JP3520874B1 - Colorimetric method - Google Patents
Colorimetric methodInfo
- Publication number
- JP3520874B1 JP3520874B1 JP2003380986A JP2003380986A JP3520874B1 JP 3520874 B1 JP3520874 B1 JP 3520874B1 JP 2003380986 A JP2003380986 A JP 2003380986A JP 2003380986 A JP2003380986 A JP 2003380986A JP 3520874 B1 JP3520874 B1 JP 3520874B1
- Authority
- JP
- Japan
- Prior art keywords
- polymer
- reagent
- creatinine
- colorimetric
- substance
- 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 - Lifetime
Links
- 238000004737 colorimetric analysis Methods 0.000 title claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 97
- 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 84
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 claims abstract description 70
- 238000000034 method Methods 0.000 claims abstract description 60
- 238000006243 chemical reaction Methods 0.000 claims abstract description 54
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 48
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 claims abstract description 42
- 229920000642 polymer Polymers 0.000 claims abstract description 40
- 229940109239 creatinine Drugs 0.000 claims abstract description 35
- 238000005259 measurement Methods 0.000 claims abstract description 25
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 claims abstract description 23
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229940116269 uric acid Drugs 0.000 claims abstract description 23
- 229960003624 creatine Drugs 0.000 claims abstract description 21
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- 238000006911 enzymatic reaction Methods 0.000 claims abstract description 11
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims abstract description 10
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- 125000004432 carbon atom Chemical group C* 0.000 claims description 4
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- LDXJRKWFNNFDSA-UHFFFAOYSA-N 2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]ethanone Chemical compound C1CN(CC2=NNN=C21)CC(=O)N3CCN(CC3)C4=CN=C(N=C4)NCC5=CC(=CC=C5)OC(F)(F)F LDXJRKWFNNFDSA-UHFFFAOYSA-N 0.000 description 3
- AJTVSSFTXWNIRG-UHFFFAOYSA-N 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid Chemical compound OCC[NH+](CCO)CCS([O-])(=O)=O AJTVSSFTXWNIRG-UHFFFAOYSA-N 0.000 description 3
- RZQXOGQSPBYUKH-UHFFFAOYSA-N 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]azaniumyl]-2-hydroxypropane-1-sulfonate Chemical compound OCC(CO)(CO)NCC(O)CS(O)(=O)=O RZQXOGQSPBYUKH-UHFFFAOYSA-N 0.000 description 3
- QZRAABPTWGFNIU-UHFFFAOYSA-N 3-[dimethyl(octyl)azaniumyl]propane-1-sulfonate Chemical compound CCCCCCCC[N+](C)(C)CCCS([O-])(=O)=O QZRAABPTWGFNIU-UHFFFAOYSA-N 0.000 description 3
- CNHVXRAZMXBDIT-UHFFFAOYSA-N 4-[3-methyl-n-(4-sulfobutyl)anilino]butane-1-sulfonic acid Chemical compound CC1=CC=CC(N(CCCCS(O)(=O)=O)CCCCS(O)(=O)=O)=C1 CNHVXRAZMXBDIT-UHFFFAOYSA-N 0.000 description 3
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- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- HMZGPNHSPWNGEP-UHFFFAOYSA-N octadecyl 2-methylprop-2-enoate Chemical compound CCCCCCCCCCCCCCCCCCOC(=O)C(C)=C HMZGPNHSPWNGEP-UHFFFAOYSA-N 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- ZPCAZHPYLUKSMY-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methylanilino)-2-hydroxypropane-1-sulfonate;dihydrate Chemical compound O.O.[Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(C)=C1 ZPCAZHPYLUKSMY-UHFFFAOYSA-M 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
【要約】
【課題】H2O2を介する定量において検体中のビリルビン
等の還元物質の影響の少ない比色定量方法、測定試薬安
定化方法、ビリルビン影響抑制方法、及び当該定量のた
めの長期間保存可能な試薬。
【解決手段】検体中のクレアチニン、クレアチン又は尿
酸を酵素反応により過酸化水素に導きパーオキシダーゼ
の存在下で発色色素を生成させる比色定量方法で、反応
系に式(I)：
【化１】
(R:H又はメチル基)及び式(II)：
【化２】
(R:H又はメチル基。X:C12〜20アルキル。)の単量体に基
く単位からなる分子量5,000〜5,000,000の重合体を存在
させる比色定量方法、保存安定化方法及びビリルビン影
響抑制方法、並びに該重合体を含む試薬。Kind Code: A1 Abstract: A method for colorimetric quantification, a method for stabilizing a measurement reagent, a method for suppressing the effect of bilirubin, and a long-term method for the quantification, which are less affected by a reducing substance such as bilirubin in a sample in quantification via H 2 O 2. A storable reagent. Kind Code: A1 A colorimetric method in which creatinine, creatine or uric acid in a sample is led to hydrogen peroxide by an enzymatic reaction to generate a coloring dye in the presence of peroxidase, wherein the reaction system is represented by the formula (I): (R: H or methyl group) and formula (II): (R: H or a methyl group. X: C12-20 alkyl.) A colorimetric method, a storage stabilization method, and a method for suppressing the effect of bilirubin, in which a polymer having a molecular weight of 5,000 to 5,000,000 consisting of units based on a monomer is present. And a reagent containing the polymer.
Description
【０００１】本発明は、臨床診断等のための比色定量方
法に関する。The present invention relates to a colorimetric method for clinical diagnosis and the like.
【０００２】臨床検査における生化学項目の測定方法に
は大きく分けると化学法と酵素法が存在する。このうち
酵素法には、例えば、デヒドロゲナーゼを用いてNADH等
の補酵素を検出する方法と、オキシダーゼを用いて測定
対象物質から定量的に発生させた過酸化水素を検出する
方法がある。オキシダーゼを用いる方法は、他法より高
感度であるため微量の物質を測定するのに適するが、過
酸化水素を介する測定系であるため、試料中に還元作用
を有する物質が存在すると、それらにより過酸化水素が
還元され、測定結果に影響を及ぼし、正確な測定を妨害
するという問題点がある(以下、このように定量のため
のものではない還元作用を示す物質を、単に「還元物
質」という)。The methods for measuring biochemical items in clinical tests are roughly divided into chemical methods and enzyme methods. Among them, the enzyme method includes, for example, a method of detecting a coenzyme such as NADH using dehydrogenase, and a method of detecting hydrogen peroxide quantitatively generated from a substance to be measured using an oxidase. The method using oxidase is more sensitive than other methods and is suitable for measuring a small amount of substance.However, since it is a measurement system mediated by hydrogen peroxide, if a substance having a reducing action exists in the sample, There is a problem that hydrogen peroxide is reduced, which affects the measurement results and interferes with accurate measurement (hereinafter, a substance that exhibits a reducing action that is not for quantitative determination is simply referred to as a “reducing substance”). That).
【０００３】前記還元物質としては、具体的には採血時
の溶血により遊離するヘモグロビンや、肝疾患患者の血
中に高濃度で存在するビリルビン、あるいは栄養剤とし
て服用されるアスコルビン酸、肝臓や赤血球、白血球等
の溶液に存在する還元型グルタチオン(GSH)等が挙げら
れる。Specific examples of the reducing substance include hemoglobin released by hemolysis during blood collection, bilirubin present at a high concentration in blood of patients with liver disease, or ascorbic acid taken as a nutritional supplement, liver or red blood cells. , Reduced glutathione (GSH) existing in a solution of white blood cells and the like.
【０００４】これらの還元物質は、比較的高濃度の対象
物質の濃度を測定する場合にはあまり問題とはならない
が、低濃度の対象物質の濃度を測定する場合には多大な
測定誤差を生じる原因となる。換言すればこれらの還元
物質により消去される過酸化水素が一定量である場合、
測定対象物質の濃度が低いほど大きな影響を受ける。These reducing substances are not a serious problem when measuring the concentration of a target substance having a relatively high concentration, but cause a large measurement error when measuring the concentration of a target substance having a low concentration. Cause. In other words, if the amount of hydrogen peroxide eliminated by these reducing substances is constant,
The lower the concentration of the substance to be measured, the greater the effect.
【０００５】例えば腎機能のマーカーであるクレアチニ
ンは正確な測定が要求される測定項目の1つであるが、
他の生化学項目と比較して検体中の存在量は微量であ
る。このため還元物質によって過酸化水素が僅かに消去
されても測定値に著しい影響を与える。しかしクレアチ
ニンはその臨床上の意義から正確な測定が求められるた
め、還元物質による影響を極力回避して低濃度おいても
正確に測定することが求められる。[0005] For example, creatinine, which is a marker of renal function, is one of the measurement items that requires accurate measurement.
The abundance in the sample is very small compared to other biochemical items. Therefore, even if hydrogen peroxide is slightly erased by the reducing substance, the measured value is significantly affected. However, since creatinine is required to be accurately measured because of its clinical significance, it is necessary to avoid the influence of reducing substances as much as possible and accurately measure even at low concentrations.
【０００６】過酸化水素を介する測定系において還元物
質の影響を回避する手段としては、カチオン性界面活性
剤又は両性界面活性剤を試薬に添加する方法(特許文献
4)、及びベタイン系界面活性剤を試薬に添加する方法
(特許文献5)が提案されている。しかしながら、これら
の方法においてもビリルビン等の還元物質の影響を完全
に解決しているとは言い難く、特にクレアチニンの測定
においては依然として十分であるとは言い難い。As a means for avoiding the influence of reducing substances in a measurement system mediated by hydrogen peroxide, a method of adding a cationic surfactant or an amphoteric surfactant to a reagent (Patent Document)
4), and a method of adding a betaine surfactant to the reagent
(Patent Document 5) has been proposed. However, it is hard to say that these methods completely solve the influence of reducing substances such as bilirubin, and it is hard to say that they are still sufficient especially for the measurement of creatinine.
【０００７】さらにイオン性界面活性剤は、酵素等の他
の成分と共に溶液とした場合、当該他の成分を経時的に
変性、若しくは沈殿させる性質があるため、性能に対す
る影響、特に他の成分の保存安定性に対する影響が大き
い。このことは、臨床検査用の上記反応のための試薬の
大半が液状試薬として提供されている現状では大きな問
題となる。還元物質の影響を回避するのに十分な量のイ
オン性界面活性剤を添加すると、それだけ酵素等の反応
を妨げることになる。この問題を解決する手段として、
酵素等の反応に関与する成分を過剰量配合することが考
えられるが、その場合製造コストがかかるという問題点
がある。Further, when an ionic surfactant is made into a solution together with other components such as an enzyme, it has a property of denaturing or precipitating the other components over time, so that the influence on the performance, especially of other components, It has a great impact on storage stability. This poses a serious problem in the present situation where most of the reagents for the above-mentioned reactions for clinical tests are provided as liquid reagents. If the ionic surfactant is added in an amount sufficient to avoid the influence of the reducing substance, the reaction of the enzyme or the like will be hindered. As a means to solve this problem,
It is conceivable to add an excessive amount of a component such as an enzyme involved in the reaction, but in that case, there is a problem that the manufacturing cost is high.
【０００８】ところで、臨床診断において、(メタ)アク
リロイルホスホリルコリンに基づく構成単位を有する重
合体を共存させて測定する方法は、例えば特許文献1〜3
に開示されている。特許文献1には、抗原-抗体反応、酵
素-基質反応、DNA-DNA反応等における反応促進剤が開示
されている。特許文献2には、ラテックスを用いる凝集
方法において、免疫学的な凝集促進剤としての(メタ)ア
クリロイルホスホリルコリンの用途が開示されている。
特許文献3には、(メタ)アクリロイルホスホリルコリン
等の重合体を用いて、ラテックスを用いる測定で希釈す
ることなくプロゾーン現象を回避してC-反応性蛋白質を
測定する方法が開示されている。[0008] By the way, in clinical diagnosis, a method of coexisting and measuring a polymer having a structural unit based on (meth) acryloylphosphorylcholine is disclosed in Patent Documents 1 to 3, for example.
