JPS6352060A - Method and kit for quantitative determination of indirect bilirubin - Google Patents
Method and kit for quantitative determination of indirect bilirubinInfo
- Publication number
- JPS6352060A JPS6352060A JP8776586A JP8776586A JPS6352060A JP S6352060 A JPS6352060 A JP S6352060A JP 8776586 A JP8776586 A JP 8776586A JP 8776586 A JP8776586 A JP 8776586A JP S6352060 A JPS6352060 A JP S6352060A
- Authority
- JP
- Japan
- Prior art keywords
- bilirubin
- reaction
- indirect
- reagent
- indirect bilirubin
- 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
- 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 title claims abstract description 92
- 238000000034 method Methods 0.000 title claims description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 34
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 29
- 108010015428 Bilirubin oxidase Proteins 0.000 claims abstract description 14
- 125000000664 diazo group Chemical group [N-]=[N+]=[*] 0.000 claims abstract description 14
- 239000000872 buffer Substances 0.000 claims abstract description 11
- 238000008789 Direct Bilirubin Methods 0.000 claims abstract description 9
- 239000000987 azo dye Substances 0.000 claims description 2
- 238000011002 quantification Methods 0.000 claims description 2
- 239000012954 diazonium Substances 0.000 abstract description 11
- 150000001989 diazonium salts Chemical class 0.000 abstract description 10
- 238000010521 absorption reaction Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 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 abstract 2
- 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 abstract 2
- 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 abstract 2
- 239000012295 chemical reaction liquid Substances 0.000 abstract 1
- 150000001875 compounds Chemical class 0.000 description 10
- 239000000243 solution Substances 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 5
- 239000004094 surface-active agent Substances 0.000 description 5
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000008050 Total Bilirubin Reagent Methods 0.000 description 2
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 239000007979 citrate buffer Substances 0.000 description 2
- YGSZNSDQUQYJCY-UHFFFAOYSA-L disodium;naphthalene-1,5-disulfonate Chemical compound [Na+].[Na+].C1=CC=C2C(S(=O)(=O)[O-])=CC=CC2=C1S([O-])(=O)=O YGSZNSDQUQYJCY-UHFFFAOYSA-L 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- SJBYGFDYNQPADT-UHFFFAOYSA-N 2,3-dichlorobenzenediazonium Chemical class ClC1=CC=CC([N+]#N)=C1Cl SJBYGFDYNQPADT-UHFFFAOYSA-N 0.000 description 1
- 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
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- LPHGQDQBBGAPDZ-UHFFFAOYSA-N Isocaffeine Natural products CN1C(=O)N(C)C(=O)C2=C1N(C)C=N2 LPHGQDQBBGAPDZ-UHFFFAOYSA-N 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 229960001948 caffeine Drugs 0.000 description 1
- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- -1 daphyllin Chemical compound 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 125000005843 halogen group Chemical group 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- XTEGVFVZDVNBPF-UHFFFAOYSA-L naphthalene-1,5-disulfonate(2-) Chemical compound C1=CC=C2C(S(=O)(=O)[O-])=CC=CC2=C1S([O-])(=O)=O XTEGVFVZDVNBPF-UHFFFAOYSA-L 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- KVMLCRQYXDYXDX-UHFFFAOYSA-M potassium;chloride;hydrochloride Chemical compound Cl.[Cl-].[K+] KVMLCRQYXDYXDX-UHFFFAOYSA-M 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- YGSDEFSMJLZEOE-UHFFFAOYSA-M salicylate Chemical compound OC1=CC=CC=C1C([O-])=O YGSDEFSMJLZEOE-UHFFFAOYSA-M 0.000 description 1
- 229960001860 salicylate Drugs 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
１童業上の利用分野
本発明は間接ビリルビンの定量法及びそれに用いるキッ
トに関する。DETAILED DESCRIPTION OF THE INVENTION 1. Field of Use in Childcare The present invention relates to a method for quantifying indirect bilirubin and a kit used therefor.
