Method for measuring cholesterol in low density lipoprotein, measuring reagent and measuring kit

Abstract

The present invention provides a method for easily and accurately measuring low-density lipoprotein cholesterol (LDL-C) in a sample without using a surfactant having an alkylphenol structure, in consideration of the environment. A sample is mixed with (i) a combination of cholesterol esterase and cholesterol oxidase, or (ii) a combination of cholesterol esterase, an oxidized coenzyme, and cholesterol dehydrogenase, [a] one or more surfactants selected from the group consisting of polyoxyethylene alkyl ethers and polyoxyethylene polyoxypropylene alkyl ethers; and [b] Polyoxyethylene polyoxypropylene condensate and reacting the reactant in an aqueous medium containing the compound (I) and the compound (II) but not containing a surfactant having an alkylphenol structure, and measuring a substance produced or consumed in the reaction.

Description

[Technical field]

[0001] The present invention relates to a method, a reagent and a kit for measuring cholesterol (hereinafter abbreviated as LDL-C) in low-density lipoprotein (hereinafter abbreviated as LDL) contained in a specimen. This application claims priority based on Japanese Patent Application No. 2017-168235, filed on September 1, 2017, the contents of which are incorporated herein by reference. [Background technology]

[0002] LDL plays a role in supplying cholesterol to peripheral cells and is a direct factor in various arteriosclerosis including coronary artery sclerosis. Increased LDL-C is one of the major risk factors for arteriosclerosis, and differential quantification is clinically useful.

[0003] Conventional methods for quantifying LDL-C include ultracentrifugation, electrophoresis, and a calculation method using the Friedewald formula. Ultracentrifugation is a method that utilizes the difference in specific gravity of lipoproteins, separates LDL using an ultracentrifuge, and then measures its cholesterol content (Non-Patent Document 1). However, the separation procedure using ultracentrifugation is complicated and has drawbacks in terms of speed and simplicity. Electrophoresis includes a method that utilizes the difference in charge of lipoproteins and separates them using agarose gel or the like as a support, and a method that utilizes the difference in particle size of lipoproteins and separates them using polyacrylamide gel as a support. However, electrophoresis is poor in quantitative determination and has problems in terms of simplicity and economy. In the Friedewald method, the amount of LDL-C is calculated from the measured values ​​of total cholesterol (hereinafter abbreviated as TC), cholesterol in high density lipoprotein (hereinafter abbreviated as HDL-C), and total triglyceride (hereinafter abbreviated as T-TG) according to the following formula (Non-Patent Document 2). However, this method has problems with accuracy because it is affected by the T-TG content in serum and diet. (LDL-C)=(TC)-(HDL-C)-(T-TG) / 5

[0004] In recent years, methods for quantifying LDL-C have also been reported that do not require separation procedures such as ultracentrifugation and can be installed on general-purpose automatic analyzers. Among these, the following methods are known for quantifying LDL-C. A method for quantifying LDL-C in a test sample (Patent Document 1) comprises a first step of eliminating cholesterol from each of the lipoproteins, namely HDL, very low density lipoprotein (hereinafter referred to as VLDL), and chylomicrons, in the test sample by reacting cholesterol esterase and cholesterol oxidase in the presence of a surfactant that acts on lipoproteins other than LDL and eliminating the hydrogen peroxide produced, and a second step of quantifying the remaining cholesterol in the test sample. A method for quantifying LDL-C in which a surfactant selected from polyoxyethylene alkylene phenyl ether and polyoxyethylene alkylene tribenzyl phenyl ether, and an enzyme reagent for measuring cholesterol are added to serum, and cholesterol in HDL and VLDL among the cholesterol in lipoproteins is preferentially reacted, and then the amount of reaction of the remaining cholesterol is measured (Patent Document 2). A method in which a polyoxyethylene derivative, a polyoxyethylene-polyoxypropylene copolymer, and an enzyme for measuring cholesterol are added to a biological sample, and LDL-C, which is one of the cholesterol levels in lipoproteins, is selectively measured (Patent Document 3).

[0005] A method for measuring LDL-C, characterized in that the measurement is performed on a biological sample in the presence of dimethyl-α-cyclodextrin and / or poly-β-cyclodextrin (Patent Document 4). A method for directly and selectively measuring cholesterol in a sample containing one or more of chylomicrons, HDL, LDL, and VLDL, which quantifies LDL-C in the sample in the presence of phospholipids or a compound containing a phospholipid-like group (Patent Document 5). A sample and a cholesterol measuring enzyme are [a] Polyoxyethylene polyoxyalkylene alkyl aryl ether; [b] one or more surfactants selected from the group consisting of polyoxyethylene-polyoxyalkylene condensates, polyoxyethylene alkenyl ethers, polyoxyethylene branched alkyl ethers, and polyoxyethylene-polyoxyalkylene branched alkyl ethers; [c] one or more surfactants selected from the group consisting of primary amines, secondary amines, tertiary amines, and quaternary ammonium; and [d] Polyanion and measuring substances produced or consumed in the reaction (Patent Document 6).

[0006] In recent years, it has been reported that alkylphenols such as octylphenol have endocrine disrupting effects, and due to environmental considerations, the production of surfactants having an alkylphenol structure, such as polyoxyethylene alkylphenyl ether, and their use in reagents have come to be avoided.

[0007] In clinical testing, not only normal samples but also various abnormal samples are often used as samples. One such abnormal sample is a sample rich in lipids, which causes a problem of lipid-induced turbidity that affects the measurement. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Publication No. 10-38888 [Patent Document 2] Japanese Patent Application Publication No. 9-313200 [Patent Document 3] WO2000 / 17388 Brochure [Patent Document 4] Japanese Patent Application Publication No. 11-30617 [Patent Document 5] JP 2002-202314 A [Patent Document 6] WO2010 / 55916 Brochure [Non-patent literature]

[0009] [Non-Patent Document 1] Adv. Lipid Res., Vol. 6, p. 1, 1968 [Non-Patent Document 2] Clin. Chem., Vol. 18, p. 499, 1972 Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a method, a reagent, and a kit for simply and accurately measuring LDL-C in a sample without using a surfactant having an alkylphenol structure, such as polyoxyethylene alkylphenyl ether, in consideration of the environment. Furthermore, an object of the present invention is to provide a method, a reagent, and a kit for simply and accurately measuring LDL-C in a sample without using a surfactant having an alkylphenol structure, such as polyoxyethylene alkylphenyl ether, and without being affected by turbidity due to lipids. [Means for solving the problem]

[0011] As a result of extensive investigations into methods for measuring LDL-C, the inventors have found that by reacting a sample with a combination of cholesterol esterase and cholesterol oxidase, or a combination of cholesterol esterase, oxidized coenzyme and cholesterol dehydrogenase, in an aqueous medium containing a specific combination of surfactants other than surfactants having an alkylphenol structure, but not containing surfactants having an alkylphenol structure, it is possible to simply and accurately measure LDL-C in a sample without eliminating cholesterol in lipoproteins other than LDL or physically fractionating the lipoproteins, and have completed the present invention.

[0012] Furthermore, the present inventors have conducted extensive research into methods for measuring LDL-C, and as a result have found that by reacting a sample with a combination of cholesterol esterase and cholesterol oxidase, or a combination of cholesterol esterase, oxidized coenzyme and cholesterol dehydrogenase in an aqueous medium containing a specific combination of surfactants other than those having an alkylphenol structure, and a polyoxypropylene derivative not containing polyoxyethylene in the molecule, but not containing a surfactant having an alkylphenol structure, it is possible to simply and accurately measure LDL-C in a sample without eliminating cholesterol in lipoproteins other than LDL, without physically fractionating the lipoproteins, and without being affected by turbidity due to lipids, and have completed the present invention.

[0013] That is, the present invention relates to the following (1) to (24). (1) A sample is mixed with (i) a combination of cholesterol esterase and cholesterol oxidase, or (ii) a combination of cholesterol esterase, an oxidized coenzyme, and cholesterol dehydrogenase, [a] one or more surfactants selected from the group consisting of polyoxyethylene alkyl ethers (hereinafter abbreviated as POE alkyl ethers) and polyoxyethylene polyoxypropylene alkyl ethers (hereinafter abbreviated as POEPOP alkyl ethers); and [b] Polyoxyethylene polyoxypropylene condensate (hereinafter abbreviated as POEPOP condensate) A method for measuring LDL-C in a sample, comprising reacting the above-mentioned components in an aqueous medium containing the above-mentioned components and not containing a surfactant having an alkylphenol structure, and measuring a substance produced or consumed in the reaction. (2) [a] The method according to (1), wherein the alkyl in the POE alkyl ether and the POEPOP alkyl ether is an alkyl having 8 to 20 carbon atoms. (3) The method according to (1) or (2), wherein the aqueous medium further contains [c] a polyoxypropylene derivative that does not contain polyoxyethylene in the molecule. (4) [c] The method according to (3), wherein the polyoxypropylene derivative not containing polyoxyethylene in the molecule is polypropylene glycol or polyoxypropylene glyceryl ether. (5) The method according to any one of (1) to (4), wherein the substance produced is hydrogen peroxide. (6) The method according to (5), wherein the measurement of hydrogen peroxide is carried out using a reagent for measuring hydrogen peroxide. (7) The method according to any one of (1) to (4), wherein the substance produced is a reduced coenzyme. (8) The method according to (7), wherein the measurement of reduced coenzymes is carried out using a reagent for measuring reduced coenzymes.

[0014] (9) [a] one or more surfactants selected from the group consisting of POE alkyl ethers and POEPOP alkyl ethers; [b] POEPOP condensates; and A reagent for measuring LDL-C in a specimen, comprising cholesterol esterase and cholesterol oxidase, and not comprising a surfactant having an alkylphenol structure. (10) The reagent according to (9), further comprising a reagent for measuring a substance produced by the reaction of cholesterol esterase and cholesterol oxidase with the sample. (11) The reagent according to (10), wherein a substance produced by the reaction of cholesterol esterase and cholesterol oxidase with the sample is hydrogen peroxide. (12) [a] one or more surfactants selected from the group consisting of POE alkyl ethers and POEPOP alkyl ethers; [b] POEPOP condensates; and A reagent for measuring cholesterol in low-density lipoprotein in a specimen, comprising cholesterol esterase, oxidized coenzyme and cholesterol dehydrogenase. (13) The reagent according to (12), further comprising a reagent for measuring a substance produced by a reaction between cholesterol esterase, oxidized coenzyme and cholesterol dehydrogenase and a specimen. (14) The reagent according to (13), wherein a substance produced by the reaction of cholesterol esterase, oxidized coenzyme, and cholesterol dehydrogenase with the sample is a reduced coenzyme. (15)[a] The reagent according to any one of (9) to (14), wherein the alkyl in the POE alkyl ether and the POEPOP alkyl ether is an alkyl having 8 to 20 carbon atoms. (16) The reagent according to any one of (9) to (15), further comprising [c] a polyoxypropylene derivative that does not contain polyoxyethylene in the molecule. (17)[c] The reagent according to (16), wherein the polyoxypropylene derivative not containing polyoxyethylene in the molecule is polypropylene glycol or polyoxypropylene glyceryl ether.