Is disclosed in. Patent Document 1 discloses a reaction accelerator in an antigen-antibody reaction, an enzyme-substrate reaction, a DNA-DNA reaction and the like. Patent Document 2 discloses the use of (meth) acryloylphosphorylcholine as an immunological aggregation promoter in an aggregation method using latex.
Patent Document 3 discloses a method of measuring a C-reactive protein by using a polymer such as (meth) acryloylphosphorylcholine and avoiding the prozone phenomenon without dilution in a measurement using a latex.
【０００９】しかしながら、検体中の測定対象物質をオ
キシダーゼにより過酸化水素に導き、これをパーオキシ
ダーゼの存在下で発色色素を生成させる比色定量方法に
おいて、ビリルビン等の還元物質の影響を抑制して、測
定対象物質が低濃度であっても高精度の測定を可能とす
る方法は開示されていない。However, in a colorimetric method in which a substance to be measured in a sample is introduced into hydrogen peroxide by an oxidase, and this is formed into a coloring dye in the presence of peroxidase, the influence of a reducing substance such as bilirubin is suppressed. However, there is no disclosure of a method that enables highly accurate measurement even if the substance to be measured has a low concentration.
【特許文献１】特開2002-22740号公報(第2頁)[Patent Document 1] Japanese Unexamined Patent Publication No. 2002-22740 (page 2)
【特許文献２】特開2002-365296号公報(第2頁)[Patent Document 2] Japanese Patent Laid-Open No. 2002-365296 (page 2)
【特許文献３】特開2001-318099号公報(第2頁)[Patent Document 3] Japanese Patent Laid-Open No. 2001-318099 (page 2)
【特許文献４】特開平3-10696号公報[Patent Document 4] Japanese Patent Laid-Open No. 3-10696
【特許文献５】特開平7-39394号公報[Patent Document 5] JP-A-7-39394
【００１０】本発明の目的は、過酸化水素を介する測定
対象物質の定量において、検体中に含まれるビリルビン
等の還元物質の存在による影響の少ない比色定量方法、
測定試薬の安定化方法又はビリルビンの影響を抑制する
方法を提供することにある。An object of the present invention is to provide a colorimetric quantification method which is less affected by the presence of a reducing substance such as bilirubin contained in a sample in quantifying a substance to be measured through hydrogen peroxide,
It is intended to provide a method for stabilizing a measurement reagent or a method for suppressing the influence of bilirubin.
【００１１】本発明の別の目的は、過酸化水素を介する
測定対象物質の定量に用いる試薬であって、長期間保存
することができ、酵素の反応性、特異性、安定性等を損
なうことなく、且つ検体中に含まれるビリルビン等の還
元物質の存在による影響の少ない定量を行なうことがで
きる試薬を提供することにある。Another object of the present invention is a reagent used for quantifying a substance to be measured through hydrogen peroxide, which can be stored for a long period of time and impairs reactivity, specificity, stability, etc. of an enzyme. It is an object of the present invention to provide a reagent which can be quantified without the presence of a reducing substance such as bilirubin contained in a sample.
【００１２】本発明者らは、前記の問題点に鑑み鋭意検
討した結果、特定の重合体を反応系に存在させると、ビ
リルビン等の還元物質が混在していてもその影響を抑制
することができることを見出し、本発明を完成するに至
った。The inventors of the present invention have made extensive studies in view of the above-mentioned problems, and as a result, when a specific polymer is present in the reaction system, even if a reducing substance such as bilirubin is mixed, its influence can be suppressed. They have found that they can do so and have completed the present invention.
【００１３】すなわち、本発明によれば、検体中のクレ
アチニン、クレアチン又は尿酸を測定対象物質とし、酵
素反応により過酸化水素を導き、パーオキシダーゼの存
在下で発色色素を生成させるクレアチニン、クレアチン
又は尿酸の比色定量方法において、反応系に下記式
(I)：That is, according to the present invention, creatinine, creatine or uric acid which is a creatinine, creatine or uric acid in a sample as a substance to be measured and leads hydrogen peroxide by an enzymatic reaction to form a coloring pigment in the presence of peroxidase. In the colorimetric method of
(I):
【００１４】[0014]
【化７】 [Chemical 7]
【００１５】(ここで、Rは水素原子またはメチル基を示
す。)及び式(II)：(Wherein R represents a hydrogen atom or a methyl group) and the formula (II):
【００１６】[0016]
【化８】
(ここで、Rは水素原子又はメチル基を示す。Xは、炭素
数12〜20のアルキル基を示す。)で表される単量体に基
く単位からなる、重量平均分子量5,000〜5,000,000の重
合体(以下、重合体Aという。)を存在させることを特徴
とする、クレアチニン、クレアチン又は尿酸の比色定量
方法が提供される。[Chemical 8] (Wherein R represents a hydrogen atom or a methyl group, X represents an alkyl group having 12 to 20 carbon atoms), and a weight-average molecular weight of 5,000 to 5,000,000 There is provided a method for colorimetric determination of creatinine, creatine or uric acid, characterized in that a combined product (hereinafter referred to as polymer A) is present.
【００１７】さらに、本発明によれば、前記比色定量の
ための比色定量試薬であって、溶媒と、酵素と、前記重
合体Aとを含む比色定量試薬が提供される。Further, according to the present invention, there is provided a colorimetric assay reagent for the colorimetric assay, which comprises a solvent, an enzyme and the polymer A.
【００１８】さらに、本発明によれば、ビリルビンを含
む検体中のクレアチニン、クレアチン又は尿酸を測定対
象とし、酵素反応により過酸化水素を導き、パーオキシ
ダーゼの存在下で発色色素を生成させる、クレアチニ
ン、クレアチン又は尿酸の比色定量において検体中のビ
リルビンの影響を抑制する方法であって、反応系に前記
重合体Aを存在させることを特徴とする抑制方法が提供
される。Further, according to the present invention, creatinine, creatine or uric acid in a sample containing bilirubin is measured, and hydrogen peroxide is introduced by an enzymatic reaction to produce a coloring pigment in the presence of peroxidase, creatinine, There is provided a method for suppressing the influence of bilirubin in a sample in the colorimetric determination of creatine or uric acid, wherein the method comprises the presence of the polymer A in a reaction system.
【００１９】さらに、本発明によれば、検体中のクレア
チニン、クレアチン又は尿酸を測定対象とし、酵素反応
により過酸化水素を導き、パーオキシダーゼの存在下で
発色色素を生成させるクレアチニン、クレアチン又は尿
酸の比色定量における測定試薬の保存安定化方法であっ
て、反応系に前記重合体Aを存在させることを特徴とす
る保存安定化方法が提供される。Further, according to the present invention, creatinine, creatine or uric acid in a sample is measured, and creatinine, creatine or uric acid which leads to hydrogen peroxide by an enzymatic reaction and produces a coloring dye in the presence of peroxidase is produced. There is provided a method for storage stabilization of a measuring reagent in colorimetric quantification, which comprises allowing the polymer A to be present in a reaction system.
【００２０】本発明の比色定量方法、本発明の安定化方
法及び本発明の抑制方法では、反応系に特定の重合体を
存在させることにより、酵素の反応性、特異性、安定性
等を損なうことなく、還元物質の影響を軽減することが
できる。例えば、本来は50%以上受ける負の影響を5%以
下に抑えうる。したがって、本発明の比色定量方法で
は、検体中の測定対象物質が低濃度である場合であって
も、真値に近い正確な定量を簡便に行なうことができ
る。In the colorimetric quantification method of the present invention, the stabilization method of the present invention and the suppression method of the present invention, the reactivity, specificity, stability, etc. of the enzyme can be improved by allowing a specific polymer to be present in the reaction system. The effect of the reducing substance can be reduced without impairing it. For example, it is possible to reduce the negative impact of 50% or more to 5% or less. Therefore, according to the colorimetric quantification method of the present invention, accurate quantification close to the true value can be easily performed even when the measurement target substance in the sample has a low concentration.
【００２１】本発明の比色定量試薬は、酵素等の成分に
加えて特定の重合体を含むので、長期間保存することが
でき、酵素の反応性、特異性、安定性等を損なうことな
く、検体中の測定対象物質が低濃度である場合であって
も、真値に近い正確な定量を簡便に行なうことを可能と
する。Since the colorimetric assay reagent of the present invention contains a specific polymer in addition to components such as an enzyme, it can be stored for a long period of time without impairing the reactivity, specificity, stability, etc. of the enzyme. Even if the substance to be measured in the sample has a low concentration, it is possible to easily perform accurate quantification close to the true value.
【００２２】本発明の比色定量方法、本発明の安定化方
法又は本発明の抑制方法では、反応系に特定の重合体
(重合体A)を存在させることを特徴とする。重合体Aは、
前記式(I)で表される単量体(以下、単量体A1という。)
及び前記式(II)で表される単量体(以下、単量体A2とい
う。)に基く単位からなる。具体的には、下記式(III)で
表わされる単位及び(IV)で表される単位を含むことがで
きる。下記式(III)及び(IV)において、R及びXは、式(I)
及び(II)中のものと同じものを示す。In the colorimetric quantification method of the present invention, the stabilization method of the present invention or the suppression method of the present invention, a polymer specific to the reaction system is used.
(Polymer A) is present. Polymer A is
The monomer represented by the formula (I) (hereinafter referred to as the monomer A1)
And a unit based on the monomer represented by the formula (II) (hereinafter referred to as the monomer A2). Specifically, it may include a unit represented by the following formula (III) and a unit represented by (IV). In the following formulas (III) and (IV), R and X are represented by the formula (I)
And the same as in (II).
【００２３】[0023]
【化９】 [Chemical 9]
【００２４】[0024]
【化１０】 [Chemical 10]
【００２５】式(II)又は(IV)において、Xで表されるア
ルキル基を炭素数12以上のものとすることにより、反応
系中に存在する還元物質の影響を良好に軽減することが
できる。単量体A2としては、具体的には例えば、ラウリ
ル(メタ)アクリレート及びステアリル(メタ)アクリレー
ト等の直鎖又は分岐アルキル(メタ)アクリレート等が挙
げられる。重合体Aにおいて、式(III)の単位及び式(IV)
の単位は、ランダム重合、交互重合、ブロック共重合等
のいずれの形態をとって共重合していてもよい。In the formula (II) or (IV), the influence of the reducing substance present in the reaction system can be satisfactorily reduced by making the alkyl group represented by X have a carbon number of 12 or more. . Specific examples of the monomer A2 include linear or branched alkyl (meth) acrylates such as lauryl (meth) acrylate and stearyl (meth) acrylate. In the polymer A, the unit of the formula (III) and the formula (IV)
The units may be copolymerized in any form such as random polymerization, alternating polymerization and block copolymerization.
【００２６】重合体Aは、単量体A1及びA2を通常のラジ
カル共重合等により共重合し、さらに必要に応じて溶媒
等に溶解し精製することにより製造することができる。
具体的には、特許文献1〜3記載の方法に準じて製造する
ことができる。The polymer A can be produced by copolymerizing the monomers A1 and A2 by an ordinary radical copolymerization, etc., and further dissolving them in a solvent or the like as necessary for purification.
Specifically, it can be produced according to the methods described in Patent Documents 1 to 3.