ビリルビンは、赤血球の代謝産物で生体には不要な成分
である。これは肝臓から排出され、ウロピリンや、ステ
ルコピリンとなって体外に排出される。血液中のビリル
ビンの量を知ることによって肝機能の障害の程度を知る
ことができる。ビリルビンは血清中で蛋白質と結合して
いるが、ゆるく結合している直接ビリルビンと、固く結
合している間接ビリルビンの２つの型があり、これらの
量を知ることによって肝疾患の種類を知ることができる
。Bilirubin is a metabolite of red blood cells and is an unnecessary component for living organisms. This is excreted from the liver and excreted from the body as uropilin or stercopyrine. By knowing the amount of bilirubin in the blood, the degree of liver function impairment can be determined. Bilirubin is bound to proteins in serum, and there are two types: direct bilirubin, which is loosely bound, and indirect bilirubin, which is tightly bound.By knowing these amounts, it is possible to know the type of liver disease. Can be done.
従来の技術
ビリルビンとジアゾ試薬とを界面活性剤等の反応促進剤
の存在下に反応させて生成するアゾ色素を定量すること
によって、間接ビリルビン及び直接ビリルビンの合計ｌ
を定量する方法は公知である。Conventional technology By reacting bilirubin and a diazo reagent in the presence of a reaction accelerator such as a surfactant and quantifying the azo dye produced, the total amount of indirect bilirubin and direct bilirubin can be determined.
Methods for quantifying are known.
反応促進剤の非存在下に上記反応を行わせることによっ
て、直接ビリルビンのみを定量することは公知である。It is known to directly quantify only bilirubin by carrying out the above reaction in the absence of a reaction promoter.
両者の差を求めることによって間接ビリルビンの量を知
ることができる。The amount of indirect bilirubin can be determined by finding the difference between the two.
又、ビリルビン・オキシダーゼを用いてビリルビンの黄
色色調の減少を測定する方法は公知である（特開昭５６
−２７６５６）。Furthermore, a method for measuring the decrease in the yellow tone of bilirubin using bilirubin oxidase is known (Japanese Patent Application Laid-Open No. 1983-1991).
-27656).
さらに、ビリルビン・オキシダーゼの至適ｐＨより低い
ｐＨ条件下ではビリルビン・オキシダーゼの作用によっ
て直接ビリルビンのみ分解し、間接ビリルビンは分解し
ないことを利用する直接ビリルビンの定量法は公知であ
る（特開昭５９−１２５８９９）。総ビリルビン量から
これを差引くことによって間接ビリルビンを定量できる
。Furthermore, there is a known method for directly quantifying bilirubin that utilizes the fact that under pH conditions lower than the optimum pH of bilirubin oxidase, only direct bilirubin is degraded by the action of bilirubin oxidase, but indirect bilirubin is not. -125899). Indirect bilirubin can be quantified by subtracting it from the total bilirubin amount.
発明が解決しようとする問題点
ジアゾ試薬を用いる方法は、感度が小さく微量の直接ビ
リルビン定量には適切でない。また、血清試料中には脂
質等の反応液を混濁させる成分を含有する。これを溶解
するのに好適な界面活性剤を用いると、間接ビリルビン
もジアゾ化し、直接ビリルビンのみを定量できない。Problems to be Solved by the Invention The method using a diazo reagent has low sensitivity and is not suitable for directly quantifying trace amounts of bilirubin. Furthermore, the serum sample contains components such as lipids that cloud the reaction solution. If a surfactant suitable for dissolving this is used, indirect bilirubin will also be diazotized, making it impossible to quantify only direct bilirubin.
一方、ビリルビン・オキシダーゼを用いる方法は、酵素
反応の前後における測定が必要であり、試料を多量に要
する。又、ビリルビン自体による吸光度の変化を測定す
るための感度がジアゾ試薬を用いる方法より低い。On the other hand, the method using bilirubin oxidase requires measurement before and after the enzymatic reaction, and requires a large amount of sample. In addition, the sensitivity for measuring changes in absorbance due to bilirubin itself is lower than the method using a diazo reagent.