[0015] (18) A method for detecting cholesterol oxidase comprising the steps of: (a) detecting cholesterol oxidase in a blood sample; [a] one or more surfactants selected from the group consisting of POE alkyl ethers and POEPOP alkyl ethers; [b] POEPOP condensates; and A kit for measuring LDL-C in a sample, characterized in that a first reagent, a second reagent, or both contain cholesterol esterase, and neither the first reagent nor the second reagent contains a surfactant having an alkylphenol structure. (19) The kit according to (18), further comprising a reagent for measuring hydrogen peroxide in the second reagent. (20) A method for detecting cholesterol dehydrogenase comprising: [a] one or more surfactants selected from the group consisting of POE alkyl ethers and POEPOP alkyl ethers; [b] POEPOP condensates; and A kit for measuring LDL-C in a sample, characterized in that a first reagent, a second reagent, or both contain cholesterol esterase, and neither the first reagent nor the second reagent contains a surfactant having an alkylphenol structure. (21) The kit according to (20), further comprising a reagent for measuring a reduced coenzyme in either or both of the first and second reagents. (22)[a] The kit according to any one of (18) to (21), wherein the alkyl in the POE alkyl ether and the POEPOP alkyl ether is an alkyl having 8 to 20 carbon atoms. (23) The kit according to any one of (18) to (22), further comprising [c] a polyoxypropylene derivative not containing polyoxyethylene in the molecule in either or both of the first reagent and the second reagent. (24)[c] The kit according to (23), wherein the polyoxypropylene derivative not containing polyoxyethylene in its molecule is polypropylene glycol or polyoxypropylene glyceryl ether. Effect of the Invention

[0016] The present invention provides a method, a reagent and a kit for simply and accurately measuring LDL-C in a sample without using a surfactant having an alkylphenol structure such as polyoxyethylene alkylphenyl ether. Furthermore, the present invention provides a method, reagent, and kit for simply and accurately measuring LDL-C in a sample without using a surfactant having an alkylphenol structure, such as polyoxyethylene alkylphenyl ether, and without being affected by turbidity due to lipids. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] 1. How to measure LDL-C The method for measuring LDL-C of the present invention is a method for measuring LDL-C in a specimen using a cholesterol measuring enzyme, and is a method for measuring LDL-C in a specimen without using a surfactant having an alkylphenol structure. The method for measuring LDL-C of the present invention is also a method for measuring LDL-C in a specimen without requiring a lipoprotein fractionation operation by a physical method such as centrifugation, and without eliminating cholesterol in lipoproteins other than LDL in the specimen prior to measuring LDL-C. Here, the enzyme for measuring cholesterol means cholesterol esterase, cholesterol oxidase, oxidized coenzyme, and cholesterol dehydrogenase.

[0018] The method for measuring LDL-C of the present invention comprises: mixing a sample with (i) a combination of cholesterol esterase and cholesterol oxidase, or (ii) a combination of cholesterol esterase, oxidized coenzyme, and cholesterol dehydrogenase; [a] one or more surfactants selected from the group consisting of POE alkyl ethers and POEPOP alkyl ethers; and [b] POEPOP condensation product and reacting the reactant in an aqueous medium containing no surfactant having an alkylphenol structure, and measuring a substance produced or consumed in the reaction, which method comprises the steps of:

[0019] [1] A method for treating a specimen with (i) a combination of cholesterol esterase and cholesterol oxidase, or (ii) a combination of cholesterol esterase, oxidized coenzyme, and cholesterol dehydrogenase, [a] one or more surfactants selected from the group consisting of POE alkyl ethers and POEPOP alkyl ethers; and [b] POEPOP condensation product reacting the compound in an aqueous medium containing no surfactant having an alkylphenol structure; [2] A step of measuring the substances produced or consumed in the step [1] above; [3] A step of correlating the measured value in [2] with a calibration curve showing the relationship between the LDL-C concentration and the amount of information derived from the produced or consumed substance, the calibration curve having a known LDL-C concentration prepared by carrying out the steps [1] and [2] above; and [4] Determining the LDL-C concentration in the sample.

[0020] Furthermore, the method for measuring LDL-C of the present invention includes mixing a sample with (i) a combination of cholesterol esterase and cholesterol oxidase, or (ii) a combination of cholesterol esterase, oxidized coenzyme and cholesterol dehydrogenase, [a] one or more surfactants selected from the group consisting of POE alkyl ethers and POEPOP alkyl ethers; [b] POEPOP condensates; and [c] Polyoxypropylene derivatives that do not contain polyoxyethylene and reacting the reactant in an aqueous medium containing no surfactant having an alkylphenol structure, and measuring a substance produced or consumed in the reaction, which method comprises the steps of:

[0021] [1] A method for treating a specimen with (i) a combination of cholesterol esterase and cholesterol oxidase, or (ii) a combination of cholesterol esterase, oxidized coenzyme, and cholesterol dehydrogenase, [a] one or more surfactants selected from the group consisting of POE alkyl ethers and POEPOP alkyl ethers; [b] POEPOP condensates; and [c] Polyoxypropylene derivatives that do not contain polyoxyethylene reacting the compound in an aqueous medium containing no surfactant having an alkylphenol structure; [2] A step of measuring the substances produced or consumed in the step [1] above; [3] A step of correlating the measured value in [2] with a calibration curve showing the relationship between the LDL-C concentration and the amount of information derived from the produced or consumed substance, the calibration curve having a known LDL-C concentration prepared by carrying out the steps [1] and [2] above; and [4] Determining the LDL-C concentration in the sample.

[0022] The aqueous medium used in the present invention is not particularly limited as long as it is an aqueous medium that enables the LDL-C measurement method of the present invention. Examples include deionized water, distilled water, and buffer solutions, with buffer solutions being preferred. The pH in the method for measuring LDL-C of the present invention may be any pH that allows the method for measuring LDL-C of the present invention, and may be, for example, pH 4 to 10. When a buffer solution is used as the aqueous medium, it is preferable to use a buffering agent according to the pH to be set. Examples of the buffering agent used in the buffer solution include tris(hydroxymethyl)aminomethane buffering agent, phosphate buffering agent, borate buffering agent, Good's buffering agent, etc.

[0023] Examples of Good's buffers include 2-morpholinoethanesulfonic acid (MES), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), N-(2-acetamido)iminodiacetic acid (ADA), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPS), and O), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), N-[ Tris(hydroxymethyl)methyl]-2-hydroxy-3-aminopropanesulfonic acid (TAPSO), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]-2-hydroxypropanesulfonic acid (HEPPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid [(H)EPPS], N-[tris(hydroxymethyl)methyl]-2-hydroxy-3-aminopropanesulfonic acid N,N-bis(2-hydroxyethyl)glycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3-amino-2-hydroxypropanesulfonic acid (CAPSO), and N-cyclohexyl-3-aminopropanesulfonic acid (CAPS).

[0024] The concentration of the buffer solution is not particularly limited as long as it is a concentration suitable for the measurement, but is preferably 0.001 to 2.0 mol / L, more preferably 0.005 to 1.0 mol / L.

[0025] In the method for measuring LDL-C of the present invention, examples of substances generated by the reaction of a specimen with a combination of (i) cholesterol esterase and cholesterol oxidase include hydrogen peroxide, and examples of substances generated by the reaction of a specimen with a combination of (ii) cholesterol esterase, oxidized coenzyme, and cholesterol dehydrogenase include reduced coenzyme. Examples of substances consumed by the reaction of a specimen with a combination of (i) cholesterol esterase and cholesterol oxidase include oxygen molecules, and examples of substances consumed by the reaction of a specimen with a combination of (ii) cholesterol esterase, oxidized coenzyme, and cholesterol dehydrogenase include oxidized coenzyme. The hydrogen peroxide generated can be measured, for example, using a hydrogen peroxide electrode or a reagent for measuring hydrogen peroxide described below. Examples of the reduced coenzyme generated can be measured by a method for measuring the absorbance of reduced coenzyme or a method for measuring reduced coenzyme described below. The oxygen molecules consumed can be measured, for example, using an oxygen electrode. The amount of oxidized coenzyme consumed can be measured, for example, by measuring the absorbance at the absorption wavelength of the oxidized coenzyme.

[0026] The specimen used in the method for measuring LDL-C of the present invention includes, for example, whole blood, plasma, serum, etc., and preferably plasma and serum. In addition, in the method for measuring LDL-C of the present invention, not only specimens from healthy subjects but also specimens rich in lipids can be used. Examples of specimens rich in lipids include high triglyceride (TG) specimens.

[0027] The cholesterol ester hydrolase in the present invention is not particularly limited as long as it is an enzyme capable of hydrolyzing cholesterol ester. For example, cholesterol esterase and lipoprotein lipase derived from animals, plants, or microorganisms, as well as cholesterol esterase and lipoprotein lipase produced by genetic engineering techniques, can be used. As the cholesterol esterase, in addition to unmodified cholesterol esterase, chemically modified cholesterol esterase can be used. Furthermore, commercially available cholesterol esterase can also be used. Commercially available cholesterol esterases include cholesterol esterase (COE-311; manufactured by Toyobo Co., Ltd.), lipoprotein lipase (LPL-311; manufactured by Toyobo Co., Ltd.), cholesterol esterase "Amano" 2 (CHE-2; manufactured by Amano Enzyme Inc.), etc. In the present invention, two or more types of cholesterol esterases can be used in combination.

[0028] In the chemical modification of cholesterol esterase, examples of the group (chemical modification group) that modifies the enzyme include a group mainly composed of polyethylene glycol, a group mainly composed of polypropylene glycol, a group having a copolymer of polypropylene glycol and polyethylene glycol, a group containing a water-soluble polysaccharide, a sulfopropyl group, a sulfobutyl group, a polyurethane group, a group having a chelating function, etc., and the group mainly composed of polyethylene glycol is preferred. Examples of the water-soluble polysaccharide include dextran, pullulan, soluble starch, etc. Reagents (chemical modifiers) for chemically modifying cholesterol esterase include compounds having both the above-mentioned chemical modifying group and a functional group or structure that can react with an amino group, a carboxyl group, a sulfhydryl group, etc., of the enzyme. Examples of functional groups or structures that can react with an amino group in the enzyme include a carboxyl group, an active ester group (such as an N-hydroxysuccinimide group), an acid anhydride, an acid chloride, an aldehyde, an epoxide group, 1,3-propane sultone, and 1,4-butane sultone. Examples of functional groups or structures that can react with a carboxyl group in the enzyme include an amino group. Examples of groups or structures that are reactive with a sulfhydryl group in the enzyme include a maleimide group, a disulfide, an α-haloester (such as an α-iodoester), and the like.

