Methods to suppress the effects of coexisting substances

Abstract

This invention provides a method for suppressing the influence of coexisting substances contained in a biological sample during measurement, and a biological sample measurement reagent utilizing this method. [Solution] In a biological sample measurement reagent composed of multiple reagents, a reagent containing at least one oxidase selected from the group consisting of cholesterol oxidase, glucose oxidase, uricase, amino acid oxidase, alcohol oxidase, xanthine oxidase, glycerophosphate oxidase, choline oxidase, pyruvate oxidase, fructosyl amino acid oxidase, and lactate oxidase is mixed with the sample to be measured first. In a biological sample measurement reagent utilizing this invention, the influence of coexisting substances contained in the sample to be measured can be suppressed.

Description

【Technical Field】 【0001】 The present invention relates to a biological sample measurement reagent composed of a plurality of reagents, which contains oxidase to suppress the influence of coexisting substances, and the reagent containing oxidase is the reagent first mixed with the measurement sample. The present invention also relates to a method for suppressing the influence of coexisting substances in a biological sample measurement reagent, which is characterized in that the reagent containing oxidase is first mixed with the measurement sample in the biological sample measurement reagent composed of a plurality of reagents. The present invention is particularly useful in the fields of chemistry, life science, analytical science, clinical examination, and the like. 【Background Art】 【0002】 Various lipids such as cholesterol exist in the living body, and each exhibits diverse functions. Therefore, measuring the lipid concentration in a biological sample and observing its fluctuations are essential for disease diagnosis, treatment, early detection, and prevention, and are widely carried out in hospitals, inspection facilities, and the like. Cholesterol and the like exist as lipoproteins in the blood, and are called chylomicron, very low density lipoprotein (hereinafter sometimes abbreviated as VLDL), intermediate density lipoprotein (hereinafter sometimes abbreviated as IDL), low density lipoprotein (hereinafter sometimes abbreviated as LDL), and high density lipoprotein (hereinafter sometimes abbreviated as HDL) in order of increasing specific gravity. Cholesterol in LDL is sometimes called LDL-cholesterol (hereinafter sometimes abbreviated as LDL-C), and cholesterol in HDL is sometimes called HDL-cholesterol (hereinafter sometimes abbreviated as HDL-C). In a blood test, it is known that an increase in the LDL-C concentration is a risk factor for atherosclerotic diseases, and conversely, a decrease in the HDL-C concentration is a risk factor for atherosclerotic diseases. Thus, the significance of cholesterol measurement varies depending on the type of lipoprotein, and it is necessary to accurately separate each lipoprotein. 【0003】 While methods using ultracentrifugation and electrophoresis are known for measuring LDL-C and HDL-C, these methods are cumbersome and time-consuming to measure many samples. Therefore, measurements using biological sample measurement reagents, which can be used with general-purpose automated analyzers, are widely used in hospital laboratories. There are various measurement principles for biological sample measurement reagents, but for example, by reacting lipoproteins contained in the sample with cholesterol esterase and cholesterol oxidase in the biological sample measurement reagent to generate hydrogen peroxide, and then reacting this hydrogen peroxide with the chromogen, coupler, and peroxidase in the biological sample measurement reagent to form a dye, the concentration of LDL-C and HDL-C can be measured by measuring the absorbance derived from this dye. 【0004】 Incidentally, when using biological materials as samples for measurements, the sample may contain many substances that are not the target of measurement. These substances that are not the target of measurement are sometimes called coexisting substances, and there is a concern that they may adversely affect the measurement. If such adverse effects occur, problems such as the inability to accurately measure the concentration of the target substance may arise. Such effects are called the effects of coexisting substances, and bilirubin is known to be one such substance. To prevent such effects, methods for avoiding these effects are known, such as performing the measurement in the presence of at least one polyoxyethylene alkyl nitrogen-containing compound selected from polyoxyethylene alkylamine compounds and polyoxyethylene alkylamide compounds (see Patent Document 1), or using amphoteric surfactants (see Patent Document 2). [Prior art documents] [Patent Documents] 【0005】 [Patent Document 1] Japanese Patent Publication No. 2013-141453 [Patent Document 2] Japanese Patent Application Publication No. 07-039394 [Overview of the Initiative] [Problems that the invention aims to solve] 【0006】 In biological sample measurement reagents, the addition of surfactants is considered to suppress the influence of coexisting substances. However, due to their surfactant and denaturing effects, surfactants can inhibit the reaction that the biological sample measurement reagent is originally intended to perform in order to measure the target substance contained in the sample, or they can denature the components contained in the biological sample measurement reagent or the target substance in the sample. In such cases, there are problems such as the inability to obtain accurate measurement values ​​or the inability to perform measurement at all. In particular, when measuring lipids, it is necessary to accurately separate each lipoprotein, and various measures have been taken regarding the type and concentration of surfactants contained in biological sample measurement reagents for this purpose, so it is expected that it will be difficult to add further surfactants to suppress the influence of coexisting substances. 