JP2005034132A - Method for colorimetric determination - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for colorimetric determination influenced by less effects from reducing substances such as bilirubin in a specimen, a method for stabilizing measuring reagents, a method for inhibiting the effect with bilirubin, and to obtain reagents used for the determination, capable of being preserved for a long period, or the like, in a determination mediated by H<SB>2</SB>O<SB>2</SB>. <P>SOLUTION: The method for a colorimetric determination inducing hydrogen peroxide from creatinine, creatine or uric acid in a specimen by an enzymatic reaction and producing a color-developing pigment in the presence of a peroxidase is provided by coexisting a polymer consisting of units based on monomers expressed by formula (I) (R is H or methyl) or formula (II) (R is H or methyl; and X is a 12-20C alkyl) and having 5,000-5,000,000 molecular weight in the reaction system. The methods for stabilizing the preservation and inhibiting the bilirubin are provided by coexisting the polymer in the reaction system. The reagent contains the polymer. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a colorimetric method for clinical diagnosis and the like.

  There are two methods for measuring biochemical items in clinical tests: chemical methods and enzymatic methods. Among these methods, for example, there are 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 oxidase. The method using oxidase is more sensitive than other methods because it is more sensitive, so it is suitable for measuring trace amounts of substances.However, because it is a measurement system that uses hydrogen peroxide, if there is a substance having a reducing action in the sample, There is a problem that hydrogen peroxide is reduced, which affects measurement results and interferes with accurate measurement (hereinafter, substances that exhibit a reducing action that is not intended for quantification are simply referred to as `` reducing substances ''. Called).

  Specific examples of the reducing substance include hemoglobin released by hemolysis at the time of blood collection, bilirubin present in a high concentration in the blood of patients with liver disease, ascorbic acid taken as a nutrient, liver, red blood cells, white blood cells, etc. And reduced glutathione (GSH) present in the above solution.

  These reducing substances are not so much a problem when measuring the concentration of a relatively high concentration of the target substance, but cause a large measurement error when measuring the concentration of a low concentration of the target substance. . In other words, when a certain amount of hydrogen peroxide is erased by these reducing substances, the lower the concentration of the substance to be measured, the greater the influence.

  For example, creatinine, which is a marker for renal function, is one of the measurement items that require accurate measurement, but its amount in the sample is very small compared to other biochemical items. For this reason, even if hydrogen peroxide is slightly erased by the reducing substance, the measured value is significantly affected. However, since creatinine requires accurate measurement because of its clinical significance, it is required to accurately measure even at low concentrations while avoiding the effects of reducing substances as much as possible.

  As a means to avoid the influence of reducing substances in the measurement system via hydrogen peroxide, a method of adding a cationic surfactant or an amphoteric surfactant to the reagent (Patent Document 4), and a betaine surfactant as a reagent A method of adding (Patent Document 5) has been proposed. However, it cannot be said that these methods completely solve the influence of a reducing substance such as bilirubin, and it is still not sufficient in the measurement of creatinine.

  In addition, ionic surfactants, when made into a solution together with other components such as enzymes, have the property of denaturing or precipitating other components over time, so they affect performance, especially the storage stability of other components. The impact on is great. This is a big problem at present when most of the reagents for the above-mentioned reaction for clinical examination are provided as liquid reagents. If a sufficient amount of the ionic surfactant is added to avoid the influence of the reducing substance, the reaction of the enzyme or the like is prevented accordingly. As a means for solving this problem, it is conceivable to add an excessive amount of a component involved in the reaction such as an enzyme. However, in this case, there is a problem that a manufacturing cost is required.

  By the way, in clinical diagnosis, methods for measuring in the presence of a polymer having a structural unit based on (meth) acryloylphosphorylcholine are disclosed in Patent Documents 1 to 3, for example. Patent Document 1 discloses a reaction accelerator for antigen-antibody reaction, enzyme-substrate reaction, 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 for measuring a C-reactive protein by using a polymer such as (meth) acryloylphosphorylcholine and avoiding the prozone phenomenon without diluting by measurement using latex.

However, in the colorimetric determination method in which the substance to be measured in the sample is led to hydrogen peroxide by oxidase and this is generated in the presence of peroxidase, the influence of the reducing substance such as bilirubin is suppressed and the object to be measured There is no disclosure of a method that enables highly accurate measurement even at low concentrations of substances.
JP 2002-22740 (2nd page) JP 2002-365296 A (page 2) JP 2001-318099 (2nd page) Japanese Patent Laid-Open No. 3-10696 Japanese Unexamined Patent Publication No. 7-39394

  The object of the present invention is to provide a colorimetric determination method, a method for stabilizing a measurement reagent, or an effect of bilirubin that is less affected by the presence of a reducing substance such as bilirubin contained in a sample in the determination of a measurement target substance through hydrogen peroxide It is in providing the method of suppressing.

