Reagent Compositions and Kits

Abstract

This reagent composition is used as a first reagent composition for a method for quantifying small dense LDL cholesterol (sdLDL-C) in a sample, the method comprising: a step for applying a first reagent composition to the sample; and a step for, after the step for applying the first reagent composition to the sample, applying a second reagent composition for quantifying the sdLDL-C to quantify cholesterol in a remaining lipoprotein, wherein the reagent composition has one or two or more activities selected from the group consisting of cholesterol esterase activity, cholesterol oxidase activity, and sphingomyelinase activity, and contains a polyoxyethylene monostyrenated phenyl ether.

Description

[Technical field]

[0001] The present invention relates to reagent compositions and kits. [Background technology]

[0002] As a technique for measuring LDL cholesterol, there is a technique described in Patent Document 1 (JP Patent Publication 2000-325097 A). The document describes a method for measuring lipoprotein cholesterol, which includes a first step of selectively enzymatically reacting HDL cholesterol by adding an enzyme and a first surfactant to a sample containing lipoprotein, a second step of selectively enzymatically reacting LDL cholesterol by adding a second surfactant, and measuring a compound consumed or produced by the reaction with the enzyme in the first or second step to measure HDL cholesterol and / or LDL cholesterol (Claim 1). According to this method, a lipoprotein agglutinant that increases the turbidity of the reaction solution is not required, there is no restriction on the enzyme to be used, and there is no need to add a new enzyme in the step of reacting LDL cholesterol, and LDL cholesterol can be quantified simply and inexpensively. In addition, a measurement method and a measurement reagent can be provided that can accurately and inexpensively measure HDL cholesterol as needed without forming lipoprotein aggregates that interfere with optical measurement, and therefore the method is considered to be particularly useful in the field of clinical testing for arteriosclerosis, etc. (paragraph 0055). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2000-325097 A Summary of the Invention [Problem to be solved by the invention]

[0004] The present inventors have investigated the measurement of small dense LDL cholesterol (sdLDL-C) among LDL cholesterol, and have found that the technique described in Patent Document 1 has room for improvement in terms of measurement accuracy.

[0005] The present invention provides a technique for measuring sdLDL-C with excellent accuracy. [Means for solving the problem]

[0006] According to the present invention, the following reagent compositions and kits are provided. [1] A step of applying a first reagent composition to a sample; After the step of applying the first reagent composition to the sample, a step of applying a second reagent composition for quantifying small dense LDL cholesterol (sdLDL-C) to quantify cholesterol in the remaining lipoproteins; A reagent composition used as the first reagent composition in the method for quantifying sdLDL-C in the sample, comprising: having one or more activities selected from the group consisting of cholesterol esterase activity, cholesterol oxidase activity and sphingomyelinase activity; A reagent composition comprising polyoxyethylene monostyrenated phenyl ether. [2] The reagent composition described in [1], wherein the degree of polymerization n of polyoxyethylene in the polyoxyethylene monostyrenated phenyl ether is 5 or more and 80 or less. [3] The reagent composition according to [1] or [2], wherein the content of the polyoxyethylene monostyrenated phenyl ether in the reagent composition is 0.05% (w / v) or more and 0.6% (w / v) or less based on the total amount of the reagent composition. [4] The reagent composition described in any one of [1] to [3], further having at least one activity selected from the group consisting of peroxidase activity and catalase activity. [5] The reagent composition described in any one of [1] to [4], wherein the reagent composition contains either a hydrogen donor or a coupler. [6] A first reagent composition comprising the reagent composition according to any one of [1] to [5]; A second reagent composition for quantifying sdLDL-C; A kit for use in quantifying the sdLDL-C in the sample, comprising: [7] The kit described in [6], wherein the second reagent composition has peroxidase activity. [8] The first reagent composition contains either a hydrogen donor or a coupler but not the other, The kit according to [6] or [7], wherein the second reagent composition does not contain one of the hydrogen donor and the coupler but contains the other. Effect of the Invention

[0007] According to the present invention, a technique for measuring sdLDL-C with excellent accuracy can be provided. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows the results of evaluation of the accuracy of sdLDL-C measurement values. [Diagram 2] FIG. 1 shows the results of evaluation of the accuracy of sdLDL-C measurement values. [Diagram 3] FIG. 1 shows the results of evaluation of the accuracy of sdLDL-C measurement values. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Hereinafter, an embodiment will be described. In this embodiment, a composition such as a measurement reagent may contain each component alone or in combination of two or more. In addition, in this specification, a numerical range "x to y" means "not less than x and not more than y", and includes both the lower limit x and the upper limit y.