【0007】 The inability to obtain accurate measurements, or even the inability to perform measurements at all, is a serious problem that can lead to misdiagnosis or delays in disease diagnosis. In the field of biological sample measurement reagents, there is a need for reagents that can provide accurate measurements. Therefore, there is a demand for biological sample measurement reagents that do not exhibit the effects of coexisting substances. [Means for solving the problem] 【0008】 The inventors of this invention conducted intensive research on the above-mentioned problems and their solutions, and as a result, discovered that the influence of coexisting substances can be suppressed by first mixing certain enzymes with the sample before measuring the target substance, thus completing the present invention. 【0009】 In other words, the present invention is as follows: (1) A biological sample measurement reagent comprising multiple reagents, characterized in that, in order to suppress the influence of coexisting substances, it contains at least one oxidase selected from the group consisting of cholesterol oxidase, glucose oxidase, uricase, amino acid oxidase, alcohol oxidase, xanthine oxidase, glycerophosphate oxidase, choline oxidase, pyruvate oxidase, fructosyl amino acid oxidase, and lactate oxidase, and the reagent containing the oxidase is the reagent that is first mixed with the sample to be measured. (2) The biological sample measurement reagent described in (1) above, characterized in that the reagent initially mixed with the sample to be measured also contains peroxidase. (3) A biological sample measurement reagent as described in (1) or (2) above, wherein the biological sample measurement reagent is a biochemical test reagent. (4) The biological sample measurement reagent according to (1) or (2) above, wherein the biological sample measurement reagent is an HDL-C measurement reagent, an LDL-C measurement reagent, a creatinine measurement reagent, or a uric acid measurement reagent. (5) A method for suppressing the influence of coexisting substances in a biological sample measurement reagent, characterized in that, in a biological sample measurement reagent composed of multiple reagents, a reagent containing at least one oxidase selected from the group consisting of cholesterol oxidase, glucose oxidase, uricase, amino acid oxidase, alcohol oxidase, xanthine oxidase, glycerophosphate oxidase, choline oxidase, pyruvate oxidase, fructosyl amino acid oxidase, and lactate oxidase is mixed with the measurement sample first in order to suppress the influence of coexisting substances. (6) The method according to (5) above, characterized in that the reagent to be initially mixed with the sample to be measured also contains peroxidase. (7) The method according to (5) or (6) above, wherein the biological sample measurement reagent is a biochemical test reagent. (8) The method according to (5) or (6) above, wherein the biological sample measurement reagent is an HDL-C measurement reagent, an LDL-C measurement reagent, a creatinine measurement reagent, or a uric acid measurement reagent. [Effects of the Invention] 【0010】 This invention makes it possible to suppress the influence of coexisting substances. Therefore, accurate measurement values ​​of the target substance can be obtained. [Modes for carrying out the invention] 【0011】 The present invention will now be described in detail. The following embodiments are illustrative for illustrating the present invention and are not intended to limit the present invention to these embodiments only. The present invention can be implemented in various forms without departing from its spirit. 【0012】 1. Overview When using biological materials as samples for measurements, the samples may contain numerous substances that are not the target of measurement. These untargeted substances are sometimes called coexisting substances, and there is a concern that they may adversely affect the measurement. If such adverse effects occur, problems such as the inability to accurately measure the concentration of the target substance may arise. This type of effect is called the effect of coexisting substances, and bilirubin is a known substance that can have such an effect. In biological sample measurement reagents, the addition of surfactants is considered to suppress the influence of coexisting substances. However, due to their surfactant and denaturing effects, surfactants can inhibit the reaction that the biological sample measurement reagent is originally intended to perform in order to measure the target substance contained in the sample, or they can denature the components contained in the biological sample measurement reagent or the target substance in the sample. In such cases, there are problems such as the inability to obtain accurate measurement values ​​or the possibility that measurement itself may not be possible. In particular, when measuring lipids, it is necessary to accurately separate each lipoprotein, and various measures have been taken regarding the type and concentration of surfactants contained in biological sample measurement reagents for this purpose, so it is expected that it will be difficult to add further surfactants to suppress the influence of coexisting substances. The inability to obtain accurate measurements, or even the inability to perform measurements at all, is a serious problem that can lead to misdiagnosis or delays in disease diagnosis. In the field of biological sample measurement reagents, there is a need for reagents that can provide accurate measurements. Therefore, there is a demand for biological sample measurement reagents that do not exhibit the effects of coexisting substances. As a result of diligent research into the above-mentioned problems and their solutions, the inventors have discovered that by first mixing a certain enzyme with the sample before measuring the target substance, the influence of coexisting substances can be suppressed. This invention was made based on these findings. 