JPH03175997A - Determination of total bilirubin and reagent used therefor - Google Patents
Determination of total bilirubin and reagent used thereforInfo
- Publication number
- JPH03175997A JPH03175997A JP31423289A JP31423289A JPH03175997A JP H03175997 A JPH03175997 A JP H03175997A JP 31423289 A JP31423289 A JP 31423289A JP 31423289 A JP31423289 A JP 31423289A JP H03175997 A JPH03175997 A JP H03175997A
- Authority
- JP
- Japan
- Prior art keywords
- group
- bilirubin
- carbon atoms
- hydrogen atom
- alkyl group
- 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.)
- Granted
Links
- 238000008050 Total Bilirubin Reagent Methods 0.000 title claims abstract description 20
- 239000003153 chemical reaction reagent Substances 0.000 title claims description 47
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 claims abstract description 114
- 108010015428 Bilirubin oxidase Proteins 0.000 claims abstract description 38
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims abstract description 14
- 150000002989 phenols Chemical class 0.000 claims abstract description 13
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 12
- 150000001448 anilines Chemical class 0.000 claims abstract description 12
- 150000001875 compounds Chemical class 0.000 claims abstract description 9
- 125000003545 alkoxy group Chemical group 0.000 claims abstract description 8
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 8
- 125000005843 halogen group Chemical group 0.000 claims abstract description 8
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 8
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 125000002768 hydroxyalkyl group Chemical group 0.000 claims abstract description 4
- 125000004964 sulfoalkyl group Chemical group 0.000 claims abstract description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 21
- 239000007853 buffer solution Substances 0.000 claims description 17
- 125000004432 carbon atom Chemical group C* 0.000 claims description 14
- 238000004445 quantitative analysis Methods 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 238000012802 pre-warming Methods 0.000 claims description 2
- 229940124530 sulfonamide Drugs 0.000 claims description 2
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 claims 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical group OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims 1
- 238000006911 enzymatic reaction Methods 0.000 abstract description 28
- 230000001737 promoting effect Effects 0.000 abstract description 6
- 238000006243 chemical reaction Methods 0.000 description 26
- 239000000243 solution Substances 0.000 description 23
- 238000002835 absorbance Methods 0.000 description 20
- 238000005259 measurement Methods 0.000 description 20
- 239000000872 buffer Substances 0.000 description 15
- 210000002966 serum Anatomy 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 239000007864 aqueous solution Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 8
- 238000010586 diagram Methods 0.000 description 7
- 125000000664 diazo group Chemical group [N-]=[N+]=[*] 0.000 description 6
- 230000035484 reaction time Effects 0.000 description 6
- 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 5
- TUAMRELNJMMDMT-UHFFFAOYSA-N 3,5-xylenol Chemical compound CC1=CC(C)=CC(O)=C1 TUAMRELNJMMDMT-UHFFFAOYSA-N 0.000 description 5
- BPYKTIZUTYGOLE-UHFFFAOYSA-N billirubin-IXalpha Natural products N1C(=O)C(C)=C(C=C)C1=CC1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(C=C3C(=C(C=C)C(=O)N3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-UHFFFAOYSA-N 0.000 description 3
- 208000019423 liver disease Diseases 0.000 description 3
- 230000005976 liver dysfunction Effects 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 210000000941 bile Anatomy 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- -1 di-substituted phenol Chemical class 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- HFZWRUODUSTPEG-UHFFFAOYSA-N 2,4-dichlorophenol Chemical compound OC1=CC=C(Cl)C=C1Cl HFZWRUODUSTPEG-UHFFFAOYSA-N 0.000 description 1
- CDGBQMHYFARRCC-UHFFFAOYSA-N 3-(n-ethyl-3,5-dimethylanilino)-2-hydroxypropane-1-sulfonic acid Chemical compound OS(=O)(=O)CC(O)CN(CC)C1=CC(C)=CC(C)=C1 CDGBQMHYFARRCC-UHFFFAOYSA-N 0.000 description 1
- 241000222519 Agaricus bisporus Species 0.000 description 1
- 108010088751 Albumins Chemical group 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- GWZYPXHJIZCRAJ-UHFFFAOYSA-N Biliverdin Natural products CC1=C(C=C)C(=C/C2=NC(=Cc3[nH]c(C=C/4NC(=O)C(=C4C)C=C)c(C)c3CCC(=O)O)C(=C2C)CCC(=O)O)NC1=O GWZYPXHJIZCRAJ-UHFFFAOYSA-N 0.000 description 1
- RCNSAJSGRJSBKK-NSQVQWHSSA-N Biliverdin IX Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(\C=C/2C(=C(C)C(=C/C=3C(=C(C=C)C(=O)N=3)C)/N\2)CCC(O)=O)N1 RCNSAJSGRJSBKK-NSQVQWHSSA-N 0.000 description 1
- 238000008789 Direct Bilirubin Methods 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 241000219745 Lupinus Species 0.000 description 1
- 241000223251 Myrothecium Species 0.000 description 1
- 244000236580 Psidium pyriferum Species 0.000 description 1
- 235000013929 Psidium pyriferum Nutrition 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical group O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 1
- QBUVFDKTZJNUPP-UHFFFAOYSA-N biliverdin-IXalpha Natural products N1C(=O)C(C)=C(C=C)C1=CC1=C(C)C(CCC(O)=O)=C(C=C2C(=C(C)C(C=C3C(=C(C=C)C(=O)N3)C)=N2)CCC(O)=O)N1 QBUVFDKTZJNUPP-UHFFFAOYSA-N 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 230000027721 electron transport chain Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- PZLXYMQOCNYUIO-UHFFFAOYSA-N lithium;hydrochloride Chemical compound [Li].Cl PZLXYMQOCNYUIO-UHFFFAOYSA-N 0.000 description 1
- 150000004994 m-toluidines Chemical class 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野１
本発明はビリルビンを酵素的に定量する方法及び、それ
に用いる定量用試薬に関し、特に血清中等の総ビリルビ
ンの定量方法に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field 1] The present invention relates to a method for enzymatically quantifying bilirubin and a quantitative reagent used therefor, and particularly relates to a method for quantifying total bilirubin in serum and the like.
［従来技術］
ビリルビンは胆汁中に最も多く存在する色素で、主とし
て老廃赤血球の崩壊により生成される血色素から作られ
、血清中では主にグルクロン酸と結合した抱合型ビリル
ビン（直接型どりルピン）またはアルブミンと結合した
非抱合型ビリルビン（間接型ビリルビン）として存在す
る。抱合型ビリルビンと非抱合型ビリルビンを合わせた
ものを総ビリルビンと呼んでいる。[Prior art] Bilirubin is the most abundant pigment in bile, and is mainly produced from hemoglobin produced by the breakdown of old red blood cells.In serum, it is mainly produced as conjugated bilirubin (direct lupine) bound to glucuronic acid or albumin. It exists as unconjugated bilirubin (indirect bilirubin) bound to The sum of conjugated and unconjugated bilirubin is called total bilirubin.