[0010] First, lipoproteins will be described. Lipoproteins are roughly divided into Very Low Density Lipoprotein (VLDL), Low Density Lipoprotein (LDL) and High Density Lipoprotein (HDL), and LDL is further divided into small dense LDL (sdLDL) and other subfractions. sdLDL is also called small particle LDL, small LDL (SLDL), dense LDL, or small, dense LDL, and other LDLs are also called large LDL (L LDL), light LDL, or large buoyant LDL (lbLDL).

[0011] These lipoprotein fractions and subfractions can be distinguished by particle size or density. The particle size diameters of lipoproteins vary depending on the reporter, but for example, VLDL is 30 nm to 80 nm (or 30 nm to 75 nm), LDL is 22 nm to 28 nm (or 19 nm to 30 nm), and HDL is 7 to 10 nm. The density of lipoproteins, for example, is 1.006 g / cm for VLDL. 3 Below, LDL is 1.019-1.063g / cm 3 , HDL is 1.063-1.21g / cm 3 It is.

[0012] Among lipoproteins, the particle diameter of LDL can be measured, for example, by gradient gel electrophoresis (GGE) (JAMA, 260, p.1917-21, 1988) or NMR (HANDBOOK OF LIPOPROTEIN TESTING 2nd Edition, edited by Nader Rifai et al., p.609-623, AACC PRESS: The Fats of Life Summer 2002, LVDD 15 YEAR ANNIVERSARY ISSUE, Volume AVI No.3, p.15-16). In addition, the density can be determined, for example, based on analysis by ultracentrifugation (Atherosclerosis, 106, p.241-253, 1994: Atherosclerosis, 83, p.59, 1990).

[0013] In this embodiment, the sdLDL to be measured is generally a subfraction of the LDL fraction having a diameter of about 22.0 to about 25.5 nm, or a density of 1.040 to 1.063 g / cm. 3 This refers to a subfraction of . LDL is divided into subfractions based on size because small particle diameter LDL is highly arteriosclerotic and has a higher malignancy than other LDLs, so it was necessary to measure small LDL separately. The diameter and density distributions within LDL are continuous, and it is not possible to clearly distinguish between LDLs with a certain density or higher that are particularly malignant. Therefore, the above density of 1.040 to 1.063 g / cm 3 This value is not an established characteristic of sdLDL, but the LDL density range of 1.019 to 1.063 g / cm is widely used and can be considered an established value. 3 For example, another report on the density of sdLDL is 1.044 to 1.060 g / cm 3 (Atherosclerosis:106 241-253 1994). The range of density of sdLDL varies slightly depending on the author, but in all cases, the presence of sdLDL when separated within that range is related to clinical malignancy.

[0014] In this specification, the term sdLDL specifically refers to LDL with a high density and clinically more atherosclerosis-inducing property than other LDL. In addition, sdLDL preferably belongs to a density range higher than the midpoint of the LDL density range, more preferably has a density of 1.044 to 1.063 g / cm. 3 Lipoproteins other than LDL include VLDL and HDL, and may also include chylomicrons, IDL (intermediate density lipoprotein), and VHDL (very high density lipoprotein).

[0015] The present inventors have studied to improve the accuracy of quantifying sdLDL-C in a sample. As a result, it has been found that when quantifying sdLDL-C by a method including a step (first step) of applying a first reagent composition to a sample, and then a step (second step) of applying a second reagent composition for quantifying sdLDL cholesterol (sdLDL-C) to quantify cholesterol in remaining lipoproteins, it is important to highly control the selectivity of the first reagent composition's action on LDL other than sdLDL in the first step. More specifically, it is important that the first reagent composition selectively acts on LDL other than sdLDL in the first step, and if the selectivity is too low, that is, if the first reagent composition is a reagent that easily acts on the sdLDL to be measured, there is a concern that the accuracy of the quantification of sdLDL-C in a sample in the second step will decrease. Therefore, the present inventors conducted further studies to improve the selectivity of the action of the first reagent composition and found that by configuring the first reagent composition to have a specific enzymatic activity and to contain a specific surfactant, a first reagent composition that acts with high selectivity on LDL other than sdLDL can be stably obtained. Furthermore, it is known that polyoxyethylene styrenated phenyl ether derivatives or polyoxyethylene distyrenated phenyl ether can be used as surfactants. However, the present inventors have newly discovered through their intensive research that the presence of polyoxyethylene monostyrenated phenyl ether among polyoxyethylene styrenated phenyl derivatives increases the selectivity for L LDL when the sample reacts with the first reagent composition, and as a result, sdLDL-C can be accurately measured. Each reagent composition will now be described in more detail.

