JPS627840B2 - - Google Patents
Info
- Publication number
- JPS627840B2 JPS627840B2 JP57022649A JP2264982A JPS627840B2 JP S627840 B2 JPS627840 B2 JP S627840B2 JP 57022649 A JP57022649 A JP 57022649A JP 2264982 A JP2264982 A JP 2264982A JP S627840 B2 JPS627840 B2 JP S627840B2
- Authority
- JP
- Japan
- Prior art keywords
- reagent
- cholesterol
- peroxidase
- mixed
- sample
- 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
- 239000003153 chemical reaction reagent Substances 0.000 claims description 41
- 102000003992 Peroxidases Human genes 0.000 claims description 10
- 239000002738 chelating agent Substances 0.000 claims description 10
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 10
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- OHJMTUPIZMNBFR-UHFFFAOYSA-N biuret Chemical compound NC(=O)NC(N)=O OHJMTUPIZMNBFR-UHFFFAOYSA-N 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 102000004316 Oxidoreductases Human genes 0.000 claims description 3
- 108090000854 Oxidoreductases Proteins 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 44
- 235000012000 cholesterol Nutrition 0.000 description 22
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 5
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 229940116269 uric acid Drugs 0.000 description 5
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 3
- 238000004040 coloring Methods 0.000 description 3
- 229910001431 copper ion Inorganic materials 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000001590 oxidative effect Effects 0.000 description 3
- 150000003626 triacylglycerols Chemical class 0.000 description 3
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 2
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229910001447 ferric ion Inorganic materials 0.000 description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 2
- 108010089254 Cholesterol oxidase Proteins 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- 108010055297 Sterol Esterase Proteins 0.000 description 1
- 102000000019 Sterol Esterase Human genes 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910001256 stainless steel alloy Inorganic materials 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本発明は、複数項目分析方法に係り、特に自動
分析装置を用いて異種の複数の分析項目を分析す
る方法に関する。
総コレステロール、遊離コレステロール、トリ
グリセライド、尿酸等の分析項目を自動化学分析
装置で測定する場合には、特定の物質に酵素を作
用させて過酸化水素（H2O2）を生成させ、この生
成されたH2O2によつてパーオキシダーゼの存在
下で色原体（例えば４―アミノアンチピリン）を
酸化して発色させ、その発色液を光度計で測定す
るのが一般的である。
たとえば、コレステロールを例にして説明する
と、コレステロール測定に用いられている従来の
試薬は、コレステロールエステラーゼ（総コレス
テロールのみ）、コレステロールオキシダーゼ、
パーオキシダーゼ（酸化還元酵素）、３―メチル
―Ｎ―エチル―Ｎ―（β―ヒドロキシエチル）―
アニリン、および４―アミノアンチピリン（色原
体）を含む。
これら従来の試薬を自動分析装置に応用して長
時間にわたる再現性を検討した場合、大部分の測
定値は、±２mg／ｄの範囲に入り良好な再現性
を示す。しかし、100回のテストに１回程度の割
合で20mg／ｄ以上も高い測定値が得られる場合
がある。
発明者らが、この点について実験を進めた結
果、同じ試薬分注器で異なつた分析項目の試薬を
次々に分注する形式の小型多項目自動分析装置に
おいて著しい測定誤差が生ずることを見い出し
た。さらに、この種自動分析装置に上述の試薬を
適用した場合、銅イオンを含むビウレツト試薬に
よつて蛋白質を測定した直後に総コレステロール
あるいは遊離コレステロールを測定すると、特異
的に著しい正誤差を生ずることが判明した。トリ
グリセライドおよび尿酸についても同様な現象が
見い出された。
本発明の目的は、一連の試料への試薬分配系を
簡素化するために同じ試薬分注器で複数種の試薬
を添加する場合でも、パーオキシダーゼを含む試
薬を用いる分析項目を、高精度に分析し得る複数
項目分析方法を提供することにある。
本発明では、自動分析装置を用い液体試料につ
いて異種の複数の分析項目を分析する方法におい
て、第１の試料にビウレツト試薬を混合して第１
の分析項目である蛋白質を測定すること、第２の
試料にパーオキシダーゼを含む試薬を混合し色原
体を過酸化水素で酸化して発色させる反応を利用
して第２の分析項目を測定すること、上記パーオ
キシダーゼを含む試薬中にはキレート剤を含有さ
せておくこと、および上記ビユレツト試薬を添加
するのと同じ試薬分注器を用いて上記パーオキシ
ダーゼを含む試薬を添加すること、を含むことを
特徴とする。
正誤差の原因をさらに究明した結果、そのほと
んどは、金属イオンによる色原体の酸化発色反応
の促進であることが判明した。本発明はキレート
剤がコレステロール、トリグリセライド、尿酸等
の各測定反応を妨害しないことを実験により試認
したことに基づいてなされている。
以下実施例を用いて本発明を詳細に説明する。
原因究明のため、前述のビウレツト試薬の各成
分を総コレステロール試薬に添加して測定値に対
する影響を調べた。その結果、硫酸銅の添加によ
つて総コレステロールの測定値が10％程度高値と
なることがわかつた。10％の誤差は、重大であ
る。また、銅イオンの他の第二鉄イオンによつて
も正誤差が生ずることも判明した。
コレステロールの発色反応は、次式の様に示さ
れる。反応により生じた過酸化水素を、酸化還元
酵素であるパーオキシダーゼと共役させて、色原
体を酸化し、この結果生じた色を比色している。