抱合型ビリルビン、非抱合型ビリルビンの血清中存在量
から、肝機能障害の分別診断および肝機能障害の程度を
知ることが可能であるため、ビリルビンの定量は臨床的
に極めて重要である。Quantification of bilirubin is clinically extremely important because it is possible to differentially diagnose liver dysfunction and determine the degree of liver dysfunction from the amount of conjugated bilirubin and unconjugated bilirubin present in serum.
従来、ビリルビンの定量法としては、ビリルビンとジア
ゾ試薬の反応で生成するアゾビリルビンを比色定量する
方法（金井 泉：臨床検査法提要金属出版 ページ Ｘ
ｌｌ−２４昭和５３年）などの化学的測定法が主に用い
られている。しかし、これらの方法は、ジアゾ試薬が検
体中に存在するビリルビン以外の生体成分と反応するた
め正確性に欠けるなど問題がある。Conventionally, the method for quantifying bilirubin is to colorimetrically quantify azobilirubin, which is produced by the reaction of bilirubin and a diazo reagent (Izumi Kanai: Clinical Test Method Required Metals Publishing Page X
Chemical measurement methods such as ll-24 (1978) are mainly used. However, these methods have problems such as a lack of accuracy because the diazo reagent reacts with biological components other than bilirubin present in the specimen.
一方、酵素を用いたビリルビンの定量法としては、タイ
−ウィング・つらによる菌茸由来のどリルビンオキシタ
ーゼを用いビリルビンを酸化させ、このときの吸光度の
減少を測定し定量する方法（特開昭５６−２７６５６）
や、付属らによるミロセシウム属（Ｍｙｒｏｔｈｅｃｉ
ｕｍ ）の微生物由来のビリルビンオキシダーゼを用い
同様にビリルビンの吸光度の減少を測定し定量する方法
（特開昭５７−１５９４８７ ）などが報告されている
。On the other hand, as a method for quantifying bilirubin using enzymes, bilirubin is oxidized using the fungal mushroom-derived throat oxidase produced by Tie-Wing Tsura, and the decrease in absorbance at this time is measured and quantified (Japanese Patent Application Laid-Open No. 1986-1999). 27656)
Myrothecium (Myrotheci) by Attachment et al.
A method for similarly measuring and quantifying the decrease in the absorbance of bilirubin using bilirubin oxidase derived from a microorganism (Japanese Patent Application Laid-Open No. 159487/1983) has been reported.
［発明が解決しようとする課題］
このような酵素を用いたビリルビンの定量法は前記ジア
ゾ試薬を用いる方法に比べ、正確性、分析精度などの点
で優れたものであるが、多量の酵素を必要とするため試
薬の価格が高く、また長い反応時間（１５〜３０分間程
度）が必要であるため測定の自動化が困難であるなどの
問題があった。[Problems to be Solved by the Invention] The method for quantifying bilirubin using such an enzyme is superior to the method using the diazo reagent in terms of accuracy and analytical precision, but it requires the use of a large amount of enzyme. Since the reagents are required, they are expensive, and the long reaction time (approximately 15 to 30 minutes) is required, making it difficult to automate the measurement.
一方、上記酵素反応による定量法の改良方法も研究され
ており、例えば、コール酸ナトリウム、サルファ剤等の
反応促進物質をビリルビンオキシダーゼと併用すること
により、ビリルビンの酵素反応性を向上させる（特公昭
６２−３３８８０他）ことが知られている。しかしなが
ら、これらによる反応促進効果は不十分であり、若干の
反応時間の短縮が可能であるものの、自動化に適応する
までには至っていなかった（１０分程度の反応時間が必
要）。On the other hand, methods for improving the quantitative method using the enzymatic reaction described above are also being studied. For example, the enzymatic reactivity of bilirubin is improved by using reaction accelerators such as sodium cholate and sulfa drugs together with bilirubin oxidase (Japanese Patent Publication No. 62 -33880, etc.). However, the effect of promoting the reaction by these is insufficient, and although it is possible to shorten the reaction time to some extent, it has not reached the point where it can be adapted to automation (reaction time of about 10 minutes is required).
本発明は上記従来技術の実情に鑑み成されたものであり
、酵素反応に基づく総ビリルビンの定量を短時間にかつ
正確に行うことのできる方法及び試薬を提供するもので
ある。The present invention has been made in view of the above-mentioned state of the prior art, and provides a method and reagent that can accurately quantify total bilirubin based on an enzyme reaction in a short time.
［課題を解決するための手段］
本発明者らは、前記の問題点を克服するために、鋭意研
究を行った結果 ビリルビンオキシダーゼに特定のフェ
ノール類または／およびアニリン類を併用することによ
り、酵素反応を著しく促進させることができることを見
出し本発明を完成したのである。[Means for Solving the Problems] In order to overcome the above-mentioned problems, the present inventors have conducted extensive research and found that the enzyme They discovered that the reaction could be significantly accelerated and completed the present invention.
すなわち、本発明は、ビリルビン含有水性検体にビリル
ビンオキシダーゼを作用させ、それにより生ずるビリル
ビンの変化を光学的に測定することにより該検体中の総
ビリルビンを定量する方法において、ビリルビンオキシ
ダーゼによる酵素反応を、一般式（Ｉ）で表わされるフ
ェノール類および一般式（ＩＩ ）で表わされるアニリ
ン類から成る群より選ばれる１種又は２種以上の化合物
により促進させることを特徴とする総ビリルビンの定量
方法、
Ｒ＠ Ｒ４
（式中、Ｒｌ、 Ｒ８はそれぞれ独立に、水素原子、ハ
ロゲン原子、炭素数１〜４のアルキル基、炭素数１〜４
のアルコキシル基、水酸基又はカルボキシル基を示す。That is, the present invention provides a method for quantifying total bilirubin in a bilirubin-containing aqueous sample by allowing bilirubin oxidase to act on the sample and optically measuring the resulting change in bilirubin. R @R4 (In the formula, Rl and R8 each independently represent a hydrogen atom, a halogen atom, an alkyl group having 1 to 4 carbon atoms, and 1 to 4 carbon atoms.
represents an alkoxyl group, hydroxyl group or carboxyl group.
但し、Ｒ３がカルボキシル基および水酸基であるものを
除く。）
（式中、
Ｒ６およびＲ７はそれぞれ独立に、
水素原
子、又は炭素数１〜４のアルキル基、ヒドロキシアルキ
ル基、スルホアルキル基、ヒドロキシスルホアルキル基
若しくはスルホナミドアルキル基を示す。Ｒ８およびＲ
９はそれぞれ独立に、水素原子、ハロゲン原子、炭素数
１〜４のアルキル基又は炭素数１〜４のアルコキシル基
を示す。）及び、上記で示されるフェノール類およびア
ニリン類から成る群より選ばれる１種又は２種以上の化
合物とビリルビンオキシダーゼとを少なくとも組み合わ
せて成るビリルビン含有水性検体中の総ビリルビン比色
定量用試薬である。However, those in which R3 is a carboxyl group or a hydroxyl group are excluded. ) (In the formula, R6 and R7 each independently represent a hydrogen atom, or an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group, a sulfoalkyl group, a hydroxysulfoalkyl group, or a sulfonamide alkyl group. R8 and R
9 each independently represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 4 carbon atoms, or an alkoxyl group having 1 to 4 carbon atoms. ), and a reagent for colorimetric determination of total bilirubin in a bilirubin-containing aqueous sample, which comprises at least a combination of one or more compounds selected from the group consisting of phenols and anilines shown above and bilirubin oxidase. .