【0013】 2. Effects of coexisting substances In this invention, the influence of coexisting substances refers to the fact that when a sample containing a large amount of coexisting substances is measured using a biological sample measurement reagent, the measured value of the target substance will differ from the expected value, including both cases where the measured value is higher or lower than the expected value. Therefore, suppressing the influence of coexisting substances means reducing or eliminating such discrepancies in measured values. There are various possible reasons why the measured value may differ from the expected value due to the influence of coexisting substances. For example, the oxidation-reduction action of coexisting substances may change or denature the target substance or substances contained in the biological sample measurement reagent, or inhibit or promote the measurement reaction that should occur. The color of coexisting substances may also change the attenuation of transmitted or scattered light. Furthermore, coexisting substances may enter between the light source and the light receiving unit, obstructing the passage of light. 【0014】 Substances that may cause the influence of coexisting substances are not particularly limited as long as they possess such properties, but examples include free bilirubin, conjugated bilirubin, hemoglobin, triglycerides, ascorbic acid, and drugs administered to the subject from whom the biological sample was collected. 【0015】 3. Oxidase The oxidase in the present invention is an enzyme that promotes an oxidation reaction and has the effect of suppressing the influence of coexisting substances, and examples thereof include cholesterol oxidase (hereinafter sometimes abbreviated as COD), glucose oxidase (hereinafter sometimes abbreviated as GOD), uricase (hereinafter sometimes abbreviated as UOD), amino acid oxidase, alcohol oxidase, xanthine oxidase, glycerophosphate oxidase, choline oxidase, pyruvate oxidase, fructosyl amino acid oxidase, lactate oxidase. Among them, in particular, COD, GOD, and UOD are preferable. In addition, oxidase may be contained for purposes other than obtaining the effects of the above actions. Further, oxidase having no such action may be contained. 【0016】 In order to suppress the influence of coexisting substances, at least one kind of oxidase may be contained in the biological sample measurement reagent, and two or more kinds may be contained. 【0017】 The origin of the oxidase to be contained in the biological sample measurement reagent is not particularly limited as long as it is a concentration that exhibits the effect of suppressing the influence of coexisting substances. For example, forms such as purified products from animal blood, organs, etc. or purified products from genetically modified Escherichia coli can be mentioned. 【0018】 The concentration of oxidase to be included in the biological sample measurement reagent is not particularly limited as long as it exhibits an effect of suppressing the influence of coexisting substances. The concentration of oxidase when mixed with the biological sample is, for example, in the concentration ranges of 0.01 - 3 kU / L, 0.1 - 3 kU / L, 0.5 - 3 kU / L, 1 - 3 kU / L, 0.01 - 5 kU / L, 0.1 - 5 kU / L, 0.5 - 5 kU / L, 1 - 5 kU / L, 3 - 5 kU / L, 0.01 - 10 kU / L, 0.1 - 10 kU / L, 0.5 - 10 kU / L, 1 - 10 kU / L, 3 - 10 kU / L, 5 - 10 kU / L, 0.01 - 50 kU / L, 0.1 - 50 kU / L, 0.5 - 50 kU / L, 1 - 50 kU / L, 3 - 50 kU / L, 5 - 50 kU / L, 10 - 50 kU / L, 0.01 - 100 kU / L, 0.1 - 100 kU / L, 0.5 - 100 kU / L, 1 - 100 kU / L, 3 - 100 kU / L, 5 - 100 kU / L, 10 - 100 kU / L or 50 - 100 kU / L. 【0019】 When the biological sample measurement reagent is composed of a plurality of reagents, the oxidase to be included for the purpose of suppressing the influence of coexisting substances may be included in at least the reagent that is first mixed with the measurement sample. In this case, oxidase may also be included in reagents other than the reagent that is first mixed with the measurement sample. It is preferable to include peroxidase (hereinafter sometimes abbreviated as POD) in the reagent that is first mixed with the measurement sample. 【0020】 4. Biological sample measurement reagent In the present invention, the biological sample measurement reagent refers to something that can measure enzyme activity, substance concentration, etc. in a biological sample, and is not particularly limited as long as it is such a thing. Biological sample measurement reagents include biochemical biological sample measurement reagents that utilize biochemical reactions, immunological biological sample measurement reagents that utilize antigen - antibody reactions, genetic biological sample measurement reagents that utilize gene analysis techniques, etc. In the present invention, biochemical biological sample measurement reagents are preferred. Examples of biochemical biological sample measurement reagents include HDL - C measurement reagents, LDL - C measurement reagents, creatinine (hereinafter sometimes abbreviated as CRE) measurement reagents, urea (hereinafter sometimes abbreviated as UA) measurement reagents, etc. 【0021】 The biological sample measurement reagent of the present invention may be used for measurement by the endpoint method or by the rate method, and the method may be selected as appropriate. 【0022】 The measurement reagent of the present invention may be a two-reagent method in which the measurement sample and two reagents are mixed simultaneously or in an appropriate order for measurement, or a multi-reagent method in which the measurement sample and three or more reagents are mixed simultaneously or in an appropriate order for measurement, and the appropriate method may be selected as appropriate. 【0023】 In the biological sample measurement reagent of the present invention, the measurement may be performed by a manual method, or it may be performed using a device such as a general-purpose automated analyzer. 【0024】 The biological sample measurement reagent of the present invention may contain all or part of its constituent reagents as liquid reagents. 