【００２７】単量体A1及び単量体A2を共重合させる際の
これらの比は、特に限定されないが、通常、モル比で単
量体A1/単量体A2=99/1〜10/90、好ましくは90/10〜20/8
0、さらに好ましくは80/20〜30/70とすることができ
る。単量体A1及び単量体A2の合計に対する単量体Aの割
合を10モル%以上とすることにより、ホスホリルコリン
基による効果を十分に発揮することができる。The ratio of these in copolymerizing the monomer A1 and the monomer A2 is not particularly limited, but is usually monomer A1 / monomer A2 = 99/1 to 10/90 in molar ratio. , Preferably 90/10 to 20/8
It can be 0, more preferably 80/20 to 30/70. When the ratio of the monomer A to the total of the monomer A1 and the monomer A2 is 10 mol% or more, the effect of the phosphorylcholine group can be sufficiently exhibited.
【００２８】重合体Aの分子量は、重量平均分子量とし
て5,000〜5,000,000の範囲であり、10,000〜1,000,000
の範囲であることが好ましい。重合体Aの分子量を5,000
以上とすることにより十分に生化学的反応を促進するこ
とでき、5,000,000以下とすることにより重合体Aの粘性
による生化学的反応の阻害を避けることができる。The molecular weight of the polymer A is in the range of 5,000 to 5,000,000 as a weight average molecular weight, and 10,000 to 1,000,000.
It is preferably in the range of. Polymer A has a molecular weight of 5,000
By setting the above, the biochemical reaction can be sufficiently promoted, and by setting it to be 5,000,000 or less, the inhibition of the biochemical reaction due to the viscosity of the polymer A can be avoided.
【００２９】本発明の比色定量方法、本発明の安定化方
法又は本発明の抑制方法では、前記の重合体Aの存在
下、検体中の測定対象物質を酵素反応により過酸化水素
に導き、これからパーオキシダーゼの存在下で発色色素
を生成させる。In the colorimetric quantification method of the present invention, the stabilization method of the present invention or the suppression method of the present invention, in the presence of the polymer A, the substance to be measured in the sample is introduced into hydrogen peroxide by an enzymatic reaction, From this, a coloring pigment is produced in the presence of peroxidase.
【００３０】前記測定対象物質は、クレアチニン、クレ
アチン又は尿酸である。The substance to be measured is creatinine, creatine or uric acid.
【００３１】前記酵素反応としては、前記測定対象物質
から過酸化水素を導く反応を適宜選択することができ
る。前記酵素反応は、１段階の反応でもよく、複数段階
の反応でもよい。前記酵素反応は、その少なくとも１段
階において、前記測定対象物質又は前記測定対象物質と
他の物質との反応物から過酸化水素を発生させる反応を
含む。当該過酸化水素を発生させる反応としては、具体
的には例えば、ウリカーゼ、ザルコシンオキシダーゼ等
のオキシダーゼによる反応を挙げることができる。As the enzyme reaction, a reaction for introducing hydrogen peroxide from the substance to be measured can be appropriately selected. The enzymatic reaction may be a one-step reaction or a multi-step reaction. The enzyme reaction includes, in at least one stage thereof, a reaction of generating hydrogen peroxide from the measurement target substance or a reaction product of the measurement target substance and another substance. Specific examples of the reaction for generating hydrogen peroxide include reactions with oxidases such as uricase and sarcosine oxidase.
【００３２】前記パーオキシダーゼは、過酸化水素を水
に還元し発色色素を生成させうるものであれば特に限定
されないが、具体的には例えば、西洋ワサビ由来、又は
大豆由来のパーオキシダーゼを用いることができる。The peroxidase is not particularly limited as long as it can reduce hydrogen peroxide to water to form a coloring pigment. Specifically, for example, horseradish-derived or soybean-derived peroxidase is used. You can
【００３３】前記発色色素は、パーオキシダーゼにより
過酸化水素量に応じて発生することができるものであれ
ば特に限定されず、各種のキノン系色素等を発生させる
ことができる。前記発色色素は、パーオキシダーゼと組
み合わせて過酸化水素量に応じて前記発色色素を発生さ
せる発色色素生成物質を反応系に存在させることにより
発生させることができる。前記発色色素生成物質として
は、ロイコ体又は各種のトリンダー試薬等を用いること
ができる。前記トリンダー試薬は、4-アミノアンチピリ
ン等の発色色素生成を補助する物質と組み合わせて用い
ることができる。前記発色色素生成物質のうちの前記ロ
イコ体としては、具体的には例えば、N-(カルボキシメ
チルアミノカルボニル)-4,4'-ビス(ジメチルアミノ)-ジ
フェニルアミン、及び10-(カルボキシメチルアミノカル
ボニル)-3,7'-ビス(ジメチルアミノ)-フェノシアジン等
が挙げられる。また前記発色色素生成物質のうちの前記
トリンダー試薬としては、具体的には例えば、N-エチル
-N-(2-ヒドロキシ-3-スルホプロピル)-3-メチルアニリ
ン、N-エチル-N-(2-ヒドロキシ-3-スルホプロピル)-3-
メトキシアニリン、N-エチル-N-(2-ヒドロキシ-3-スル
ホプロピル)-3,5-ジメトキシアニリン、N-(2-ヒドロキ
シ-3-スルホプロピル)-3,5-ジメトキシアニリン、3-ヒ
ドロキシ-2,4,6-トリヨード安息香酸等が挙げられる。The coloring dye is not particularly limited as long as it can be generated by peroxidase depending on the amount of hydrogen peroxide, and various quinone dyes can be generated. The color-developing dye can be generated by allowing a color-developing dye-forming substance, which is combined with peroxidase to generate the color-developing dye depending on the amount of hydrogen peroxide, to be present in the reaction system. As the color-forming dye-forming substance, a leuco body or various Trinder's reagents can be used. The Trinder reagent can be used in combination with a substance such as 4-aminoantipyrine which assists in the formation of a coloring dye. The leuco form of the color-forming dye-forming substance, specifically, for example, N- (carboxymethylaminocarbonyl) -4,4'-bis (dimethylamino) -diphenylamine, and 10- (carboxymethylaminocarbonyl) ) -3,7'-bis (dimethylamino) -phenocyanazine and the like. Further, as the Trinder reagent of the color forming dye-forming substance, specifically, for example, N-ethyl
-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-
Methoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, 3-hydroxy -2,4,6-triiodobenzoic acid and the like can be mentioned.
【００３４】本発明の比色定量の測定対象物質及びその
測定に用いる酵素の組み合わせの具体例を下記表１に示
す。Specific examples of the combination of the substance to be measured for colorimetric determination of the present invention and the enzyme used for the measurement are shown in Table 1 below.
【００３５】[0035]
【表１】 [Table 1]
【００３６】本発明の比色定量方法は、測定対象物質、
各種酵素、発色色素生成物質及び重合体A等を水等の溶
媒中において混合し、反応させることにより行なうこと
ができる。具体的には例えば、測定対象物質を含む検体
と、各種酵素、発色色素生成物質及び重合体A等を含む
溶液とを混合することにより行なうことができる。各種
酵素、発色色素生成物質及び重合体Aを含む溶液は、こ
れらを別々に含む2以上の溶液として別々に調製し、反
応を開始するにあたりそれらを混合することにより反応
を行なうことができる。また、かかる溶液は、必要に応
じて、前記発色色素の生成を補助する物質、緩衝剤等の
他の物質を適宜含むことができる。The colorimetric quantification method of the present invention comprises a substance to be measured,
It can be carried out by mixing various enzymes, a color-developing dye forming substance, polymer A and the like in a solvent such as water and reacting them. Specifically, for example, it can be performed by mixing a sample containing the substance to be measured with a solution containing various enzymes, a color-pigment forming substance, polymer A and the like. A solution containing various enzymes, a color-developing dye-forming substance and polymer A can be prepared separately as two or more solutions containing them separately, and the reaction can be carried out by mixing them when starting the reaction. In addition, such a solution can appropriately contain other substances such as a substance that assists the formation of the color forming dye and a buffering agent, if necessary.
【００３７】本発明の比色定量方法における反応系中の
各成分の濃度は、測定対象物質、各種酵素、発色色素生
成物質及び重合体Aを含む混合液中の終濃度として、以
下の濃度とすることができる。測定対象物質の濃度は、
例えばクレアチニンの場合、0.0008〜1.6mg/dL、尿酸の
場合0.002〜2.0mg/dLとすることができるが、特に、ク
レアチニンの場合0.016mg/dL(約1.4μmol/L)以下、尿酸
の場合0.06mg/dL(3.6μmol/L)以下程度の低濃度である
場合でも、還元物質の影響を受けずに正確な測定ができ
る。オキシダーゼの濃度は1〜100u/mL、好ましくは5〜2
0u/mLとすることができる。パーオキシダーゼの濃度は1
〜100u/mL、好ましくは5〜20u/mLとすることができる。
オキシダーゼの添加割合に対するパーオキシダーゼの添
加割合は、オキシダーゼ1uに対してパーオキシダーゼ0.
3〜100uとすることができる。発色色素生成物質の濃度
は0.5〜10mmol/L、好ましくは1〜5mmol/Lとすることが
できる。さらに、発色色素生成物質と共に4-アミノアン
チピリン等の発色色素の生成を補助する物質を用いる場
合、その濃度は、0.5〜10mmol/L、好ましくは1〜5mmol/
Lとすることができる。The concentration of each component in the reaction system in the colorimetric determination method of the present invention is the following concentration as the final concentration in the mixed solution containing the substance to be measured, various enzymes, the color-pigment forming substance and the polymer A. can do. The concentration of the substance to be measured is
For example, in the case of creatinine, it can be 0.0008 to 1.6 mg / dL, and in the case of uric acid it can be 0.002 to 2.0 mg / dL, but especially 0.016 mg / dL (about 1.4 μmol / L) or less in the case of creatinine, 0.06 in the case of uric acid. Even when the concentration is as low as mg / dL (3.6 μmol / L) or less, accurate measurement can be performed without being affected by the reducing substance. The concentration of oxidase is 1 to 100 u / mL, preferably 5 to 2
It can be 0 u / mL. Peroxidase concentration is 1
It can be -100 u / mL, preferably 5-20 u / mL.
The addition ratio of peroxidase to the addition ratio of oxidase is 0.
It can be 3-100u. The concentration of the color-forming dye forming substance can be 0.5 to 10 mmol / L, preferably 1 to 5 mmol / L. Furthermore, when a substance that assists the production of a color-forming dye such as 4-aminoantipyrine is used together with the color-forming dye forming substance, the concentration thereof is 0.5 to 10 mmol / L, preferably 1 to 5 mmol / L.
It can be L.
【００３８】反応系中の重合体Aの濃度は、反応系に含
まれる測定対象物質、還元物質及び他の試薬の量に応じ
て適宜選択することができるが、具体的には例えば終濃
度として概ね0.05〜0.75w/v%であることが好ましい。The concentration of the polymer A in the reaction system can be appropriately selected according to the amounts of the substance to be measured, the reducing substance and the other reagents contained in the reaction system. It is preferably about 0.05 to 0.75 w / v%.