本発明によれば、従来知られていた酵素による総ビリル
ビン定量法に比べ、短時間に安定して正確な総ビリルビ
ン（以下、特に記載がなければビリルビンと称す。）量
を測定することができ、測定の自動化を容易に図ること
が可能となる。According to the present invention, the amount of total bilirubin (hereinafter referred to as bilirubin unless otherwise specified) can be measured stably and accurately in a short time compared to the conventional enzyme-based total bilirubin quantitative method. , it becomes possible to easily automate the measurement.
一般式（Ｉ）で示されるフェノール類及び一般式（ＩＩ
）で示されるアニリン類はビリルビンオキシダーゼによ
る酵素反応を促進する化合物（以下、酵素反応促進剤と
略称する。）であるが、これらは公知のコール酸ナトリ
ウム等の直接化剤とは異なり、直接型ビリルビンに対し
ても有効な酵素反応促進効果を奏する。その作用機序に
ついては不明であるが、前記フェノール類又はアニリン
類は電子供与体としての性質があり、ビリルビンオキシ
ダーゼの電子伝達系に作用し、その結果、ビリルビンの
酸化反応を促進するものと考えられる。Phenols represented by general formula (I) and general formula (II)
) are compounds that promote the enzymatic reaction by bilirubin oxidase (hereinafter referred to as enzymatic reaction promoters), but unlike known direct-acting agents such as sodium cholate, these are compounds that promote the enzymatic reaction by bilirubin oxidase. It also has an effective enzyme reaction promoting effect on bilirubin. Although the mechanism of action is unknown, the phenols or anilines have properties as electron donors and are thought to act on the electron transport chain of bilirubin oxidase, thereby promoting the oxidation reaction of bilirubin. It will be done.
本発明で用いることのできる酵素反応促進剤は一般式（
Ｉ）および（ＩＩ）で示されるフェノール類およびアニ
リン類であるが、特に、モノまたはジ置換フェノール誘
導体、フェノール、モノまたはジ置換アニリン誘導体及
びアニリンでは良好な効果を得ることができる。これら
の化合物の例としては、以下の表に示すものを挙げるこ
とができる。The enzyme reaction promoter that can be used in the present invention has the general formula (
Among the phenols and anilines represented by I) and (II), particularly good effects can be obtained with mono- or di-substituted phenol derivatives, phenol, mono- or di-substituted aniline derivatives, and aniline. Examples of these compounds include those shown in the table below.
フェノール類 表中、 記載がない場合は水素である。Phenols In the table, If not specified, it is hydrogen.
アニリン類 表中、記載が無い場合は水素である。Anilines In the table, if there is no description, it is hydrogen.
次に、本発明のビリルビン定量方法について説明する。Next, the method for quantifying bilirubin of the present invention will be explained.
本発明において、ビリルビンの定量を行う検体としては
、−Ｍにビリルビンを含有する水性検体であり、例えば
、血清、血漿、胆汁等が挙げられ、これらは必要により
希釈されたり、前処理されていてもよい。In the present invention, the specimen for quantifying bilirubin is an aqueous specimen containing bilirubin in -M, such as serum, plasma, bile, etc., which may be diluted or pretreated as necessary. Good too.
又、用いることのできるビリルビンオキシダーゼとして
はミロセシウム属微生物由来のものの他、アガリカスビ
スポーラス由来のもの等その由来に関係なく、一般にビ
リルビンオキシダーゼとして用いられる酵素であればい
ずれでもよいが、ミロセシウム属微生物由来のものは入
手し易く、取扱い易い。Bilirubin oxidases that can be used include those derived from microorganisms of the genus Myrocesium, those derived from Agaricus bisporus, and any enzyme that is generally used as bilirubin oxidase regardless of its origin; The origin is easy to obtain and easy to handle.
本発明のビリルビン定量方法は基本的にビリルビン含有
水性検体（以下、単に検体と略称す。）にビリルビンオ
キシダーゼを作用させ、酵素反応促進剤で該オキシダー
ゼで起る酵素反応を促進し、所定時間経過後に吸光度変
化を測定することにより総ビリルビンを定量するという
ものである。これは酵素反応によりビリルビンが酸化を
受はビリベルジンに変化し、吸光特性が変化することに
基づいている。The method for quantifying bilirubin of the present invention basically involves treating a bilirubin-containing aqueous sample (hereinafter simply referred to as the sample) with bilirubin oxidase, promoting the enzymatic reaction caused by the oxidase with an enzymatic reaction promoter, and elapsed for a predetermined period of time. Total bilirubin is then quantified by measuring changes in absorbance. This is based on the fact that bilirubin undergoes oxidation and changes to biliverdin through an enzymatic reaction, which changes its light absorption properties.
−ｉ的にビリルビンオキシダーゼの至適ｐＨは６〜７、
至適温度は４０℃付近であるが、本発明の促進剤を用い
ることにより、ｐＨ５〜１１で顕著な反応促進効果を得
ることができる。一方、検体中のビリルビンはｐＨによ
り吸光特性が変化するため、測定中は反応液のｐＨは一
定に保つことが必要である。このため、一般に酵素反応
によりビリルビンを定量する場合は、緩衝液を用いる。-i-wise, the optimum pH of bilirubin oxidase is 6 to 7,
Although the optimum temperature is around 40°C, by using the accelerator of the present invention, a remarkable reaction accelerating effect can be obtained at pH 5 to 11. On the other hand, since the light absorption characteristics of bilirubin in the sample change depending on the pH, it is necessary to keep the pH of the reaction solution constant during measurement. For this reason, a buffer solution is generally used when bilirubin is quantified by enzymatic reaction.
本発明で用いることのできる緩衝液としてはｐＨが５〜
１１程度のものであればいずれでもよく、例えばトリス
−塩酸緩衝液、リン酸緩衝液等を用いることができる。Buffers that can be used in the present invention have a pH of 5 to 5.
Any buffer having a concentration of about 11 may be used, and for example, Tris-HCl buffer, phosphate buffer, etc. can be used.
すなわち、総ビリルビンを定量するには、直接型及び間
接型ビリルビンの両者を定量しなければならないが、間
接型ビリルビンは低いｐＨ領域（例えばｐＨ４以下）で
はほとんど酵素反応を受けなくなるため、反応液のｐＨ
は５〜１１、好ましくは７〜１０程度に設定しておくと
よい。In other words, to quantify total bilirubin, it is necessary to quantify both direct and indirect bilirubin, but indirect bilirubin hardly undergoes enzymatic reaction in a low pH range (for example, pH 4 or less), so pH
is preferably set to about 5 to 11, preferably about 7 to 10.