【0025】 The biological sample measurement reagent of the present invention can be sold on its own or used for measuring target substances in biological samples. 【0026】 The biological sample measurement reagent of the present invention can also be sold or used for measuring target substances in biological samples in combination with other reagents other than the biological sample measurement reagent described above. Examples of other reagents other than the biological sample measurement reagent described above include buffer solutions, sample diluents, reagent diluents, reagents containing substances for calibration, or reagents containing substances for quality control. 【0027】 The biological sample measurement reagent of the present invention may be a biological sample measurement reagent kit consisting of a first reagent, a second reagent, or a plurality of other reagents. 【0028】 The pH of the biological sample measurement reagent of the present invention can be appropriately selected considering the stability of the contained components during storage, the reaction rate during biological sample measurement, etc. Furthermore, if the biological sample measurement reagent is composed of multiple reagents, the pH of each reagent constituting the biological sample measurement reagent can be appropriately selected considering the stability of the contained components during storage, the reaction rate during sample measurement, etc. 【0029】 For example, in the case of HDL-C measuring reagents, the lower limit of the pH when measuring the HDL-C concentration in the sample is preferably 4.0, and particularly preferably 5.0. Furthermore, the upper limit is preferably 8.0, and particularly preferably 7.0. Regarding the pH of this HDL-C measuring reagent, for example, if the lower limit is 4.0, then a range of 4.0 to 7.0 or 4.0 to 8.0 can be given, and if the lower limit is 5.0, then a range of 5.0 to 7.0 or 5.0 to 8.0 can be given. 【0030】 The biological sample measurement reagent of the present invention may contain, as necessary, known surfactants, buffers, enzymes, coenzymes, reaction substrates, pH adjusters, preservatives, etc., in addition to organic acids and surfactants for dissolving organic acids. The concentration of each of these components can be appropriately selected considering the stability of the contained components during storage and the reaction rate during biological sample measurement. Furthermore, when the biological sample measurement reagent is composed of multiple reagents, the concentration of each of these components in each reagent constituting the biological sample measurement reagent can be appropriately selected considering the stability of the contained components during storage and the reaction rate during biological sample measurement.The buffering agent is not particularly limited, but examples include acetic acid, citric acid, tartaric acid, carbonic acid, boric acid, phosphoric acid, trishydroxymethylaminomethane, imidazole, glycylglycine, bistris, tricine, bicine, bistrispropane (hereinafter sometimes abbreviated as BTP), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (hereinafter sometimes abbreviated as TES), 2-morpholinoethanesulfonic acid (hereinafter sometimes abbreviated as MES), N-(2-acetamide)iminodiacetic acid (hereinafter sometimes abbreviated as ADA), N-(2-acetamide)-2- Aminoethanesulfonic acid (hereinafter sometimes abbreviated as ACES), piperazine-1,4-bis(2-ethanesulfonic acid) (hereinafter sometimes abbreviated as PIPES), 2-hydroxy-3-morpholinopropanesulfonic acid (hereinafter sometimes abbreviated as MOPSO), 3-morpholinopropane-1-sulfonic acid (hereinafter sometimes abbreviated as MOPS), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (hereinafter sometimes abbreviated as BES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (hereinafter sometimes abbreviated as HEPES) (Also available), 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (hereinafter sometimes abbreviated as DIPSO), 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (hereinafter sometimes abbreviated as TAPSO), piperazine-1,4-bis(2-hydroxypropanesulfonic acid) (hereinafter sometimes abbreviated as POPSO), N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid) (hereinafter sometimes abbreviated as HEPPS), 4-(2-hydroxyethyl)piperazine Examples include -1-(2-hydroxypropane-3-sulfonic acid) (sometimes abbreviated as HEPPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (sometimes abbreviated as TAPS), 2-cyclohexylaminoethanesulfonic acid (sometimes abbreviated as CHES), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (sometimes abbreviated as CAPSO), or N-cyclohexyl-3-aminopropanesulfonic acid (sometimes abbreviated as CAPS), or salts thereof. 【0031】 5. Biological samples In the present invention, a sample refers to an object that is the subject of measurement, such as enzyme activity or substance concentration, and a biological sample refers to an object contained in a living body that is the subject of measurement, such as enzyme activity or substance concentration. The biological sample is not particularly limited as long as it is one of these types of objects. Examples of such biological samples include human or animal blood, serum, plasma, urine, breast milk, feces, semen, cerebrospinal fluid, saliva, sweat, tears, ascites, amniotic fluid, organs such as the brain, and tissues and cells such as hair, skin, nails, muscles, or nerves. 【0032】 The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Examples] 【0033】 [Example 1] (Measurement of sample using HDL-C measuring reagent) Using the HDL-C measurement reagent of the present invention, a sample containing bilirubin as a coexisting substance was measured. 