[0016] (Reagent composition (first reagent composition)) In this embodiment, the reagent composition is used as a first reagent composition in a method for quantifying sdLDL-C in a sample, the method including the steps of: applying a first reagent composition to the sample; and, after the step of applying the first reagent composition to the sample, applying a second reagent composition for quantifying sdLDL-C to quantify cholesterol in remaining lipoproteins. Specifically, the first reagent composition is liquid. Hereinafter, the reagent composition used as the first reagent composition will also be simply referred to as the "first reagent composition." The first reagent composition has one or more activities selected from the group consisting of cholesterol esterase activity, cholesterol oxidase activity, and sphingomyelinase activity, and contains polyoxyethylene monostyrenated phenyl ether.

[0017] In this embodiment, since the first reagent composition contains a specific nonionic surfactant and has a specific enzyme activity, when the first reagent composition is added to a sample, it can stably act on and eliminate LDL other than sdLDL in the sample with high selectivity. Also, cholesterol in LDL other than sdLDL can be stably led out of the reaction system with high selectivity.

[0018] Here, "the surfactant acts (reacts)" means that the surfactant breaks down lipoproteins and releases cholesterol from the lipoproteins. For example, in the case of "a surfactant acting (reacting) on ​​lipoproteins other than sdLDL", it is not required that the surfactant does not act on sdLDL at all, but rather that it acts mainly on lipoproteins other than sdLDL. Similarly, in the case of "a surfactant acting (reacting) on ​​LDL other than sdLDL", it is required that the surfactant acts mainly on LDL other than sdLDL. "Elimination" means that a substance in a test sample is decomposed so that the decomposition product is not detected in the next step. In other words, "eliminating cholesterol in lipoproteins other than sdLDL" means that lipoproteins other than sdLDL in a test sample are decomposed so that the cholesterol in these lipoproteins, which is the decomposition product, is not detected in the subsequent step. Similarly, "eliminating cholesterol in LDL other than sdLDL" means that cholesterol in LDL other than sdLDL in a test sample is not detected in the subsequent step.

[0019] Specifically, "guiding out of the reaction system" refers to eliminating or agglutinating cholesterol contained in HDL, VLDL, L LDL, etc., or inhibiting it from reacting in a subsequent step so that the cholesterol contained in HDL, VLDL, L LDL, etc. does not affect the quantification of sdLDL-C. The components contained in the first reagent composition will be described in more detail below.

[0020] (Polyoxyethylene monostyrenated phenyl ether) The first reagent composition contains polyoxyethylene monostyrenated phenyl ether (POE monostyrenated phenyl ether). POE monostyrenated phenyl ether may be composed of a plurality of compounds having different polymerization degrees of the POE portion. In this case, the polymerization degree n of polyoxyethylene in POE monostyrenated phenyl ether is preferably 5 or more and 80 or less from the viewpoint of improving the accuracy in the quantification of sdLDL-C. In addition, the average polymerization degree of oxyethylene is, for example, more than 1, preferably 5 or more, more preferably 10 or more, and for example, 100 or less, preferably 80 or less, more preferably 50 or less, even more preferably 40 or less, and may be, for example, 30 or less, from the viewpoint of improving the accuracy in the quantification of sdLDL-C. Here, the average degree of polymerization is determined from the intensity of the oxyethylene signal of POE monostyrenated phenyl ether measured by NMR. For example, in the NMR spectrum of the TMS derivative of POE monostyrenated phenyl ether, it can be calculated from the ratio of the signal intensity of the methylene protons (4) of the POE part at 3.5 ppm to the intensity of the protons (9) of the TMS group at 0.08 ppm.