問題点を解決するための手段
本発明によれば、直接ビリルビン及び間接ビリルビンを
含有する試料中の直接ビリルビンを、ビリルビン・オキ
シダーゼの作用によってビルベルジンに変換し、次いで
間接ビリルビンとジアゾ試薬とをジアゾ反応促進剤の存
在下に反応させて色素を生成させ、これを定量すること
によって、間接ビリルビンを定量できる。Means for Solving the Problems According to the present invention, direct bilirubin in a sample containing direct and indirect bilirubin is converted to bilverdin by the action of bilirubin oxidase, and then indirect bilirubin and a diazo reagent are subjected to a diazo reaction. Indirect bilirubin can be quantified by reacting in the presence of an accelerator to produce a dye and quantifying this.
本発明を実施するに際しては、ビリルビンを含有する試
料にビリルビン・オキシダーゼを加えて適当な緩衝液中
で反応を行わせ（以下第一反応という）次いで、これに
ジアゾ試薬としてジアゾニウム塩を間接ビリルビンの等
モル以上、通常１０〜１０００倍モル及び反応促進剤を
加えて反応させる（以下第２反応という）。When carrying out the present invention, bilirubin oxidase is added to a sample containing bilirubin and a reaction is carried out in an appropriate buffer (hereinafter referred to as the first reaction).Next, a diazonium salt is added as a diazo reagent to the sample to indirectly react with bilirubin. The reaction is carried out by adding equimolar or more, usually 10 to 1000 times the molar amount, and a reaction accelerator (hereinafter referred to as the second reaction).
第１反応は１５〜４５℃、好ましくは３０〜４０℃で行
われ、２〜３０分間、通常約５分で完了する。The first reaction is carried out at 15-45°C, preferably 30-40°C, and is completed in 2-30 minutes, usually about 5 minutes.
第２反応は５〜５０℃、好ましくは２０〜４０℃で行わ
れ、２〜３０分間、通常約５分で完了する。The second reaction is carried out at 5-50°C, preferably 20-40°C, and is completed in 2-30 minutes, usually about 5 minutes.
反応後着色した反応液の吸収を生成色素の極大吸収波長
（λｍａｘ ）で試薬ブランクを対照として測定し、予
め既知量についての試験から求めた検量線を利用して試
料中の間接ビリルビンを定量できる。After the reaction, the absorption of the colored reaction solution is measured at the maximum absorption wavelength (λmax) of the dye produced, using a reagent blank as a reference, and the indirect bilirubin in the sample can be quantified using a calibration curve determined in advance from tests on known amounts. .
第１反応で用いられるビリルビン・オキシダーゼは、ミ
ロセシウム属、トラキデルマ属等の微生物由来の酵素が
容易に入手でき、これらは０，１〜１０１１／ｍｌで用
いられる。As the bilirubin oxidase used in the first reaction, enzymes derived from microorganisms such as Myrocesium and Trachyderma are easily available, and these are used at 0.1 to 1011/ml.
第１反応、第２反応で用いられる緩衝剤としてはｐＨ１
〜１１のもの、例えば塩酸−塩化カリウム。The buffer used in the first and second reactions has a pH of 1
~11, such as hydrochloric acid-potassium chloride.
クエン酸、酢酸、リン酸、乳酸、酒石酸、トリス−塩酸
、コハク酸、シュウ酸、Ｇｏｏｄ’ｓ等が０．００５〜
２Ｍ／Ｌで用いられる。Citric acid, acetic acid, phosphoric acid, lactic acid, tartaric acid, Tris-hydrochloric acid, succinic acid, oxalic acid, Good's, etc. from 0.005 to
Used at 2M/L.