[0029] Commercially available chemical modifying agents can also be used. Commercially available chemical modifying agents include Sunbright VFM-4101, Sunbright ME-050AS, and Sunbright DE-030AS (all manufactured by NOF Corp.), which have a group mainly composed of polyethylene glycol and an N-hydroxysuccinimide group, Sunbright AKM series (e.g., Sunbright AKM-1510, etc.), Sunbright ADM series, and Sunbright ACM series (all manufactured by NOF Corp.), which have a group mainly composed of polyalkylene glycol and an acid anhydride structure, EPOX-3400 and M-EPOX-5000 (all manufactured by Shearwater Polymers), which have a group mainly composed of polyethylene glycol and an epoxide group, and diethylenetriamine-N,N,N',N'',N''-pentadiacetic anhydride (DTPA anhydride; manufactured by Dojindo Laboratories), which have a group having a chelating function and an acid anhydride structure. Chemical modification of cholesterol esterase can be carried out, for example, by the following method, but is not limited to this method. First, cholesterol esterase is dissolved in a buffer solution of pH 8.0 or more (e.g., HEPES buffer solution), and 0.01 to 500 times the molar amount of a chemical modifier is added at 0 to 55° C., followed by stirring for 5 minutes to 5 hours. In the enzymatic reaction, not only the reaction solution itself but also a solution from which unreacted chemical modifiers, etc. have been removed by an ultrafiltration membrane or the like, as necessary, can be used as the chemically modified cholesterol esterase.

[0030] The concentration of cholesterol esterase in the method for measuring LDL-C of the present invention is not particularly limited as long as it is a concentration that enables the method for measuring LDL-C of the present invention, and the concentration in the reaction solution is usually 0.001 to 800 kU / L, and preferably 0.01 to 300 kU / L.

[0031] The cholesterol oxidase in the present invention is not particularly limited as long as it is an enzyme capable of oxidizing cholesterol to generate hydrogen peroxide, and for example, cholesterol oxidase derived from animals, plants, or microorganisms, as well as cholesterol oxidase produced by genetic engineering techniques, can be used, and commercially available products such as cholesterol oxidase (CHODI; manufactured by Kikkoman Corporation), cholesterol oxidase (CHO-CE; manufactured by Kikkoman Corporation), cholesterol oxidase (COO-321; manufactured by Toyobo Co., Ltd.) can also be used. In the present invention, two or more types of cholesterol oxidases can also be used in combination. The cholesterol oxidase may be an unmodified enzyme or a chemically modified enzyme. The chemically modified cholesterol oxidase may be prepared by the above-mentioned chemical modification method using, for example, the above-mentioned chemical modifying agent.

[0032] The concentration of cholesterol oxidase in the LDL-C measurement method of the present invention is not particularly limited as long as it is a concentration that enables the LDL-C measurement method of the present invention, and the concentration in the reaction solution is usually 0.001 to 800 kU / L, and preferably 0.01 to 300 kU / L.

[0033] The cholesterol dehydrogenase in the present invention is not particularly limited as long as it is an enzyme capable of oxidizing cholesterol in the presence of an oxidized coenzyme to generate a reduced coenzyme. For example, cholesterol dehydrogenase derived from animals, plants, or microorganisms, as well as cholesterol dehydrogenase produced by genetic engineering techniques, can be used. Commercially available products such as cholesterol dehydrogenase "Amano" 5 (CHDH-5; manufactured by Amano Enzyme Co., Ltd.) can also be used. In addition, in the present invention, two or more types of cholesterol dehydrogenases can be used in combination. The cholesterol dehydrogenase may be an unmodified enzyme or a chemically modified enzyme. The chemically modified cholesterol dehydrogenase can be prepared by the above-mentioned chemical modification method, for example, using the above-mentioned chemical modifying agent.

[0034] The concentration of cholesterol dehydrogenase in the LDL-C measurement method of the present invention is not particularly limited as long as it is a concentration that enables the LDL-C measurement method of the present invention, and the concentration in the reaction solution is usually 0.001 to 800 kU / L, and preferably 0.01 to 300 kU / L.

[0035] In the method of the present invention using cholesterol dehydrogenase, an oxidized coenzyme is used. Examples of the oxidized coenzyme include NAD, NADP, thio-NAD, and thio-NADP. The concentration of the oxidized coenzyme is not particularly limited as long as it allows the method of the present invention to measure LDL-C, and is preferably 0.01 to 10 g / L.

[0036] Examples of the surfactant having an alkylphenol structure in the present invention include polyoxyethylene alkylphenyl ether (hereinafter, abbreviated as POE alkylphenyl ether), polyoxyethylene polyoxypropylene alkylphenyl ether (hereinafter, abbreviated as POEPOP alkylphenyl ether), etc. Examples of the alkyl in POE alkylphenyl ether and POEPOP alkylphenyl ether include octyl, nonyl, etc.

[0037] A specific example of the POE alkyl phenyl ether is Triton X-100 (POE octyl phenyl ether; manufactured by Sigma-Aldrich). Specific examples of POEPOP alkyl phenyl ethers include Emulgen L40 (manufactured by Kao Corporation) and Acronesis KP189R (manufactured by NOF Corporation).

[0038] Examples of the alkyl in the POE alkyl ether and POEPOP alkyl ether used in the present invention include alkyl having 8 to 20 carbon atoms, preferably alkyl having 9 to 18 carbon atoms, more preferably alkyl having 9 to 16 carbon atoms, and particularly preferably alkyl having 10 to 13 carbon atoms. Examples of the alkyl having 8 to 20 carbon atoms include octyl, isooctyl, nonyl, isononyl, decyl, isodecyl, undecyl, isoundecyl, dodecyl (lauryl), isododecyl, tridecyl, isotridecyl, tetradecyl (myristyl), isotetradecyl, pentadecyl, isopentadecyl, hexadecyl (cetyl), isohexadecyl, heptadecyl, isoheptadecyl, octadecyl (stearyl), isooctadecyl, nonadecyl, isononadecyl, icosyl, isoicosyl, and the like. Examples of the alkyl having 9 to 18 carbon atoms include nonyl, isononyl, decyl, isodecyl, undecyl, isoundecyl, dodecyl (lauryl), isododecyl, tridecyl, isotridecyl, tetradecyl (myristyl), isotetradecyl, pentadecyl, isopentadecyl, hexadecyl (cetyl), isohexadecyl, heptadecyl, isoheptadecyl, octadecyl (stearyl), isooctadecyl, etc. Examples of the alkyl having 9 to 16 carbon atoms include nonyl, isononyl, decyl, isodecyl, undecyl, isoundecyl, dodecyl (lauryl), isododecyl, tridecyl, isotridecyl, tetradecyl (myristyl), isotetradecyl, pentadecyl, isopentadecyl, hexadecyl (cetyl), isohexadecyl, etc. Examples of the alkyl having 10 to 13 carbon atoms include decyl, isodecyl, undecyl, isoundecyl, dodecyl (lauryl), isododecyl, tridecyl, and isotridecyl. The polymerization mode of POEPOP in the POEPOP alkyl ether is not particularly limited, and examples thereof include block polymerization and random polymerization modes. Examples of the block polymerization mode include diblock copolymer, triblock copolymer, and tetrablock copolymer.

[0039] Specific examples of POE alkyl ethers include FINESURF D-35, FINESURF D-45, FINESURF D-60, FINESURF D-65, FINESURF D-85, SAFETYCUT ID-1033, SAFETYCUT ID-1087 (all POE isodecyl ether; manufactured by Aoki Oil Industries Co., Ltd.), FINESURF TD-30, FINESURF TD-50, FINESURF TD-70, FINESURF TD-80, FINESURF TD-85, FINESURF TD-90, FINESURF TD-100, FINESURF TD-120, FINESURF TD-150, FINESURF TD-200 (all POE tridecyl ether; manufactured by Aoki Oil Industries Co., Ltd.), NIKKOL BL-2, NIKKOL BL-4.2, NIKKOL BL-9EX, NIKKOL BL-21, NIKKOL BL-25 (POE lauryl ether; manufactured by Nikko Chemicals), NIKKOL BC-2, NIKKOL BC-5.5, NIKKOL BC-7, NIKKOL BC-10, NIKKOL BC-15, NIKKOL BC-20, NIKKOL BC-23, NIKKOL BC-25, NIKKOL BC-30, NIKKOL BC-40 (above, POE cetyl ether; manufactured by Nikko Chemicals Co., Ltd.), EMALEX 703, EMALEX 705, EMALEX 707, EMALEX 709, EMALEX 710, EMALEX 712, EMALEX 715, EMALEX 720, EMALEX 725, EMALEX 730, EMALEX 750 (above, POE lauryl ether; manufactured by Nippon Emulsion Co., Ltd.), EMALEX 102, EMALEX 103, EMALEX 104, EMALEX 105, EMALEX 107, EMALEX Examples of such tertiary esters include EMALEX 112, EMALEX 115, EMALEX 117, EMALEX 120, EMALEX 125, and EMALEX 130 (all POE cetyl ether; manufactured by Nippon Emulsion Co., Ltd.), Nonion ID-203, Nonion ID-206, and Nonion ID-209 (all POE isodecyl ether; manufactured by NOF Corporation), Nonion K-204, Nonion K-220, and Nonion K-230 (all POE lauryl ether; manufactured by NOF Corporation), Nonion P-208, Nonion P-210, and Nonion P-213 (all POE cetyl ether; manufactured by NOF Corporation).

[0040] Specific examples of POEPOP alkyl ethers include WONDASURF NDR-800, WONDASURF NDR-1000, WONDASURF NDR-1400 (all of which are POEPOP decyl ether; manufactured by Aoki Oil Industries Co., Ltd.), WONDASURF ID-50, WONDASURF ID-70, WONDASURF ID-90, FINESURF IDEP-608, FINESURF IDEP-604, FINESURF IDEP-802 (all of which are POEPOP isodecyl ether; manufactured by Aoki Oil Industries Co., Ltd.), WONDASURF RL-80, WONDASURF RL-100, WONDASURF RL-140, WONDASURF 80, WONDASURF 100, WONDASURF 140 (all of which are POEPOP lauryl ether; manufactured by Aoki Oil Industries Co., Ltd.), WONDASURF S-800, WONDASURF S-1000, WONDASURF S-1400, FINESURF TDP-0633K, FINESURF TDE-1033, FINESURF TDE-1055 (all POEPOP tridecyl ether; manufactured by Aoki Oil Industries Co., Ltd.), NIKKOL PBC-31, NIKKOL PBC-33, NIKKOL PBC-34, NIKKOL PBC-44 (all POEPOP cetyl ether; manufactured by Nikko Chemicals Co., Ltd.), EMALEX DAPE-0203, EMALEX DAPE-0205, EMALEX DAPE-0207, EMALEX DAPE-0210, EMALEX DAPE-0212, EMALEX DAPE-0215, EMALEX DAPE-0220, EMALEX DAPE-0230 (all POEPOP decyl ether; manufactured by Nippon Emulsion Co., Ltd.), and the like.

[0041] The concentration of POE alkyl ether in the LDL-C measurement method of the present invention is not particularly limited as long as it is a concentration that enables the LDL-C measurement method of the present invention, and its concentration in the reaction solution is usually 0.0001 to 20% (w / v), preferably 0.001 to 5% (w / v).