[0021] In addition, from the viewpoint of more stably improving the accuracy in the quantification of sdLDL-C, it is preferable to include one or more types selected from the group consisting of compounds in which the average degree of polymerization n of oxyethylene in POE monostyrenated phenyl ether is n = 10 to 20. Here, the degree of polymerization of oxyethylene in the components constituting POE monostyrenated phenyl ether is determined by structural analysis using liquid chromatography mass spectrometry. For example, when POE monostyrenated phenyl ether in the first reagent composition is separated by liquid chromatography (LC) and then introduced into a quadrupole time-of-flight mass spectrometer (QTOF mass spectrometer), a mass spectrum consisting of peaks spaced 44 amu apart is obtained due to the difference in the degree of polymerization of oxyethylene in each component. The degree of polymerization of oxyethylene in each component can be calculated by subtracting the mass number (MW 198) of monostyrenated phenol from the molecular weight estimated from the m / z of each peak and dividing by 44. Furthermore, the chemical composition (C 14 H 14 The degree of polymerization of oxyethylene in each component can be analyzed by subtracting 1O) or by performing a product ion scan and performing detailed structural analysis.

[0022] From the viewpoint of improving the accuracy in the quantification of sdLDL-C, the content of POE monostyrenated phenyl ether in the first reagent composition is preferably 0.01% (w / v) or more, more preferably 0.03% (w / v) or more, even more preferably 0.05% (w / v) or more, and even more preferably 0.1% (w / v) or more, based on the entire first reagent composition. From the same viewpoint, the content of POE monostyrenated phenyl ether in the first reagent composition is preferably 0.6% (w / v) or less, more preferably 0.4% (w / v) or less, even more preferably 0.35% (w / v) or less, and even more preferably 0.3% (w / v) or less, based on the entire first reagent composition.

[0023] Here, POE monostyrenated phenyl ether is available as an industrial raw material or a research reagent, and for example, a commercially available product can be used. Alternatively, for example, POE monostyrenated phenyl ether may be produced and used. Specifically, it can be produced by the following method and conditions. That is, POE monostyrenated phenyl ether is obtained by adding a predetermined amount of ethylene oxide to monostyrenated phenol in the presence of a basic catalyst. Examples of the basic catalyst include sodium hydroxide, potassium hydroxide, and alcoholates of alkali metals. The addition reaction temperature is preferably 120 to 200°C. On the other hand, styrenated phenol can be obtained by alkylating phenol and styrene in the presence of an acid catalyst at temperatures between 70°C and 200°C. Examples of acid catalysts that can be used include inorganic acids such as sulfuric acid and phosphoric acid, organic acids such as p-toluenesulfonic acid and methanesulfonic acid, and Lewis acids such as boron trifluoride diethyl ether complex. In this reaction, by controlling the conditions such as the equivalent ratio of phenol to styrene, the type of acid catalyst, and the reaction temperature, it is possible to obtain monostyrenated phenol, in which one styrene is bonded to the phenol and benzene ring. Monostyrenated phenol can be synthesized, for example, by adding styrene dropwise to phenol in the presence of a phosphoric acid catalyst to carry out an alkylation reaction, and then adding a sulfuric acid or magnesium sulfate catalyst to remove unreacted phenol and styrene to complete the alkylation reaction. In this case, the amount of phosphoric acid catalyst used is preferably 0.001 to 0.01 equivalents relative to phenol. The amount of sulfuric acid or magnesium sulfate catalyst used is preferably 2% by mass to 10% by mass based on the mass of the phosphoric acid catalyst. The equivalent ratio of phenol to styrene is preferably 0.9 to 1.3 in terms of the equivalent ratio of styrene to phenol. Subsequently, after the alkylation reaction is completed, an aqueous solution of sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide or the like is added to neutralize the product of the alkylation reaction, and the resulting neutralized salt is removed by filtration, whereby monostyrenated phenol can be recovered.

[0024] The obtained monostyrenated phenol and ethylene oxide are subjected to an addition reaction under heating and pressure in a pressure-resistant vessel such as an autoclave using potassium hydroxide as a catalyst, thereby obtaining POE monostyrenated phenyl ether. The heating and pressure conditions are, for example, a pressure of 1.5 kg / cm. 3The temperature is about 130° C. In addition, the number of repeating oxyethylene units, that is, the degree of polymerization of oxyethylene, can be adjusted by adjusting the molar ratio of ethylene oxide to 1 mole of monostyrenated phenol.