【0034】 1. Preparation of reagents 【0035】 (1) HDL-C measurement reagent First reagent Preparation of reagent 1-1 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the total concentration was 375 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was then dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated. The pH was adjusted to 6.0 (20°C) to prepare Reagent 1-1, the first reagent of the HDL-C measurement reagent. In this reagent, the concentration is 3 g / L of KF-354L and 0.4 mM of flufenamic acid. Bis-Tris 25mM (Actec) MES monohydrate 10mM (Actec) N-(2-carboxyethyl)-N-ethyl-m-toluidine hydrochloride (hereinafter sometimes abbreviated as CEMB) 1 mM (Dojin Chemical Research Institute) Sodium chloride 50mM (Kanto Chemical) (2) HDL-C measurement reagents: First reagent, preparation of reagents 1-2 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the total concentration was 375 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was then dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare Reagent 1-2 of the HDL-C measurement reagent. In this reagent, the concentration is 3 g / L of KF-354L and 0.4 mM of flufenamic acid. Bis-Tris 25mM (Actec) MES monohydrate 10mM (Actec) CEMB 1mM (Dojindo Chemical Research Institute) Sodium chloride 50mM (Kanto Chemical) COD 0.5kU / L (Toyobo) (3) HDL-C measurement reagents: First reagent, preparation of reagents 1-3 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the total concentration was 375 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was then dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent 1-3 of the HDL-C measurement reagent. In this reagent, the concentration is 3 g / L of KF-354L and 0.4 mM of flufenamic acid. Bis-Tris 25mM (Actec) MES monohydrate 10mM (Actec) CEMB 1mM (Dojindo Chemical Research Institute) Sodium chloride 50mM (Kanto Chemical) COD 1kU / L (Toyobo) (4) HDL-C measurement reagents: First reagent, preparation of reagents 1-4 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the total concentration was 375 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was then dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent 1-4 of the first reagent for HDL-C measurement. In this reagent, the concentration is 3 g / L of KF-354L and 0.4 mM of flufenamic acid. Bis-Tris 25mM (Actec) MES monohydrate 10mM (Actec) CEMB 1mM (Dojindo Chemical Research Institute) Sodium chloride 50mM (Kanto Chemical) COD 3kU / L (Toyobo) (5) HDL-C measurement reagents: First reagent, preparation of reagents 1-5 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the total concentration was 375 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was then dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent 1-5 of the HDL-C measurement reagent. In this reagent, the KF-354L concentration is 3 g / L and the flufenamic acid concentration is 0.4 mM. Bis-Tris 25mM (Actec) MES monohydrate 10mM (Actec) CEMB 1mM (Dojindo Chemical Research Institute) Sodium chloride 50mM (Kanto Chemical) GOD 1kU / L (Oriental Yeast Co., Ltd.) (6) HDL-C measurement reagents: First reagent, preparation of reagents 1-6 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the total concentration was 375 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was then dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare Reagent 1-6 of the HDL-C measurement reagent. In this reagent, the concentration is 3 g / L of KF-354L and 0.4 mM of flufenamic acid. Bis-Tris 25mM (Actec) MES monohydrate 10mM (Actec) CEMB 1mM (Dojindo Chemical Research Institute) Sodium chloride 50mM (Kanto Chemical) UOD 1kU / L (Toyobo) (7) HDL-C measurement reagents: First reagent, preparation of reagents 1-7 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the total concentration was 375 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was then dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent 1-7 of the first reagent for HDL-C measurement. In this reagent, the concentration is 3 g / L of KF-354L and 0.4 mM of flufenamic acid. Bis-Tris 25mM (Actec) MES monohydrate 10mM (Actec) CEMB 1mM (Dojindo Chemical Research Institute) Sodium chloride 50mM (Kanto Chemical) COD 0.5kU / L (Toyobo) POD 0.5kU / L (Toyobo) (8) HDL-C measurement reagents: First reagent, preparation of reagents 1-8 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the total concentration was 375 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was then dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare Reagent 1-8, the first reagent of the HDL-C measurement reagent. In this reagent, the concentration is 3 g / L of KF-354L and 0.4 mM of flufenamic acid. Bis-Tris 25mM (Actec) MES monohydrate 10mM (Actec) CEMB 1mM (Dojindo Chemical Research Institute) Sodium chloride 50mM (Kanto Chemical) COD 1kU / L (Toyobo) POD 0.5kU / L (Toyobo) (9) HDL-C measurement reagents: First reagent, preparation of reagents 1-9 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the total concentration was 375 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was then dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent 1-9 of the first reagent for HDL-C measurement. In this reagent, the concentration is 3 g / L of KF-354L and 0.4 mM of flufenamic acid. Bis-Tris 25mM (Actec) MES monohydrate 10mM (Actec) CEMB 1mM (Dojindo Chemical Research Institute) Sodium chloride 50mM (Kanto Chemical) COD 3kU / L (Toyobo) POD 0.5kU / L (Toyobo) (10) HDL-C measurement reagents: First reagent, preparation of reagents 1-10 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the total concentration was 375 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was then dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent 1-10, the first reagent of the HDL-C measurement reagent. In this reagent, the KF-354L concentration is 3 g / L and the flufenamic acid concentration is 0.4 mM. Bis-Tris 25mM (Actec) MES monohydrate 10mM (Actec) CEMB 1mM (Dojindo Chemical Research Institute) Sodium chloride 50mM (Kanto Chemical) GOD 1kU / L (Oriental Yeast Co., Ltd.) POD 0.5kU / L (Toyobo) (11) HDL-C measurement reagents: First reagent, preparation of reagents 1-11 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the total concentration was 375 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was then dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare Reagent 1-11, the first reagent of the HDL-C measurement reagent. In this reagent, the KF-354L concentration is 3 g / L and the flufenamic acid concentration is 0.4 mM. Bis-Tris 25mM (Actec) MES monohydrate 10mM (Actec) CEMB 1mM (Dojindo Chemical Research