The present invention relates to a method for analyzing multiple items, and more particularly to a method for analyzing a plurality of different types of analysis items using an automatic analyzer. When measuring analytical items such as total cholesterol, free cholesterol, triglycerides, and uric acid using an automatic chemical analyzer, an enzyme is applied to a specific substance to generate hydrogen peroxide (H 2 O 2 ), and this generated Generally, a chromogen (for example, 4-aminoantipyrine) is oxidized with H 2 O 2 in the presence of peroxidase to develop a color, and the coloring solution is measured with a photometer. For example, using cholesterol as an example, conventional reagents used for cholesterol measurement include cholesterol esterase (total cholesterol only), cholesterol oxidase,
Peroxidase (oxidoreductase), 3-methyl-N-ethyl-N-(β-hydroxyethyl)-
Contains aniline, and 4-aminoantipyrine (chromogen). When these conventional reagents are applied to an automatic analyzer and their reproducibility over a long period of time is examined, most of the measured values fall within the range of ±2 mg/d, indicating good reproducibility. However, in about 1 out of every 100 tests, a measured value higher than 20 mg/d may be obtained. As a result of conducting experiments on this point, the inventors discovered that a significant measurement error occurred in a compact multi-item automatic analyzer that dispenses reagents for different analysis items one after another using the same reagent dispenser. . Furthermore, when the above-mentioned reagents are applied to this type of automatic analyzer, if total cholesterol or free cholesterol is measured immediately after protein is measured using Biuret's reagent containing copper ions, a significant error may specifically occur. found. Similar phenomena were found for triglycerides and uric acid. The purpose of the present invention is to simplify the reagent distribution system for a series of samples, so that analysis items using reagents containing peroxidase can be performed with high precision even when multiple types of reagents are added using the same reagent dispenser. The purpose of the present invention is to provide a method for analyzing multiple items. In the present invention, in a method of analyzing a plurality of different analysis items for a liquid sample using an automatic analyzer, a Biuret reagent is mixed with a first sample,
The second analysis item is measured by mixing a reagent containing peroxidase with the second sample and oxidizing the chromogen with hydrogen peroxide to develop color. , containing a chelating agent in the reagent containing the peroxidase, and adding the reagent containing the peroxidase using the same reagent dispenser used to add the Buillet reagent. It is characterized by As a result of further investigation into the cause of the error, it was found that most of it was due to promotion of the oxidative coloring reaction of the chromogen by metal ions. The present invention was made based on the experimental confirmation that chelating agents do not interfere with the reactions for measuring cholesterol, triglyceride, uric acid, and the like. The present invention will be explained in detail below using Examples. In order to investigate the cause, each component of the Biuret reagent mentioned above was added to the total cholesterol reagent and the effect on the measured value was investigated. As a result, it was found that the addition of copper sulfate increased the measured value of total cholesterol by about 10%. A 10% error is significant. It was also found that ferric ions other than copper ions also caused positive errors. The coloring reaction of cholesterol is shown by the following formula. Hydrogen peroxide produced by the reaction is conjugated with peroxidase, an oxidoreductase, to oxidize the chromogen, and the resulting color is compared.