[0025] In addition, in the above-mentioned method, a mixture of monostyrenated phenol, distyrenated phenol in which two styrene units are bonded, and tristyrenated phenol in which three styrene units are bonded can be obtained as the styrenated phenol, and monostyrenated phenol can be separated and purified from the mixture for use.

[0026] The POE monostyrenated phenyl ether in the first reagent composition can be confirmed, for example, by an analytical method that combines IR, NMR, LC-MS, etc. Examples of a method for determining the structure of the POE monostyrenated phenyl ether in the first reagent composition include an analytical method using LC / MS / MS and NMR. The concentration of POE monostyrenated phenyl ether in the first reagent composition can be calculated, for example, by HPLC or LC / MS measurement. The concentration of POE monostyrenated phenyl ether in the first reagent composition can also be obtained by NMR measurement.

[0027] (enzyme) The first reagent composition has one or more activities selected from the group consisting of cholesterol esterase (CHE) activity, cholesterol oxidase (COO) activity, and sphingomyelinase activity. From the viewpoint of allowing the first reagent composition to act more stably on lipoproteins other than sdLDL and directing the cholesterol out of the reactive form, the first reagent composition preferably further has at least one activity selected from the group consisting of peroxidase activity (POD) and catalase activity.

[0028] Here, "having cholesterol esterase activity" specifically means that an enzyme (e.g., cholesterol esterase) capable of hydrolyzing a substrate, cholesterol ester, exists and a reaction catalyzed by cholesterol esterase can occur. The same applies to other enzyme activities such as cholesterol oxidase activity. "Having sphingomyelinase activity" means that an enzyme capable of decomposing the substrate sphingomyelin exists, and a reaction catalyzed by the enzyme can occur. Enzymes with sphingomyelinase activity include sphingomyelinase C, sphingomyelinase D, and phospholipase C and phospholipase D, which are capable of decomposing sphingomyelin.

[0029] Moreover, the first reagent composition specifically contains at least one enzyme selected from the group consisting of an enzyme having cholesterol esterase activity, an enzyme having cholesterol oxidase activity, and an enzyme having sphingomyelinase activity, and more specifically contains at least one selected from the group consisting of cholesterol esterase, cholesterol oxidase, and sphingomyelinase. From the viewpoint of allowing the first reagent composition to act more stably on lipoproteins other than sdLDL and remove the cholesterol from the reactive form, the first reagent composition preferably further contains at least one enzyme selected from the group consisting of enzymes having peroxidase activity and enzymes having catalase activity, and more specifically, it is preferable that the first reagent composition contains at least one of peroxidase and catalase. Cholesterol esterase, cholesterol oxidase, sphingomyelinase, peroxidase and catalase that can be used are, for example, those derived from bacteria or fungi, or those derived from plants.

[0030] The cholesterol esterase activity of the first reagent composition, from the viewpoint of more stably directing cholesterol in lipoproteins other than sdLDL out of the reaction system, is preferably 50 U / L or more, more preferably 100 U / L or more, and also preferably 3000 U / L or less, more preferably 2500 U / L or less, even more preferably 2000 U / L or less, even more preferably 1000 U / L or less, and may be, for example, 1800 U / L or less, or, for example, 1500 U / L or less.

[0031] The cholesterol oxidase activity of the first reagent composition, from the viewpoint of more stably directing cholesterol in lipoproteins other than sdLDL out of the reaction system, is preferably 100 U / L or more, more preferably 150 U / L or more, and also preferably 800 U / L or less, more preferably 750 U / L or less, even more preferably 700 U / L or less, even more preferably 650 U / L or less, and still more preferably 600 U / L or less.

[0032] The sphingomyelinase activity of the first reagent composition is preferably 100 U / L or more, more preferably 200 U / L or more, from the viewpoint of more stably removing cholesterol in lipoproteins other than sdLDL from the reaction system, and is preferably 3000 U / L or less, more preferably 2800 U / L or less.

[0033] From the viewpoint of more stably directing cholesterol in lipoproteins other than sdLDL out of the reaction system, the peroxidase activity of the first reagent composition is preferably 200 U / L or more, more preferably 300 U / L or more, and also preferably 3000 U / L or less, more preferably 2500 U / L or less, and also preferably, for example, 2000 U / L or less. The peroxidase activity of the first reagent composition may be, for example, 5000 U / L or less, or, for example, 4000 U / L or less.