[0042] The concentration of POEPOP alkyl ether in the LDL-C measurement method of the present invention is not particularly limited as long as it is a concentration that enables the LDL-C measurement method of the present invention, and the concentration in the reaction solution is usually 0.0001 to 20% (w / v), preferably 0.001 to 5% (w / v).

[0043] The polymerization mode of POEPOP in the POEPOP condensate is not particularly limited, and examples thereof include block polymerization and random polymerization modes. Examples of block polymerization modes include diblock copolymers, triblock copolymers, and tetrablock copolymers. The molecular weight of POP is 500 to 7000, and preferably 1000 to 6000. The molecular weight of POEPOP condensate is 600 to 12000, and preferably 1500 to 8000.

[0044] Specific examples of POEPOP condensates include Pluronic L-101, Pluronic L-121, Pluronic P-103, Pluronic F-108 (all manufactured by ADEKA Corporation), Pronon 201, Pronon 204, Pronon 208 (all manufactured by NOF Corporation), and the like.

[0045] The concentration of POEPOP condensate in the LDL-C measurement method of the present invention is not particularly limited as long as it is a concentration that enables the LDL-C measurement method of the present invention, and its concentration in the reaction solution is usually 0.0001 to 20% (w / v), preferably 0.001 to 5% (w / v).

[0046] The polyoxypropylene derivative not containing polyoxyethylene in the molecule in the present invention is not particularly limited as long as it is a polyoxypropylene derivative not containing polyoxyethylene in the molecule that enables the LDL-C measurement method of the present invention, and examples thereof include polypropylene glycol, polyoxypropylene glyceryl ether, etc. The molecular weight of the polyoxypropylene derivative not containing polyoxyethylene in the molecule in the present invention is not particularly limited as long as it is a molecular weight that enables the LDL-C measurement method of the present invention, and is usually 500 to 4000, preferably 1000 to 3500, and particularly preferably 1200 to 3000.

[0047] Specific examples of polyoxypropylene derivatives not containing polyoxyethylene in the molecule include Uniol D-700, Uniol D-1000, Uniol D-1200, Uniol D-2000, Uniol D-4000 (all polypropylene glycols; manufactured by NOF Corporation), Uniol TG-1000R, Uniol TG-3000 (polyoxypropylene glyceryl ethers; manufactured by NOF Corporation), polypropylene glycol diol type 1,000, polypropylene glycol diol type 2,000, polypropylene glycol diol type 3,000 (all polypropylene glycols; manufactured by Wako Pure Chemical Industries, Ltd.), and the like.

[0048] In the LDL-C measurement method of the present invention, the concentration of the polyoxypropylene derivative that does not contain polyoxyethylene in the molecule is not particularly limited as long as it is a concentration that enables the LDL-C measurement method of the present invention, and its concentration in the reaction solution is usually 0.0001 to 5% (w / v), preferably 0.001 to 1% (w / v).

[0049] The reaction temperature in the method for measuring LDL-C of the present invention is not particularly limited as long as it is a temperature that enables the method for measuring LDL-C of the present invention, but is preferably 10 to 50° C., more preferably 30 to 40° C. The reaction temperature set in a general-purpose automatic analyzer is usually 37° C.

[0050] The reaction time in the LDL-C measurement method of the present invention is not particularly limited as long as it is a reaction time that enables the LDL-C measurement method of the present invention, but is preferably 1 to 60 minutes, more preferably 2 to 30 minutes.

[0051] In the method for measuring LDL-C of the present invention, LDL-C can be measured, for example, by measuring the amount of hydrogen peroxide or reduced coenzyme produced by the reaction, or by measuring the amount of oxygen consumed in the reaction. The amount of hydrogen peroxide generated can be measured, for example, using a hydrogen peroxide electrode or a hydrogen peroxide measuring reagent. The hydrogen peroxide measuring reagent is a reagent for converting the generated hydrogen peroxide into a detectable substance. Examples of the detectable substance include dyes and luminescence, with dyes being preferred. When the detectable substance is a dye, the hydrogen peroxide measuring reagent contains a peroxidatively active substance such as an oxidative color-developing chromogen and peroxidase. Examples of the oxidative color-developing chromogen include the oxidative color-developing chromogens described below. When the detectable substance is luminescence, the hydrogen peroxide measuring reagent contains a chemiluminescent substance. Examples of the chemiluminescent substance include luminol, isoluminol, lucigenin, acridinium ester, and the like.

[0052] When a reagent containing an oxidative color-developing chromogen and a peroxidatively active substance such as peroxidase is used as a reagent for measuring hydrogen peroxide, hydrogen peroxide can be measured by reacting with the oxidative color-developing chromogen in the presence of the peroxidatively active substance to generate a pigment and measuring the generated pigment. When a reagent for measuring hydrogen peroxide containing a chemiluminescent substance is used, hydrogen peroxide can be measured by reacting with the chemiluminescent substance to generate photons and measuring the generated photons.

[0053] Examples of the oxidative color-developing chromogen include leuco type chromogens and oxidative coupling color-developing chromogens. Leuco chromogens are substances that are converted to dyes by themselves in the presence of peroxidatively active substances such as hydrogen peroxide and peroxidase. Specific examples include tetramethylbenzidine, o-phenylenediamine, 10-N-carboxymethylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine (MCDP), N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine sodium salt (DA-64), 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt (DA-67), 4,4'-bis(dimethylamino)diphenylamine, and bis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine (BCMA).

[0054] Oxidative coupling color-developing chromogens are substances that generate dyes by oxidative coupling of two compounds in the presence of hydrogen peroxide and a peroxidative substance such as peroxidase. Examples of combinations of two compounds include a combination of a coupler and an aniline, a combination of a coupler and a phenol, etc. Examples of the coupler include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazone.

[0055] Anilines include N-(3-sulfopropyl)aniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS), N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N,N-dimethyl-3-methylaniline, N,N-di(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-(3-sulfopropyl)aniline, N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline. toxaniline, N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine (EMSE), N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-4-fluoro-3,5-dimethoxyaniline (F-DAOS), N-[2-(succinylamino)ethyl]-2-methoxy-5-methylaniline (MASE), N-ethyl-N-[2-(succinylamino)ethyl]-2-methoxy-5-methylaniline (Et-MASE), and the like. Examples of phenols include phenol, 4-chlorophenol, 3-methylphenol, and 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB).

[0056] In the measurement of hydrogen peroxide, the concentration of the peroxidatively active substance is not particularly limited as long as it is a concentration that enables the LDL-C measurement method of the present invention, but when peroxidase is used as the peroxidatively active substance, it is preferably 1 to 100 kU / L.In addition, the concentration of the oxidative color-developing chromogen is not particularly limited as long as it is a concentration that enables the LDL-C measurement method of the present invention, but it is preferably 0.01 to 10 g / L.

[0057] Examples of the method for measuring reduced coenzymes include a method for measuring the absorbance of the generated reduced coenzyme and a method using a reagent for measuring reduced coenzyme. In the method for measuring the absorbance of reduced coenzyme, the absorbance is preferably 300 to 500 nm, more preferably 330 to 400 nm, and particularly preferably around 340 nm. The reagent for measuring reduced coenzyme is a reagent for converting the generated reduced coenzyme into a detectable substance. Examples of the detectable substance include dyes. Examples of the reagent for measuring reduced coenzyme when the detectable substance is a dye include a reagent containing diaphorase, an electron carrier, and a reduced color-developing chromogen. Examples of the electron carrier include 1-methoxy-5-methylphenadium methyl sulfate. When a reagent containing diaphorase, an electron carrier, and a reduced color-developing chromogen is used as the reagent for measuring reduced coenzyme, the reduced coenzyme can be quantified by quantifying the pigment generated by conversion of the reduced color-developing chromogen.

[0058] Examples of the reductive color-developing chromogen include 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1), 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-3), etc. The concentration of the reductive color-developing chromogen is not particularly limited as long as it is a concentration that enables the LDL-C measurement method of the present invention, and is preferably 0.01 to 10 g / L.

[0059] 2. LDL-C measurement reagents and LDL-C measurement kits The LDL-C measurement reagent of the present invention is a reagent used in the LDL-C measurement method of the present invention. The LDL-C measurement reagent of the present invention can be in the form of a kit suitable for storage, distribution and use. The LDL-C measurement kit of the present invention is a kit used in the LDL-C measurement method of the present invention. The LDL-C measurement kit of the present invention can be in the form of, for example, a two-reagent kit or a three-reagent kit, and is preferably a two-reagent kit consisting of a first reagent and a second reagent. When measuring LDL-C in a sample using a two-reagent kit as the LDL-C measurement kit of the present invention, first, the sample and the first reagent are added to a reaction cell and a reaction (primary reaction) is carried out for a certain period of time at a certain temperature, and then the second reagent is added and a reaction (secondary reaction) is carried out for a certain period of time at a certain temperature, and LDL-C can be measured by measuring the substances produced or consumed in the secondary reaction.

[0060] The LDL-C measuring reagent of the present invention may be in a freeze-dried state or in a dissolved state in an aqueous medium. When measuring LDL-C in a sample using a freeze-dried LDL-C measuring reagent, the reagent is dissolved in an aqueous medium beforehand and used for measuring LDL-C. Examples of the aqueous medium include the above-mentioned aqueous medium.

[0061] Examples of the cholesterol esterase, cholesterol oxidase, oxidized coenzyme, cholesterol dehydrogenase, surfactant having an alkylphenol structure, POE alkyl ether, POEPOP alkyl ether, POEPOP condensate, polyoxypropylene derivative not containing polyoxyethylene in the molecule, reagent for measuring hydrogen peroxide, and reagent for measuring reduced coenzyme in the LDL-C measurement reagent and measurement kit of the present invention include the above-mentioned cholesterol esterase, cholesterol oxidase, oxidized coenzyme, cholesterol dehydrogenase, surfactant having an alkylphenol structure, POE alkyl ether, POEPOP alkyl ether, POEPOP condensate, polyoxypropylene derivative not containing polyoxyethylene in the molecule, reagent for measuring hydrogen peroxide, and reagent for measuring reduced coenzyme, respectively.

[0062] In a two-reagent LDL-C measurement kit consisting of a first reagent and a second reagent, a surfactant having an alkylphenol structure is not contained in either the first reagent or the second reagent, and cholesterol esterase is contained in either the first reagent or the second reagent or both. In a two-reagent LDL-C measurement kit used for LDL-C measurement using cholesterol esterase and cholesterol oxidase, cholesterol oxidase is not contained in the first reagent but is contained in the second reagent. In a two-reagent LDL-C measurement kit used for LDL-C measurement using cholesterol esterase, cholesterol dehydrogenase and oxidized coenzyme, cholesterol dehydrogenase is not contained in the first reagent but is contained in the second reagent, and oxidized coenzyme is contained in at least the first reagent.