【表】
一般に、パーオキシダーゼを用いる方法は、還
元物質による負の影響を受けやすいと同様に、酸
化物質による正の影響も受けると考えられる。上
式の反応から考えて、ビウレツト試薬中に含まれ
ている銅イオンや、血清ピペツテイング系、試薬
分注系などの自動分析装置のステンレス合金に由
来する第二鉄イオによつて、色原体の酸化が促進
されるために、発色率が増大すると推定される。
以上の観点から、本発明においては、蛋白質を
測定する場合のビウレツト試薬に由来する重金属
イオンをコレステロール等の測定時にはマスキン
グするように、コレステロール等の試薬のキレー
ト剤を含有させた。
表１に、総コレステロールを測定する合の試薬
組成例を示す。キレート剤としてエチレンジアミ
ン四酢酸ニナトリウム塩（EDTA・2Na）を用い
ている。[Table] In general, methods using peroxidase are considered to be susceptible to negative effects from reducing substances as well as positive effects from oxidizing substances. Considering the reaction in the above equation, the chromogen is produced by the copper ions contained in Biuret's reagent and the ferric ions derived from the stainless steel alloy of automatic analyzers such as serum pipetting systems and reagent dispensing systems. It is presumed that the rate of color development increases because the oxidation of In view of the above, in the present invention, a chelating agent for reagents such as cholesterol is included so as to mask heavy metal ions derived from Biuret's reagent when measuring proteins. Table 1 shows an example of a reagent composition for measuring total cholesterol. Ethylenediaminetetraacetic acid disodium salt (EDTA・2Na) is used as a chelating agent.
【表】
従来の試薬組成は、表１のEDTA・2Naを含ま
ない組成で表わされる。
本実施例の試薬と従来の試薬を比較するため
に、両試薬を用いて、前述の小型多項目自動分析
装置によつて、総蛋白と総コレステロールを同一
の試薬分注器を用い同時測定を行なつた。結果を
表２に示す。本実施例の試薬では、総蛋白を測定
した直後の総コレステロール測定値の増加は認め
られない。これに対して、従来の試薬では、10％
程度の増加が認められる。[Table] The conventional reagent composition is shown in Table 1, which does not contain EDTA/2Na. In order to compare the reagent of this example with a conventional reagent, total protein and total cholesterol were measured simultaneously using the same reagent dispenser using both reagents using the above-mentioned compact multi-item automatic analyzer. I did it. The results are shown in Table 2. With the reagent of this example, no increase in the measured value of total cholesterol was observed immediately after measuring total protein. In contrast, with conventional reagents, 10%
An increase in the degree was observed.
【表】
次に、キレート剤であるEDTA・2Naを総コレ
ステロール試薬に添加したことによる総コレステ
ロール測定値に対する影響をみた。EDTA・2Na
添加（本発明で用いる試薬）と、無添加（従来の
試薬）を用いて同一装置で相関実験を行なつた。
この結果を第１図に示した。キレート剤の添加に
よる悪影響は認められなかつた。
また、EDTA・2Naの添加量を検討した。この
結果10-4M以上の濃度で、誤差除去効果が認めら
れた。さらに、EDTA・2Na以外に、GEDTA
（グリコールエーテルジアミン四酢酸）、DPTA―
OH（ジアミンプロパノール四酢酸）などのキレ
ート剤についても同様の効果が認められた。
遊離コレステロール、トリグリセライドおよび
尿酸についても、従来試薬にキレート剤を添加す
ることによつて、蛋白質と同時分析しても上記の
総コレステロールと同様の効果が認められた。
以上のように、本発明では、試薬中に適量のキ
レート剤を混在させることによつて、多項目自動
分析装置による、総コレステロール、遊離コレス
テロール、トリグリセリド、尿酸等の測定におけ
る異常な正誤差を防止できた。[Table] Next, we looked at the effect of adding EDTA/2Na, a chelating agent, to the total cholesterol reagent on total cholesterol measurements. EDTA・2Na
Correlation experiments were conducted using the same apparatus using additives (reagents used in the present invention) and non-additives (conventional reagents).