本発明において、検体中のビリルビン量を定量するのに
用いる試薬は、基本的に（ｉ）緩衝液（ｉｉ）酵素反応
促進剤（ｉ ｉ ｉ）ビリルビンオキシダーゼの３つで
構成される。In the present invention, the reagent used to quantify the amount of bilirubin in a specimen is basically composed of three components: (i) a buffer, (ii) an enzyme reaction promoter, and (ii) bilirubin oxidase.
上記３つの試薬をどのような手順及び形態で検体に加え
るかは、特に制限はないが、酵素反応促進剤とビリルビ
ンオキシダーゼとが長時間接触すると該促進剤が酵素反
応により酸化され着色したりするため、前記２つの試薬
は検体に投入される前までは分離しておく必要がある。There are no particular restrictions on the procedure and form in which the above three reagents are added to the sample, but if the enzyme reaction accelerator and bilirubin oxidase come into contact for a long time, the accelerator will be oxidized and colored by the enzymatic reaction. Therefore, the two reagents need to be separated before being added to the sample.
本発明において上記試薬を用いる態様としては次に示す
ものがある。Examples of embodiments in which the above reagents are used in the present invention include the following.
（１）検体に、酵素反応促進剤を含有した緩衝液を加え
予備加温後、ビリルビンオキシダーゼ溶液を加える（２
試薬系）。(1) Add a buffer solution containing an enzyme reaction promoter to the sample, pre-warm it, and then add bilirubin oxidase solution (2
reagent system).
（２）検体に５緩衝液を加え予備加温後、酵素反応促進
剤溶液とビリルビンオキシダーゼ溶液を同時に加える（
３試薬系）。(2) Add 5 buffer solution to the sample and prewarm it, then add the enzyme reaction promoter solution and bilirubin oxidase solution at the same time (
3 reagent system).
特に、自動分析装置によりビリルビンを定量する場合に
は、２試薬系である（１）が好適であり、測定の迅速化
を図ることができる。ここで、酵素反応に要求されるｐ
Ｈ領域は、総ビリルビンの場合、５〜１１であるので緩
衝液にる緩衝作用は反応液（すなわち、すべての必要な
試薬を検体に加えたもの）でも維持されている必要があ
る。このため、酵素反応促進剤又はビリルビンオキシダ
ーゼを緩衝液とは別に加えかつそれらが溶液として量的
に多い場合は、所定のｐＨ領域を維持することができな
くなるので、この場合は、必要により前記成分を適当な
緩衝液に溶解して加えるとよい。いずれにせよ、酵素反
応促進剤は反応系で溶解している必要があり、反応液の
ｐ）Ｉ帯等により適宜選択して用いる。又、ビリルビン
オキシダーゼは水溶液でもよく、緩衝液を用いる場合は
ｐ！１６〜９程度の範囲とするとよい。Particularly, when bilirubin is to be quantified using an automatic analyzer, the two-reagent system (1) is preferable and can speed up the measurement. Here, the p required for the enzymatic reaction is
Since the H region is 5 to 11 for total bilirubin, the buffering effect of the buffer must be maintained in the reaction solution (ie, all necessary reagents added to the sample). For this reason, if an enzyme reaction promoter or bilirubin oxidase is added separately from the buffer solution and they are present in large amounts as a solution, it will not be possible to maintain a predetermined pH range. It is recommended to dissolve it in an appropriate buffer and add it. In any case, the enzyme reaction promoter needs to be dissolved in the reaction system, and is appropriately selected and used depending on the p)I band of the reaction solution. In addition, bilirubin oxidase may be in an aqueous solution, and if a buffer solution is used, p! It is preferable to set it in the range of about 16 to 9.
総ビリルビンの定量を行う場合の、フェノール類の用量
は、酵素反応の促進効果を奏する量であればよいが、反
応液中の濃度（最終濃度）として通常０．１〜５０ｍＭ
、好ましくは１〜１５ｍＭがよい。アニリン類は同様に
０．００１〜５０ｍＭ、好ましくは０．０５〜１０ｍＭ
がよい。When quantifying total bilirubin, the dose of phenols may be any amount that has the effect of promoting the enzyme reaction, but the concentration in the reaction solution (final concentration) is usually 0.1 to 50 mM.
, preferably 1 to 15 mM. Anilines are similarly 0.001-50mM, preferably 0.05-10mM
Good.
ビリルビンオキシダーゼの使用量は、基本的に、検体中
に存在するビリルビンの量により異なるが、通常最終濃
度として０．０５〜１．、ＯＵ／ｍ１使用するのが好ま
しい。The amount of bilirubin oxidase used basically varies depending on the amount of bilirubin present in the sample, but the final concentration is usually 0.05 to 1. , OU/m1 is preferably used.
上記それぞれの最終濃度は、反応液中での濃度であり、
試薬溶液中の濃度及び試薬溶液の量は特に限定されない
。ビリルビンオキシダーゼは水性溶液に相当量溶解する
ため（例えば１００ Ｕ／ｍ１以上）、溶解性からの制
約はなく、この意味で各試薬溶液の割合は、所望のｐＨ
領域になれば、測定の操作上の便宜により設定すること
ができる。自動分析装置を用いる場合は、前記（１）の
２試薬系を用い、酵素反応促進剤含有緩衝液とビリルビ
ンオキシダーゼ溶液との比は、通常、５：１から１＝１
程度が実用的である。The final concentration of each of the above is the concentration in the reaction solution,
The concentration in the reagent solution and the amount of the reagent solution are not particularly limited. Since bilirubin oxidase is dissolved in a considerable amount in an aqueous solution (for example, 100 U/ml or more), there is no restriction from solubility, and in this sense, the proportion of each reagent solution is adjusted to the desired pH.
Once the range is reached, it can be set according to the operational convenience of measurement. When using an automatic analyzer, the two-reagent system described in (1) above is used, and the ratio of the enzyme reaction promoter-containing buffer to the bilirubin oxidase solution is usually 5:1 to 1=1.
The degree is practical.
尚、本発明においては、反応液中に公知の直接化剤等反
応促進剤を加えてもよい。例えば、コール酸ナトリウム
、ドデシル硫酸ナトリウム等を緩衝液に存在させておく
ことができる。これらの反応促進剤を用いる場合は通常
０．１〜１％程度でよい。In the present invention, a reaction accelerator such as a known directing agent may be added to the reaction solution. For example, sodium cholate, sodium dodecyl sulfate, etc. can be present in the buffer solution. When using these reaction accelerators, the amount may be generally about 0.1 to 1%.