  Another object of the present invention is a reagent used for the determination of a substance to be measured via hydrogen peroxide, which can be stored for a long period of time without impairing the reactivity, specificity, stability, etc. of the enzyme, and An object of the present invention is to provide a reagent capable of performing quantification with little influence by the presence of a reducing substance such as bilirubin contained in a specimen.

  As a result of intensive studies in view of the above problems, the present inventors have found that when a specific polymer is present in the reaction system, the influence can be suppressed even if a reducing substance such as bilirubin is present. The present invention has been completed.

  That is, according to the present invention, creatinine, creatine or uric acid in a specimen is used as a substance to be measured, hydrogen peroxide is induced by an enzymatic reaction, and a chromogenic dye is produced in the presence of peroxidase. In the quantification method, the reaction system has the following formula (I):

(Wherein R represents a hydrogen atom or a methyl group) and formula (II):

(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。

(Wherein R represents a hydrogen atom or a methyl group, X represents an alkyl group having 12 to 20 carbon atoms), and a unit having 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 by the presence of a polymer (hereinafter referred to as polymer A).

  Furthermore, according to the present invention, there is provided a colorimetric reagent for colorimetric determination, which comprises a solvent, an enzyme, and the polymer A.

  Furthermore, according to the present invention, creatinine, creatine, or uric acid, which is targeted for measurement of creatinine, creatine, or uric acid in a sample containing bilirubin, induces hydrogen peroxide by an enzymatic reaction and generates a coloring pigment in the presence of peroxidase. There is provided a method for suppressing the influence of bilirubin in a sample in the colorimetric determination of the method, wherein the polymer A is present in a reaction system.

  Furthermore, according to the present invention, colorimetric determination of creatinine, creatine, or uric acid, which uses creatinine, creatine, or uric acid in a sample as a measurement target, induces hydrogen peroxide by an enzymatic reaction, and generates a coloring pigment in the presence of peroxidase. A method for stabilizing the storage of a measuring reagent in the above-mentioned method, wherein the polymer A is present in a reaction system is provided.

  In the colorimetric determination method of the present invention, the stabilization method of the present invention, and the suppression method of the present invention, the presence of a specific polymer in the reaction system does not impair the reactivity, specificity, stability, etc. of the enzyme. The effect of reducing substances can be reduced. For example, it is possible to suppress the negative effect of 50% or more to 5% or less. Therefore, in the colorimetric determination method of the present invention, accurate determination close to the true value can be easily performed even when the measurement target substance in the sample has a low concentration.

  Since the colorimetric reagent of the present invention contains a specific polymer in addition to the components such as the enzyme, it can be stored for a long period of time, and it does not impair the reactivity, specificity, stability, etc. of the enzyme. Even when the measurement target substance has a low concentration, accurate quantification close to the true value can be easily performed.

  The colorimetric determination method of the present invention, the stabilization method of the present invention, or the suppression method of the present invention is characterized in that a specific polymer (polymer A) is present in the reaction system. The polymer A includes a monomer represented by the formula (I) (hereinafter referred to as monomer A1) and a monomer represented by the formula (II) (hereinafter referred to as monomer A2). It consists of units based on Specifically, a unit represented by the following formula (III) and a unit represented by (IV) can be included. In the following formulas (III) and (IV), R and X are the same as those in the formulas (I) and (II).

In the formula (II) or (IV), when the alkyl group represented by X has 12 or more carbon atoms, the influence of the reducing substance present in the reaction system can be reduced well.
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 unit of the formula (IV) may be copolymerized in any form such as random polymerization, alternating polymerization, block copolymerization and the like.

  The polymer A can be produced by copolymerizing the monomers A1 and A2 by ordinary radical copolymerization or the like, and further dissolving and purifying as necessary in a solvent or the like. Specifically, it can be produced according to the methods described in Patent Documents 1 to 3.

  These ratios when copolymerizing the monomer A1 and the monomer A2 are not particularly limited, but are usually monomer A1 / monomer A2 = 99/1 to 10/90 in molar ratio, preferably 90/10 to 20/80, more preferably 80/20 to 30/70. By setting the ratio of the monomer A to the sum of the monomers A1 and A2 to 10 mol% or more, the effect of the phosphorylcholine group can be sufficiently exerted.

  The molecular weight of the polymer A is in the range of 5,000 to 5,000,000 as the weight average molecular weight, and preferably in the range of 10,000 to 1,000,000. When the molecular weight of the polymer A is 5,000 or more, the biochemical reaction can be sufficiently promoted, and when it is 5,000,000 or less, the inhibition of the biochemical reaction due to the viscosity of the polymer A can be avoided.

  In the colorimetric determination 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 led to hydrogen peroxide by an enzymatic reaction, and then peroxidase In the presence of the chromogenic dye.