【００３９】反応系に重合体Aを存在させることによ
り、還元物質による影響を抑制することができる。重合
体Aは、生体膜の成分であるリン脂質と類似の構造を持
ち、タンパク質を変性、失活、沈殿させる等の悪影響が
少なく、したがって酵素に対する影響が少なく酵素の反
応性、特異性、安定性を損なうことが無い。そのため、
酵素を含む反応系に高濃度添加することも可能であり、
ビリルビンの影響回避に十分な濃度を添加することが可
能である。また予め調製した比色定量用の試薬に重合体
Aを配合してから長期間経過しても試薬の劣化が少ない
ので、予め調製した試薬による簡便な定量の操作を行な
うことができる。The presence of the polymer A in the reaction system can suppress the influence of the reducing substance. Polymer A has a structure similar to that of phospholipid, which is a component of biological membrane, and has less adverse effects such as denaturation, inactivation, and precipitation of proteins, and therefore has less effect on enzymes, reactivity, specificity, and stability of enzymes. There is no loss of sex. for that reason,
It is also possible to add a high concentration to the reaction system containing the enzyme,
It is possible to add a concentration sufficient to avoid the effects of bilirubin. In addition, the polymer was added to the previously prepared colorimetric reagent.
Since the reagent does not deteriorate much even after a long period of time since A was added, a simple quantitative operation can be performed using the reagent prepared in advance.
【００４０】本発明の比色定量方法の好ましい例とし
て、還元物質としてビリルビンを含む検体中のクレアチ
ニン、クレアチン又は尿酸を測定する方法が挙げられ
る。本発明によるクレアチニン、クレアチン及び尿酸の
比色定量は、それぞれ下記反応式(i)〜(iii)に示される
反応に基いて行なうことができる：A preferred example of the colorimetric quantification method of the present invention is a method for measuring creatinine, creatine or uric acid in a sample containing bilirubin as a reducing substance. The colorimetric determination of creatinine, creatine and uric acid according to the present invention can be carried out based on the reactions shown in the following reaction formulas (i) to (iii):
【００４１】[0041]
【化１１】 [Chemical 11]
【００４２】[0042]
【化１２】 [Chemical 12]
【００４３】[0043]
【化１３】 [Chemical 13]
【００４４】略号は次のものを示す。 CRN：クレアチニナーゼ CR：クレアチナーゼ SOD：ザルコシンオキシダーゼ POD：パーオキシダーゼ TODB：N,N-ビス(4-スルホブチル)-3-メチルアニリン 4AAP：4-アミノアンチピリンThe abbreviations indicate the following. CRN: Creatininase CR: Creatinase SOD: Sarcosine oxidase POD: Peroxidase TODB: N, N-bis (4-sulfobutyl) -3-methylaniline 4AAP: 4-aminoantipyrine
【００４５】上記反応による比色定量は、各成分を適宜
混合することにより発色させ、吸光度の変化を測定する
ことにより行なうことができる。各成分の混合の手順は
特に限定されないが、具体的には例えばクレアチニンの
定量の場合、以下の手順により行なうことができる：The colorimetric determination by the above reaction can be carried out by appropriately mixing the respective components to develop the color and measuring the change in the absorbance. The procedure for mixing the components is not particularly limited, but specifically, for example, in the case of quantifying creatinine, the following procedure can be performed:
【００４６】(1) N,N-ビス(4-スルホブチル)-3-メチル
アニリン、クレアチナーゼ及びザルコシンオキシダーゼ
を含む試薬1と、4-アミノアンチピリン、クレアチニナ
ーゼ及びパーオキシダーゼを含む試薬2とを調製する。
試薬1又は2のいずれか一方又は両方に重合体Aを配合す
る。
(2) クレアチニンとビリルビン等の還元物質とを含む検
体と、試薬1とを混合し、37℃で5分間保温する。
(3) (2)の混合物に試薬2を添加する。
(4) (3)の混合物中で発色したキノン色素の生成量を、5
00〜600nmの吸光度変化として測定することによりクレ
アチニンの濃度を定量する。(1) A reagent 1 containing N, N-bis (4-sulfobutyl) -3-methylaniline, creatinase and sarcosine oxidase and a reagent 2 containing 4-aminoantipyrine, creatininase and peroxidase were prepared. Prepare.
Polymer A is blended with either or both of Reagents 1 and 2. (2) Reagent 1 is mixed with a sample containing creatinine and a reducing substance such as bilirubin and incubated at 37 ° C for 5 minutes. (3) Add Reagent 2 to the mixture of (2). (4) The amount of quinone dye formed in the mixture of (3) was adjusted to 5
The creatinine concentration is quantified by measuring the change in absorbance from 00 to 600 nm.
【００４７】前記試薬1及び試薬2中の各成分の配合割合
は、試薬1、試薬2及び検体との混合物中の終濃度とし
て、クレアチニナーゼは20〜400u/mL、クレアチナーゼ
は50〜100u/mL、ザルコシンオキシダーゼは1〜20u/mL、
パーオキシダーゼは1〜100u/mL、好ましくは5〜20u/m
L、N,N-ビス(4-スルホブチル)-3-メチルアニリン及び4-
アミノアンチピリンはそれぞれ0.5〜10mmol/L、好まし
くは1〜5mmol/Lとすることができる。重合体Aの配合割
合は、例えば検体中に50mg/dLのビリルビンが存在し、
検体と試薬1及び2の合計との量比がおよそ1:60(体積比)
の場合、重合体Aは反応系中に、終濃度として概ね0.05
〜0.75w/v%含まれていることが好ましい。The mixing ratio of each component in the reagent 1 and the reagent 2 is such that the final concentration in the mixture of the reagent 1, reagent 2 and the sample is 20 to 400 u / mL for creatininase and 50 to 100 u / mL for creatinase. mL, 1-20 u / mL for sarcosine oxidase,
Peroxidase is 1-100u / mL, preferably 5-20u / m
L, N, N-bis (4-sulfobutyl) -3-methylaniline and 4-
The amount of aminoantipyrine can be 0.5 to 10 mmol / L, preferably 1 to 5 mmol / L. The blending ratio of the polymer A is, for example, 50 mg / dL of bilirubin is present in the sample,
Approximately 1:60 (volume ratio) between the sample and the total of Reagents 1 and 2
In the case of, the polymer A has a final concentration of about 0.05 in the reaction system.
It is preferable that the content is 0.75 w / v%.
【００４８】前記試薬1及び試薬2には、必要に応じて緩
衝剤を配合し所望のpHに維持することができる。緩衝剤
は特に限定されず通常の比色定量において用いられてい
るものを適宜選択することができる。具体的には例え
ば、2-ヒドロキシ-N-トリス(ヒドロキシメチル)メチル-
3-アミノプロパンスルホン酸(以下「TAPSO」という。)
又はN-トリス(ヒドロキシメチル)メチル-3-アミノプロ
パンスルホン酸(以下「TAPS」という。)を10〜1000mmol
/Lの範囲内で配合し、さらに塩酸、水酸化ナトリウム等
で所望のpHに調整することができる。A buffer may be added to the above-mentioned reagent 1 and reagent 2, if necessary, to maintain a desired pH. The buffer is not particularly limited, and those used in ordinary colorimetric determination can be appropriately selected. Specifically, for example, 2-hydroxy-N-tris (hydroxymethyl) methyl-
3-Aminopropanesulfonic acid (hereinafter referred to as "TAPSO")
Alternatively, 10-1000 mmol of N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (hereinafter referred to as “TAPS”).
It can be mixed within the range of / L and further adjusted to a desired pH with hydrochloric acid, sodium hydroxide or the like.
【００４９】本発明の比色定量試薬は、前記本発明の比
色定量方法のための試薬であって、溶媒と、酵素と、重
合体Aとを含む。当該溶媒としては、水等を挙げること
ができる。当該酵素としては、オキシダーゼ、パーオキ
シダーゼ、測定対象物質を基質としてオキシダーゼによ
り過酸化水素を定量的に発生させうる物質を生成しうる
酵素、又はこれらの組み合わせを含むことができる。ま
た、必要に応じて、上記の緩衝剤、発色色素生成物質、
発色色素生成を補助する物質等を含むことができる。よ
り具体的には例えば、上記試薬1及び2のように、2液以
上を混合することにより本発明における反応を行なえる
ようにした複数の試薬の組み合わせにおいて、それら複
数の試薬の1以上において重合体Aを含んだ試薬とするこ
とができる。The colorimetric assay reagent of the present invention is a reagent for the colorimetric assay method of the present invention, and comprises a solvent, an enzyme and a polymer A. Water etc. can be mentioned as the said solvent. The enzyme may include oxidase, peroxidase, an enzyme capable of producing a substance capable of quantitatively generating hydrogen peroxide by the oxidase using a substance to be measured as a substrate, or a combination thereof. In addition, if necessary, the above-mentioned buffering agent, color-forming pigment-forming substance,
A substance or the like that assists the formation of a coloring pigment can be included. More specifically, for example, as in the case of reagents 1 and 2 described above, in a combination of a plurality of reagents capable of carrying out the reaction in the present invention by mixing two or more liquids, one or more of the plurality of reagents do not overlap. It can be a reagent containing the combination A.
【００５０】以下、実施例に基づいて、本発明をさらに
詳細に説明するが、本発明はこれらに限定されない。な
お、実施例中、同一の略語で示された試薬等は、別に断
らない限り同一の製品を用いた。The present invention will be described in more detail based on the following examples, but the invention is not intended to be limited thereto. In the examples, the same products were used for reagents and the like indicated by the same abbreviations unless otherwise specified.
【００５１】合成例 1
(PC重合体-1の合成)
メタクリロイルホスホリルコリン(MPC) 29.1g及びステ
アリルメタクリレート(C18MA)8.4gをエタノール204gに
溶解し4つ口フラスコに入れ、30分間窒素を吹き込んだ
後に、60℃でアゾビスイソブチロニトリル(以下AIBNと
略す)0.67gを加えて8時間重合反応させた。反応終了
後、反応液を3Lのジエチルエーテル中に撹拌しながら滴
下し、析出した沈殿を濾過し、48時間室温で真空乾燥を
行って、PC重合体-1 30.1gを粉末として得た。 Synthesis Example 1 ( Synthesis of PC Polymer-1) 29.1 g of methacryloylphosphorylcholine (MPC) and 8.4 g of stearyl methacrylate (C 18 MA) were dissolved in 204 g of ethanol, placed in a 4-neck flask, and blown with nitrogen for 30 minutes. After that, 0.67 g of azobisisobutyronitrile (hereinafter abbreviated as AIBN) was added at 60 ° C. and a polymerization reaction was carried out for 8 hours. After completion of the reaction, the reaction solution was dropped into 3 L of diethyl ether while stirring, and the deposited precipitate was filtered and vacuum dried at room temperature for 48 hours to obtain 30.1 g of PC polymer-1 as a powder.
【００５２】合成例 2
(PC重合体-2の合成)
MPC50.0gをエタノール100gに溶解し4つ口フラスコに入
れ、30分間窒素を吹き込んだ後に、60℃でAIBN 0.24gを
加えて8時間重合反応させた。反応終了後、反応液を3L
のジエチルエーテル中に撹拌しながら滴下し、析出した
沈殿を濾過し、48時間室温で真空乾燥を行って、PC重合
体-2 29.6gを粉末として得た。 Synthesis Example 2 ( Synthesis of PC Polymer-2) MPC (50.0 g) was dissolved in ethanol (100 g), placed in a four-necked flask, blown with nitrogen for 30 minutes, and then AIBN (0.24 g) was added at 60 ° C. for 8 hours. A polymerization reaction was carried out. After the reaction is complete, add 3 L of the reaction solution.
Was added dropwise to diethyl ether with stirring, the deposited precipitate was filtered, and vacuum dried at room temperature for 48 hours to obtain 29.6 g of PC polymer-2 as a powder.