上述測定方法の具体的操作としては、前記（１）の２試
薬系の場合、ビリルビン含有水性検体に酵素反応促進剤
含有緩衝液を所定量加え、予備加温後（例えば３７℃、
３分間）、所定量のビリルビンオキシダーゼ溶液を加え
、反応液のｐＨを５〜１１の範囲に保って検体中に存在
していたビリルビンを酵素反応により酸化する。この際
、反応液中のビリルビンの減少を比色定量すれば、結果
的に検体中のビリルビン量を測定することができる。本
発明の方法によればビリルビンオキシダーゼ装入後、１
〜２分間程度で正確な定量が可能となる。In the case of the two-reagent system described in (1) above, the specific operation of the above measurement method is to add a predetermined amount of an enzyme reaction promoter-containing buffer to a bilirubin-containing aqueous sample, and after prewarming (for example, 37°C,
3 minutes), add a predetermined amount of bilirubin oxidase solution, and maintain the pH of the reaction solution in the range of 5 to 11 to oxidize bilirubin present in the sample by enzymatic reaction. At this time, by colorimetrically quantifying the decrease in bilirubin in the reaction solution, the amount of bilirubin in the sample can be measured as a result. According to the method of the present invention, after charging bilirubin oxidase, 1
Accurate quantification is possible in about 2 minutes.
尚、間接型ビリルビンの安定性はｐＨにより異なり低い
ｐＨでは酵素反応を受けにくくなるため、アニリン類、
特にｍ−トルイジン誘導体（前記の表、Ａｔ、Ａ５．Ａ
ＩＯ，Ａ１８）を用い、ｐＨを３．０〜４．０に調節す
ることにより、直接型ビリルビンの分別測定に対しても
、非常に有効な反応促進剤となり得る。Note that the stability of indirect bilirubin varies depending on the pH and is less susceptible to enzymatic reactions at low pH.
In particular m-toluidine derivatives (above table, At, A5.A
By using IO, A18) and adjusting the pH to 3.0 to 4.0, it can become a very effective reaction accelerator even for the fractional measurement of direct bilirubin.
本発明のビリルビン定量用試薬を自動分析装置等に適用
するには、キットとして取扱うと実用上非常に便利であ
る。すなわち、前記一般式（Ｉ）及び（ＩＩ）で表わさ
れるフェノール類およびアニリン類から成る群より選ば
れる１種又は２種以上の化合物を少なくとも溶解してい
る緩衝液を第１試薬、ビリルビンオキシダーゼ溶液を第
２試薬としたビリルビン定量用キットを用いると操作を
簡単化することができる。尚、ビリルビンオキシダーゼ
は安定性が比較的低いため、溶液状態で保存することは
好ましくなく、測定の直前に調製するとよい。この場合
の溶液は前述したようにｐＨ６〜９程度の緩衝液か水溶
液でもよい。又、各成分はそれらの最終濃度が前述した
範囲内になるように調整し、かつ緩衝液のｐＨが所定の
範囲内になるようにする。例えば、検体１０〜１００μ
ｔに対して、第１試薬としてフェノール類を０．２〜１
００ ｍＭ又は／及びアニリン類を０．００２〜１００
ｍＭ含有しているｐＨ５〜１１の緩衝液２ｍ１（さらに
コール酸ナトリウム等を含有していてもよい）、第２試
薬としてビリルビンオキシダーゼを０．３〜６Ｕ／ｍｌ
含有している水溶液０．３ｍｌ〜２ｍｌ程度を用いると
よい。ビリルビンオキシダーゼの量は検体中に存在する
ビリルビン量により調整すればよいが、血清等を検体と
する場合には通常、上記範囲内の程度でよい。In order to apply the bilirubin quantitative reagent of the present invention to an automatic analyzer or the like, it is practically very convenient to handle it as a kit. That is, a buffer solution in which at least one or more compounds selected from the group consisting of phenols and anilines represented by the general formulas (I) and (II) is dissolved is used as the first reagent and a bilirubin oxidase solution. The operation can be simplified by using a kit for quantifying bilirubin in which the second reagent is . In addition, since bilirubin oxidase has relatively low stability, it is not preferable to store it in a solution state, and it is preferable to prepare it immediately before measurement. The solution in this case may be a buffer solution or an aqueous solution having a pH of about 6 to 9, as described above. Further, each component is adjusted so that its final concentration is within the range described above, and the pH of the buffer solution is adjusted within a predetermined range. For example, sample 10-100μ
t, the amount of phenols as the first reagent is 0.2 to 1
00 mM or/and anilines from 0.002 to 100
2 ml of pH 5-11 buffer containing mM (may also contain sodium cholate etc.), 0.3-6 U/ml of bilirubin oxidase as a second reagent.
It is preferable to use about 0.3 ml to 2 ml of the aqueous solution containing the liquid. The amount of bilirubin oxidase may be adjusted depending on the amount of bilirubin present in the sample, but when a sample such as serum is used, it is usually within the above range.
尚、比色定量は常法によって行うことができ、例えば、
市販の分光光度計（例えば日立Ｕ−３２００形自記分光
光度計）を用い、波長４００〜４８０ｎｍにて試薬盲検
を対照にして吸光度を測定する。この際、対照として既
知濃度のビリルビン含有血清（以下、標準という）を用
い、同様に吸光度を測定し、さらに検体盲検としてこれ
ら検体血清および標準にビリルビンオキシダーゼを含ま
ない緩衝液を添加したものについて同様に吸光度を測定
し、これらに基づいて下記式よりビリルビン濃度（ｍｇ
／ｄｉ ）を算出すれば、ビリルビンを定量することが
できる。In addition, colorimetric determination can be carried out by a conventional method, for example,
Using a commercially available spectrophotometer (for example, Hitachi U-3200 self-recording spectrophotometer), the absorbance is measured at a wavelength of 400 to 480 nm using a reagent blind test as a control. At this time, the absorbance was measured in the same manner using serum containing bilirubin at a known concentration (hereinafter referred to as standard) as a control, and as a sample blind test, a buffer solution containing no bilirubin oxidase was added to the sample serum and standard. Measure the absorbance in the same way, and based on these, calculate the bilirubin concentration (mg
/di), bilirubin can be quantified.
：検体血清の吸光度（検体盲検） ：検体血清の吸光度（酵素反応した もの） Ａｓｓ ：標準の吸光度（検体盲検） ＡＣ？ ：標準の吸光度（酵素反応したもの） 式中、Ａ。: Absorbance of sample serum (sample blinded) : Absorbance of sample serum (enzyme-reacted thing) Ass: Standard absorbance (sample blinded) AC? : Standard absorbance (enzymatically reacted) In the formula, A.
Ａｔ
Ｘ ：標準中のビリルビン濃度（ｍｇ／旧）〔実施例
〕
次に実施例を挙げて本発明を更に詳しく説明する。AtX: Bilirubin concentration in standard (mg/old) [Example] Next, the present invention will be explained in more detail with reference to Examples.