  The substance to be measured is creatinine, creatine or uric acid.

  As the enzyme reaction, a reaction for deriving hydrogen peroxide from the substance to be measured can be appropriately selected. The enzyme reaction may be a single-stage reaction or a multi-stage reaction. The enzyme reaction includes, in at least one stage, a reaction for 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.

  The peroxidase is not particularly limited as long as it can reduce hydrogen peroxide to water to produce a coloring pigment. Specifically, for example, peroxidase derived from horseradish or soybean can be used.

  The coloring dye is not particularly limited as long as it can be generated according to the amount of hydrogen peroxide by peroxidase, and various quinone dyes and the like can be generated. The coloring dye can be generated by combining a coloring agent-forming substance that generates the coloring dye according to the amount of hydrogen peroxide in combination with peroxidase in the reaction system. As the coloring dye-forming substance, a leuco body or various Trinder reagents can be used. The Trinder reagent can be used in combination with a substance that assists the production of a coloring dye such as 4-aminoantipyrine. Specific examples of the leuco form of the coloring dye-forming substance include N- (carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) -diphenylamine and 10- (carboxymethylaminocarbonyl). ) -3,7'-bis (dimethylamino) -phenocyanazine and the like. In addition, the Trinder reagent of the coloring dye-forming substance specifically includes, 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.

  Specific examples of the measurement target substances for colorimetric determination of the present invention and combinations of enzymes used for the measurement are shown in Table 1 below.

  The colorimetric determination method of the present invention can be carried out by mixing and reacting a substance to be measured, various enzymes, a coloring pigment forming substance, polymer A and the like in a solvent such as water. Specifically, for example, it can be carried out by mixing a specimen containing the substance to be measured with a solution containing various enzymes, a coloring dye-forming substance, polymer A and the like. A solution containing various enzymes, a coloring dye-forming substance, and a polymer A can be prepared by separately preparing them as two or more solutions containing these separately, and mixing them before starting the reaction. In addition, such a solution can appropriately contain other substances such as a substance that assists the production of the coloring dye and a buffer as necessary.

  In the colorimetric determination method of the present invention, the concentration of each component in the reaction system may be the following concentration as the final concentration in the mixed solution containing the substance to be measured, various enzymes, the coloring pigment-forming substance, and the polymer A. it can. The concentration of the substance to be measured can be, for example, 0.0008 to 1.6 mg / dL in the case of creatinine and 0.002 to 2.0 mg / dL in the case of uric acid, but in particular 0.0116 mg / dL (about 1.4 μmol / L in the case of creatinine. ) Hereinafter, in the case of uric acid, even when the concentration is as low as 0.06 mg / dL (3.6 μmol / L) or less, accurate measurement can be performed without being affected by the reducing substance. The concentration of the oxidase can be 1 to 100 u / mL, preferably 5 to 20 u / mL. The concentration of peroxidase can be 1 to 100 u / mL, preferably 5 to 20 u / mL. The peroxidase addition ratio relative to the oxidase addition ratio can be 0.3 to 100 u peroxidase per oxidase 1u. The concentration of the color forming substance can be 0.5 to 10 mmol / L, preferably 1 to 5 mmol / L. Further, when a substance that assists the production of a coloring dye such as 4-aminoantipyrine is used together with the coloring dye-forming substance, the concentration thereof can be 0.5 to 10 mmol / L, preferably 1 to 5 mmol / L.

  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 other reagents contained in the reaction system. Specifically, for example, the final concentration is generally 0.05 to It is preferably 0.75 w / v%.

  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 phospholipids, which are components of biological membranes, and has little adverse effects such as denaturation, inactivation, and precipitation, and therefore has little effect on enzymes, and the reactivity, specificity, and stability of enzymes. There is no loss of sex. Therefore, it is possible to add a high concentration to the reaction system containing the enzyme, and it is possible to add a concentration sufficient to avoid the influence of bilirubin. Further, since the deterioration of the reagent is small even after a long period of time since the polymer A is blended with the colorimetric reagent prepared in advance, a simple quantitative operation with the reagent prepared in advance can be performed.

  A preferred example of the colorimetric determination method of the present invention is a method of measuring creatinine, creatine or uric acid in a specimen 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), respectively:

Abbreviations indicate the following:
CRN: Creatininase
CR: creatinase
SOD: Sarcosine oxidase
POD: Peroxidase
TODB: N, N-bis (4-sulfobutyl) -3-methylaniline
4AAP: 4-aminoantipyrine

  The colorimetric determination by the above reaction can be performed by coloring each component appropriately and measuring the change in absorbance. The procedure for mixing each component is not particularly limited, and specifically, for example, in the case of quantification of creatinine, it can be performed by the following procedure:

(1) Reagent 1 containing N, N-bis (4-sulfobutyl) -3-methylaniline, creatinase and sarcosine oxidase and Reagent 2 containing 4-aminoantipyrine, creatininase and peroxidase are prepared. Polymer A is blended in either or both of Reagents 1 and 2.
(2) A sample containing creatinine and a reducing substance such as bilirubin and reagent 1 are mixed and incubated at 37 ° C. for 5 minutes.
(3) Add Reagent 2 to the mixture of (2).
(4) The concentration of creatinine is quantified by measuring the amount of quinone dye produced in the mixture of (3) as a change in absorbance at 500 to 600 nm.