The results are shown in FIG. No adverse effects were observed due to the addition of the chelating agent. Additionally, the amount of EDTA/2Na added was investigated. As a result, an error removal effect was observed at a concentration of 10 -4 M or higher. Furthermore, in addition to EDTA/2Na, GEDTA
(Glycol ether diamine tetraacetic acid), DPTA-
A similar effect was observed with chelating agents such as OH (diaminepropanoltetraacetic acid). For free cholesterol, triglyceride, and uric acid, the same effects as for total cholesterol were observed when analyzed simultaneously with protein by adding a chelating agent to the conventional reagent. As described above, the present invention prevents abnormal errors in the measurement of total cholesterol, free cholesterol, triglycerides, uric acid, etc. using a multi-item automatic analyzer by mixing an appropriate amount of chelating agent in the reagent. did it.
第１図は、従来組成の試薬と、本発明を適用し
た実施例の試薬を用いた場合の総コレステロール
測定の相関図である。
FIG. 1 is a correlation diagram of total cholesterol measurements when using a reagent having a conventional composition and a reagent according to an example to which the present invention is applied.
Claims (1)
複数の分析項目を分析する方法において、第１試
料にビウレツト試薬を混合して第１の分析項目で
ある蛋白質を測定すること、第２の試料にパーオ
キシダーゼを含む試薬を混合した色原体を過酸化
水素で酸化して発色させる反応を利用して第２の
分析項目を測定すること、上記パーオキシダーゼ
を含む試薬中にはキレート剤を含有させておくこ
と、および上記ビユレツト試薬を添加すると同じ
試薬分注器を用いて上記パーオキシダーゼを含む
試薬を添加すること、を含むことを特徴とする複
数項目分析方法。1 In a method of analyzing multiple different analysis items for a liquid sample using an automatic analyzer, the first sample is mixed with Biuret's reagent to measure protein, which is the first analysis item, and the second sample is mixed with protein. The second analysis item is measured using a reaction in which a chromogen mixed with a reagent containing oxidase is oxidized with hydrogen peroxide to develop color, and the reagent containing peroxidase contains a chelating agent. and adding the peroxidase-containing reagent using the same reagent dispenser used to add the Buillet reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2264982A JPS58141797A (en) | 1982-02-17 | 1982-02-17 | Reagent for automatic analysis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2264982A JPS58141797A (en) | 1982-02-17 | 1982-02-17 | Reagent for automatic analysis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS58141797A JPS58141797A (en) | 1983-08-23 |
| JPS627840B2 true JPS627840B2 (en) | 1987-02-19 |
Family
ID=12088693
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2264982A Granted JPS58141797A (en) | 1982-02-17 | 1982-02-17 | Reagent for automatic analysis |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS58141797A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS62261962A (en) * | 1986-05-08 | 1987-11-14 | Chemo Sero Therapeut Res Inst | Liquid control serum for lipid component |
| WO2009037784A1 (en) * | 2007-09-21 | 2009-03-26 | Tya K. K. | Analytical instrument for body fluid component |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5342886A (en) * | 1976-09-30 | 1978-04-18 | Wako Pure Chem Ind Ltd | Measuring reagents for acid phosphatase in body fluids |
| JPS548318A (en) * | 1977-06-22 | 1979-01-22 | Hitachi Ltd | Truck for car |
| JPS58898A (en) * | 1981-06-23 | 1983-01-06 | Shinotesuto Kenkyusho:Kk | Quantitative analysis of hydrogen peroxide |
| JPS58899A (en) * | 1981-06-23 | 1983-01-06 | ベ−リンガ−・マンハイム・ゲゼルシヤフト・ミツト・ベシユレンクテル・ハフツング | Stabilized reagent for detecting h2o2 or h2o2 donor system |
-
1982
- 1982-02-17 JP JP2264982A patent/JPS58141797A/en active Granted
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5342886A (en) * | 1976-09-30 | 1978-04-18 | Wako Pure Chem Ind Ltd | Measuring reagents for acid phosphatase in body fluids |
| JPS548318A (en) * | 1977-06-22 | 1979-01-22 | Hitachi Ltd | Truck for car |
| JPS58898A (en) * | 1981-06-23 | 1983-01-06 | Shinotesuto Kenkyusho:Kk | Quantitative analysis of hydrogen peroxide |
| JPS58899A (en) * | 1981-06-23 | 1983-01-06 | ベ−リンガ−・マンハイム・ゲゼルシヤフト・ミツト・ベシユレンクテル・ハフツング | Stabilized reagent for detecting h2o2 or h2o2 donor system |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS58141797A (en) | 1983-08-23 |
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