【００５３】合成例 3
(PC重合体-3の合成)
MPC 35.7g及びブチルメタクリレート(BMA)4.3gをエタノ
ール160gに溶解し4つ口フラスコに入れ、30分間窒素を
吹き込んだ後に、60℃でAIBN 0.82gを加えて8時間重合
反応させた。反応終了後、反応液を3Lのジエチルエーテ
ル中に撹拌しながら滴下し、析出した沈殿を濾過し、48
時間室温で真空乾燥を行って、PC重合体-3 29.6gを粉末
として得た。 Synthesis Example 3 ( Synthesis of PC Polymer-3) MPC (35.7 g) and butyl methacrylate (BMA) (4.3 g) were dissolved in ethanol (160 g), placed in a four-necked flask, and blown with nitrogen for 30 minutes, then at 60 ° C. AIBN 0.82g was added and the polymerization reaction was carried out for 8 hours. After completion of the reaction, the reaction solution was added dropwise to 3 L of diethyl ether while stirring, and the deposited precipitate was filtered,
After vacuum drying at room temperature for 2 hours, 29.6 g of PC polymer-3 was obtained as a powder.
【００５４】合成例 4
(PC重合体-4の合成)
MPC 23.0g及び式CH2=C(CH3)-CO-(OCH2CH2)4-OHで表され
るメトキシポリ(エチレンオキシド)モノメタクリレート
7.7gをエタノール160gに溶解し4つ口フラスコに入れ、3
0分間窒素を吹き込んだ後に、60℃でAIBN 0.82gを加え
て8時間重合反応させた。反応終了後、反応液を3Lのジ
エチルエーテル中に撹拌しながら滴下し、析出した沈殿
を濾過し、48時間室温で真空乾燥を行って、PC重合体-4
19.8gを粉末として得た。 Synthesis Example 4 ( Synthesis of PC polymer-4) MPC 23.0 g and methoxypoly (ethylene oxide) monomethacrylate represented by the formula CH 2 = C (CH 3 ) -CO- (OCH 2 CH 2 ) 4- OH.
Dissolve 7.7 g in 160 g of ethanol and put in a four-necked flask.
After bubbling nitrogen for 0 minutes, 0.82 g of AIBN was added at 60 ° C. to carry out a polymerization reaction for 8 hours. After completion of the reaction, the reaction solution was added dropwise to 3 L of diethyl ether with stirring, the deposited precipitate was filtered, and vacuum dried at room temperature for 48 hours to obtain PC polymer-4.
19.8 g was obtained as a powder.
【００５５】合成例 5
(PC重合体-5の合成)
MPC 12.0g及びメタクリル酸8.0gを水40gに溶解し4つ口
フラスコに入れ、30分間窒素を吹き込んだ後に、60℃で
コハク酸パーオキサイド 0.82gを加えて8時間重合反応
させた。反応終了後、反応液を3Lのジエチルエーテル中
に撹拌しながら滴下し、析出した沈殿を濾過し、48時間
室温で真空乾燥を行って、PC重合体-5 19.8gを粉末とし
て得た。 Synthesis Example 5 ( Synthesis of PC Polymer-5) 12.0 g of MPC and 8.0 g of methacrylic acid were dissolved in 40 g of water, placed in a 4-necked flask, and blown with nitrogen for 30 minutes, and then at 60 ° C. 0.82 g of oxide was added and a polymerization reaction was carried out for 8 hours. After completion of the reaction, the reaction solution was added dropwise to 3 L of diethyl ether while stirring, and the deposited precipitate was filtered and vacuum dried at room temperature for 48 hours to obtain 19.8 g of PC polymer-5 as a powder.
【００５６】合成例 6
(PC重合体-6の合成)
MPC 13.4gと、2-ヒドロキシ-3-メタクリロイルオキシプ
ロピルトリメチルアンモニウムクロライド50w/w%含有水
溶液9.2gとを水77gに溶解し4つ口フラスコに入れ、30分
間窒素を吹き込んだ後に、60℃でコハク酸パーオキサイ
ド 0.15gを加えて8時間重合反応させた。反応終了後、
反応液を3Lのジエチルエーテル中に撹拌しながら滴下
し、析出した沈殿を濾過し、48時間室温で真空乾燥を行
って、PC重合体-6 14.8gを粉末として得た。 Synthesis Example 6 ( Synthesis of PC Polymer-6) MPC (13.4 g) and 2-hydroxy-3-methacryloyloxypropyltrimethylammonium chloride (50 w / w% aqueous solution 9.2 g) were dissolved in water (77 g) to give a four-port mixture. The mixture was placed in a flask, and after blowing nitrogen for 30 minutes, 0.15 g of succinic acid peroxide was added at 60 ° C. and a polymerization reaction was carried out for 8 hours. After the reaction,
The reaction solution was added dropwise to 3 L of diethyl ether with stirring, the deposited precipitate was filtered, and vacuum dried at room temperature for 48 hours to obtain 14.8 g of PC polymer-6 as a powder.
【００５７】合成例1〜5で得たPC重合体-1〜6の分子量
をゲルパーミエーションクロマトグラフィー(GPC)によ
り分析した。分析条件は20mMリン酸緩衝液(pH7.4)を溶
離液とし、ポリエチレングリコールを標準物質とし、共
重合体の溶出を屈折率により検出した。結果を表２に示
す。また、これらの重合体の共重合組成比を1H-NMRによ
り分析した。結果を表２に示す。The molecular weights of the PC polymers-1 to 6 obtained in Synthesis Examples 1 to 5 were analyzed by gel permeation chromatography (GPC). The analysis conditions were 20 mM phosphate buffer (pH 7.4) as the eluent, polyethylene glycol as the standard substance, and the elution of the copolymer was detected by the refractive index. The results are shown in Table 2. Further, the copolymerization composition ratio of these polymers was analyzed by 1 H-NMR. The results are shown in Table 2.
【００５８】[0058]
【表２】 [Table 2]
【００５９】実施例 1-1 〜 1-4 及び比較例 1
(ビリルビン存在下及び非存在下におけるクレアチニン
濃度測定の対比；その1)
TAPSO 50mmo1/L、TODB 1mmo1/L、CR(キッコーマン株式
会社製)30u/mL及びSOD(キッコーマン株式会社製)10u/m
L、ならびに各種濃度のPC重合体-1を含む水溶液を調製
し、1mo1/Lの塩酸または水酸化ナトリウムでpH8.0に調
整し、試薬1-1を得た。なお、試薬1-1中のPC重合体-1の
濃度は、後述する試薬1-2及び血清検体1又は2と混合し
た際の終濃度が0.00w/v%(比較例1)、0.15w/v%(実施例1-
1)、0.30w/v%(実施例1-2)、0.45w/v%(実施例1-3)又は0.
75w/v%(実施例1-4)となる濃度とした。 Examples 1-1 to 1-4 and Comparative Example 1 ( Comparison of Creatinine Concentration Measurement in the Presence and Absence of Bilirubin; Part 1) TAPSO 50mmo1 / L, TODB 1mmo1 / L, CR (manufactured by Kikkoman Corporation) ) 30u / mL and SOD (manufactured by Kikkoman Corporation) 10u / m
An aqueous solution containing L and PC polymer-1 at various concentrations was prepared and adjusted to pH 8.0 with 1 mol / L hydrochloric acid or sodium hydroxide to obtain reagent 1-1. The concentration of PC polymer-1 in the reagent 1-1, the final concentration when mixed with the reagent 1-2 and serum sample 1 or 2 described later 0.00w / v% (Comparative Example 1), 0.15w / v% (Example 1-
1), 0.30 w / v% (Example 1-2), 0.45 w / v% (Example 1-3) or 0.
The concentration was 75 w / v% (Example 1-4).
【００６０】一方、TAPSO 50mmo1/L、4AAP 1mmo1/L、CR
N(キッコーマン株式会社製)400u/mL及びPOD(天野エンザ
イム株式会社製)10u/mLを含む水溶液を調製し、lmo1/L
の塩酸または水酸化ナトリウムでpH8.0に調整し、試薬1
-2を得た。On the other hand, TAPSO 50mmo1 / L, 4AAP 1mmo1 / L, CR
Prepare an aqueous solution containing N (manufactured by Kikkoman Corporation) 400u / mL and POD (manufactured by Amano Enzyme Inc.) 10u / mL, lmo1 / L
Adjust the pH to 8.0 with hydrochloric acid or sodium hydroxide, and then use reagent 1
I got -2.
【００６１】クレアチニン0.85mg/dL、尿酸3.1mg/dL及
びクレアチン1.0mg/dLを含有するヒト血清(商品名「ス
イトロールN」、日本水産株式会社製、ベース血清)(血
清検体1)、及びそれにビリルビンを50mg/dL添加したも
の(血清検体2)を調製した。Human serum containing creatinine 0.85 mg / dL, uric acid 3.1 mg / dL and creatine 1.0 mg / dL (trade name "Sitolol N", Nippon Suisan Co., Ltd., base serum) (serum sample 1), and Bilirubin (50 mg / dL) was added (serum sample 2).
【００６２】5μLの血清検体1又は2に、試薬1-1を240μ
L添加し、37℃で5分間加温後、更に試薬1-2を80μL加え
て、546nmの吸光度変化を自動分析装置H7170型((株)日
立製作所製)を用いて、反応系内の温度を37℃に保って
測定した。測定方法にはエンドポイント法を適用した。240 μl of reagent 1-1 was added to 5 μl of serum sample 1 or 2.
After adding L and heating at 37 ° C for 5 minutes, 80 μL of reagent 1-2 was further added, and the change in absorbance at 546 nm was measured using an automatic analyzer H7170 type (manufactured by Hitachi, Ltd.) and the temperature in the reaction system. Was kept at 37 ° C. for measurement. The endpoint method was applied as the measurement method.
【００６３】血清検体1における吸光度変化に対する、
血清検体2における吸光度変化を、相対感度(%)として求
めた。結果を表３に示す。With respect to the change in absorbance in the serum sample 1,
The change in absorbance in serum sample 2 was determined as relative sensitivity (%). The results are shown in Table 3.
【００６４】[0064]
【表３】 [Table 3]
【００６５】PC重合体-1を添加しなかった比較例1で
は、ビリルビンに基く-56%の負の影響を認めたが、PC重
合体-1を添加した実施例1-1〜1-4においてはビリルビン
の影響が軽減し、-4〜-2%の負の影響に止まった。よっ
てPC重合体-1にビリルビンの影響を軽減する効果がある
ことがわかる。以上より、本発明の比色定量方法及び比
色定量試薬を用いることにより、還元物質であるビリル
ビンの影響を軽減し、クレアチニン濃度を正確に定量で
きることがわかった。In Comparative Example 1 in which PC Polymer-1 was not added, a negative effect of -56% based on bilirubin was recognized, but in Examples 1-1 to 1-4 in which PC Polymer-1 was added, , The effect of bilirubin was reduced, and the negative effect remained at -4 to -2%. Therefore, it is understood that PC polymer-1 has an effect of reducing the influence of bilirubin. From the above, it was found that the use of the colorimetric assay method and colorimetric assay reagent of the present invention can reduce the influence of bilirubin, which is a reducing substance, and accurately quantify the creatinine concentration.
【００６６】実施例 2-1 〜 2-5 及び比較例 2
(ビリルビン存在下及び非存在下におけるクレアチニン
濃度測定の対比；その2)
TAPS 50mmo1/L、TODB 1mmo1/L、CR 50u/mL及びSOD 15u/
mLを含む水溶液を調製し、1mo1/L塩酸または水酸化ナト
リウムでpH8.0に調整し、試薬2-1を得た。 Examples 2-1 to 2-5 and Comparative Example 2 ( Comparison of creatinine concentration measurement in the presence and absence of bilirubin; Part 2) TAPS 50mmo1 / L, TODB 1mmo1 / L, CR 50u / mL and SOD 15u /
An aqueous solution containing mL was prepared, and the pH was adjusted to 8.0 with 1mo1 / L hydrochloric acid or sodium hydroxide to obtain Reagent 2-1.