  The mixing ratio of each component in Reagent 1 and Reagent 2 is 20 to 400 u / mL for creatininase, 50 to 100 u / mL for creatinase, and colander as final concentrations in the mixture of Reagent 1, Reagent 2 and the specimen. Cosin oxidase is 1 to 20 u / mL, peroxidase is 1 to 100 u / mL, preferably 5 to 20 u / mL, and N, N-bis (4-sulfobutyl) -3-methylaniline and 4-aminoantipyrine are each 0.5 to It can be 10 mmol / L, preferably 1 to 5 mmol / L. The blending ratio of polymer A is, for example, when 50 mg / dL of bilirubin is present in the specimen, and when the quantity ratio of the specimen and the total of reagents 1 and 2 is approximately 1:60 (volume ratio), polymer A reacts. It is preferable that the final concentration is generally 0.05 to 0.75 w / v% in the system.

  The reagent 1 and the reagent 2 can be mixed with a buffer as necessary to maintain a desired pH. The buffer is not particularly limited, and a buffer used in normal colorimetric determination can be appropriately selected. Specifically, for example, 2-hydroxy-N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (hereinafter referred to as “TAPSO”) or N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid ( (Hereinafter referred to as “TAPS”) within a range of 10 to 1000 mmol / L, and further adjusted to a desired pH with hydrochloric acid, sodium hydroxide or the like.

  The colorimetric reagent of the present invention is a reagent for the colorimetric method of the present invention, and includes a solvent, an enzyme, and a polymer A. Examples of the solvent include water. Examples of the enzyme include oxidase, peroxidase, an enzyme that can generate a substance capable of quantitatively generating hydrogen peroxide by oxidase using a substance to be measured as a substrate, or a combination thereof. Further, if necessary, the above-mentioned buffering agent, a coloring dye-forming substance, a substance for assisting the coloring dye generation, and the like can be included. More specifically, for example, in the combination of a plurality of reagents capable of performing the reaction in the present invention by mixing two or more liquids, such as the above-described reagents 1 and 2, in one or more of the plurality of reagents, A reagent containing the combined A can be obtained.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to these. In the examples, the same products were used for the reagents indicated by the same abbreviations unless otherwise noted.

Synthesis example 1
(Synthesis of PC polymer-1)
29.1 g of methacryloyl phosphorylcholine (MPC) and 8.4 g of stearyl methacrylate (C ₁₈ MA) are dissolved in 204 g of ethanol, put into a four-necked flask, blown with nitrogen for 30 minutes, and then azobisisobutyronitrile (hereinafter referred to as AIBN) at 60 ° C. And 0.67 g 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 with 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.

Synthesis example 2
(Synthesis of PC polymer-2)
MPC (50.0 g) was dissolved in ethanol (100 g), placed in a four-necked flask, and nitrogen was blown for 30 minutes. Then, AIBN (0.24 g) 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 with stirring, and 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.

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 nitrogen was blown for 30 minutes. After completion of the reaction, the reaction solution was added dropwise to 3 L of diethyl ether with stirring, and the deposited precipitate was filtered and vacuum dried at room temperature for 48 hours to obtain 29.6 g of PC polymer-3 as a powder.

Synthesis example 4
(Synthesis of PC polymer-4)
MPC 23.0 g and the formula CH ₂ ═C (CH ₃ ) —CO— (OCH ₂ CH ₂ ) ₄ —OH methoxypoly (ethylene oxide) monomethacrylate 7.7 g was dissolved in ethanol 160 g and placed in a four-necked flask, After nitrogen was blown for 30 minutes, 0.82 g of 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 added dropwise to 3 L of diethyl ether with stirring, and the deposited precipitate was filtered and vacuum dried at room temperature for 48 hours to obtain 19.8 g of PC polymer-4 as a powder.

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, put into a four-necked flask, and nitrogen was blown for 30 minutes. Then, 0.82 g of succinic acid peroxide was added at 60 ° C., and a polymerization reaction was performed for 8 hours. After completion of the reaction, the reaction solution was added dropwise to 3 L of diethyl ether with 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.