第２反応で用いられるジアゾ試薬としては、間接とリル
ビンと反応して安定な色素を定量的に生成するものであ
ればいずれも用いろるが、例えばＰ−スルホベンゼンジ
アゾニウム塩、２．４−ジクロロフェニルジアゾニウム
塩、２．５−ジクロロフェニルジアゾニウム塩、一般式
（１）
（式中、Ｘはハロゲン例えばＦ、ＣＩ、Ｂを示す）で表
される化合物〔以下化合物（１）という〕等のジアゾニ
ウム塩が用いられる。As the diazo reagent used in the second reaction, any diazo reagent that can quantitatively produce a stable dye by reacting indirectly with rilubin can be used, such as P-sulfobenzenediazonium salt, 2.4- Diazonium salts such as dichlorophenyldiazonium salts, 2.5-dichlorophenyldiazonium salts, compounds represented by general formula (1) (in the formula, X represents a halogen such as F, CI, B) [hereinafter referred to as compound (1)], etc. is used.
これらのジアゾニウム塩としては安定化された塩、例え
ば１，５−ナフタレンジスルホン酸塩が好ましく用いら
れる。As these diazonium salts, stabilized salts such as 1,5-naphthalenedisulfonate are preferably used.
反応促進剤として例えば、カフェイン、ダイフィリン、
安息香酸塩、ジメチルスルホキシド、酢酸塩、尿素、ア
セトアマイド、サリチル酸塩、トＩＪ ）ンＸ−１００
等の界面活性剤等が０．１〜１００ｍｇ／ｍｌ含有さ卓
る。Examples of reaction accelerators include caffeine, daphyllin,
Benzoate, dimethyl sulfoxide, acetate, urea, acetamide, salicylate, Tonne X-100
Contains 0.1 to 100 mg/ml of surfactants such as.
実施に際しては、ビリルビン・オキシダーゼを緩衝剤に
溶解した第１試薬液及びジアゾニウム塩及び反応促進剤
を溶解した緩衝剤に溶解した第２試薬液を用意しておい
て反応を行わせるのが便利である。When carrying out the reaction, it is convenient to prepare a first reagent solution in which bilirubin oxidase is dissolved in a buffer and a second reagent solution in which a diazonium salt and a reaction accelerator are dissolved in a buffer. be.
本発明によれば間接ビリルビン定量用キットが提供され
る。このキットは、ビリルビン・オキシダーゼと緩衝剤
の組合せからなる第１試薬及びジアゾニウム塩１反応促
進剤と緩衝剤の組合せからなる第２試薬からなる。第２
試薬のジアゾニウム塩には不安定なものがあり、その際
にはジアゾニウム塩の代わりにジアゾニウム塩を調製す
るための化合物を組合せることができる。According to the present invention, a kit for indirect bilirubin quantification is provided. This kit consists of a first reagent consisting of a combination of bilirubin oxidase and a buffer, and a second reagent consisting of a combination of a diazonium salt 1 reaction promoter and a buffer. Second
Some diazonium salts of reagents are unstable, and in that case, a compound for preparing a diazonium salt can be combined in place of the diazonium salt.
ジアゾニウム塩調製用化合物としては、２．４−ジクロ
ロアニリン、２．５−ジクロロアニリン、式（Ｘは前記
と同義を示す）で表される化合物〔以下化合物（ｎ）と
いう〕と、ジアゾ化のための亜硝酸ソーダ及び酸、例え
ば塩酸、硫酸が組合わされる。Compounds for preparing diazonium salts include 2.4-dichloroaniline, 2.5-dichloroaniline, a compound represented by the formula (X has the same meaning as above) [hereinafter referred to as compound (n)], and a diazotized compound. Sodium nitrite and acids such as hydrochloric acid and sulfuric acid are combined.
これらのキットにおける各化合物は、前記反応で用いら
れる濃度の範囲となるように組合わされる。The compounds in these kits are combined to provide the range of concentrations used in the reactions described above.
本発明方法によれば、第２反応で反応促進剤を多量に用
いることができるので、試料中の混濁成分を溶解するに
充分な量の界面活性剤を加えることができ、試薬ブラン
クのテストの必要がなく、試料も少量で測定でき且つ簡
便である。According to the method of the present invention, since a large amount of reaction accelerator can be used in the second reaction, a sufficient amount of surfactant can be added to dissolve the cloudy components in the sample, and it is possible to add a sufficient amount of surfactant to dissolve the cloudy components in the sample. It is not necessary, the measurement can be performed with a small amount of sample, and it is simple.