[0063] The POE alkyl ether may be contained in either or both of the first and second reagents, but an embodiment in which it is contained in the second reagent is preferred. The POEPOP alkyl ether may be contained in either or both of the first and second reagents, but is preferably contained in the second reagent.

[0064] The polyoxypropylene derivative not containing polyoxyethylene in the molecule may be contained in either or both of the first and second reagents, but is preferably contained in the second reagent.

[0065] The reagent for measuring hydrogen peroxide may be contained in either or both of the first and second reagents, but is preferably contained in at least the first reagent. When the reagent for measuring hydrogen peroxide contains an oxidative coupling type chromogen, it is preferable that the two compounds of the oxidative coupling type chromogen, i.e., the coupler and the anilines, or the coupler and the phenols, are contained in separate reagents. The reagent for measuring a reducing coenzyme may be contained in either or both of the first and second reagents, but is preferably contained in both the first and second reagents.

[0066] When the LDL-C measurement kit of the present invention is a kit consisting of three or more reagents, none of the reagents constituting the kit contains a surfactant having an alkylphenol structure.

[0067] The LDL-C measuring reagent and measuring kit of the present invention may contain an aqueous medium, a stabilizer, a preservative, an agent for avoiding the influence of interfering substances, a reaction promoter, etc., as necessary. Examples of the aqueous medium include the above-mentioned aqueous medium, etc. Examples of the stabilizer include ethylenediaminetetraacetic acid (EDTA), sucrose, calcium chloride, etc. Examples of the preservative include sodium azide, antibiotics, etc. Examples of the agent for avoiding the influence of interfering substances include ascorbic acid oxidase for avoiding the influence of ascorbic acid, potassium ferrocyanide for avoiding the influence of bilirubin, etc. Examples of the reaction promoter include enzymes such as colipase and phospholipase, salts such as sodium sulfate and sodium chloride, etc.

[0068] Specific embodiments of the LDL-C measurement reagent of the present invention are described below, but the LDL-C measurement reagent of the present invention is not limited to these. Reagent 1 A reagent containing POE alkyl ether, POEPOP condensation product, cholesterol esterase, and cholesterol oxidase, but not containing a surfactant having an alkylphenol structure. Reagent 2 A reagent containing POE alkyl ether, POEPOP condensation product, cholesterol esterase, cholesterol oxidase and a reagent for measuring hydrogen peroxide, and not containing a surfactant having an alkylphenol structure. Reagent 3 A reagent containing a POEPOP alkyl ether, a POEPOP condensate, cholesterol esterase, and cholesterol oxidase, but not containing a surfactant having an alkylphenol structure. Reagent 4 A reagent containing a POEPOP alkyl ether, a POEPOP condensate, cholesterol esterase, cholesterol oxidase and a reagent for measuring hydrogen peroxide, and not containing a surfactant having an alkylphenol structure. Reagent 5 A reagent containing POE alkyl ether, POEPOP alkyl ether, POEPOP condensate, cholesterol esterase, and cholesterol oxidase, but not containing a surfactant having an alkylphenol structure. Reagent 6 A reagent containing POE alkyl ether, POEPOP alkyl ether, POEPOP condensate, cholesterol esterase, cholesterol oxidase and a reagent for measuring hydrogen peroxide, and not containing a surfactant having an alkylphenol structure.

[0069] Reagent 7 A reagent that contains POE alkyl ether, POEPOP condensation product, polyoxypropylene derivative that does not contain polyoxyethylene in the molecule, cholesterol esterase and cholesterol oxidase, but does not contain a surfactant having an alkylphenol structure. Reagent 8 A reagent containing POE alkyl ether, POEPOP condensation product, polyoxypropylene derivative not containing polyoxyethylene in the molecule, cholesterol esterase, cholesterol oxidase and a reagent for measuring hydrogen peroxide, and not containing a surfactant having an alkylphenol structure. Reagent 9 A reagent containing a POEPOP alkyl ether, a POEPOP condensate, a polyoxypropylene derivative not containing polyoxyethylene in the molecule, cholesterol esterase and cholesterol oxidase, but not containing a surfactant having an alkylphenol structure. 10 Reagents A reagent containing a POEPOP alkyl ether, a POEPOP condensate, a polyoxypropylene derivative not containing polyoxyethylene in the molecule, cholesterol esterase, cholesterol oxidase and a reagent for measuring hydrogen peroxide, and not containing a surfactant having an alkylphenol structure. Reagent 11 A reagent containing POE alkyl ether, POEPOP alkyl ether, POEPOP condensate, a polyoxypropylene derivative not containing polyoxyethylene in the molecule, cholesterol esterase and cholesterol oxidase, and not containing a surfactant having an alkylphenol structure. Reagent 12 A reagent containing POE alkyl ether, POEPOP alkyl ether, POEPOP condensation product, polyoxypropylene derivative not containing polyoxyethylene in the molecule, cholesterol esterase, cholesterol oxidase and a reagent for measuring hydrogen peroxide, and not containing a surfactant having an alkylphenol structure.

[0070] Reagent 13 A reagent that contains POE alkyl ether, POEPOP condensation product, cholesterol esterase, oxidized coenzyme and cholesterol dehydrogenase, but does not contain a surfactant having an alkylphenol structure. Reagent 14 A reagent containing POE alkyl ether, POEPOP condensation product, cholesterol esterase, oxidized coenzyme, cholesterol dehydrogenase and a reagent for measuring reduced coenzyme, and not containing a surfactant having an alkylphenol structure. Reagent 15 A reagent containing POEPOP alkyl ether, POEPOP condensation product, cholesterol esterase, oxidized coenzyme and cholesterol dehydrogenase, but not containing a surfactant having an alkylphenol structure. Reagent 16 A reagent containing a POEPOP alkyl ether, a POEPOP condensate, cholesterol esterase, an oxidized coenzyme, cholesterol dehydrogenase and a reagent for measuring reduced coenzyme, and not containing a surfactant having an alkylphenol structure. Reagent 17 A reagent containing POE alkyl ether, POEPOP alkyl ether, POEPOP condensation product, cholesterol ester hydrolase, oxidized coenzyme and cholesterol dehydrogenase, but not containing a surfactant having an alkylphenol structure. Reagents 18 A reagent containing a POE alkyl ether, a POEPOP alkyl ether, a POEPOP condensation product, cholesterol esterase, an oxidized coenzyme, cholesterol dehydrogenase and a reagent for measuring reduced coenzymes, and not containing a surfactant having an alkylphenol structure.

[0071] Reagents 19 A reagent that contains POE alkyl ether, POEPOP condensation product, polyoxypropylene derivative that does not contain polyoxyethylene in the molecule, cholesterol ester hydrolase, oxidized coenzyme and cholesterol dehydrogenase, but does not contain a surfactant having an alkylphenol structure. 20 Reagents A reagent containing POE alkyl ether, POEPOP condensation product, polyoxypropylene derivative not containing polyoxyethylene in the molecule, cholesterol ester hydrolase, oxidized coenzyme, cholesterol dehydrogenase and a reagent for measuring reduced coenzyme, and not containing a surfactant having an alkylphenol structure. Reagent 21 A reagent containing a POEPOP alkyl ether, a POEPOP condensate, a polyoxypropylene derivative not containing polyoxyethylene in the molecule, cholesterol ester hydrolase, an oxidized coenzyme and cholesterol dehydrogenase, but not containing a surfactant having an alkylphenol structure. Reagents 22 A reagent containing a POEPOP alkyl ether, a POEPOP condensate, a polyoxypropylene derivative not containing polyoxyethylene in the molecule, cholesterol ester hydrolase, an oxidized coenzyme, cholesterol dehydrogenase and a reagent for measuring reduced coenzyme, and not containing a surfactant having an alkylphenol structure. Reagents 23 A reagent containing POE alkyl ether, POEPOP alkyl ether, POEPOP condensation product, polyoxypropylene derivative not containing polyoxyethylene in the molecule, cholesterol ester hydrolase, oxidized coenzyme and cholesterol dehydrogenase, but not containing a surfactant having an alkylphenol structure. Reagents 24 A reagent containing POE alkyl ether, POEPOP alkyl ether, POEPOP condensation product, polyoxypropylene derivative not containing polyoxyethylene in the molecule, cholesterol ester hydrolase, oxidized coenzyme, cholesterol dehydrogenase and a reagent for measuring reduced coenzyme, and not containing a surfactant having an alkylphenol structure.

[0072] The content of cholesterol esterase in the LDL-C measurement reagent of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that gives a concentration of 0.001 to 800 kU / L when dissolved in an aqueous medium, and preferably a content that gives a concentration of 0.01 to 300 kU / L. The content of cholesterol oxidase in the LDL-C measurement reagent of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that gives a concentration of 0.001 to 800 kU / L when dissolved in an aqueous medium, and preferably a content that gives a concentration of 0.01 to 300 kU / L. The content of cholesterol dehydrogenase in the LDL-C measurement reagent of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that results in a concentration of 0.001 to 800 kU / L when dissolved in an aqueous medium, and preferably a content that results in a concentration of 0.01 to 300 kU / L.

[0073] The content of POE alkyl ether in the LDL-C measurement reagent of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that results in a concentration of 0.0001 to 20% (w / v) when dissolved in an aqueous medium, and preferably a content that results in a concentration of 0.001 to 5% (w / v). The content of POEPOP alkyl ether in the LDL-C measurement reagent of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that results in a concentration of 0.0001 to 20% (w / v) when dissolved in an aqueous medium, preferably a content that results in a concentration of 0.001 to 5% (w / v). The content of POEPOP condensate in the LDL-C measurement reagent of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that results in a concentration of 0.0001 to 20% (w / v) when dissolved in an aqueous medium, preferably 0.001 to 5% (w / v). The content of the polyoxypropylene derivative that does not contain polyoxyethylene in the molecule in the LDL-C measurement reagent of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that results in a concentration of 0.0001 to 5% (w / v) when dissolved in an aqueous medium, and preferably a content that results in a concentration of 0.001 to 1% (w / v).

[0074] Specific embodiments of the LDL-C measurement kit of the present invention are described below, but the LDL-C measurement kit of the present invention is not limited to these. Kit 1 First Reagent POE alkyl ether, POEPOP condensate Second Reagent Cholesterol esterase, cholesterol oxidase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 2 First Reagent POE alkyl ether, POEPOP condensation product, hydrogen peroxide measuring reagent Second Reagent Cholesterol esterase, cholesterol oxidase, and hydrogen peroxide measuring reagents In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 3 First Reagent Hydrogen peroxide measuring reagent Second Reagent POE alkyl ether, POEPOP condensation product, cholesterol esterase, cholesterol oxidase, hydrogen peroxide measuring reagent In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 4 First Reagent POEPOP alkyl ether, POEPOP condensate Second Reagent Cholesterol esterase, cholesterol oxidase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 5 First Reagent POEPOP alkyl ether, POEPOP condensation product, hydrogen peroxide measuring reagent Second Reagent Cholesterol esterase, cholesterol oxidase, and hydrogen peroxide measuring reagents In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 6 First Reagent Hydrogen peroxide measuring reagent Second Reagent POEPOP alkyl ether, POEPOP condensation product, cholesterol esterase, cholesterol oxidase, hydrogen peroxide measuring reagent In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent.