Synthesis example 6
(Synthesis of PC polymer-6)
13.4 g of MPC and 9.2 g of 2-hydroxy-3-methacryloyloxypropyltrimethylammonium chloride 50 w / w% aqueous solution were dissolved in 77 g of water, placed in a four-necked flask, blown with nitrogen for 30 minutes, and then heated at 60 ° C. 0.15 g of succinic acid peroxide 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 with stirring, and 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.

The molecular weights of 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 eluent, polyethylene glycol as a standard substance, and elution of the copolymer was detected by refractive index. The results are shown in Table 2. Further, the copolymer composition ratio of these polymers was analyzed by ¹ H-NMR. The results are shown in Table 2.

Examples 1-1 to 1-4 and Comparative Example 1
(Contrast of creatinine concentration measurement in the presence and absence of bilirubin; part 1)
Prepare an aqueous solution containing TAPSO 50mmo1 / L, TODB 1mmo1 / L, CR (manufactured by Kikkoman Corporation) 30u / mL and SOD (manufactured by Kikkoman Corporation) 10u / mL, and various concentrations of PC polymer-1. The pH was adjusted to 8.0 with L hydrochloric acid or sodium hydroxide to obtain Reagent 1-1. The concentration of the PC polymer-1 in the reagent 1-1 is 0.00w / v% (Comparative Example 1), 0.15w at the final concentration when mixed with the reagent 1-2 and the serum sample 1 or 2 described later. / v% (Example 1-1), 0.30 w / v% (Example 1-2), 0.45 w / v% (Example 1-3) or 0.75 w / v% (Example 1-4) Concentration.

  Meanwhile, an aqueous solution containing TAPSO 50mmo1 / L, 4AAP 1mmo1 / L, CRN (Kikkoman Co., Ltd.) 400u / mL and POD (Amano Enzyme Co., Ltd.) 10u / mL was prepared, and lmo1 / L hydrochloric acid or sodium hydroxide was prepared. To pH 8.0 to obtain Reagent 1-2.

  Human serum containing creatinine 0.85mg / dL, uric acid 3.1mg / dL and creatine 1.0mg / dL (trade name `` Suitrol N '', manufactured by Nihon Suisan Co., Ltd., base serum) (serum sample 1), and 50 mg of bilirubin A sample to which / dL was added (serum sample 2) was prepared.

  To 5 μL of serum sample 1 or 2, add 240 μL of reagent 1-1, warm at 37 ° C. for 5 minutes, then add 80 μL of reagent 1-2, and change the absorbance at 546 nm using the automatic analyzer H7170 The temperature in the reaction system was kept at 37 ° C. The endpoint method was applied as the measurement method.

  The change in absorbance in serum sample 2 relative to the change in absorbance in serum sample 1 was determined as relative sensitivity (%). The results are shown in Table 3.

  In Comparative Example 1 in which PC polymer-1 was not added, a negative effect of -56% based on bilirubin was observed, but in Examples 1-1 to 1-4 in which PC polymer-1 was added, bilirubin was added. The effect of 軽 減 was reduced, and the negative effect was -4 to -2%. Therefore, it can be seen that PC polymer-1 has an effect of reducing the influence of bilirubin. From the above, it was found that by using the colorimetric determination method and colorimetric reagent of the present invention, the influence of bilirubin as a reducing substance can be reduced and the creatinine concentration can be accurately determined.

Examples 2-1 to 2-5 and Comparative Example 2
(Contrast of creatinine concentration measurement in the presence and absence of bilirubin; Part 2)
An aqueous solution containing TAPS 50mmo1 / L, TODB 1mmo1 / L, CR 50u / mL and SOD 15u / mL was prepared, and adjusted to pH 8.0 with 1mo1 / L hydrochloric acid or sodium hydroxide to obtain Reagent 2-1.

  On the other hand, TAPS 100mmo1 / L, 4AAP 3mmo1 / L, CRN 450u / mL and POD 15u / mL, and aqueous solutions containing various concentrations of PC polymer-1 were prepared, and pH 8 with 1mo1 / L hydrochloric acid or sodium hydroxide. Adjustment to 0 gave reagent 2-2. The concentration of PC polymer-1 in Reagent 2-2 was 0.00% (Comparative Example 2), 0.05% (Example 2-) when mixed with Reagent 2-1 and serum sample 1 or 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) concentration It was.

  Add 240 μL of reagent 2-1 to 5 μL of serum sample 1 or 2 and warm at 37 ° C. for 5 minutes, then add 80 μL of reagent 2-2 and change the absorbance at 546 nm using automatic analyzer H7170 It was measured. The endpoint method was applied as the measurement method.

  The change in absorbance in serum sample 2 relative to the change in absorbance in serum sample 1 was determined as relative sensitivity (%). The results are shown in Table 4.