【００６７】一方、TAPS 100mmo1/L、4AAP 3mmo1/L、CR
N 450u/mL及びPOD 15u/mL、並びに各種濃度のPC重合体-
1を含む水溶液を調製し、1mo1/Lの塩酸または水酸化ナ
トリウムでpH8.0に調整し、試薬2-2を得た。なお、試薬
2-2中のPC重合体-1の濃度は、試薬2-1及び血清検体1又
は2と混合した際の終濃度が0.00%(比較例2)、0.05%(実
施例2-1)、0.10%(実施例2-2)、0.15%(実施例2-3)、0.20
%(実施例2-4)又は0.25%(実施例2-5)(W/V)となる濃度と
した。On the other hand, TAPS 100mmo1 / L, 4AAP 3mmo1 / L, CR
N 450u / mL and POD 15u / mL, and PC polymers of various concentrations-
An aqueous solution containing 1 was prepared and adjusted to pH 8.0 with 1 mol / L hydrochloric acid or sodium hydroxide to obtain a reagent 2-2. In addition, the reagent
The concentration of PC polymer-1 in 2-2, the final concentration when mixed with reagent 2-1 and serum sample 1 or 2 0.00% (Comparative Example 2), 0.05% (Example 2-1), 0.10% (Example 2-2), 0.15% (Example 2-3), 0.20
% (Example 2-4) or 0.25% (Example 2-5) (W / V).
【００６８】5μLの血清検体1又は2に、試薬2-1を240μ
L添加し、37℃で5分間加温後、更に試薬2-2を80μL加え
て、546nmの吸光度変化を自動分析装置H7170型を用いて
測定した。測定方法にはエンドポイント法を適用した。240 μl of reagent 2-1 was added to 5 μl of serum sample 1 or 2.
After adding L and heating at 37 ° C. for 5 minutes, 80 μL of reagent 2-2 was further added, and the change in absorbance at 546 nm was measured using an automatic analyzer H7170 type. The endpoint method was applied as the measurement method.
【００６９】血清検体1における吸光度変化に対する、
血清検体2における吸光度変化を、相対感度(%)として求
めた。結果を表４に示す。With respect to the change in absorbance in the serum sample 1,
The change in absorbance in serum sample 2 was determined as relative sensitivity (%). The results are shown in Table 4.
【００７０】[0070]
【表４】 [Table 4]
【００７１】PC重合体-1を添加しなかった比較例2で
は、ビリルビンに基く-56%の負の影響を認めたが、PC重
合体-1を添加した実施例2-1〜2-5においてはビリルビン
の影響が軽減し、-17〜-5%の負の影響に止まった。よっ
てPC重合体-1にビリルビンの影響を軽減する効果がある
ことがわかる。以上より、本発明の比色定量方法及び比
色定量試薬を用いることにより、還元物質であるビリル
ビンの影響を軽減し、クレアチニン濃度を正確に定量で
きることがわかった。In Comparative Example 2 in which PC Polymer-1 was not added, a negative effect of -56% based on bilirubin was observed, but in Examples 2-1 to 2-5 in which PC Polymer-1 was added, , The effect of bilirubin was reduced, and the negative effect remained at -17 to -5%. Therefore, it is understood that PC polymer-1 has an effect of reducing the influence of bilirubin. From the above, it was found that the use of the colorimetric assay method and colorimetric assay reagent of the present invention can reduce the influence of bilirubin, which is a reducing substance, and accurately quantify the creatinine concentration.
【００７２】また実施例1及び2の結果より、PC重合体-1
は、クレアチナーゼを含む試薬(試薬1-1又は2-1)とクレ
アチニナーゼを含む試薬(試薬1-2又は試薬2-2)とを混合
する反応系において、そのいずれの試薬に添加してもビ
リルビンの影響の軽減に有効であることがわかる。From the results of Examples 1 and 2, PC polymer-1
Is a reaction system in which a reagent containing creatinase (reagent 1-1 or 2-1) and a reagent containing creatininase (reagent 1-2 or reagent 2-2) are mixed, and added to any of the reagents. It can be seen that is also effective in reducing the effect of bilirubin.
【００７３】実施例 3
(他のベタイン型界面活性剤を含む反応系との対比)
TAPS 50mmo1/L、TODB 1mmo1/L、CR 50u/mL、SOD 15u/m
L、及びPC重合体-1 0.3w/v%を含む水溶液を調製し、1mo
1/Lの塩酸または水酸化ナトリウムでpH7.9に調整し、試
薬3-1を得た。 Example 3 (Comparison with Reaction System Containing Other Betaine Surfactant) TAPS 50mmo1 / L, TODB 1mmo1 / L, CR 50u / mL, SOD 15u / m
Prepare an aqueous solution containing L and PC polymer-1 0.3 w / v% and
The pH was adjusted to 7.9 with 1 / L hydrochloric acid or sodium hydroxide to obtain Reagent 3-1.
【００７４】一方、TAPS 100mmo1/L、4AAP 3mmo1/L、CR
N 450u/mL及びPOD 15u/mLを含む水溶液を調製し、1mo1/
Lの塩酸または水酸化ナトリウムでpH8.3に調整し、試薬
3-2を得た。On the other hand, TAPS 100mmo1 / L, 4AAP 3mmo1 / L, CR
Prepare an aqueous solution containing N 450u / mL and POD 15u / mL, and add 1mo1 /
Adjust to pH 8.3 with L hydrochloric acid or sodium hydroxide, and then
Got 3-2.
【００７５】5μLの血清検体1又は2に、試薬3-1を240μ
L添加し、37℃で5分間加温後、更に試薬3-2を80μL加え
て、546nmの吸光度変化を自動分析装置H7170型を用いて
測定した。測定方法にはエンドポイント法を適用した。To 5 μL of serum sample 1 or 2, 240 μL of reagent 3-1
After adding L and heating at 37 ° C. for 5 minutes, 80 μL of reagent 3-2 was further added, and the change in absorbance at 546 nm was measured using an automatic analyzer H7170 type. The endpoint method was applied as the measurement method.
【００７６】血清検体1における吸光度変化に対する、
血清検体2における吸光度変化を、相対感度(%)として求
めた。結果を表５に示す。For the change in absorbance in the serum sample 1,
The change in absorbance in serum sample 2 was determined as relative sensitivity (%). The results are shown in Table 5.
【００７７】比較例 3-1
PC重合体-1 0.3w/v%を添加しなかった他は、実施例3と
同様に操作し、試薬を調製し、反応させ、吸光度変化を
測定し、血清検体1における吸光度変化に対する、血清
検体2における吸光度変化を、相対感度(%)として求め
た。結果を表５に示す。 Comparative Example 3-1 PC Polymer-1 In the same manner as in Example 3 except that 0.3 w / v% was not added, the reagents were prepared and reacted, the change in absorbance was measured, and the serum was measured. The change in absorbance of serum sample 2 with respect to the change in absorbance of sample 1 was determined as relative sensitivity (%). The results are shown in Table 5.
【００７８】比較例 3-2 〜 3-12
PC重合体-1 0.3w/v%の代わりに、アンヒトール24B(商品
名、花王株式会杜製、ラウリルベタイン)(比較例3-2)、
アンヒトール20BS(商品名、花王株式会杜製、ラウリル
ベタイン)(比較例3-3)、AM-3130N(商品名、日光ケミカ
ルズ株式会杜製、ヤシ油脂肪酸アミドプロピルジメチル
アミノ酢酸ベタインR-CO-NH-(CH2)3-N+(CH3)2-CH2COO-)
(比較例3-4)、AM-301(商品名、日光ケミカルズ株式会
杜製、ラウリルジメチルアミノ酢酸ベタインR-N+(CH3)2
-CH2-COO-)(比較例3-5)、SB3-8(商品名、3-(N,N-ジメチ
ルオクチルアンモニオ)プロパンスルホン酸、シグマ社
製)(比較例3-6)、SB3-12(商品名、3-(ドデシルジメチル
アンモニオ)プロパンスルホン酸、シグマ社製)(比較例3
-7)、又はPC重合体-2〜6(比較例3-8〜3-12)を0.3w/v%添
加した他は、実施例3と同様に操作し、試薬を調製し、
反応させ、吸光度変化を測定し、血清検体1における吸
光度変化に対する、血清検体2における吸光度変化を、
相対感度(%)として求めた。結果を表５に示す。 Comparative Examples 3-2 to 3-12 PC Polymer-1 0.3 w / v% was replaced with Amphitol 24B (trade name, manufactured by Kao Shikaikai Co., Ltd., lauryl betaine) (Comparative Example 3-2),
Amphitol 20BS (trade name, manufactured by Kao Co., Ltd., lauryl betaine) (Comparative Example 3-3), AM-3130N (trade name, manufactured by Nikko Chemicals Co., Ltd., coconut oil fatty acid amide propyl dimethylamino acetic acid betaine R-CO- NH- (CH 2) 3 -N + (CH 3) 2 -CH 2 COO -)
(Comparative Example 3-4), AM-301 (trade name, manufactured by Nikko Chemicals Co., Ltd., lauryl dimethylamino acetic acid betaine RN + (CH 3 ) 2
-CH 2 -COO -) (Comparative Example 3-5), SB3-8 (trade name, 3- (N, N-dimethyl octyl ammonio) propane sulfonic acid, Sigma) (Comparative Example 3-6), SB3-12 (trade name, 3- (dodecyldimethylammonio) propanesulfonic acid, manufactured by Sigma) (Comparative Example 3
-7), or PC polymer-2 to 6 (Comparative Examples 3-8 to 3-12) except that 0.3w / v% was added, the same operation as in Example 3, to prepare a reagent,
React, measure the absorbance change, for the absorbance change in the serum sample 1, the absorbance change in the serum sample 2,
It was calculated as relative sensitivity (%). The results are shown in Table 5.
【００７９】[0079]
【表５】 [Table 5]
【００８０】各試薬でのビリルビンの影響は、PC重合体
-1の場合、-1%；アンヒトール24Bの場合、-8%；アンヒ
トール20BSの場合、-7%；AM-3130Nの場合、-6%、AM-301
の場合、-19%；SB3-8の場合、-9%；SB3-12の場合、-9
%；PC重合体-2〜6の場合、概ね-50%であった。この結果
よりPC重合体-1は、他のベタイン型界面活性剤と比較し
て、ビリルビンの影響をおよそ1/5以下、また他のPC重
合体と比較して1/50以下とすることができ、ビリルビン
の影響を軽減する効果が著しいことがわかる。以上よ
り、ベタイン系界面活性剤(比較例3-2〜比較例3-7)、及
び重合体A以外のPC重合体(比較例3-8〜比較例3-12)を用
いた場合と比較すると、本発明の比色定量方法及び比色
定量試薬を用いることにより、還元物質であるビリルビ
ンの影響を軽減し、クレアチニン濃度を正確に定量でき
ることがわかった。The influence of bilirubin on each reagent was
In the case of -1, -1%; in the case of Amphitol 24B -8%; in the case of Amphitol 20BS -7%; In the case of AM-3130N -6%, AM-301
In case of, -19%; In case of SB3-8, -9%; In case of SB3-12, -9
%; In the case of PC polymer-2 to 6, it was about -50%. From this result, PC polymer-1, compared with other betaine type surfactants, the effect of bilirubin is about 1/5 or less, and compared with other PC polymers 1/50 or less. It can be seen that the effect of reducing the effect of bilirubin is remarkable. From the above, compared with the case of using a betaine surfactant (Comparative Example 3-2 ~ Comparative Example 3-7), and a PC polymer other than the polymer A (Comparative Example 3-8 ~ Comparative Example 3-12). Then, it was found that by using the colorimetric method and colorimetric assay reagent of the present invention, the effect of bilirubin, which is a reducing substance, can be reduced and the creatinine concentration can be accurately quantified.