実施例１
ビリルビン溶液（メルク社製結晶ビリルビン１５ ｍｇ
／ｄｌ） ５０μｍに２．４−ジクロロフェノール５ｍ
Ｍを含有するＢｒ １ｔｔｏｎ−Ｒｏｂ １ｎｓｏｎの
広域緩衝液（第１試薬：ｐＨ５，６，７，８，９，１０
または１１）２ｍｌを加え３分間予備加温後、これに３
Ｕ／ｍｌのビリルビンオキシダーゼ水溶液（第２試薬
）０．５ｍｌを加え３７℃で波長４５０ｎｍにおける反
応曲線の測定を行った。また、この際２．４−ジクロロ
フエノールを含まないＢｒ１ｔｔｏｎ−Ｒｏｂｉｎｓｏ
ｎの広域緩衝液を用いた場合についても同様に測定を行
い、反応時間を比較した。得られた反応曲線を第１図（
ａ）〜（ｇ）に示す。Example 1 Bilirubin solution (crystalline bilirubin manufactured by Merck & Co., Ltd. 15 mg
/dl) 2.4-dichlorophenol 5m to 50μm
Br 1tton-Robinson broad range buffer containing M (first reagent: pH 5, 6, 7, 8, 9, 10
Or 11) Add 2ml and prewarm for 3 minutes, then add 3ml
0.5 ml of a U/ml bilirubin oxidase aqueous solution (second reagent) was added, and a reaction curve was measured at 37° C. at a wavelength of 450 nm. In addition, at this time, Br1tton-Robinso which does not contain 2,4-dichlorophenol
Measurements were made in the same manner when a wide range buffer solution of n was used, and the reaction times were compared. The obtained reaction curve is shown in Figure 1 (
Shown in a) to (g).
２．４−ジクロロフェノールはｐ）１５〜１１において
反応促進効果を示し、特にｐＨ７〜１０において顕著で
あった。2.4-dichlorophenol showed a reaction accelerating effect at p)15-11, especially at pH7-10.
実施例２
検体血清５０μｍに３．５−ジメチルフェノール５ｍＭ
および０．５％コール酸ナトリウムを含有する０、２ｍ
Ｍ ）リス−塩酸緩衝液（第１試薬：ｐｔ１７．８、ま
たは９）２ｍｌを加え３分間予備加温後、これに３０／
ｍｌビリルビンオキシダーゼ水溶液（第２試薬）０．５
ｍｌを加え３７℃で波長４５０ｎｍにおける反応曲線の
測定を行った。また、この際３．５−ジメチルフエノー
ルを含まない緩衝液についても同様に測定を行い、反応
時間を比較した。得られた反応曲線を第２図（ａ）〜（
Ｃ）に示す。Example 2 5mM of 3.5-dimethylphenol in 50μm of sample serum
and 0.2m containing 0.5% sodium cholate
M) Add 2 ml of lithium-hydrochloric acid buffer (first reagent: pt17.8 or 9) and preheat for 3 minutes, then add 30%
ml bilirubin oxidase aqueous solution (second reagent) 0.5
ml was added and the reaction curve was measured at 37° C. at a wavelength of 450 nm. At this time, measurements were also made in the same manner using a buffer solution that did not contain 3,5-dimethylphenol, and the reaction times were compared. The reaction curves obtained are shown in Figure 2 (a) to (
Shown in C).
実施例１と同様の効果が得られた。The same effects as in Example 1 were obtained.
実施例３
検体血清５０μｍにＮ−エチル−Ｎ−（２−ヒドロキシ
−３−スルホプロピル）−３，５−ジメチルアニリン５
ｍＭおよび０．５％コール酸ナトリウムを含有する０、
２Ｍ トリス−塩酸緩衝液（第１試薬：ｐ１１７．８
、または９）２ｍｌを加え３分間予備加温後、これに３
０／ｍｌビリルビンオキシダーゼ水溶液（第２試薬）０
．５ｍｌを加えて３７℃で波長４５０ｎｍにおける反応
曲線の測定を行った。また、この際Ｎ−エチル−Ｎ−（
２−ヒドロキシ−３−スルホプロピル）−３，５−ジメ
チルアニリンを含まない緩衝液についても同様に測定を
行い、反応時間を比較した。得られた反応曲線を第３図
（ａ）〜（ｃ）に示す。実施例１と同様の効果が得られ
た。Example 3 N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline 5 was added to 50 μm of sample serum.
0, containing mM and 0.5% sodium cholate.
2M Tris-HCl buffer (1st reagent: p117.8
, or 9) Add 2 ml and prewarm for 3 minutes, then add 3 ml to this.
0/ml bilirubin oxidase aqueous solution (second reagent) 0
．． 5 ml was added and the reaction curve was measured at 37° C. at a wavelength of 450 nm. In addition, at this time, N-ethyl-N-(
Measurements were made in the same manner using a buffer solution that did not contain 2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline, and the reaction times were compared. The obtained reaction curves are shown in FIGS. 3(a) to (c). The same effects as in Example 1 were obtained.
実施例４
下記の第１試薬、第２試薬を用い、標準液および高ビリ
ルビン血清を５段希釈した液を各々検体として測定し、
試薬の直線性を検討した。Example 4 Using the following first reagent and second reagent, a standard solution and a 5-step dilution of high bilirubin serum were each used as specimens to measure,
The linearity of the reagent was investigated.
（３式薬系且成）
第１試薬：０．１Ｍトリス−塩酸緩衝剤（ｐｉ４８．５
）５ｍＭ３．５−ジメチルフェノール、
０．５％コール酸ナトリウム含有。(Type 3 drug system composition) First reagent: 0.1M Tris-HCl buffer (pi48.5
) Contains 5mM 3.5-dimethylphenol, 0.5% sodium cholate.
第２試薬：３Ｕ／ｍｌビリルビンオキシダーゼ水溶液
（測定および結果）
検体１４μ工に第１試薬４００μ■を加え３７℃で５分
間反応させる。５分後に吸光度を測定する（Ａ、）。次
に、該当反応液に第２試薬１００μｌを加え、３７℃で
５分間反応させ、吸光度を測定する（Ａ：）。吸光度差
＝Ａ； Ａ；を求め、標準液（ビリルビン既知濃度）
との吸光度差と比較してビリルビン濃度を求めた。なお
、測定には日立７０５形自動分析装置（日立製作所）を
使用し、測定波長は主波長４５０ｎｍ、副波長６６０ｎ
ｍの２波長で行った。Second reagent: 3U/ml bilirubin oxidase aqueous solution (measurement and results) Add 400μ of the first reagent to a 14μ sample and allow to react at 37°C for 5 minutes. Measure the absorbance after 5 minutes (A,). Next, 100 μl of the second reagent is added to the corresponding reaction solution, reacted at 37° C. for 5 minutes, and the absorbance is measured (A:). Determine the absorbance difference = A; A; and use the standard solution (known concentration of bilirubin).
The bilirubin concentration was determined by comparing the absorbance difference between the two. In addition, a Hitachi 705 type automatic analyzer (Hitachi, Ltd.) was used for the measurement, and the measurement wavelength was a main wavelength of 450 nm and a sub wavelength of 660 nm.