  In Comparative Example 2 where PC polymer-1 was not added, a negative effect of -56% based on bilirubin was observed, but in Examples 2-1 to 2-5 where PC polymer-1 was added, bilirubin was added. The effect of 軽 減 was reduced, and the negative effect was -17 to -5%. Therefore, it can be seen that PC polymer-1 has an effect of reducing the influence of bilirubin. From the above, it was found that by using the colorimetric determination method and colorimetric reagent of the present invention, the influence of bilirubin as a reducing substance can be reduced and the creatinine concentration can be accurately determined.

  Further, from the results of Examples 1 and 2, PC polymer-1 is a reagent containing creatinase (reagent 1-1 or 2-1) and a reagent containing creatininase (reagent 1-2 or reagent 2-2). It can be seen that, in a reaction system in which the mixture is added, it is effective to reduce the influence of bilirubin even if it is added to any of the reagents.

Example 3
(Comparison with other reaction systems containing betaine surfactants)
Prepare an aqueous solution containing TAPS 50mmo1 / L, TODB 1mmo1 / L, CR 50u / mL, SOD 15u / mL, and PC polymer-1 0.3w / v%, pH 7 with 1mo1 / L hydrochloric acid or sodium hydroxide. Adjustment to 9 gave reagent 3-1.

  Meanwhile, prepare an aqueous solution containing TAPS 100mmo1 / L, 4AAP 3mmo1 / L, CRN 450u / mL and POD 15u / mL, adjust the pH to 8.3 with 1mo1 / L hydrochloric acid or sodium hydroxide, and add reagent 3-2. Obtained.

  Add 240 μL of reagent 3-1 to 5 μL of serum sample 1 or 2 and warm at 37 ° C. for 5 minutes, then add 80 μL of reagent 3-2, and change the absorbance at 546 nm using automatic analyzer H7170 It was measured. The endpoint method was applied as the measurement method.

  The change in absorbance in serum sample 2 relative to the change in absorbance in serum sample 1 was determined as relative sensitivity (%). The results are shown in Table 5.

Comparative Example 3-1
PC polymer-1 The procedure was the same as in Example 3 except that 0.3 w / v% was not added, and the reagent was prepared, reacted, the change in absorbance was measured, and the change in absorbance in serum sample 1 was measured. The change in absorbance in Sample 2 was determined as relative sensitivity (%). The results are shown in Table 5.

Comparative Examples 3-2 to 3-12
PC polymer-1 Instead of 0.3 w / v%, Amphitol 24B (trade name, manufactured by Kao Corporation, lauryl betaine) (Comparative Example 3-2), Amphitol 20BS (trade name, manufactured by Kao Corporation, lauryl) Betaine) (Comparative Example 3-3), AM-3130N (trade name, manufactured by Nikko Chemicals Co., Ltd., palm oil fatty acid amidopropyldimethylaminoacetic acid betaine R-CO-NH- (CH ₂ ) ₃ -N ⁺ (CH ₃ ) ₂ -CH ₂ COO ^-) (Comparative Example 3-4), AM-301 (trade name, Nikko Chemicals stock meeting Mori made, lauryl betaine ^{_{RN + (CH 3) 2 -CH}} 2 -COO -) ( Comparative Example 3-5), SB3 -8 (trade name, 3- (N, N-dimethyloctylammonio) propanesulfonic acid, manufactured by Sigma) (Comparative Example 3-6), SB3-12 (trade name, 3- (dodecyldimethylammonio) propane Except for adding 0.3 w / v% of sulfonic acid (manufactured by Sigma) (Comparative Example 3-7) or PC polymer-2 to 6 (Comparative Examples 3-8 to 3-12), the same as Example 3. The reagent was prepared and reacted, the change in absorbance was measured, and the change in absorbance in serum sample 2 relative to the change in absorbance in serum sample 1 was determined as relative sensitivity (%). The results are shown in Table 5.

  The effect of bilirubin on each reagent is as follows: -1% for PC polymer-1; -8% for Amphital 24B; -7% for Amphital 20BS; -6% for AM-3130N; In the case of -301, it was -19%; in the case of SB3-8, -9%; in the case of SB3-12, -9%; From this result, PC polymer-1 has a bilirubin effect of about 1/5 or less compared to other betaine surfactants, and 1/50 or less compared to other PC polymers. It can be seen that the effect of reducing the influence of bilirubin is remarkable. From the above, compared with the case of using a betaine surfactant (Comparative Example 3-2 to Comparative Example 3-7) and a PC polymer other than Polymer A (Comparative Example 3-8 to Comparative Example 3-12) Then, it was found that by using the colorimetric determination method and colorimetric reagent of the present invention, the influence of the reducing substance bilirubin can be reduced and the creatinine concentration can be accurately determined.

Example 4
(Reagent storage stability; Part 1)
Prepare an aqueous solution containing TAPS 50mmo1 / L, TODB 1mmo1 / L, CR 50u / mL, SOD 15u / mL, and PC polymer-1 0.3w / v%, pH 7 with 1mo1 / L hydrochloric acid or sodium hydroxide. Adjustment to 9 gave reagent 4-1.