【００８１】実施例 4
(試薬の保存安定性;その1)
TAPS 50mmo1/L、TODB 1mmo1/L、CR 50u/mL、SOD 15u/m
L、及びPC重合体-1 0.3w/v%を含む水溶液を調製し、1mo
1/Lの塩酸または水酸化ナトリウムでpH7.9に調整し、試
薬4-1を得た。 Example 4 (Storage Stability of Reagent; Part 1) TAPS 50mmo1 / L, TODB 1mmo1 / L, CR 50u / mL, SOD 15u / m
Prepare an aqueous solution containing L and PC polymer-1 0.3 w / v% and
The pH was adjusted to 7.9 with 1 / L hydrochloric acid or sodium hydroxide to obtain reagent 4-1.
【００８２】一方、TAPS 100mmo1/L、4AAP 3mmo1/L、CR
N 450u/mL及びPOD 15u/mLを含む水溶液を調製し、1mo1/
Lの塩酸または水酸化ナトリウムでpH8.3に調整し、試薬
4-2を得た。On the other hand, TAPS 100mmo1 / L, 4AAP 3mmo1 / L, CR
Prepare an aqueous solution containing N 450u / mL and POD 15u / mL, and add 1mo1 /
Adjust to pH 8.3 with L hydrochloric acid or sodium hydroxide, and then
Got 4-2.
【００８３】さらに、検体として、クレアチニンの5mg/
dL水溶液(検体3)を調製した。Further, as a sample, 5 mg of creatinine /
A dL aqueous solution (Sample 3) was prepared.
【００８４】試薬4-1を、調製後直ちに(実施例4A)又は
調製後37℃で1週間保存後(実施例4B)、5μLの検体3に24
0μL添加し、37℃で5分間加温後、更に試薬4-2を80μL
加えて、546nmの吸光度変化を自動分析装置H7170型を用
いて測定した。吸光度変化は、試薬4-1を添加した時点
を0分として、経時的に測定した。結果を図1に示す。図
1中、白抜き円及び中黒円のプロットは、それぞれ実施
例4A及び実施例4Bの結果をそれぞれ示す。Reagent 4-1 was added to 5 μL of sample 3 immediately after preparation (Example 4A) or after storage at 37 ° C. for 1 week (Example 4B).
Add 0 μL and warm at 37 ° C for 5 minutes, then add 80 μL of Reagent 4-2.
In addition, the change in absorbance at 546 nm was measured using an automatic analyzer H7170 type. The change in absorbance was measured over time, with 0 minute being the time when reagent 4-1 was added. The results are shown in Figure 1. Figure
Plots of white circles and black circles in 1 show the results of Example 4A and Example 4B, respectively.
【００８５】図1の結果より、PC重合体-1を添加した試
薬を用いた反応系においては、37℃で1週間保存した後
にも反応速度の低下が見られず、PC重合体-1による試薬
の安定性の低下がないことがわかる。From the results of FIG. 1, in the reaction system using the reagent to which PC polymer-1 was added, the reaction rate did not decrease even after storage for 1 week at 37 ° C. It can be seen that there is no decrease in reagent stability.
【００８６】比較例 4-1
PC重合体-1 0.3w/v%を添加しなかった他は、実施例4A及
び4Bと同様に操作し、試薬を調製し、反応させ、吸光度
変化を経時的に測定した。結果を図2に示す。図2中、白
抜き円及び中黒円のプロットは、それぞれ試薬4-1を調
製後直ちに用いた場合(比較例4-1A)又は調製後37℃で1
週間保存後に用いた場合(比較例4-1B)の結果をそれぞれ
示す。 Comparative Example 4-1 PC Polymer-1 In the same manner as in Examples 4A and 4B except that 0.3 w / v% was not added, the reagents were prepared and reacted, and the change in absorbance with time Measured. The result is shown in figure 2. In FIG. 2, the plots of the open circles and the filled circles represent the cases where the reagent 4-1 was used immediately after preparation (Comparative Example 4-1A) or 1 at 37 ° C. after preparation.
The results when used after storage for a week (Comparative Example 4-1B) are shown.
【００８７】比較例 4-2 〜 4-3
PC重合体-1 0.3w/v%の代わりに、アンヒトール24B(比較
例4-2)、又はAM-301(比較例4-3)を0.3w/v%添加した他
は、実施例4A及び4Bと同様に操作し、試薬を調製し、反
応させ、吸光度変化を経時的に測定した。比較例4-2及
び4-3の結果をそれぞれ図3及び図4に示す。図3及び図4
中、白抜き円及び中黒円のプロットは、それぞれ試薬4-
1を調製後直ちに用いた場合(比較例4-2A、4-3A)又は調
製後37℃で1週間保存後に用いた場合(比較例4-2B、4-3
B)の結果をそれぞれ示す。 Comparative Examples 4-2 to 4-3 PC Polymer-1 0.3 w / v% was replaced by 0.3 w of Amphitol 24B (Comparative Example 4-2) or AM-301 (Comparative Example 4-3). A reagent was prepared and reacted in the same manner as in Examples 4A and 4B except that / v% was added, and changes in absorbance were measured over time. The results of Comparative Examples 4-2 and 4-3 are shown in FIGS. 3 and 4, respectively. 3 and 4
The plots of the middle circles, open circles, and middle circles are for reagent 4-
When 1 was used immediately after preparation (Comparative Examples 4-2A, 4-3A) or after storage for 1 week at 37 ° C. after preparation (Comparative Examples 4-2B, 4-3)
The results of B) are shown respectively.
【００８８】実施例4及び比較例4-1〜4-3において、各
反応系中のクレアチニン濃度の指標として、試薬4-2を
添加した後35秒から71秒間の吸光度(mAbs)の変化量を算
出した。実施例4Aの吸光度変化量に対する実施例4Bの吸
光度変化量の割合を、保存安定性(%)として求めた。ま
た、比較例4-1〜4-3についても、同様に保存安定性を求
めた。求めた値を表６に示す。また、これらの結果と並
べて、実施例3、比較例3-1、アンヒトール24Bを用いた
比較例3-2、及びAM-201を用いた比較例3-5におけるビリ
ルビンに対する効果についての実験結果を、併せて表６
に示す。In Example 4 and Comparative Examples 4-1 to 4-3, as an index of the creatinine concentration in each reaction system, the amount of change in absorbance (mAbs) from 35 seconds to 71 seconds after addition of the reagent 4-2. Was calculated. The ratio of the amount of change in absorbance of Example 4B to the amount of change in absorbance of Example 4A was determined as the storage stability (%). Further, also in Comparative Examples 4-1 to 4-3, storage stability was similarly obtained. Table 6 shows the obtained values. In addition, side by side with these results, Example 3, Comparative Example 3-1, Comparative Example 3-2 using Amphitol 24B, and the experimental results for the effect on bilirubin in Comparative Example 3-5 using AM-201. , Together with Table 6
Shown in.
【００８９】[0089]
【表６】 [Table 6]
【００９０】表６より、本願発明の比色定量試薬は、そ
の保存安定性に優れることが分かった。更に、表3、表
4、表5及び表6より、本発明の比色定量方法及び比色定
量試薬を用いることにより、還元物質であるビリルビン
の影響を軽減しクレアチニン濃度を正確に定量すること
ができ、且つ試薬の保存安定性を維持できることがわか
った。From Table 6, it was found that the colorimetric reagent of the present invention has excellent storage stability. Furthermore, Table 3, Table
4, From Table 5 and Table 6, by using the colorimetric method and colorimetric reagent of the present invention, it is possible to accurately quantify the creatinine concentration by reducing the effect of bilirubin, which is a reducing substance, and It was found that storage stability can be maintained.
【００９１】実施例 5
(尿酸の測定)
水に、N,N-ビス(2-ヒドロキシエチル)-2-アミノエタン
スルホン酸(以下BESという。)50mmol/L、TODB 2mmol/L
及びPOD 5u/mL、ならびにPC重合体-1を溶解し、1mol/L
の塩酸又は水酸化ナトリウムでpHを7.5に調整し、試薬5
-1を調製した。なお、試薬5-1中のPC重合体-1の濃度
は、後述する試薬5-2及び血清検体1又は2と混合した際
の終濃度が0.4w/v%となる濃度とした。 Example 5 (Measurement of uric acid) In water, N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (hereinafter referred to as BES) 50 mmol / L, TODB 2 mmol / L
, POD 5u / mL, and PC polymer-1 dissolved, 1mol / L
Adjust the pH to 7.5 with hydrochloric acid or sodium hydroxide, and
-1 was prepared. The concentration of the PC polymer-1 in the reagent 5-1 was set to a concentration at which the final concentration when mixed with the reagent 5-2 and the serum sample 1 or 2 described later was 0.4 w / v%.
【００９２】一方、水に、BES 50mmol/L、4AAP 2mmol/L
及びウリカーゼ(東洋紡績株式会社製) 1u/mLを溶解し、
1mol/Lの塩酸又は水酸化ナトリウムでpHを7.5に調整
し、試薬5-2を調製した。On the other hand, in water, BES 50 mmol / L, 4AAP 2 mmol / L
And uricase (manufactured by Toyobo Co., Ltd.) 1u / mL is dissolved,
Reagent 5-2 was prepared by adjusting the pH to 7.5 with 1 mol / L hydrochloric acid or sodium hydroxide.
【００９３】5μLの血清検体1又は2に、試薬5-1を240μ
L添加し、37℃で5分間加温後、更に試薬5-2を60μL加え
て、546nmの吸光度を自動分析装置H7170型((株)日立製
作所製)を用いて、反応系内の温度を37℃に保って測定
した。測定方法にはエンドポイント法を適用した。240 μl of reagent 5-1 was added to 5 μl of serum sample 1 or 2.
After adding L and heating at 37 ° C for 5 minutes, 60 μL of reagent 5-2 was further added, and the absorbance at 546 nm was measured using an automatic analyzer H7170 type (manufactured by Hitachi, Ltd.) to measure the temperature in the reaction system. The measurement was performed by keeping at 37 ° C. The endpoint method was applied as the measurement method.
【００９４】血清検体1における吸光度変化に対する、
血清検体2における吸光度変化を、相対感度(%)として求
めた。結果を表７に示す。With respect to the change in absorbance in the serum sample 1,
The change in absorbance in serum sample 2 was determined as relative sensitivity (%). The results are shown in Table 7.
【００９５】比較例 5
試薬5-1にPC重合体-1を添加しなかった他は、実施例5と
同様に操作し、吸光度変化を測定した。結果を表７に示
す。 Comparative Example 5 The procedure of Example 5 was repeated except that the PC polymer-1 was not added to the reagent 5-1 and the change in absorbance was measured. The results are shown in Table 7.
【００９６】[0096]
【表７】 [Table 7]
【００９７】PC重合体-1を添加しなかった比較例5にお
いては、ビリルビンに基づく-73%の負の影響を認めた
が、PC重合体-1を添加した実施例5においてはビリルビ
ンの影響が軽減し、-5％の負の影響に止まった。以上よ
り、本発明の比色定量方法及び比色定量試薬を用いるこ
とにより、還元物質であるビリルビンの影響を軽減し、
尿酸濃度を正確に定量できることがわかった。In Comparative Example 5 in which PC Polymer-1 was not added, a negative effect of -73% based on bilirubin was observed, but in Example 5 in which PC Polymer-1 was added, the effect of bilirubin was observed. Has been reduced and stopped at a negative impact of -5%. From the above, by using the colorimetric method and colorimetric reagent of the present invention, reduce the effect of bilirubin is a reducing substance,
It was found that the uric acid concentration can be accurately quantified.