以下に本発明の態様を示す実施例を示す。Examples illustrating aspects of the present invention are shown below.
実施例１゜
第一試薬として、０．１Ｍクエン酸緩衝液（ｐＨ４，０
）１００ｍｌにビリルビン・オキシダーゼ〔トラキデル
マ属微生物由来、宝酒造四社製〕を１００単位溶解した
ものを調製する。第二試薬として、０、０５ Ｍフタル
酸緩衝液（ｐＨ３，０）１０ Ｑｍｌにトリトンｘ−ｉ
ｏｏを１ｇ、アセトアマイドを２５０■、およびジアゾ
ニウム塩として、化合物（１）（Ｘ＝ＣＩを示す）を５
０ｍｇ溶解したものを調製する。Example 1゜As the first reagent, 0.1M citrate buffer (pH 4,0
) Prepare a solution by dissolving 100 units of bilirubin oxidase (derived from a microorganism of the genus Trachyderma, manufactured by Takara Shuzo Shisha) in 100 ml. As a second reagent, add Triton x-i to 10 Qml of 0.05 M phthalate buffer (pH 3.0).
Compound (1) (representing
Prepare a solution containing 0 mg.
第一試薬にビリルビン標準液（オメガビリルビンコント
ロール）を添加し、３７℃で５分加温後、第二試薬を添
加する。３７℃で５分加温後、その吸光度を試薬盲検を
対照としてλｍａｘ５４０ｎｍにて測定する。間接ビリ
ルビンの濃度と吸光度の関係を第１表に示す。Bilirubin standard solution (omega bilirubin control) is added to the first reagent, and after heating at 37°C for 5 minutes, the second reagent is added. After heating at 37° C. for 5 minutes, the absorbance is measured at λmax 540 nm using a reagent blind as a control. Table 1 shows the relationship between the concentration of indirect bilirubin and the absorbance.
第 １ 表
間接ビリルビンの濃度と吸光度との関係は直線的比例関
係にあることが第１表から明らかである。Table 1 It is clear from Table 1 that the relationship between the concentration of indirect bilirubin and absorbance is linearly proportional.
実施例２゜
第１試薬として０．１ Ｍクエン酸緩衝液（ｐＨ４，０
）１０ Ｑｍｌにビリルビンオキシダーゼ〔トラキデル
マ属微生物由来、宝酒造■社製〕を１００単位溶解した
ものを調製する。第二試薬として０．０５ Ｍフタル酸
緩衝液（ｐＨ３，０）１０ ＱｍｌにトリトンＸ−１０
０をＩｇ、アセトアマイドを２５０ｍｇおよびアジゾニ
ウム塩として、■Ｐ−スルホベンゼンジアゾニウム、１
．５−ナフタレンジスルホン酸ナトリウム、■２．４−
ジクロロフェニルジアゾニウム、１．５−ナフタレンジ
スルホン酸ナトリウム。Example 2 0.1 M citrate buffer (pH 4,0
) Prepare a solution by dissolving 100 units of bilirubin oxidase (derived from a microorganism of the genus Trachyderma, manufactured by Takara Shuzo ■) in 10 Qml. As a second reagent, add Triton
0 as Ig, acetamide as 250 mg and azizonium salt, ■P-sulfobenzenediazonium, 1
．． 5-Sodium naphthalenedisulfonate, ■2.4-
Dichlorophenyldiazonium, sodium 1,5-naphthalenedisulfonate.
■２．５−ジクロロフェニルジアゾニウム、１．５−ナ
フタレンジスルホン酸ナトリウム、■化合物Ｉ（Ｘ＝（
Ｊ）を各々５０ｍｇずつ溶解したものを調製する。■2.5-dichlorophenyldiazonium, sodium 1.5-naphthalenedisulfonate, ■Compound I (X=(
Prepare a solution of 50 mg of each of J).