  Meanwhile, prepare an aqueous solution containing TAPS 100mmo1 / L, 4AAP 3mmo1 / L, CRN 450u / mL and POD 15u / mL, adjust the pH to 8.3 with 1mo1 / L hydrochloric acid or sodium hydroxide, and add reagent 4-2. Obtained.

  Furthermore, a 5 mg / dL aqueous solution of creatinine (Sample 3) was prepared as a sample.

  Reagent 4-1 immediately after preparation (Example 4A) or after preparation and storage at 37 ° C. for 1 week (Example 4B), 240 μL is added to 5 μL of specimen 3, heated at 37 ° C. for 5 minutes, and further reagent 80 μL of 4-2 was added, and the change in absorbance at 546 nm was measured using an automatic analyzer H7170. The change in absorbance was measured over time with the time when reagent 4-1 was added as 0 minutes. The results are shown in Figure 1. In FIG. 1, the white circle and the black circle plots show the results of Example 4A and Example 4B, respectively.

  From the results shown in FIG. 1, in the reaction system using the reagent to which PC polymer-1 was added, there was no decrease in the reaction rate even after storage at 37 ° C. for 1 week, and the stability of the reagent by PC polymer-1 It can be seen that there is no decline in sex.

Comparative Example 4-1
PC Polymer-1 A reagent was prepared and reacted in the same manner as in Examples 4A and 4B, except that 0.3 w / v% was not added, and the change in absorbance was measured over time. The result is shown in figure 2. In FIG. 2, the white circles and the black circles are plotted when the reagent 4-1 is used immediately after preparation (Comparative Example 4-1A) or when it is used after storage at 37 ° C. for 1 week (Comparative Example). The results of 4-1B) are shown respectively.

Comparative Examples 4-2 to 4-3
PC polymer-1 Instead of 0.3 w / v%, Example 4A and Example 4A except that Amphital 24B (Comparative Example 4-2) or AM-301 (Comparative Example 4-3) was added in an amount of 0.3 w / v%. By operating in the same manner as in 4B, reagents were prepared and reacted, 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. In FIG. 3 and FIG. 4, the white circle and the black circle plots are stored when reagent 4-1 is used immediately after preparation (Comparative Example 4-2A, 4-3A) or stored at 37 ° C. for 1 week, respectively. The results when used later (Comparative Examples 4-2B and 4-3B) are shown respectively.

  In Example 4 and Comparative Examples 4-1 to 4-3, as an index of creatinine concentration in each reaction system, the amount of change in absorbance (mAbs) from 71 seconds to 35 seconds after the addition of reagent 4-2 was calculated. . The ratio of the absorbance change amount of Example 4B to the absorbance change amount of Example 4A was determined as storage stability (%). Moreover, the storage stability was similarly determined for Comparative Examples 4-1 to 4-3. Table 6 shows the obtained values. In addition to these results, the experimental results on the effect on bilirubin in Example 3, Comparative Example 3-1, Comparative Example 3-2 using Amphital 24B, and Comparative Example 3-5 using AM-201 are shown. These are also shown in Table 6.

  From Table 6, it was found that the colorimetric reagent of the present invention is excellent in storage stability. Furthermore, from Table 3, Table 4, Table 5, and Table 6, by using the colorimetric determination method and colorimetric reagent of the present invention, the effect of the reducing substance bilirubin is reduced and the creatinine concentration is accurately determined. And the storage stability of the reagent can be maintained.

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 and POD 5 u / mL, and PC polymer-1 were dissolved. Reagent 5-1 was prepared by adjusting the pH to 7.5 with 1 mol / L hydrochloric acid or sodium hydroxide. The concentration of PC polymer-1 in reagent 5-1 was such that the final concentration when mixed with later-described reagent 5-2 and serum sample 1 or 2 was 0.4 w / v%.

  On the other hand, BES 50 mmol / L, 4AAP 2 mmol / L and uricase (Toyobo Co., Ltd.) 1 u / mL are dissolved in water, and the pH is adjusted to 7.5 with 1 mol / L hydrochloric acid or sodium hydroxide. 2 was prepared.

  To 5 μL of serum sample 1 or 2, add 240 μL of reagent 5-1 and warm at 37 ° C. for 5 minutes, then add 60 μL of reagent 5-2, and measure the absorbance at 546 nm with automatic analyzer H7170 type (Corporation) (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.

  The change in absorbance in serum sample 2 relative to the change in absorbance in serum sample 1 was determined as relative sensitivity (%). The results are shown in Table 7.

Comparative Example 5
The change in absorbance was measured in the same manner as in Example 5 except that PC polymer-1 was not added to reagent 5-1. The results are shown in Table 7.