[0034] The catalase activity of the first reagent composition is preferably 100 KU / L or more, more preferably 200 KU / L or more, and also preferably 2000 KU / L or less, more preferably 1500 KU / L or less, from the viewpoint of stably removing hydrogen peroxide generated when the first reagent composition is applied to a sample, and from the viewpoint of more stably directing cholesterol in lipoproteins other than sdLDL in a sample out of the reaction system.

[0035] The cholesterol oxidase activity, cholesterol esterase activity, sphingomyelinase activity, peroxidase activity and calatase activity of the first reagent composition can be measured, for example, by the following methods.

[0036] In the measurement of cholesterol oxidase activity, a 6 mM cholesterol solution (dissolved in isopropanol) is used as a substrate solution. A diluent (0.1 M phosphate buffer, TritonX100, pH 7.0) is added to the measurement target so that the measurement target is 2 to 4 U / mL, and 3 mL of the diluted solution is heated at 37°C for 5 minutes, after which 0.05 mL of substrate solution is added. The mixture is then reacted at 37°C to measure the amount of absorbance change at a wavelength of 240 nm. After the reaction at 37°C, the amount of absorbance change from 2 to 7 minutes is measured, and the cholesterol oxidase activity is calculated. For example, if the amount of absorbance change is 3 U / L or more, it can be said that the measurement target has cholesterol oxidase activity, and more specifically, that it contains cholesterol oxidase.

[0037] The cholesterol esterase activity was measured using a substrate (0.04% cholesterol linoleate, 1% TritonX100, 0.6% sodium cholate solution), 300 U / mL cholesterol oxidase solution, and enzyme diluent (20 mM phosphate buffer, 0.5 mM EDTA 2Na, 2 mM MgCl 2, 0.2% bovine serum albumin (BSA), pH 7.5) and reaction solution (0.06% 4-aminoantipyrine, 0.4% phenol, 7.5KU / L peroxidase (POD)). After mixing 1.75mL of reaction solution with 1.0mL of substrate solution, the mixture is heated at 37℃ for 5 minutes, and 0.1mL of cholesterol oxidase solution is added. After heating at 37℃ for 2 minutes, 0.1mL of the measurement target diluted with diluent is added, and the mixture is reacted at 37℃, and the amount of change in absorbance at a wavelength of 500nm is measured. After reaction at 37℃, the amount of change in absorbance from 0 to 3.5 minutes is measured, and cholesterol esterase activity is calculated. For example, if the amount of change in absorbance is 8U / L or more, it can be said that the measurement target has cholesterol esterase activity, and more specifically, that it contains cholesterol esterase.

[0038] The sphingomyelinase activity of the first reagent composition is measured, for example, by the following method. That is, a reaction solution (0.008% sphingomyelin, 0.05% TritonX100 solution, 10 U / mL alkaline phosphatase, 10 U / mL cholesterol oxidase, 2 U / mL peroxidase, 0.02% 4-aminoantipyrine, 0.02% TODB mixture), a reaction stop solution (1% sodium dodecyl sulfate solution), and a diluent (10 mM Tris buffer, 0.1% TritonX100, pH 8.0) are used. 0.08 mL of the reaction solution and 0.003 mL of the measurement target diluted with the diluent are mixed and heated at 37°C for 5 minutes, and then 0.16 mL of the reaction stop solution is added. After the reaction is stopped, the amount of absorbance change at the main wavelength of 546 nm and the sub-wavelength of 700 nm is measured, and the sphingomyelinase activity is calculated. For example, if the change in absorbance is 2 U / L or more, it can be said that the measurement target has sphingomyelinase activity, and more specifically, that it contains sphingomyelinase.