５種類の血清試料各々に第一試薬を添加し、３７℃で５
分加温後、それぞれの第二試薬を添加する。３７℃で５
分加温後、その吸光度を試薬盲検を対照としてλｍａｘ
５４Ｑｎｍにて測定し、予めビリルビン標準液を用いて
作成しておいた検量線より、血清試料中の間接ビリルビ
ン濃度を求めた。測定値を公知のジアゾ法による測定試
薬であるビリルビンＢテスト「和光」 〔和光純薬■社
製〕で求めた値と併記して第２表に示す。ただし、ビリ
ルビンＢテスト「和光」の間接ビリルビン値は総ビリル
ビン値から直接ビリルビン値を差し引いて算出した値で
ある。Add the first reagent to each of the five serum samples and incubate at 37°C for 5 minutes.
After warming for 1 minute, each second reagent is added. 5 at 37℃
After heating for minutes, measure the absorbance at λmax with the reagent blind as a control.
The indirect bilirubin concentration in the serum sample was measured at 54 Qnm and determined from a calibration curve prepared in advance using a bilirubin standard solution. The measured values are shown in Table 2 together with the values determined using the bilirubin B test "Wako" (manufactured by Wako Pure Chemical Industries, Ltd.), which is a measuring reagent using the known diazo method. However, the indirect bilirubin value of the bilirubin B test "Wako" is a value calculated by subtracting the direct bilirubin value from the total bilirubin value.
第 ２ 表
ジアゾニウム塩の種類にかかわらず、アルカリアゾビリ
ルビンブルー法を用いた「和光」キットと良い相関を示
している。Table 2 Regardless of the type of diazonium salt, it shows a good correlation with the "Wako" kit using the alkaline azobilirubin blue method.
発明の効果
本発明方法により、より正確に間接ビリルビンを定量す
ることができる。Effects of the Invention By the method of the present invention, indirect bilirubin can be determined more accurately.
Claims (2)
ビリルビンをビリルビン・オキシダーゼの作用によって
ビルベルリンに変換し、残存する間接ビリルビンとジア
ゾ試薬とを反応促進剤の存在下に反応させてアゾ色素を
生成させ、これを定量することを特徴とする間接ビリル
ビンの定量法。(1) Direct bilirubin in a sample containing direct and indirect bilirubin is converted to bilberlin by the action of bilirubin oxidase, and the remaining indirect bilirubin is reacted with a diazo reagent in the presence of a reaction accelerator to form an azo dye. A method for quantifying indirect bilirubin, which is characterized by producing and quantifying bilirubin.
からなる第一試薬及び Ｂ）ジアゾ試薬、反応促進剤及び緩衝剤の組合せからな
る第２試薬 からなる、間接ビリルビン定量用キット。(2) A kit for indirect bilirubin quantification, comprising a first reagent consisting of a combination of bilirubin oxidase and a buffer, and B) a second reagent consisting of a combination of a diazo reagent, a reaction promoter, and a buffer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61087765A JPH0675072B2 (en) | 1986-04-16 | 1986-04-16 | Indirect bilirubin assay and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61087765A JPH0675072B2 (en) | 1986-04-16 | 1986-04-16 | Indirect bilirubin assay and kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6352060A true JPS6352060A (en) | 1988-03-05 |
| JPH0675072B2 JPH0675072B2 (en) | 1994-09-21 |
Family
ID=13924055
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61087765A Expired - Lifetime JPH0675072B2 (en) | 1986-04-16 | 1986-04-16 | Indirect bilirubin assay and kit |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0675072B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0387784A2 (en) * | 1989-03-13 | 1990-09-19 | Unitika Ltd. | Quantitative determination of bilirubin and a reagent therefor |
| US5858695A (en) * | 1995-10-27 | 1999-01-12 | Kyowa Medex Co., Ltd. | Method of quantitative determination of bilirubin and a reagent therefor |
| WO2008136273A1 (en) * | 2007-04-27 | 2008-11-13 | Arkray, Inc. | Method for bilirubin determination and analytical instrument used for bilirubin determination |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5923253A (en) * | 1982-07-07 | 1984-02-06 | 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 | Direct type bilirubin and method and reagent of measuring total bilirubin |
| JPS60154162A (en) * | 1984-01-24 | 1985-08-13 | Nitsusui Seiyaku Kk | Quantitative determination of free bilirubin |