Institute) Sodium chloride 50mM (Kanto Chemical) UOD 1kU / L (Toyobo) POD 0.5kU / L (Toyobo) 【0036】 (12) HDL-C measurement reagent, second reagent, preparation of reagent 2-1 The following components were added to pure water to the concentrations indicated, and the pH was adjusted to 6.0 (20°C) to prepare Reagent 2-1, the second reagent of the HDL-C measurement reagent. Bis-Tris 10mM (Actec) MES monohydrate 40mM (Actec) POD 1kU / L (Toyobo) 4-aminoantipyrine 1 mM (Actec) Emulgen (registered trademark) B-66 10g / L (Kao Corporation) Cholesterol esterase 2.5 kU / L (Asahi Kasei Pharma) COD 3kU / L (Toyobo) (13) HDL-C measurement reagent, second reagent, preparation of reagent 2-2 The following components were added to pure water to the concentrations indicated, and the pH was adjusted to 6.0 (20°C) to prepare Reagent 2-1, the second reagent of the HDL-C measurement reagent. Bis-Tris 10mM (Actec) MES monohydrate 40mM (Actec) POD 1kU / L (Toyobo) 4-aminoantipyrine 1 mM (Actec) Emulgen B-66 10g / L (Kao) Cholesterol esterase 2.5 kU / L (Asahi Kasei Pharma) 【0037】 Kit X was created by combining reagent 1-1 and reagent 2-1. Kit A-1 was created by combining reagent 1-2 and reagent 2-2. Kit A-2 was created by combining reagent 1-3 and reagent 2-2. Kit A-3 was created by combining reagent 1-4 and reagent 2-2. Kit B was created by combining reagent 1-5 and reagent 2-1. Kit C was created by combining reagent 1-6 and reagent 2-1. Kit D-1 was created by combining reagent 1-7 and reagent 2-2. Kit D-2 was created by combining reagent 1-8 and reagent 2-2. Kit D-3 was created by combining reagent 1-9 and reagent 2-2. Kit E was created by combining reagent 1-10 and reagent 2-1. Kit F was created by combining reagent 1-11 and reagent 2-1. 【0038】 2. Preparation of the measurement sample 【0039】 Using pooled serum (GoldenWest) and Interference Check A Plus (Sysmex), pooled serum samples were prepared with conjugated bilirubin C concentrations of 0, 20, and 50 mg / dL, according to the Interference Check A Plus package insert. 【0040】 3. Measurement of the sample 【0041】 Using reagent 1, the HDL-C concentration was measured with sample 2. The HDL-C concentration of the sample with a bilirubin C concentration of 20 mg / dL was divided by the HDL-C concentration of the sample with a bilirubin C concentration of 0 mg / dL, and multiplied by 100 to obtain the influence at a bilirubin C concentration of 20 mg / dL. The influence is shown as a percentage, and the closer to 100%, the smaller the influence of bilirubin C. A similar calculation was performed for the measurement results of the sample with a bilirubin C concentration of 50 mg / dL, and the value was taken as the influence at a bilirubin C concentration of 50 mg / dL. These values ​​are shown in Table 1. The HDL-C concentration was measured as shown below. (Measurement method) A Hitachi High-Tech 7180 automated analyzer was used for the measurements. 2.0 μL of the sample was mixed with 200 μL of the first reagent from Kit X, and reacted at 37°C for 5 minutes. Then, 67 μL of the second reagent from Kit X was added and reacted at 37°C for another 5 minutes. The difference between the absorbance at the primary wavelength of 600 nm and the secondary wavelength of 700 nm at 4 minutes and 30 seconds (16th point) after the addition of the first reagent and the absorbance at the primary wavelength of 600 nm and the secondary wavelength of 700 nm at 3 minutes and 20 seconds (28th point) after the addition of the second reagent was calculated proportionally to the difference in absorbance measured when using a lipid standard serum (Sinotest) with an HDL-C concentration of 55.2 mg / dL instead of the sample, thereby determining the HDL-C concentration (mg / dL) of the sample. In addition, similar measurements were performed using kits A-1, A-2, A-3, B, C, D-1, D-2, D-3, E, and F, respectively, instead of kit X. 【0042】 [Table 1] 【0043】 4. Summary 【0044】 In Kit X, the impact at a bilirubin C concentration of 20 mg / dL was 64%, and the impact at a bilirubin C concentration of 50 mg / dL was 39%. However, in Kits A-1, A-2, A-3, B, C, D-1, D-2, D-3, E, and F, the impact at a bilirubin C concentration of 20 mg / dL was closer to 100% than 64%, and the impact at a bilirubin C concentration of 50 mg / dL was closer to 100% than 39%. Furthermore, regarding the impact of each measurement sample, the impact of Kit D-1 was closer to 100% than that of Kit A-1, the impact of Kit D-2 was closer to 100% than that of Kit A-2, the impact of Kit D-3 was closer to 100% than that of Kit A-3, the impact of Kit E was closer to 100% than that of Kit B, and the impact of Kit F was closer to 100% than that of Kit C. 【0045】 The influence of bilirubin C in kits A-1, A-2, A-3, B, C, D-1, D-2, D-3, E, and F, which utilize the present invention, was closer to 100% than the influence of bilirubin C in kit X, which does not utilize the present invention, indicating that the influence of coexisting substances is suppressed. Furthermore, regarding the influence of each measurement sample, the influence of kit D-1 was closer to 100% than that of kit A-1, the influence of kit D-2 was closer to 100% than that of kit A-2, the influence of kit D-3 was closer to 100% than that of kit A-3, the influence of kit E was closer to 100% than that of kit B, and the influence of kit F was closer to 100% than that of kit C, indicating that the influence of coexisting substances is further suppressed by including POD in the reagent that is initially mixed with the sample. 【0046】 This confirms that the reagent of the present invention has the effect of suppressing the influence of coexisting substances. 【0047】 [Example 2] (Measurement of sample using HDL-C measuring reagent) The HDL-C measurement reagent using the present invention was used to measure a sample. 【0048】 1. Preparation of reagents 【0049】 (14) Preparation of HDL-C measurement reagent In the same manner as in [Example 1], reagents 1-9 (9) of the first reagent for HDL-C measurement, reagents 1-10 (10) of the first reagent for HDL-C measurement, reagents 2-1 (12) of the second reagent for HDL-C measurement, and reagents 2-2 (13) of the second reagent for HDL-C measurement were prepared. Reagents 1-9 and 2-2 were combined to make Kit D-3. Reagents 1-10 and 2-1 were combined to make Kit E. (15) Preparation of HDL-C measurement reagents I prepared the first and second reagents for Quick Auto Neo HDL-C (Sinotest). 