、別に
濃度既知の尿酸を用い同様の操作により作成した検量線
を用い検体中の尿酸の量を算出する。(2) Take 50 μt of operation specimen serum, add 3 m of reagent, and add 370 G.
After heating for 1 minute, the absorbance is measured at a wavelength of 550 nm, and the amount of uric acid in the sample is calculated using a calibration curve prepared in a similar manner using uric acid of known concentration.
次に本発明の詳細な説明するために次の試験を行なった
。Next, the following tests were conducted to explain the present invention in detail.
すなわち試薬および対照の3−に夫々20m97dlビ
リルビン溶液50μtまたは水50μtを加えさらに2
0Tvdl尿酸水溶液50μtまたは水50μtを加え
それぞれ37℃、10分間加温したものについて波長5
50nmで吸光度を測定した。That is, 50 μt of 20 m 97 dl bilirubin solution or 50 μt of water was added to the reagent and control 3-2, respectively.
0 Tvdl uric acid aqueous solution 50 μt or water 50 μt was added and heated at 37°C for 10 minutes.
Absorbance was measured at 50 nm.
その結果を表2に示す。The results are shown in Table 2.
この表より本発明の効果が明らかである。The effect of the present invention is clear from this table.
実施例2
遊離脂肪酸測定
■、試薬
試薬1
アシルロ一式シンセターゼ0.5U/l
アデノシントリホスフエイト5.15μmol/lコエ
ンザイムAO,8μmol/を
以上を0.05mol/Lト’)ス緩衝液に溶解しpH
7,8とする。Example 2 Free fatty acid measurement ■, Reagents Reagent 1 Acyllo-synthetase 0.5 U/l Adenosine triphosphate 5.15 μmol/l Coenzyme AO, 8 μmol/l Dissolve the above in 0.05 mol/L torsion buffer. pH
7, 8.
試薬2
N−エチルアレイミド
水溶液10mol/1
試薬3
N−エチル−N−スルホプロピル−
m−)ルイジン0.5mmot/1
4−アミノアンチピリン0.2mmo、!/lアシ/1
/ニアーAオキシダーゼ0.125TJAnlベルオキ
シダーセ2000TJAnl
EDTA−鉄(1)o、75mmot/を以上を0.0
2mol/lN、N−ビス(2−ハイドロキシル)−2
−アミノエタンスルホン酸緩衝液に溶解しpH7,2と
する。Reagent 2 N-ethylaleimide aqueous solution 10 mol/1 Reagent 3 N-ethyl-N-sulfopropyl-m-)luidine 0.5 mmot/1 4-aminoantipyrine 0.2 mmo,! /l reed/1
/ Near A oxidase 0.125TJAnl Peroxidase 2000TJAnl EDTA-Iron (1) o, 75mmot/ or more 0.0
2mol/lN, N-bis(2-hydroxyl)-2
-Dissolve in aminoethanesulfonic acid buffer to pH 7.2.
たソし後述の対照の場合はEDTA−鉄(1)を含まな
いものを用いる。In the case of a control described later, a sample containing no EDTA-iron (1) is used.
2、樽作
検体血清50μtK試薬1の0.5ゴを加え37℃、1
5分間加温後試薬2の0.5mlを加え、さらに試薬3
の2−を加え37℃、10分間加温後波長550nmで
吸光度を測定し、別に濃度既知の脂肪酸を用いて同様の
操作により作成した検量線を用い検体中の遊離脂肪酸の
量を算出する。2. Add 50μt of barrel sample serum and 0.5g of K reagent 1 and heat at 37°C.
After heating for 5 minutes, add 0.5 ml of reagent 2, and then add reagent 3.
After heating at 37° C. for 10 minutes, the absorbance is measured at a wavelength of 550 nm, and the amount of free fatty acid in the sample is calculated using a calibration curve prepared in the same manner using fatty acids of known concentration.
次に本発明の詳細な説明するために次の試験を行なった
。Next, the following tests were conducted to explain the present invention in detail.
すなわち試薬および対照の夫々3rnlに20m97d
lビリルビン溶液50μtまたは水50μtを加え、さ
らに1mmol/1オレイン酸ナトリウム酸浴トリウム
水溶液50μtμtを加え37℃、15分間加温後試薬
2の0.57nlを加えさらに試薬302m1を加え3
7℃、10分間加温したものについて波長550nmで
吸光度を測定した。i.e. 20m97d in 3rnl each of reagent and control.
Add 50 μt of bilirubin solution or 50 μt of water, then add 50 μt μt of 1 mmol/1 sodium oleate acid bath thorium aqueous solution, heat at 37°C for 15 minutes, add 0.57 nl of reagent 2, and add 302 ml of reagent 3.
The absorbance was measured at a wavelength of 550 nm after heating at 7°C for 10 minutes.
その結果を表3に示す。The results are shown in Table 3.
この表より本発明の効果が明らかである。The effect of the present invention is clear from this table.
Claims (1)
水素によりペルオキシダーゼの存在下発色性水素供与体
を酸化することにより当該成分を比色定量する方法にお
いてEDTA−鉄錯体を添加することにより血清に共存
するビリルビンの干渉を回避捷だは軽減することを特徴
とする血清成分の測定力法。1. By adding an EDTA-iron complex in a method of colorimetrically quantifying serum components by applying an enzymatic reaction system to the measurement of serum components and oxidizing a chromogenic hydrogen donor in the presence of peroxidase with the generated hydrogen peroxide. A method for measuring serum components that is characterized by avoiding or reducing the interference of bilirubin coexisting in serum.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP14630980A JPS5822200B2 (en) | 1980-10-21 | 1980-10-21 | How to measure serum components |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP14630980A JPS5822200B2 (en) | 1980-10-21 | 1980-10-21 | How to measure serum components |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS5771398A JPS5771398A (en) | 1982-05-04 |
JPS5822200B2 true JPS5822200B2 (en) | 1983-05-07 |
Family
ID=15404755
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP14630980A Expired JPS5822200B2 (en) | 1980-10-21 | 1980-10-21 | How to measure serum components |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS5822200B2 (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ZA841185B (en) * | 1983-03-28 | 1984-09-26 | Miles Lab | Ascorbate interference-resistant composition,device and method for the determination of peroxidatively active substances |
US4587220A (en) * | 1983-03-28 | 1986-05-06 | Miles Laboratories, Inc. | Ascorbate interference-resistant composition, device and method for the determination of peroxidatively active substances |
JPS608750A (en) * | 1983-06-29 | 1985-01-17 | Yatoron:Kk | Measuring method of serum component |
JP6203708B2 (en) | 2012-04-27 | 2017-09-27 | 協和メデックス株式会社 | Method for measuring components to be measured in a sample |
CN103571916B (en) * | 2013-11-22 | 2016-03-16 | 重庆医科大学 | A kind of double reagent method measures the formula of uric acid content test kit |
-
1980
- 1980-10-21 JP JP14630980A patent/JPS5822200B2/en not_active Expired
Also Published As
Publication number | Publication date |
---|---|
JPS5771398A (en) | 1982-05-04 |
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