【００９８】実施例 6
(クレアチンの測定)
水に、TAPS 30mmol/L、TODB 1mmol/L及びSOD 15u/mL、
ならびにPC重合体-1を溶解し、1mol/Lの塩酸又は水酸化
ナトリウムでpHを8.2に調整し、試薬6-1を調製した。な
お、試薬6-1中のPC重合体-1の濃度は、後述する試薬6-2
及び血清検体1又は2と混合した際の終濃度が0.4w/v%と
なる濃度とした。 Example 6 (Measurement of creatine) In water, TAPS 30 mmol / L, TODB 1 mmol / L and SOD 15 u / mL,
Also, the PC polymer-1 was dissolved and the pH was adjusted to 8.2 with 1 mol / L hydrochloric acid or sodium hydroxide to prepare a reagent 6-1. The concentration of the PC polymer-1 in the reagent 6-1 is the reagent 6-2 described later.
And the final concentration when mixed with serum sample 1 or 2 was 0.4 w / v%.
【００９９】一方、水に、TAPS 30mmol/L、4AAP 2mmol/
L、CR150u/mL及びPOD 20u/mLを溶解し、1mol/Lの塩酸又
は水酸化ナトリウムでpHを8.0に調整し、試薬6-2を調製
した。On the other hand, in water, TAPS 30 mmol / L, 4AAP 2 mmol / L
Reagent 6-2 was prepared by dissolving L, CR 150 u / mL and POD 20 u / mL and adjusting the pH to 8.0 with 1 mol / L hydrochloric acid or sodium hydroxide.
【０１００】5μLの血清検体1又は2に、試薬6-1を240μ
L添加し、37℃で5分間加温後、更に試薬6-2を80μL加え
て、546nmの吸光度から600nmの吸光度を減じた値の変化
を自動分析装置H7170型((株)日立製作所製)を用いて、
反応系内の温度を37℃に保って測定した。測定方法には
エンドポイント法を適用した。To 5 μL of serum sample 1 or 2, add 240 μ of reagent 6-1.
After adding L and heating at 37 ° C for 5 minutes, 80 μL of reagent 6-2 was further added, and the change in the value obtained by subtracting the absorbance at 600 nm from the absorbance at 546 nm was changed to an automatic analyzer H7170 type (manufactured by Hitachi, Ltd.). Using,
The temperature in the reaction system was kept at 37 ° C. for measurement. The endpoint method was applied as the measurement method.
【０１０１】血清検体1における吸光度変化に対する、
血清検体2における吸光度変化を、相対感度(%)として求
めた。結果を表８に示す。With respect to the change in absorbance in the serum sample 1,
The change in absorbance in serum sample 2 was determined as relative sensitivity (%). The results are shown in Table 8.
【０１０２】比較例 6
試薬6-1にPC重合体-1を添加しなかった他は、実施例6と
同様に操作し、吸光度変化を測定した。結果を表８に示
す。 Comparative Example 6 The change in absorbance was measured in the same manner as in Example 6 except that the PC polymer-1 was not added to the reagent 6-1. The results are shown in Table 8.
【０１０３】[0103]
【表８】 [Table 8]
【０１０４】PC重合体-1を添加しなかった比較例6にお
いては、ビリルビンに基づく-72%の負の影響を認めた
が、PC重合体-1を添加した実施例6においてはビリルビ
ンの影響が軽減し、-3％の負の影響に止まった。以上よ
り、本発明の比色定量方法及び比色定量試薬を用いるこ
とにより、還元物質であるビリルビンの影響を軽減し、
クレアチン濃度を正確に定量できることがわかった。In Comparative Example 6 in which PC Polymer-1 was not added, a negative effect of -72% based on bilirubin was observed, but in Example 6 in which PC Polymer-1 was added, the effect of bilirubin was observed. Has been reduced to a negative impact of -3%. From the above, by using the colorimetric method and colorimetric reagent of the present invention, reduce the effect of bilirubin is a reducing substance,
It was found that the creatine concentration can be accurately quantified.
【０１０５】[0105]
【図１】実施例4における、反応時間と吸光度との関係
を示すグラフである。FIG. 1 is a graph showing the relationship between reaction time and absorbance in Example 4.
【図２】比較例4-1における、反応時間と吸光度との関
係を示すグラフである。FIG. 2 is a graph showing the relationship between reaction time and absorbance in Comparative Example 4-1.
【図３】比較例4-2における、反応時間と吸光度との関
係を示すグラフである。FIG. 3 is a graph showing the relationship between reaction time and absorbance in Comparative Example 4-2.
【図４】比較例4-3における、反応時間と吸光度との関
係を示すグラフである。FIG. 4 is a graph showing the relationship between reaction time and absorbance in Comparative Example 4-3.
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開2002−159299（ＪＰ，Ａ) 特開 平７−39394（ＪＰ，Ａ) 特開 平９−89867（ＪＰ，Ａ) 特開 平11−103888（ＪＰ，Ａ) 特開 平11−243993（ＪＰ，Ａ) 特開 平３−10696（ＪＰ，Ａ) 特開2002−22740（ＪＰ，Ａ) (58)調査した分野(Int.Cl.7，ＤＢ名) G01N 33/70 C12Q 1/26 C12Q 1/28 G01N 33/531 ─────────────────────────────────────────────────── --Continued from the front page (56) References JP-A 2002-159299 (JP, A) JP-A 7-39394 (JP, A) JP-A 9-89867 (JP, A) JP-A 11-103888 (JP, A) JP 11-243993 (JP, A) JP 3-10696 (JP, A) JP 2002-22740 (JP, A) (58) Fields investigated (Int. Cl. 7 , DB name) G01N 33/70 C12Q 1/26 C12Q 1/28 G01N 33/531
Claims (4)
尿酸を測定対象物質とし、酵素反応により過酸化水素を
導き、パーオキシダーゼの存在下で発色色素を生成させ
るクレアチニン、クレアチン又は尿酸の比色定量方法に
おいて、反応系に下記式(I)： 【化１】 (ここで、Rは水素原子またはメチル基を示す。) 及び式(II)： 【化２】 (ここで、Rは水素原子又はメチル基を示す。Xは、炭素
数12〜20のアルキル基を示す。)で表される単量体に基
く単位からなる、重量平均分子量5,000〜5,000,000の重
合体Aを存在させることを特徴とする、クレアチニン、
クレアチン又は尿酸の比色定量方法。1. A colorimetric quantification method for creatinine, creatine or uric acid, wherein creatinine, creatine or uric acid in a sample is used as a substance to be measured, and hydrogen peroxide is introduced by an enzymatic reaction to produce a coloring dye in the presence of peroxidase. In the reaction system, the following formula (I): (Wherein R represents a hydrogen atom or a methyl group) and the formula (II): (Wherein R represents a hydrogen atom or a methyl group, X represents an alkyl group having 12 to 20 carbon atoms), and a weight-average molecular weight of 5,000 to 5,000,000 Creatinine, characterized by the presence of coalescence A,
Colorimetric determination of creatine or uric acid.
めの比色定量試薬であって、溶媒と、酵素と、前記重合
体Aとを含む比色定量試薬。2. A colorimetric reagent for the colorimetric method according to claim 1, comprising a solvent, an enzyme, and the polymer A.
ン、クレアチン又は尿酸を測定対象物質とし、酵素反応
により過酸化水素を導き、パーオキシダーゼの存在下で
発色色素を生成させる、クレアチニン、クレアチン又は
尿酸の比色定量において検体中のビリルビンの影響を抑
制する方法であって、反応系に下記式(I)： 【化３】 (ここで、Rは水素原子またはメチル基を示す。) 及び式(II)： 【化４】 (ここで、Rは水素原子又はメチル基を示す。Xは、炭素
数12〜20のアルキル基を示す。)で表わされる単量体に
基く単位からなる、重量平均分子量5,000〜5,000,000の
重合体Aを存在させることを特徴とする抑制方法。3. A ratio of creatinine, creatine or uric acid which uses creatinine, creatine or uric acid in a sample containing bilirubin as a measurement target substance and leads hydrogen peroxide by an enzymatic reaction to form a coloring pigment in the presence of peroxidase. A method for suppressing the influence of bilirubin in a sample in color quantification, wherein the reaction system is represented by the following formula (I): (Wherein R represents a hydrogen atom or a methyl group) and the formula (II): (Here, R represents a hydrogen atom or a methyl group. X represents an alkyl group having 12 to 20 carbon atoms.) A polymer having a weight average molecular weight of 5,000 to 5,000,000, which is composed of a unit based on a monomer A method for suppressing the presence of A.
尿酸を測定対象物質とし、酵素反応により過酸化水素を
導き、パーオキシダーゼの存在下で発色色素を生成させ
るクレアチニン、クレアチン又は尿酸の比色定量におけ
る測定試薬の保存安定化方法であって、反応系に下記式
(I)： 【化５】 (ここで、Rは水素原子またはメチル基を示す。) 及び式(II)： 【化６】 (ここで、Rは水素原子又はメチル基を示す。Xは、炭素
数12〜20のアルキル基を示す。) で表される単量体に基く単位からなる、重量平均分子量
5,000〜5,000,000の重合体Aを存在させることを特徴と
する保存安定化方法。4. A method for colorimetric determination of creatinine, creatine or uric acid in which a creatinine, creatine or uric acid in a sample is used as a substance to be measured, and hydrogen peroxide is introduced by an enzymatic reaction to produce a coloring dye in the presence of peroxidase. A method for stabilizing the storage of reagents, comprising the following formula in the reaction system:
(I): [Chemical 5] (Wherein R represents a hydrogen atom or a methyl group) and the formula (II): (Here, R represents a hydrogen atom or a methyl group. X represents an alkyl group having 12 to 20 carbon atoms.) A weight average molecular weight composed of a unit based on a monomer
A storage stabilization method comprising the presence of 5,000 to 5,000,000 polymer A.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016179981A1 (en) * | 2015-05-11 | 2016-11-17 | 深圳贝申医疗技术有限公司 | Automatic detection method and system for neonatal jaundice |
| CN112525844A (en) * | 2020-11-12 | 2021-03-19 | 德莱福(重庆)医疗器械有限公司 | Method for testing urea concentration in stable dialyzer clearance rate simulation solution |
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|---|---|---|---|---|
| JP6203708B2 (en) * | 2012-04-27 | 2017-09-27 | 協和メデックス株式会社 | Method for measuring components to be measured in a sample |
| EP2972275B1 (en) | 2013-03-14 | 2019-07-10 | Instrumentation Laboratory Company | Whole blood hemolysis sensor |
| KR102077301B1 (en) * | 2018-08-30 | 2020-02-13 | 한양대학교 산학협력단 | Method, Apparatus and Composition for Detecting Uric Acid |
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2003
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016179981A1 (en) * | 2015-05-11 | 2016-11-17 | 深圳贝申医疗技术有限公司 | Automatic detection method and system for neonatal jaundice |
| CN112525844A (en) * | 2020-11-12 | 2021-03-19 | 德莱福(重庆)医疗器械有限公司 | Method for testing urea concentration in stable dialyzer clearance rate simulation solution |
| CN112525844B (en) * | 2020-11-12 | 2024-06-04 | 德莱福(重庆)医疗器械有限公司 | Stable urea concentration test method in dialyser clearance simulation liquid |
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