[0075] Kit 7 First Reagent POE alkyl ether, POEPOP alkyl ether, POEPOP condensate Second Reagent Cholesterol esterase, cholesterol oxidase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 8 First Reagent POE alkyl ether, POEPOP alkyl ether, POEPOP condensation product, hydrogen peroxide measuring reagent Second Reagent Cholesterol esterase, cholesterol oxidase, and hydrogen peroxide measuring reagents In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 9 First Reagent Hydrogen peroxide measuring reagent Second Reagent POE alkyl ether, POEPOP alkyl ether, POEPOP condensation product, cholesterol esterase, cholesterol oxidase, hydrogen peroxide measuring reagent In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 10 First Reagent POE alkyl ether, POEPOP condensate Second Reagent Polyoxypropylene derivatives that do not contain polyoxyethylene in the molecule, cholesterol esterase, cholesterol oxidase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 11 First Reagent POE alkyl ether, POEPOP condensation product, hydrogen peroxide measuring reagent Second Reagent Polyoxypropylene derivatives that do not contain polyoxyethylene in the molecule, cholesterol esterase, cholesterol oxidase, and hydrogen peroxide measuring reagents In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 12 First Reagent Hydrogen peroxide measuring reagent Second Reagent POE alkyl ether, POEPOP condensation products, polyoxypropylene derivatives that do not contain polyoxyethylene in the molecule, cholesterol esterase, cholesterol oxidase, and reagents for measuring hydrogen peroxide In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent.

[0076] Kit 13 First Reagent POEPOP alkyl ether, POEPOP condensate Second Reagent Polyoxypropylene derivatives that do not contain polyoxyethylene in the molecule, cholesterol esterase, cholesterol oxidase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 14 First Reagent POEPOP alkyl ether, POEPOP condensation product, hydrogen peroxide measuring reagent Second Reagent Polyoxypropylene derivatives that do not contain polyoxyethylene in the molecule, cholesterol esterase, cholesterol oxidase, and hydrogen peroxide measuring reagents In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 15 First Reagent Hydrogen peroxide measuring reagent Second Reagent POEPOP alkyl ether, POEPOP condensation products, polyoxypropylene derivatives not containing polyoxyethylene in the molecule, cholesterol esterase, cholesterol oxidase, reagents for measuring hydrogen peroxide In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 16 First Reagent POE alkyl ether, POEPOP alkyl ether, POEPOP condensate Second Reagent Polyoxypropylene derivatives that do not contain polyoxyethylene in the molecule, cholesterol esterase, cholesterol oxidase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 17 First Reagent POE alkyl ether, POEPOP alkyl ether, POEPOP condensation product, hydrogen peroxide measuring reagent Second Reagent Polyoxypropylene derivatives that do not contain polyoxyethylene in the molecule, cholesterol esterase, cholesterol oxidase, and hydrogen peroxide measuring reagents In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 18 First Reagent Hydrogen peroxide measuring reagent Second Reagent POE alkyl ether, POEPOP alkyl ether, POEPOP condensation product, polyoxypropylene derivatives not containing polyoxyethylene in the molecule, cholesterol esterase, cholesterol oxidase, hydrogen peroxide measuring reagent In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent.

[0077] Kit 19 First Reagent POE alkyl ether, POEPOP condensate, oxidized coenzyme Second Reagent Cholesterol esterase, cholesterol dehydrogenase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 20 First Reagent POE alkyl ether, POEPOP condensation product, oxidized coenzyme, reduced coenzyme measuring reagent Second Reagent Cholesterol esterase, cholesterol dehydrogenase, reduced coenzyme measuring reagent In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 21 First Reagent Oxidized coenzymes Second Reagent POE alkyl ether, POEPOP condensation product, cholesterol ester hydrolase, cholesterol dehydrogenase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 22 First Reagent Reagents for measuring oxidized coenzymes and reduced coenzymes Second Reagent POE alkyl ether, POEPOP condensation product, cholesterol esterase, cholesterol dehydrogenase, reduced coenzyme measuring reagent In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent.

[0078] Kit 23 First Reagent POEPOP alkyl ether, POEPOP condensation product, oxidized coenzyme Second Reagent Cholesterol esterase, cholesterol dehydrogenase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 24 First Reagent POEPOP alkyl ether, POEPOP condensation product, oxidized coenzyme, reduced coenzyme measuring reagent Second Reagent Cholesterol esterase, cholesterol dehydrogenase, reduced coenzyme measuring reagent In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 25 First Reagent Oxidized coenzymes Second Reagent POEPOP alkyl ether, POEPOP condensation product, cholesterol ester hydrolase, cholesterol dehydrogenase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 26 First Reagent Reagents for measuring oxidized coenzymes and reduced coenzymes Second Reagent POEPOP alkyl ether, POEPOP condensation product, cholesterol esterase, cholesterol dehydrogenase, reduced coenzyme measuring reagent In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent.

[0079] Kit 27 First Reagent POE alkyl ether, POEPOP alkyl ether, POEPOP condensation product, oxidized coenzyme Second Reagent Cholesterol esterase, cholesterol dehydrogenase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 28 First Reagent POE alkyl ether, POEPOP alkyl ether, POEPOP condensation product, oxidized coenzyme, reduced coenzyme measuring reagent Second Reagent Cholesterol esterase, cholesterol dehydrogenase, reduced coenzyme measuring reagent In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 29 First Reagent Oxidized coenzymes Second Reagent POE alkyl ether, POEPOP alkyl ether, POEPOP condensation product, cholesterol ester hydrolase, cholesterol dehydrogenase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 30 First Reagent Reagents for measuring oxidized coenzymes and reduced coenzymes Second Reagent POE alkyl ether, POEPOP alkyl ether, POEPOP condensation product, cholesterol esterase, cholesterol dehydrogenase, reduced coenzyme measuring reagent In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent.

[0080] Kit 31 First Reagent POE alkyl ether, POEPOP condensate, oxidized coenzyme Second Reagent Polyoxypropylene derivatives that do not contain polyoxyethylene in the molecule, cholesterol esterase, cholesterol dehydrogenase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 32 First Reagent POE alkyl ether, POEPOP condensation product, oxidized coenzyme, reduced coenzyme measuring reagent Second Reagent Polyoxypropylene derivatives that do not contain polyoxyethylene in the molecule, cholesterol esterase, cholesterol dehydrogenase, and reduced coenzyme measuring reagents In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 33 First Reagent Oxidized coenzymes Second Reagent POE alkyl ether, POEPOP condensation product, polyoxypropylene derivatives that do not contain polyoxyethylene in the molecule, cholesterol ester hydrolase, cholesterol dehydrogenase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 34 First Reagent Reagents for measuring oxidized coenzymes and reduced coenzymes Second Reagent POE alkyl ether, POEPOP condensation products, polyoxypropylene derivatives not containing polyoxyethylene in the molecule, cholesterol esterase, cholesterol dehydrogenase, reduced coenzyme measuring reagents In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent.

[0081] Kit 35 First Reagent POEPOP alkyl ether, POEPOP condensation product, oxidized coenzyme Second Reagent Polyoxypropylene derivatives that do not contain polyoxyethylene in the molecule, cholesterol esterase, cholesterol dehydrogenase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 36 First Reagent POEPOP alkyl ether, POEPOP condensation product, oxidized coenzyme, reduced coenzyme measuring reagent Second Reagent Polyoxypropylene derivatives that do not contain polyoxyethylene in the molecule, cholesterol esterase, cholesterol dehydrogenase, and reduced coenzyme measuring reagents In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 37 First Reagent Oxidized coenzymes Second Reagent POEPOP alkyl ether, POEPOP condensation product, polyoxypropylene derivatives not containing polyoxyethylene in the molecule, cholesterol ester hydrolase, cholesterol dehydrogenase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 38 First Reagent Reagents for measuring oxidized coenzymes and reduced coenzymes Second Reagent POEPOP alkyl ether, POEPOP condensation products, polyoxypropylene derivatives not containing polyoxyethylene in the molecule, cholesterol esterase, cholesterol dehydrogenase, and reduced coenzyme measuring reagents In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent.

[0082] Kit 39 First Reagent POE alkyl ether, POEPOP alkyl ether, POEPOP condensation product, oxidized coenzyme Second Reagent Polyoxypropylene derivatives that do not contain polyoxyethylene in the molecule, cholesterol esterase, cholesterol dehydrogenase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 40 First Reagent POE alkyl ether, POEPOP alkyl ether, POEPOP condensation product, oxidized coenzyme, reduced coenzyme measuring reagent Second Reagent Polyoxypropylene derivatives that do not contain polyoxyethylene in the molecule, cholesterol esterase, cholesterol dehydrogenase, and reduced coenzyme measuring reagents In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 41 First Reagent Oxidized coenzymes Second Reagent POE alkyl ether, POEPOP alkyl ether, POEPOP condensation product, polyoxypropylene derivatives not containing polyoxyethylene in the molecule, cholesterol ester hydrolase, cholesterol dehydrogenase In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent. Kit 42 First Reagent Reagents for measuring oxidized coenzymes and reduced coenzymes Second Reagent POE alkyl ether, POEPOP alkyl ether, POEPOP condensation products, polyoxypropylene derivatives not containing polyoxyethylene in the molecule, cholesterol esterase, cholesterol dehydrogenase, reduced coenzyme measuring reagent In addition, surfactants having an alkylphenol structure are not contained in either the first or second reagent.

[0083] The content of cholesterol esterase in the first reagent constituting the LDL-C measurement kit of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that gives a concentration of 0.002 to 1600 kU / L when dissolved in an aqueous medium, and preferably a content that gives a concentration of 0.02 to 600 kU / L. The content of cholesterol esterase in the second reagent constituting the LDL-C measurement kit of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that gives a concentration of 0.004 to 3200 kU / L when dissolved in an aqueous medium, and preferably a content that gives a concentration of 0.04 to 1200 kU / L. The content of cholesterol oxidase in the second reagent constituting the LDL-C measurement kit of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that results in a concentration of 0.004 to 3200 kU / L when dissolved in an aqueous medium, and preferably a content that results in a concentration of 0.04 to 1200 kU / L. The content of cholesterol dehydrogenase in the second reagent constituting the LDL-C measurement kit of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that results in a concentration of 0.004 to 3200 kU / L when dissolved in an aqueous medium, and preferably a content that results in a concentration of 0.04 to 1200 kU / L.