| JPS62105047A (en) * | 1985-10-31 | 1987-05-15 | Amano Pharmaceut Co Ltd | Method for fractionating and measuring bilirubin |
-
1986
- 1986-04-16 JP JP61087765A patent/JPH0675072B2/en not_active Expired - Lifetime
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5923253A (en) * | 1982-07-07 | 1984-02-06 | 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 | Direct type bilirubin and method and reagent of measuring total bilirubin |
| JPS60154162A (en) * | 1984-01-24 | 1985-08-13 | Nitsusui Seiyaku Kk | Quantitative determination of free bilirubin |
| JPS62105047A (en) * | 1985-10-31 | 1987-05-15 | Amano Pharmaceut Co Ltd | Method for fractionating and measuring bilirubin |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0387784A2 (en) * | 1989-03-13 | 1990-09-19 | Unitika Ltd. | Quantitative determination of bilirubin and a reagent therefor |
| US5858695A (en) * | 1995-10-27 | 1999-01-12 | Kyowa Medex Co., Ltd. | Method of quantitative determination of bilirubin and a reagent therefor |
| WO2008136273A1 (en) * | 2007-04-27 | 2008-11-13 | Arkray, Inc. | Method for bilirubin determination and analytical instrument used for bilirubin determination |
| JP4785926B2 (en) * | 2007-04-27 | 2011-10-05 | アークレイ株式会社 | Bilirubin measurement method and analytical tool used for bilirubin measurement |
| US9442122B2 (en) | 2007-04-27 | 2016-09-13 | Arkray, Inc. | Method for assaying bilirubin and assay instrument used in bilirubin assay |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0675072B2 (en) | 1994-09-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4495293A (en) | Fluorometric assay | |
| US4425427A (en) | Simultaneous, kinetic, spectrophotometric analysis of blood serum for multiple components | |
| Berti et al. | Enzymatic colorimetric method for the determination of inorganic phosphorus in serum and urine | |
| CN101498662A (en) | Reagent kit for monoamine oxidase MAO single-reagent measurement | |
| US4271265A (en) | Method and reagent for the determination of glutamate-oxalacetate transaminase and glutamate-pyruvate transaminase | |
| JPS6352060A (en) | Method and kit for quantitative determination of indirect bilirubin | |
| JP4073963B2 (en) | Ascorbic acid quantitative method and quantitative reagent | |
| JPS5845557A (en) | Reagent for quantitative determination of hydrogen peroxide | |
| JPWO2006030866A1 (en) | Method for quantitative determination of uric acid | |
| JP2994831B2 (en) | Cholesterol assay and reagents | |
| JP2516381B2 (en) | Method for quantifying hydrogen peroxide and reagent for quantifying the same | |
| US3985621A (en) | Stopping agents for enzyme reactions of dehydrogenase systems | |
| JP2590124B2 (en) | Water-soluble tetrazolium compound and method for measuring reducing substance using the compound | |
| JPH03187400A (en) | Method and reagent for optically measuring bilirubin | |
| JPS6240300A (en) | Activity measurement of adenosine deaminase in humor | |
| JP3287879B2 (en) | Determination of 1,5-anhydroglucitol | |
| JPH02142498A (en) | Determination of calcium in body fluid | |
| JPH0595798A (en) | Determination of pyruvic acid and determination of bio-component using the method | |
| JPH0638757B2 (en) | Peroxidase or H ▲ lower 2 ▼ O ▲ lower 2 ▼ measuring method | |
| JPH02145196A (en) | Method for determining magnesium | |
| JPS61174267A (en) | Novel imidazole derivative and measuring method using same as color forming component | |
| JPH0413351B2 (en) | ||
| JPS6191570A (en) | Assay of reducing type nicotinamide adenine dinucleotide and reducing type nicotinamide adenine dinucleotide phosphate | |
| JPS60225061A (en) | Reagent and assay of hydrogen peroxide and assay method | |
| JPH0599A (en) | Determination of phosphatase activity |