[0039] The peroxidase activity of the first reagent composition is measured, for example, by the following method. That is, reaction solution 1 (1.5 mM HDAOS, 0.05% TritonX100, 50 mM phosphate buffer, pH 7.0), reaction solution 2 (5 mM 4-aminoantipyrine, 0.05% TritonX100, 1% hydrogen peroxide, 50 mM phosphate buffer, pH 7.0), and diluent (50 mM phosphate buffer, pH 7.0) are used. 0.3 mL of reaction solution 1 and 0.08 mL of the measurement target diluted with diluent are mixed and heated at 37°C for 5 minutes. Then, 0.1 mL of reaction solution 2 is added, reacted at 37°C, and the amount of absorbance change at the main wavelength of 600 nm and the secondary wavelength of 700 nm is measured. After the reaction at 37°C, the amount of absorbance change from 2 minutes to 5 minutes is measured to calculate the peroxidase activity. For example, if the change in absorbance is 10 U / L or more, it can be said that the measurement target has peroxidase activity, and more specifically, that it contains peroxidase.

[0040] The catalase activity of the first reagent composition is measured, for example, by the following method. That is, in measuring catalase activity, a substrate (0.06% hydrogen peroxide, 50 mM phosphate buffer, pH 7.0) is used. 2.0 mL of the substrate solution is pre-warmed at 25°C, mixed with 0.1 mL of the measurement target, and the amount of change in absorbance at 240 nm is measured. For example, after reaction at 25°C, the amount of change in absorbance from 0 to 3 minutes is measured to calculate the catalase activity. For example, if the amount of change in absorbance is 50 U / L or more, it can be said that the measurement target has catalase activity, and more specifically, that it contains catalase.

[0041] The first reagent composition may further have other enzyme activities, specifically, it may further have one or more enzyme activities selected from the group consisting of ascorbic acid oxidase activity and lipoprotein lipase (LPL) activity. Furthermore, the first reagent composition may contain, for example, one or more enzymes having enzymatic activity selected from the group consisting of an enzyme having ascorbic acid oxidase activity and an enzyme having lipoprotein lipase activity, and more specifically, it contains one or more enzymes selected from the group consisting of ascorbic acid oxidase and lipoprotein lipase.

[0042] From the viewpoint of avoiding the effect of ascorbic acid coexisting in the sample on the accuracy of the measurement, the first reagent composition preferably further has ascorbic acid oxidase activity, preferably contains an enzyme having ascorbic acid oxidase activity, and more preferably contains ascorbic acid oxidase.

[0043] The ascorbic acid oxidase activity of the first reagent composition is preferably 0.1 U / mL or more, more preferably 0.2 U / mL or more, and is preferably 15 U / mL or less, more preferably 10 U / mL or less, from the viewpoint of avoiding the influence of ascorbic acid coexisting in the sample on the accuracy of the measurement.

[0044] The ascorbic acid oxidase activity of the first reagent composition is measured, for example, by the following method. 2 PO 4 -5 mM NaHPO 4 1 mL of the substrate was preheated at 30°C for 5 minutes, and then diluted with 90 mM NaHPO containing 0.05% BSA. 4 Add 0.1 mL of the measurement target diluted with the solution and mix to start the reaction. After 5 minutes, add 3.0 mL of reaction stop solution (0.2N HCl) to stop the reaction, measure the absorbance at 245 nm, and calculate the ascorbic acid oxidase activity. For example, if the activity is 10 U / L or more, the measurement target can be said to have ascorbic acid oxidase activity, and more specifically, to contain "ascorbic acid oxidase."

[0045] Moreover, from the viewpoint of favorably adjusting the action on various lipoproteins, the first reagent composition further has lipoprotein lipase activity, preferably contains an enzyme having lipoprotein lipase activity, and more preferably contains lipoprotein lipase.

[0046] In this embodiment, the enzymes in the first reagent composition and the second reagent composition described later can also be identified by the following method. That is, first, a sample containing the target enzyme is decomposed with trypsin to obtain fragment peptides, which are detected by a hybrid mass spectrometer. The masses of the peptides obtained by the mass spectrometer and the spectrum (MS / MS data) of the fragment ions obtained by colliding with argon gas in the mass spectrometer are searched in a database (e.g., Mascot search) to identify the protein. If the sequence of the fragment peptides derived from the amino acid sequence in the reagent composition matches the amino acid sequence registered in the database as a unique sequence, it can be considered that the target enzyme is contained.