The experiment was conducted using two wavelengths of m.
その結果第４図に示す如く、約３５ｍｇ／ｄｌまでの直
線性が明かであった。As a result, as shown in FIG. 4, linearity up to about 35 mg/dl was clear.
実施例５
本発明によるビリルビン定量値と従来法であるジアゾ法
による定量値を比較した。すなわち、検体（血清）１４
μｍに下記第１試薬４００μｍを加え３７℃で５分間反
応させる。５分後に吸光度を測定する（Ａ、）。次に該
当反応液に下記第２試薬１００μｍを加え、３７℃で５
分間反応させ、吸光度を測定する（Ａｌｌ）。吸光度差
＝ＡＢＡｓを求め、標準液（総ビリルビン既知濃度）と
の吸光度差と比較して総ビリルビン濃度を求めた。Example 5 The quantitative value of bilirubin according to the present invention was compared with the quantitative value according to the conventional diazo method. That is, sample (serum) 14
Add 400 μm of the following first reagent to μm and react at 37° C. for 5 minutes. Measure the absorbance after 5 minutes (A,). Next, add 100 μm of the following second reagent to the corresponding reaction solution, and
Let it react for a minute and measure the absorbance (All). The absorbance difference = ABAs was determined, and the total bilirubin concentration was determined by comparing the absorbance difference with a standard solution (known concentration of total bilirubin).
なお、測定には日立７０５形自動分析装置（日立製作所
）を使用し、測定波長は主波長４５０ｎｍ、副波長６６
０ｎｍの２波長で行った。A Hitachi 705 type automatic analyzer (Hitachi, Ltd.) was used for the measurement, and the measurement wavelength was 450 nm as the main wavelength and 66 nm as the sub wavelength.
The experiment was carried out using two wavelengths of 0 nm.
試薬組成
第一試薬 ０．１５Ｍ トリス−塩酸緩衝液（ｐＨ８
，５）
１０ｍＭ３．５−ジメチルフェノール
０．５％ コール酸ナトリウム
第二試薬 ２０ｍＭ トリス−塩酸緩衝液（ｐＨ９，
０）
４ ＩＩ／ｍｌ ビリルビンオキシダーゼ結果は下記
及び第５図に示すように、相関係数ｒ ＝ ０．９９７
９と極めて高い相関性が認められた。Reagent composition First reagent 0.15M Tris-HCl buffer (pH 8
,5) 10mM 3.5-dimethylphenol 0.5% sodium cholate second reagent 20mM Tris-HCl buffer (pH 9,
0) 4 II/ml Bilirubin oxidase results are shown below and in Figure 5, correlation coefficient r = 0.997
An extremely high correlation with 9 was observed.
データ数：Ｎ＝４０
相関係数： ｒ ＝ ０．９９７９
平均値；ｘ（酵素法） ＝ ３．３７８ｍｇ／ｄｌＹ（
ジアゾ法） ＝ ３．０１０ｍｇ／ｄ１回帰式；Ｙ＝Ω
、９６７Ｘ −０，２５６Ｓ ｙ ｘ ＝ ０．３７９
０ｔ（計算値）・０．２８３（有意差なし）［発明の効
果］
以上説明したように、本発明によれば、特定のフェノー
ル類又はアニリン類を用いることによりビリルビンオキ
シダーゼによる酵素反応を促進することが可能となるた
め、肝機能障害の診断に利用できる総ビリルビン量を正
確に、かつ迅速に測定することができ、測定の自動化を
容易に図ることができる。又本発明の各試薬をキットと
することで取扱いが簡単になる等、本発明は実用上非常
に有用なものである。Number of data: N = 40 Correlation coefficient: r = 0.9979 Average value; x (enzyme method) = 3.378 mg/dlY (
Diazo method) = 3.010mg/d1 regression equation; Y=Ω
, 967X −0,256S y x = 0.379
0t (calculated value)・0.283 (no significant difference) [Effect of the invention] As explained above, according to the present invention, the enzymatic reaction by bilirubin oxidase is promoted by using specific phenols or anilines. Therefore, the total amount of bilirubin that can be used for diagnosing liver dysfunction can be measured accurately and quickly, and the measurement can be easily automated. In addition, the present invention is very useful in practice, as the reagents of the present invention are made into a kit, which makes handling easier.
第１図は実施例１で行なった総ビリルビン測定結果を吸
光度の変化で示した図であり、（ａ）、（ｂ）、（Ｃ）
、（ｄ） （ｅ）、（ｆ）、（ｇ）はそれぞれ緩衝
液のｐ）ｌが５．６．７．８．９．１０．１１のときの
図、第２図は実施例２で行なった総ビリルビン測定結果
を吸光度の変化で示した図であり、（ａ）、（ｂ）、（
Ｃ）はそれぞれ緩衝液のｐＨが７．８．９のときの図、
第３図は実施例３で行なった総ビリルビン測定結果を示
した図であり、（ａ）、（ｂ）、（Ｃ）はそれぞれ緩衝
液のｐＨが７．８．９のときの図、第４図は実施例４で
行なった検体を希釈した際の測定の直線性を示す図、第
５図は実施例５で行なった従来法（ジアゾ法）と本発明
による酵素法との相関を示す図である。FIG. 1 is a diagram showing the results of total bilirubin measurement conducted in Example 1 as changes in absorbance; (a), (b), (C)
, (d) (e), (f), and (g) are diagrams when p)l of the buffer solution is 5.6.7.8.9.10.11, respectively, and Figure 2 is for Example 2. It is a diagram showing the results of total bilirubin measurement as changes in absorbance, (a), (b), (
C) is a diagram when the pH of the buffer solution is 7.8.9, respectively.
FIG. 3 is a diagram showing the results of total bilirubin measurement conducted in Example 3, and (a), (b), and (C) are diagrams when the pH of the buffer solution was 7.8.9, respectively. Figure 4 shows the linearity of the measurement when the sample was diluted in Example 4, and Figure 5 shows the correlation between the conventional method (diazo method) and the enzyme method of the present invention performed in Example 5. It is a diagram.