  In Comparative Example 5 in which PC polymer-1 was not added, a negative effect of -733% based on bilirubin was observed, but in Example 5 in which PC polymer-1 was added, the effect of bilirubin was reduced. -5% negative effect. From the above, it was found that by using the colorimetric determination method and colorimetric reagent of the present invention, the effect of bilirubin as a reducing substance can be reduced and the uric acid concentration can be accurately determined.

Example 6
(Measurement of creatine)
Dissolve TAPS 30mmol / L, TODB 1mmol / L and SOD 15u / mL, and PC polymer-1 in water, adjust the pH to 8.2 with 1mol / L hydrochloric acid or sodium hydroxide, and add reagent 6-1. Prepared. The concentration of PC polymer-1 in reagent 6-1 was such that the final concentration when mixed with reagent 6-2 and serum sample 1 or 2 described later was 0.4 w / v%.

  On the other hand, TAPS 30mmol / L, 4AAP 2mmol / L, CR150u / mL and POD 20u / mL are dissolved in water, pH is adjusted to 8.0 with 1mol / L hydrochloric acid or sodium hydroxide, and reagent 6-2 is prepared. did.

  Change of the value obtained by adding 240 μL of reagent 6-1 to 5 μL of serum sample 1 or 2 and heating at 37 ° C. for 5 minutes, then adding 80 μL of reagent 6-2, and subtracting the absorbance at 600 nm from the absorbance at 546 nm Was measured using an automatic analyzer H7170 type (manufactured by Hitachi, Ltd.) while maintaining the temperature in the reaction system at 37 ° C. The endpoint method was applied as the measurement method.

  The change in absorbance in serum sample 2 relative to the change in absorbance in serum sample 1 was determined as relative sensitivity (%). The results are shown in Table 8.

Comparative Example 6
The change in absorbance was measured in the same manner as in Example 6 except that PC polymer-1 was not added to reagent 6-1. The results are shown in Table 8.

  In Comparative Example 6 where PC polymer-1 was not added, a negative effect of -72% based on bilirubin was observed, but in Example 6 where PC polymer-1 was added, the effect of bilirubin was reduced. , Stopped at a negative effect of -3%. From the above, it was found that the use of the colorimetric determination method and colorimetric reagent of the present invention can reduce the influence of the reducing substance bilirubin and accurately determine the creatine concentration.

6 is a graph showing the relationship between reaction time and absorbance in Example 4. It is a graph which shows the relationship between reaction time and the light absorbency in the comparative example 4-1. 14 is a graph showing the relationship between reaction time and absorbance in Comparative Example 4-2. 6 is a graph showing the relationship between reaction time and absorbance in Comparative Example 4-3.

Claims (4)

In the colorimetric determination method of creatinine, creatine, or uric acid that uses creatinine, creatine, or uric acid in a sample as a substance to be measured, induces hydrogen peroxide by an enzymatic reaction, and generates a coloring dye in the presence of peroxidase, Formula (I):

(Here, R represents a hydrogen atom or a methyl group.)
And formula (II):

(Here, R represents a hydrogen atom or a methyl group. X represents an alkyl group having 12 to 20 carbon atoms.)
A method for the colorimetric determination of creatinine, creatine, or uric acid, comprising the presence of a polymer A having a weight average molecular weight of 5,000 to 5,000,000, comprising units based on the monomer represented by the formula:   A colorimetric reagent for colorimetric determination according to claim 1 or 2, comprising a solvent, an enzyme, and the polymer A. In the colorimetric determination of creatinine, creatine, or uric acid, which uses creatinine, creatine, or uric acid in a sample containing bilirubin as a substance to be measured, induces hydrogen peroxide by an enzymatic reaction, and generates a coloring pigment in the presence of peroxidase. In which the reaction system has the following formula (I):

(Here, R represents a hydrogen atom or a methyl group.)
And formula (II):

(Here, R represents a hydrogen atom or a methyl group. X represents an alkyl group having 12 to 20 carbon atoms.)
And a polymer A having a weight average molecular weight of 5,000 to 5,000,000, comprising a unit based on a monomer represented by the formula: Storage stability of measurement reagents in the colorimetric determination of creatinine, creatine, or uric acid, which uses creatinine, creatine, or uric acid in a sample as a substance to be measured, induces hydrogen peroxide by an enzymatic reaction, and produces a coloring dye in the presence of peroxidase Wherein the reaction system comprises the following formula (I):

(Here, R represents a hydrogen atom or a methyl group.)
And formula (II):

(Here, R represents a hydrogen atom or a methyl group. X represents an alkyl group having 12 to 20 carbon atoms.)
A storage stabilization method comprising the presence of a polymer A having a weight average molecular weight of 5,000 to 5,000,000, which comprises units based on a monomer represented by the formula:

Images