【0050】 2. Sample preparation 【0051】 Pooled plasma (GoldenWest) was prepared. 【0052】 3. Measurement of the sample 【0053】 The HDL-C concentration was measured using reagent 1 with sample 2. The values ​​are shown in Table 2. The HDL-C concentration was measured as follows. (Measurement method) A Hitachi High-Tech 7180 automated analyzer was used for the measurements. 2.0 μL of the sample was mixed with 200 μL of the first reagent from Kit D-3, and reacted at 37°C for 5 minutes. Then, 67 μL of the second reagent from Kit D-3 was added and reacted at 37°C for another 5 minutes. The difference between the absorbance at the primary wavelength of 600 nm and the secondary wavelength of 700 nm at 4 minutes and 30 seconds (16th point) after the addition of the first reagent and the absorbance at the primary wavelength of 600 nm and the secondary wavelength of 700 nm at 3 minutes and 20 seconds (28th point) after the addition of the second reagent was calculated proportionally to the difference in absorbance measured using a lipid standard serum (Sinotest) with an HDL-C concentration of 55.2 mg / dL instead of the sample, thereby determining the HDL-C concentration (mg / dL) of the sample. Similar measurements were also performed using Kit E and Quick Auto Neo HDL-C instead of Kit D-3. 【0054】 [Table 2] 【0055】 4. Summary 【0056】 When pooled plasma was measured using Kit D-3, the measured HDL-C concentration was 25 mg / dL. When pooled plasma was measured using Kit E, the measured HDL-C concentration was 24 mg / dL. In contrast, the Quick Auto Neo HDL-C measured 24 mg / dL. 【0057】 Since the HDL-C concentrations obtained using kits D-3 and E, which utilize the present invention, are almost the same as those obtained using Quick Auto Neo HDL-C, it can be seen that the HDL-C measuring reagents utilizing the present invention accurately measure HDL-C concentration. 【0058】 This demonstrates that the reagent of the present invention can accurately measure the concentration of the target substance. 【0059】 [Example 3] (Measurement of sample using CRE measuring reagent) Using the CRE measurement reagent of the present invention, a sample containing bilirubin as a coexisting substance was measured. 【0060】 1. Preparation of reagents 【0061】 (16) Preparation of CRE measurement reagents I prepared the first and second reagents for Cygnus Auto CRE (Sinotest). (17) Preparation of CRE measurement reagent To the first reagent of Cygnus Auto CRE, COD (Toyobo) was added at a concentration of 1 kU / L and POD (Toyobo) at a concentration of 10 kU / L. This first reagent was combined with the second reagent of Cygnus Auto CRE to create Kit G. 【0062】 2. Sample preparation 【0063】 Using pooled serum (GoldenWest) and Interference Check A Plus (Sysmex), pooled serum with conjugated bilirubin C concentrations of 0 and 20 mg / dL was prepared according to the Interference Check A Plus package insert. 【0064】 3. Measurement of the sample 【0065】 The CRE concentration was measured using reagent 1 with sample 2 as the measurement sample. The CRE concentration of the measurement sample with a bilirubin C concentration of 20 mg / dL was divided by the CRE concentration of the measurement sample with a bilirubin C concentration of 0 mg / dL, and multiplied by 100 to obtain the degree of influence at a bilirubin C concentration of 20 mg / dL. The degree of influence is shown as a percentage, and the closer to 100%, the smaller the influence of bilirubin C. The values ​​are shown in Table 3. The CRE concentration was measured as shown below. (Measurement method) A Canon Medical Systems TBA120FR Sora Edition automated analyzer was used for the measurements. 6.0 μL of the sample was mixed with 150 μL of the Cygnus Auto CRE first reagent and reacted at 37°C for 5 minutes. Then, 50 μL of the Cygnus Auto CRE second reagent was added and reacted at 37°C for another 5 minutes. The CRE concentration (mg / dL) of the sample was determined by proportionally calculating the difference between the average absorbance values ​​at the primary wavelength of 548 nm and secondary wavelength of 660 nm at 4 minutes 4 seconds (14th point), 4 minutes 22 seconds (15th point), and 4 minutes 40 seconds (16th point) after the addition of the first reagent, and the average absorbance values ​​at the primary wavelength of 548 nm and secondary wavelength of 660 nm at 4 minutes 11 seconds (31st point), 4 minutes 29 seconds (32nd point), and 4 minutes 47 seconds (33rd point) after the addition of the second reagent, and the difference in average absorbance values ​​when using a CRE standard solution (Sinotest) with a CRE concentration of 5.0 mg / dL instead of the sample. Similar measurements were also performed using Kit G instead of Cygnus Auto CRE. 【0066】 [Table 3] 【0067】 4. Summary 【0068】 In Cygnus Auto CRE, the effect at a bilirubin C concentration of 20 mg / dL was 76%, but in Kit G, the effect at a bilirubin C concentration of 20 mg / dL was closer to 100% than 76%. 【0069】 The influence of bilirubin C in Kit G, which utilizes the present invention, was closer to 100% than the influence of bilirubin C in Cygnus Auto CRE, which does not utilize the present invention, indicating that the influence of coexisting substances is suppressed. 【0070】 This confirms that the reagent of the present invention has the effect of suppressing the influence of coexisting substances. 【0071】 [Example 4] (Measurement of sample using UA measurement reagent) Using the UA measurement reagent of the present invention, a sample containing bilirubin as a coexisting substance was measured. 