[0084] The content of POE alkyl ether in the first reagent constituting the LDL-C measurement kit of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that results in a concentration of 0.0002 to 40% (w / v) when dissolved in an aqueous medium, and preferably a content that results in a concentration of 0.002 to 10% (w / v). The content of POE alkyl ether in the second reagent constituting the LDL-C measurement kit of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that results in a concentration of 0.0004 to 40% (w / v) when dissolved in an aqueous medium, and preferably a content that results in a concentration of 0.004 to 20% (w / v). The content of POEPOP alkyl ether in the first reagent constituting the LDL-C measurement kit of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that results in a concentration of 0.0002 to 40% (w / v) when dissolved in an aqueous medium, preferably a content that results in a concentration of 0.002 to 10% (w / v). The content of POEPOP alkyl ether in the second reagent constituting the LDL-C measurement kit of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that results in a concentration of 0.0004 to 40% (w / v) when dissolved in an aqueous medium, preferably a content that results in a concentration of 0.004 to 20% (w / v).

[0085] The content of POEPOP condensate in the first reagent constituting the LDL-C measurement kit of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that results in a concentration of 0.0002 to 40% (w / v) when dissolved in an aqueous medium, preferably a content that results in a concentration of 0.002 to 10% (w / v). The content of POEPOP condensate in the second reagent constituting the LDL-C measurement kit of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that results in a concentration of 0.0004 to 40% (w / v) when dissolved in an aqueous medium, preferably a content that results in a concentration of 0.004 to 20% (w / v). The content of the polyoxypropylene derivative not containing polyoxyethylene in the molecule in the first reagent constituting the LDL-C measurement kit of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that results in a concentration of 0.0002 to 10% (w / v) when dissolved in an aqueous medium, and preferably a content that results in a concentration of 0.002 to 2% (w / v). The content of the polyoxypropylene derivative not containing polyoxyethylene in the molecule in the second reagent constituting the LDL-C measurement kit of the present invention is not particularly limited as long as it is a content that enables the LDL-C measurement method of the present invention, and is usually a content that results in a concentration of 0.0004 to 20% (w / v) when dissolved in an aqueous medium, preferably 0.004 to 4% (w / v). EXAMPLES

[0086] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited thereto. In the examples and comparative examples, reagents and enzymes from the following manufacturers were used.

[0087] PIPES (Dojindo Laboratories), EMSE (Daito Chemistry), ascorbate oxidase (Asahi Kasei Pharma), 4-AA (Actec), calcium chloride (Wako Pure Chemical Industries, Ltd.), potassium ferrocyanide (Kanto Chemical), LPL-311 (cholesterol esterase; Toyobo), CHO-CE (cholesterol oxidase; Kikkoman), peroxidase (Toyobo), Pluronic L-121 (POEPOP condensate; ADEKA)

[0088] Safetycut ID-1087 (POE isodecyl ether; manufactured by Aoki Oil Industries Co., Ltd.), Finesurf TD-200 (POE tridecyl ether; manufactured by Aoki Oil Industries Co., Ltd.), Wondersurf ID-70 (POEPOP isodecyl ether; manufactured by Aoki Oil Industries Co., Ltd.), Wondersurf ID-90 (POEPOP isodecyl ether; manufactured by Aoki Oil Industries Co., Ltd.), Wondersurf S-1400 (POEPOP tridecyl ether; manufactured by Aoki Oil Industries Co., Ltd.)

[0089] Rheodol TW-L120 (polyoxyethylene sorbitan monolaurate; manufactured by Kao Corporation), Emanon 1112 (polyethylene glycol monolaurate; manufactured by Kao Corporation), Nymeen L-207 (polyoxyethylene laurylamine; manufactured by NOF Corporation), Unigly MK-278 (polyoxyethylene coconut oil fatty acid glyceryl; manufactured by NOF Corporation), Uniox HC-100 (polyoxyethylene hydrogenated castor oil; manufactured by NOF Corporation)

[0090] Uniol D-1200 (polypropylene glycol; NOF Corp.), Uniol D-2000 (polypropylene glycol; NOF Corp.), polypropylene glycol diol type 3,000 (polypropylene glycol; Wako Pure Chemical Industries, Ltd.), Uniol TG-3000 (polyoxypropylene glyceryl ether; NOF Corp.), PEG#2000 (polyethylene glycol; NOF Corp.)

[0091] Physiological saline (Otsuka Pharmaceutical Factory), Intralipos infusion 10% (refined soybean oil; Otsuka Pharmaceutical Factory), Determiner L TCII (cholesterol measurement kit; Kyowa Medex), MetaboLead LDL-C (LDL-C measurement kit; Kyowa Medex), Determiner Standard for Serum Lipid Measurement (standard product used in combination with Determiner L TCII; Kyowa Medex, hereafter abbreviated as cholesterol standard product), MetaboLead Standard for Serum HDL / LDL-C Measurement (standard product used in combination with MetaboLead LDL-C; Kyowa Medex, hereafter abbreviated as LDL-C standard product)

[0092] [Reference Example 1] Preparation of lipoprotein fractions and determination of cholesterol concentration (1) Preparation of lipoprotein fractions Whole blood collected from a healthy individual affiliated with Kyowa Medex Co., Ltd. was centrifuged at 3,000 rpm at 25°C for 20 minutes to prepare human serum. Next, VLDL (specific gravity 1.006 or less), LDL (specific gravity 1.006 to 1.063), and HDL (specific gravity 1.063 or more) were separated from the human serum according to the ultracentrifugation procedure described in "New Biochemical Experiment Course 4 Lipids I Neutral Fat and Lipoproteins" (Tokyo Kagaku Dojin, ISBN: 4-8079-1080-9), and each lipoprotein fraction was prepared.

[0093] (2) Preparation of a standard curve for determining cholesterol concentration Determiner L TCII was used as a kit for measuring cholesterol, and a cholesterol standard with a determined cholesterol concentration was used as a sample, and the absorbance of the cholesterol standard was measured according to the following procedure. The standard (2 μL) and the first reagent (150 μL) of Determiner L TCII were added to a reaction cell, and reacted at 37° C. for 5 minutes, and the absorbance (E1) of the reaction solution was measured at a main wavelength of 600 nm and a secondary wavelength of 800 nm. Next, the second reagent (50 μL) of Determiner L TCII was added to this reaction solution, and the reaction was further carried out at 37° C. for 5 minutes, and the absorbance (E2) of the reaction solution was measured at a main wavelength of 600 nm and a secondary wavelength of 800 nm. E1 was subtracted from E2, and the absorbance difference ΔE 標準品 was calculated. The absorbance difference ΔE was measured by the same method except that saline was used instead of the standard. ブランク was calculated. Absorbance difference ΔE relative to the standard 標準品 and the absorbance difference ΔE relative to saline ブランク From this, a calibration curve was created showing the relationship between cholesterol concentration (mg / dL) and absorbance.

[0094] (3) Determination of cholesterol concentration in each lipoprotein fraction Using Determiner L TCII as a cholesterol measurement kit and each lipoprotein fraction prepared in (1) as a sample, the absorbance difference for each lipoprotein fraction was calculated in the same manner as in (2). The cholesterol concentration (mg / dL) in each lipoprotein fraction was determined from the calculated absorbance difference and the calibration curve in (2).

[0095] [Example 1] Kits for measuring LDL-C (Kits 1A to 1E) consisting of the following first and second reagents were prepared. First Reagent PIPES (pH 7.0) 50mmol / L EMSE 0.3g / L Ascorbic acid oxidase 4kU / L Second Reagent PIPES (pH 7.0) 50mmol / L 4-AA 0.5g / L Calcium chloride 0.1g / L Potassium ferrocyanide 0.02g / L LPL-311 3kU / L CHO-CE 2kU / L Peroxidase 20kU / L Pluronic L-121 7g / L Surfactants A to E (see Table 1)

[0096] [Comparative Example 1] Kits for measuring LDL-C (kits 1a to 1e) consisting of the following first and second reagents were prepared. First Reagent PIPES (pH 7.0) 50mmol / L EMSE 0.3g / L Ascorbic acid oxidase 4kU / L Second Reagent PIPES (pH 7.0) 50mmol / L 4-AA 0.5g / L Calcium chloride 0.1g / L Potassium ferrocyanide 0.02g / L LPL-311 3kU / L CHO-CE 2kU / L Peroxidase 20kU / L Pluronic L-121 7g / L Surfactants a-e (see Table 1)

[0097] [Example 2] Kit 1A of Example 1 was used as an LDL-C measurement kit, and each lipoprotein fraction prepared in Reference Example 1 (1) was used as a sample, and the reaction rate of cholesterol in each lipoprotein fraction was determined according to the following procedure. Kit 1A of Example 1 and Metabolead LDL-C were also used as an LDL-C measurement kit, and 20 samples of human serum prepared in (4) described below were used as samples, and the correlation between the measurement method using Kit 1A of Example 1 and the measurement method using Metabolead LDL-C was verified according to the following procedure, and the correlation coefficient was determined.

[0098] (1) Preparation of a calibration curve for determining LDL-C concentration Kit 1A of Example 1 was used as the LDL-C measurement kit, and an LDL-C standard with a determined LDL-C concentration was used as the sample, and the absorbance of the LDL-C standard was measured according to the following procedure. The standard (2.5 μL) and the first reagent (150 μL) of Kit 1A of Example 1 were added to the reaction cell, and the reaction was allowed to proceed at 37° C. for 5 minutes, and the absorbance (E1) of the reaction solution was measured at a main wavelength of 600 nm and a secondary wavelength of 700 nm. Next, the second reagent (50 μL) of Kit 1A of Example 1 was added to the reaction solution, and the reaction was allowed to proceed for another 5 minutes at 37° C., and the absorbance (E2) of the reaction solution was measured at a main wavelength of 600 nm and a secondary wavelength of 700 nm. E1 was subtracted from E2 to obtain the absorbance difference ΔE 標準品 was calculated. The absorbance difference ΔE was measured by the same method except that saline was used instead of the standard. ブランク was calculated. Absorbance difference ΔE relative to the standard 標準品 and the absorbance difference ΔE relative to saline ブランク From this, a calibration curve was created showing the relationship between LDL-C concentration (mg / dL) and absorbance.

[0099] (2) Determination of cholesterol concentration in each lipoprotein fraction Using Kit 1A of Example 1 and each lipoprotein fraction prepared in (1) of Reference Example 1 as a sample, the absorbance difference for cholesterol in each lipoprotein fraction was calculated in the same manner as in (1). The cholesterol concentration (mg / dL) in each lipoprotein fraction was determined from the calculated absorbance difference and the calibration curve in (1).

[0100] (3) Determination of the reaction rate of cholesterol in each lipoprotein fraction From the cholesterol concentration in each lipoprotein fraction determined in (3) of Reference Example 1 and the cholesterol concentration in each lipoprotein fraction determined in (2) above, the reaction rate of cholesterol in each lipoprotein fraction in Kit 1A of Example 1 was determined according to the following formula (I).

[0101]

number

[0102] The reaction rate of cholesterol in each lipoprotein fraction was determined and expressed as "-" for 0% to less than 10%, "±" for 10% to less than 20%, "+" for 20% to less than 40%, and "++" for 40% or more. The results are shown in Table 1.