Claims (1)
を作用させ、それにより生ずるビリルビンの変化を光学
的に測定することにより該検体中の総ビリルビンを定量
する方法において、ビリルビンオキシダーゼによる酵素
反応を、一般式（ I ）で表わされるフェノール類およ
び一般式（II）で表わされるアニリン類から成る群より
選ばれる１種又は２種以上の化合物により促進させるこ
とを特徴とする総ビリルビンの定量法。 ▲数式、化学式、表等があります▼・・・（ I ） （式中、Ｒ＾１〜Ｒ＾５はそれぞれ独立に、水素原子、
ハロゲン原子、炭素数１〜４のアルキル基、炭素数１〜
４のアルコキシル基、水酸基又はカルボキシル基を示す
。但し、Ｒ＾３がカルボキシル基および水酸基であるも
のを除く。） ▲数式、化学式、表等があります▼・・・（II） （式中、Ｒ＾６およびＲ＾７はそれぞれ独立に、水素原
子、又は炭素数１〜４のアルキル基、ヒドロキシアルキ
ル基、スルホアルキル基、ヒドロキシスルホアルキル基
若しくはスルホンアミドアルキル基を示す。Ｒ＾８およ
びＲ＾９はそれぞれ独立に、水素原子、ハロゲン原子、
炭素数１〜４のアルキル基又は炭素数１〜４のアルコキ
シル基を示す。）２、ビリルビン含有水性検体に、前記
フェノール類およびアニリン類から成る群より選ばれる
１種又は２種以上の化合物を含有している緩衝液を加え
、予備加温後、ビリルビンオキシダーゼを加える請求項
１に記載の定量法。 ３、前記フェノール類がモノ又はジ置換体である請求項
１に記載の定量方法。 ４、前記アニリン類がモノ又はジ置換体である請求項１
に記載の定量法。 ５、下記で示されるフェノール類およびアニリン類から
成る群より選ばれる１種又は２種以上の化合物とビリル
ビンオキシダーゼとを少なくとも組み合せて成るビリル
ビン含有水性検体中の総ビリルビン比色定量用試薬。 ▲数式、化学式、表等があります▼ （式中、Ｒ＾１〜Ｒ＾５はそれぞれ独立に、水素原子、
ハロゲン原子、炭素数１〜４のアルキル基、炭素数１〜
４のアルコキシル基、水酸基又はカルボキシル基を示す
。但し、Ｒ＾３がカルボキシル基および水酸基であるも
のを除く。） ▲数式、化学式、表等があります▼ （式中、Ｒ＾６およびＲ＾７はそれぞれ独立に、水素原
子、又は炭素数１〜４のアルキル基、ヒドロキシアルキ
ル基、スルホアルキル基、ヒドロキシスルホアルキル基
若しくはスルホンアミドアルキル基を示す。Ｒ＾８およ
びＲ＾９はそれぞれ独立に、水素原子、ハロゲン原子、
炭素数１〜４のアルキル基又は炭素数１〜４のアルコキ
シル基を示す。）[Scope of Claims] 1. A method for quantifying total bilirubin in a bilirubin-containing aqueous sample by acting bilirubin oxidase on the sample and optically measuring the resulting change in bilirubin, which comprises: is promoted by one or more compounds selected from the group consisting of phenols represented by general formula (I) and anilines represented by general formula (II). . ▲There are mathematical formulas, chemical formulas, tables, etc.▼...(I) (In the formula, R^1 to R^5 each independently represent a hydrogen atom,
Halogen atom, alkyl group having 1 to 4 carbon atoms, 1 to 4 carbon atoms
4 represents an alkoxyl group, hydroxyl group or carboxyl group. However, those in which R^3 is a carboxyl group or a hydroxyl group are excluded. ) ▲There are mathematical formulas, chemical formulas, tables, etc.▼...(II) (In the formula, R^6 and R^7 each independently represent a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group, Represents a sulfoalkyl group, hydroxysulfoalkyl group, or sulfonamidoalkyl group.R^8 and R^9 each independently represent a hydrogen atom, a halogen atom,
It represents an alkyl group having 1 to 4 carbon atoms or an alkoxyl group having 1 to 4 carbon atoms. 2) A buffer solution containing one or more compounds selected from the group consisting of phenols and anilines is added to the bilirubin-containing aqueous sample, and after prewarming, bilirubin oxidase is added. The quantitative method described in 1. 3. The quantitative determination method according to claim 1, wherein the phenol is a mono- or di-substituted product. 4. Claim 1, wherein the aniline is mono- or di-substituted.
Quantitative method described in. 5. A reagent for colorimetric determination of total bilirubin in a bilirubin-containing aqueous sample, which comprises at least a combination of one or more compounds selected from the group consisting of phenols and anilines shown below and bilirubin oxidase. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R^1 to R^5 each independently represent a hydrogen atom,
Halogen atom, alkyl group having 1 to 4 carbon atoms, 1 to 4 carbon atoms
4 represents an alkoxyl group, hydroxyl group or carboxyl group. However, those in which R^3 is a carboxyl group or a hydroxyl group are excluded. ) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R^6 and R^7 each independently represent a hydrogen atom, a C1-C4 alkyl group, a hydroxyalkyl group, a sulfoalkyl group, a hydroxysulfonate group) Represents an alkyl group or a sulfonamide alkyl group.R^8 and R^9 each independently represent a hydrogen atom, a halogen atom,
It represents an alkyl group having 1 to 4 carbon atoms or an alkoxyl group having 1 to 4 carbon atoms. )
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP31423289A JP2880209B2 (en) | 1989-12-05 | 1989-12-05 | Determination of total bilirubin and reagents used for it |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP31423289A JP2880209B2 (en) | 1989-12-05 | 1989-12-05 | Determination of total bilirubin and reagents used for it |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03175997A true JPH03175997A (en) | 1991-07-31 |
| JP2880209B2 JP2880209B2 (en) | 1999-04-05 |
Family
ID=18050875
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP31423289A Expired - Lifetime JP2880209B2 (en) | 1989-12-05 | 1989-12-05 | Determination of total bilirubin and reagents used for it |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2880209B2 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20150087077A (en) * | 2014-01-20 | 2015-07-29 | 김길재 | Hydrogen water produce device |
| KR101598021B1 (en) * | 2014-09-11 | 2016-02-29 | 주식회사 솔고 바이오 메디칼 | Bottle being capable of keeping taste of tee |
| KR20160041550A (en) * | 2014-10-08 | 2016-04-18 | 수소비전 주식회사 | Neutral-pH Hydrogen-enriched electrolyzed water production device for portable |
| KR101643129B1 (en) * | 2015-02-04 | 2016-07-27 | 주식회사 동양이지텍 | Production device of portable type for hydrogen water |
| CN114277088A (en) * | 2021-12-02 | 2022-04-05 | 深圳市锦瑞生物科技股份有限公司 | Total bilirubin determination reagent, preparation method of reagent ball and determination chip |
-
1989
- 1989-12-05 JP JP31423289A patent/JP2880209B2/en not_active Expired - Lifetime
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20150087077A (en) * | 2014-01-20 | 2015-07-29 | 김길재 | Hydrogen water produce device |
| KR101598021B1 (en) * | 2014-09-11 | 2016-02-29 | 주식회사 솔고 바이오 메디칼 | Bottle being capable of keeping taste of tee |
| KR20160041550A (en) * | 2014-10-08 | 2016-04-18 | 수소비전 주식회사 | Neutral-pH Hydrogen-enriched electrolyzed water production device for portable |
| KR101643129B1 (en) * | 2015-02-04 | 2016-07-27 | 주식회사 동양이지텍 | Production device of portable type for hydrogen water |
| CN114277088A (en) * | 2021-12-02 | 2022-04-05 | 深圳市锦瑞生物科技股份有限公司 | Total bilirubin determination reagent, preparation method of reagent ball and determination chip |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2880209B2 (en) | 1999-04-05 |
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