【0072】 1. Preparation of reagents 【0073】 (18) Preparation of UA measurement reagent kit H The following components were added to pure water to the concentrations indicated, and the pH was adjusted to 8.0 (at 20°C) to prepare the first reagent for UA measurement. ADA 10mM (Actec) Boric acid (SSG) 50mM (Actec) CEMB 1mM (Dojindo Chemical Research Institute) Potassium chloride 50mM (Kanto Chemical) POD 10kU / L (Toyobo) Ascorbic acid oxidase 3 kU / L (Asahi Kasei Pharma) This first reagent and the second reagent of Quick Auto Neo UA (Sinotest) were combined to create Kit H. (19) Preparation of UA measurement reagent kit I The following components were added to pure water to the concentrations indicated, and the pH was adjusted to 8.0 (at 20°C) to prepare the first reagent for UA measurement. ADA 10mM (Actec) Boric acid (SSG) 50mM (Actec) CEMB 1mM (Dojindo Chemical Research Institute) Potassium chloride 50mM (Kanto Chemical) POD 10kU / L (Toyobo) Ascorbic acid oxidase 3 kU / L (Asahi Kasei Pharma) COD 1kU / L (Toyobo) This first reagent and the second reagent from Quick Auto Neo UA were combined to form Kit I. 【0074】 2. Sample preparation 【0075】 Using pooled serum (GoldenWest) and Interference Check A Plus (Sysmex), pooled serum with conjugated bilirubin C concentrations of 0 and 20 mg / dL was prepared according to the Interference Check A Plus package insert. 【0076】 3. Measurement of the sample 【0077】 The UA concentration was measured using reagent 1 with sample 2. The UA concentration of the sample with a bilirubin C concentration of 20 mg / dL was divided by the UA concentration of the sample with a bilirubin C concentration of 0 mg / dL and multiplied by 100 to obtain the degree of influence at a bilirubin C concentration of 20 mg / dL. The degree of influence is shown as a percentage, and the closer to 100%, the smaller the influence of bilirubin C. The values ​​are shown in Table 4. The UA concentration was measured as shown below. (Measurement method) A Canon Medical Systems TBA120FR Sora Edition automated analyzer was used for the measurements. 3.3 μL of the sample was mixed with 160 μL of the first reagent from Kit H, and reacted at 37°C for 5 minutes. Then, 40 μL of the second reagent from Kit H was added and reacted at 37°C for another 5 minutes. The UA concentration (mg / dL) of the sample was determined by proportionally calculating the difference between the average absorbance values ​​at the dominant wavelength of 548 nm and the secondary wavelength of 660 nm at 4 minutes 4 seconds (14th point), 4 minutes 22 seconds (15th point), and 4 minutes 40 seconds (16th point) after the addition of the first reagent, and the average absorbance values ​​at the dominant wavelength of 548 nm and the secondary wavelength of 660 nm at 4 minutes 11 seconds (31st point), 4 minutes 29 seconds (32nd point), and 4 minutes 47 seconds (33rd point) after the addition of the second reagent, and the difference in average absorbance values ​​when using a UA standard solution (Sinotest) with a UA concentration of 15 mg / dL instead of the sample. Similar measurements were also performed using Kit I instead of Kit H. 【0078】 [Table 4] 【0079】 4. Summary 【0080】 In Kit H, the effect at a bilirubin C concentration of 20 mg / dL was 84%, while in Kit I, the effect at a bilirubin C concentration of 20 mg / dL was closer to 100% than 84%. 【0081】 The influence of bilirubin C in Kit I, which utilizes the present invention, was closer to 100% than the influence of bilirubin C in Kit H, which does not utilize the present invention, indicating that the influence of coexisting substances is suppressed. 【0082】 This confirms that the reagent of the present invention has the effect of suppressing the influence of coexisting substances. In biological sample measurement reagents, mixing reagents containing oxidases such as COD, GOD, and UOD with the sample to be measured first has the effect of obtaining accurate measurement values ​​even when measuring samples containing coexisting substances.

Claims

[Claim 1] A biological sample measurement reagent comprising multiple reagents, characterized in that, in order to suppress the influence of coexisting substances, it contains at least one oxidase selected from the group consisting of cholesterol oxidase, glucose oxidase, uricase, amino acid oxidase, alcohol oxidase, xanthine oxidase, glycerophosphate oxidase, choline oxidase, pyruvate oxidase, fructosyl amino acid oxidase, and lactate oxidase, and the reagent containing the oxidase is the reagent that is first mixed with the sample to be measured. [Claim 2] The biological sample measurement reagent according to claim 1, characterized in that the reagent initially mixed with the sample to be measured also contains peroxidase. [Claim 3] The biological sample measurement reagent according to claim 1 or 2, wherein the biological sample measurement reagent is a biochemical test reagent. [Claim 4] The biological sample measurement reagent according to claim 1 or 2, wherein the biological sample measurement reagent is an HDL-C measurement reagent, an LDL-C measurement reagent, a creatinine measurement reagent, or a uric acid measurement reagent. [Claim 5] A method for suppressing the influence of coexisting substances in a biological sample measurement reagent, characterized in that, in a biological sample measurement reagent composed of multiple reagents, a reagent containing at least one oxidase selected from the group consisting of cholesterol oxidase, glucose oxidase, uricase, amino acid oxidase, alcohol oxidase, xanthine oxidase, glycerophosphate oxidase, choline oxidase, pyruvate oxidase, fructosyl amino acid oxidase, and lactate oxidase is mixed with the measurement sample first, in order to suppress the influence of coexisting substances. [Claim 6] The inhibition method according to claim 5, characterized in that the reagent initially mixed with the sample to be measured also contains peroxidase. [Claim 7] The suppression method according to claim 5 or 6, wherein the biological sample measurement reagent is a biochemical test reagent. [Claim 8] The suppression method according to claim 5 or 6, wherein the biological sample measurement reagent is an HDL-C measurement reagent, an LDL-C measurement reagent, a creatinine measurement reagent, or a uric acid measurement reagent.

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