[0103] (4) Determination of LDL-C concentration in human serum Whole blood collected from a healthy individual belonging to Kyowa Medex Co., Ltd. was centrifuged at 3,000 rpm and 25°C for 20 minutes to prepare 20 human serum samples. Next, Kit 1A of Example 1 and MetaboLead LDL-C were used as an LDL-C measurement kit, and the 20 human serum samples were used as samples. The absorbance difference for each human serum was calculated in the same manner as in (1). The LDL-C concentration (mg / dL) in each human serum was determined from the calculated absorbance difference and the calibration curve in (1).

[0104] (5) Determination of correlation coefficient between the measurement method using the measurement kit of the present invention and the measurement method using Metabolead LDL-C From the LDL-C concentration (mg / dL) in each human serum determined in (4) using Kit 1A of Example 1 as the LDL-C measurement kit and the LDL-C concentration (mg / dL) in each human serum determined in (4) using Metabolead LDL-C as the LDL-C measurement kit, the correlation between the measurement method using Kit 1A of Example 1 and the measurement method using Metabolead LDL-C was verified, and the correlation coefficient was determined. The results are shown in Table 1.

[0105] [Example 3] Except for using Kits 1B, 1C, 1D, and 1E of Example 1 as LDL-C measurement kits instead of Kit 1A of Example 1, the reaction rate of each kit to cholesterol in each lipoprotein fraction and the correlation coefficient between the measurement method using each kit and the measurement method using Metabolead LDL-C were determined in the same manner as in Example 2. The results are shown in Table 1.

[0106] [Comparative Example 2] Except for using kits 1a, 1b, 1c, 1d, and 1e of Comparative Example 1 as LDL-C measurement kits instead of kit 1A of Example 1, the reaction rate of cholesterol in each lipoprotein fraction in each kit and the correlation coefficient between the measurement method using each kit and the measurement method using Metabolead LDL-C were determined in the same manner as in Example 2. The results are shown in Table 1.

[0107] [Table 1]

[0108] As described in (1) of Reference Example 1, each lipoprotein fraction used in the measurement was prepared by separating VLDL, LDL, and HDL by ultracentrifugation. Therefore, it means that the lower the reaction rate of cholesterol in the VLDL fraction and cholesterol in the HDL fraction, and the higher the reaction rate of cholesterol in the LDL fraction, the more accurately the LDL-C in the sample can be measured with an LDL-C measurement kit. As is clear from Table 1, it was found that the reaction rate of the VLDL fraction and HDL fraction was lower and the reaction rate of the LDL fraction was higher in the measurement using each kit of Kits 1A to 1E in Example 1, compared with the measurement using each kit of Kits 1a to 1e in Comparative Example 1. Therefore, it was revealed that the LDL-C in the sample can be accurately measured by the LDL-C measurement method of the present invention using the LDL-C measurement kit of the present invention, which contains one or more surfactants selected from the group consisting of POE alkyl ethers and POEPOP alkyl ethers and a POEPOP condensate.

[0109] As is clear from Table 1, the correlation coefficient between the measurement method using each of Kits 1A to 1E in Example 1 and the measurement method using Metabolead LDL-C was 0.95 or more, and a good correlation was observed between the two measurements. On the other hand, no good correlation was observed between the measurement method using each of Kits 1a to 1e in Comparative Example 1 and the measurement method using Metabolead LDL-C. Therefore, it was revealed that LDL-C in a sample can be accurately measured by the LDL-C measurement method of the present invention using the LDL-C measurement kit of the present invention, which contains one or more surfactants selected from the group consisting of POE alkyl ethers and POEPOP alkyl ethers and a POEPOP condensate.

[0110] [Example 4] Whole blood collected from a healthy individual affiliated with Kyowa Medex Co., Ltd. was centrifuged at 3,000 rpm and 25° C. for 20 minutes to prepare five human serum specimens. Next, using Kit 1E of Example 1 and Metabolead LDL-C as an LDL-C measurement kit and the five human serum specimens as samples, the LDL-C concentration (mg / dL) in each human serum was determined in the same manner as in (1) and (2) of Example 2. The results are shown in Table 2.

[0111] [Table 2]

[0112] As is clear from Table 2, the LDL-C concentration (mg / dL) determined in the measurement using Kit 1E of Example 1 as the LDL-C measurement kit was almost the same as the LDL-C concentration (mg / dL) determined in the measurement using Metabolead LDL-C as the LDL-C measurement kit. Therefore, it was demonstrated that LDL-C in a sample can be accurately measured by the LDL-C measurement method of the present invention using the LDL-C measurement kit of the present invention containing one or more surfactants selected from the group consisting of POE alkyl ethers and POEPOP alkyl ethers and a POEPOP condensate.

[0113] [Example 5] Kits for measuring LDL-C (Kits 2A to 2D) consisting of the following first and second reagents were prepared. First Reagent PIPES (pH 7.0) 50mmol / L EMSE 0.3g / L Ascorbic acid oxidase 4kU / L Second Reagent PIPES (pH 7.0) 50mmol / L 4-AA 0.5g / L Calcium chloride 0.1g / L Potassium ferrocyanide 0.02g / L LPL-311 3kU / L CHO-CE 2kU / L Peroxidase 20kU / L Pluronic L-121 7g / L WONDASURF S-1400 4g / L Polyoxypropylene derivatives A to D that do not contain polyoxyethylene in the molecule (see Table 3)

[0114] [Comparative Example 3] An LDL-C measurement kit 2a was prepared, which consisted of the following first and second reagents. First Reagent PIPES (pH 7.0) 50mmol / L EMSE 0.3g / L Ascorbic acid oxidase 4kU / L Second Reagent PIPES (pH 7.0) 50mmol / L 4-AA 0.5g / L Calcium chloride 0.1g / L Potassium ferrocyanide 0.02g / L LPL-311 3kU / L CHO-CE 2kU / L Peroxidase 20kU / L Pluronic L-121 7g / L WONDASURF S-1400 4g / L PEG#2000 3.6g / L

[0115] The LDL-C measurement kit 2a of Comparative Example 3 is the same as the LDL-C measurement kits of Example 5 (Kits 2A to 2D) except that PEG#2000 is used instead of the polyoxypropylene derivative not containing polyoxyethylene in the molecule in the second reagent.

[0116] [Example 6] Kit 2A of Example 5 was used as the LDL-C measurement kit, and Intralipos-added serum prepared by adding Intralipos to serum, and saline-added serum prepared by adding saline to serum were used as samples. The LDL-C concentrations in the Intralipos-added serum and the saline-added serum were determined by the following procedure to evaluate the effect of Intralipos.

[0117] (1) Preparation of Intralipos-added serum and saline-added serum Human serum was mixed with 10% Intralipos infusion in a ratio of 9:1 to prepare serum supplemented with Intralipos.Similarly, human serum was mixed with saline in a ratio of 9:1 to prepare serum supplemented with saline. (2) Determination of LDL-C concentration in serum containing Intralipos and in serum containing saline Kit 2A of Example 5 was used as the LDL-C measurement kit, and the Intralipos-added serum and saline-added serum prepared in (1) were used as samples, and the LDL-C concentration (mg / dL) in the serum was determined by procedures similar to those in (1) and (2) of Example 2. (3) Evaluation of the effect of IntraLipos on LDL-C measurement The effect of Intralipos on LDL-C measurement using Kit 2A of Example 5 was evaluated based on the LDL-C concentration (mg / dL) in serum containing Intralipos determined in (2) using Kit 2A of Example 5 as an LDL-C measurement kit, and the LDL-C concentration (mg / dL) in serum containing saline, according to the following formula (II). The results are shown in Table 3.

[0118]

number

[0119] [Example 7] The effect of Intralipos on LDL-C measurement using each of the kits 2B, 2C, 2D, and 1E of Example 5 was evaluated in the same manner as in Example 6, except that each of the kits 2B, 2C, 2D, and 1E of Example 5 was used as the LDL-C measurement kit instead of the kit 2A of Example 5. The results are shown in Table 3.

[0120] [Comparative Example 4] Except for using Kit 2a of Comparative Example 3 as the LDL-C measurement kit instead of Kit 2A of Example 5, the effect of Intralipos on LDL-C measurement using each kit was evaluated in the same manner as in Example 6. The results are shown in Table 3.

[0121] [Table 3]

[0122] Intralipos is a lipid emulsion whose main component is triglyceride derived from soybean oil, and is often used to evaluate the effect of lipid turbidity in samples for clinical test reagents. Here, the closer the ratio of the LDL-C concentration in serum containing Intralipos to the LDL-C concentration in serum containing saline is to 100%, the more accurate the LDL-C measurement will be without the effect of Intralipos.

[0123] As is clear from Table 3, compared with kit 1E not containing a polyoxypropylene derivative not containing polyoxyethylene in the molecule, and kit 2a of Comparative Example 3 containing polyoxyethylene glycol instead of a polyoxypropylene derivative not containing polyoxyethylene in the molecule, the ratio of the LDL-C concentration in the serum containing intralipos to the LDL-C concentration in the serum containing physiological saline in each kit of kits 2A to 2D of Example 5 containing a polyoxypropylene derivative not containing polyoxyethylene in the molecule was high at 70% or more. Therefore, it has become clear that the LDL-C measurement method of the present invention using the LDL-C measurement kit of the present invention containing one or more surfactants selected from the group consisting of POE alkyl ethers and POEPOP alkyl ethers, POEPOP condensates, and polyoxypropylene derivatives not containing polyoxyethylene in the molecule can accurately measure LDL-C in a sample without being affected by turbidity due to lipids. [Industrial Applicability]

[0124] INDUSTRIAL APPLICABILITY The present invention provides a method for measuring LDL-C, a measuring reagent and a measuring kit, which are useful for diagnosing metabolic syndrome, arteriosclerosis and the like.

Claims

1. A sample is mixed with (i) a combination of cholesterol esterase and cholesterol oxidase, or (ii) a combination of cholesterol esterase, an oxidized coenzyme, and cholesterol dehydrogenase, [a] one or more surfactants selected from the group consisting of polyoxyethylene alkyl ethers and polyoxyethylene polyoxypropylene alkyl ethers; [b] polyoxyethylene polyoxypropylene condensates; and [c] A method for reducing the effect of turbidity due to lipids in a sample in the measurement of cholesterol in low-density lipoprotein in the sample, which comprises reacting polypropylene glycol or polyoxypropylene glyceryl ether in an aqueous medium not containing a surfactant having an alkylphenol structure, and measuring a substance produced or consumed in the reaction.

2. [a] The method according to claim 1, wherein the alkyl in the polyoxyethylene alkyl ether and the polyoxyethylene polyoxypropylene alkyl ether has 8 to 20 carbon atoms.

3. 3. The method according to claim 1 or 2, wherein the product is hydrogen peroxide.

4. 4. The method according to claim 3, wherein the measurement of hydrogen peroxide is carried out using a reagent for measuring hydrogen peroxide.

5. 3. The method according to claim 1 or 2, wherein the produced substance is a reduced coenzyme.

6. 6. The method according to claim 5, wherein the measurement of reduced coenzymes is carried out using a reagent for measuring reduced coenzymes.

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