[0047] In addition, the enzymes in the first reagent composition and the second reagent composition described later can be identified by, for example, the following quantification. That is, among the fragment peptides obtained by decomposing the target enzyme with trypsin, a peptide that is specific to the target enzyme and gives a strong signal in mass spectrometry is selected as the peptide to be quantified. For the peptide to be quantified, an unlabeled peptide and a peptide labeled with a stable isotope as an internal standard are prepared by chemical synthesis. A sample containing the target enzyme is completely digested with trypsin, a known amount of stable isotope-labeled peptide is added, and the peptides are measured in MRM mode (multiple reaction monitoring mode) using a triple quadrupole mass spectrometer (LC-MS / MS) connected to HPLC. A mixture of the unlabeled peptide of the peptide to be quantified and a known amount of stable isotope-labeled peptide is similarly measured to create a calibration curve of the concentration ratio of the internal standard and the peak area ratio, and the absolute amount of the peptide to be quantified in the sample can be calculated to quantify the target enzyme.

[0048] (Other Ingredients) The first reagent composition may contain components other than the above-mentioned components, such as a buffer solution, a salt, a protein having no enzymatic activity, a preservative, a hydrogen donor, a coupler, and a surfactant other than polyoxyethylene monostyrenated phenyl ether.

[0049] The type of buffer solution can be appropriately selected, for example. Specific examples of buffer solutions include MOPS (3-morpholinopropanesulfonic acid) buffer solution, phosphate buffer solution, Tris buffer solution, PIPES (piperazine-1,4-bis(2-ethanesulfonic acid)) buffer solution, and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution. From the viewpoint of maintaining the enzyme activity in the composition and improving the storage stability of the reagent, the concentration of the buffer is preferably 1 mM or more, more preferably 5 mM or more, even more preferably 10 mM or more, and is preferably 300 mM or less, preferably 200 mM or less, even more preferably 150 mM or less, and even more preferably 100 mM or less.

[0050] Specifically, the salt is blended as a pH adjuster or an ionic strength adjuster. Specific examples of the salt include basic substances such as sodium salts, such as sodium hydroxide and sodium sulfate; potassium salts, such as potassium hydroxide; magnesium salts, such as magnesium chloride and magnesium sulfate; and ammonium salts, such as ammonium sulfate and ammonium chloride. In addition, the first reagent composition contains at least one of monovalent cations and divalent cations or salts thereof, which makes it easier to separate sdLDL from L LDL. From the viewpoint of acting more stably as a pH adjuster or an ionic strength adjuster, the salt concentration in the first reagent composition is preferably 2 mmol / L or more, more preferably 5 mmol / L or more, and is preferably 50 mmol / L or less, more preferably 30 mmol / L or less, based on the total composition of the first reagent composition.

[0051] The pH of the first reagent composition is preferably 6.0 or more, more preferably 6.5 or more, from the viewpoint of maintaining the enzyme activity in the composition and improving the storage stability of the reagent, and is preferably 8.0 or less, more preferably 7.5 or less.

[0052] A specific example of a protein having no enzymatic activity is albumin such as bovine serum albumin (BSA). The concentration of albumin such as BSA in the first reagent composition is preferably 1 g / L or more, more preferably 2 g / L or more, and preferably 20 g / L or less, more preferably 10 g / L or less, based on the total composition of the first reagent composition, from the viewpoint of stabilizing the enzyme in the first reagent composition and from the viewpoint of stabilizing the first step of leading cholesterol in lipoproteins other than sdLDL out of the reaction system.

[0053] The first reagent composition preferably contains at least one of a hydrogen donor and a coupler, and more preferably contains either one of a hydrogen donor and a coupler. In this case, either one of the hydrogen donor and the coupler is used to guide cholesterol in lipoproteins other than sdLDL out of the reaction system in the first step. More specifically, either one of the hydrogen donor and the coupler is used to act on cholesterol in lipoproteins other than sdLDL with cholesterol esterase or cholesterol oxidase, and convert the generated hydrogen peroxide into a colorless quinone in the presence of peroxidase. In addition, from the viewpoint of improving the storage stability of the first reagent composition, the first reagent composition preferably contains either a hydrogen donor or a coupler, more preferably contains a hydrogen donor but does not contain a coupler, and even more preferably contains a hydrogen donor and peroxidase but does not contain a coupler. On the other hand, from the viewpoint of easily checking the reactivity of the first reagent composition in the first step described below, the first reagent composition preferably contains a hydrogen donor and a coupler, and more preferably contains a hydrogen donor, a coupler, and a peroxidase. From the same viewpoint, it is also preferable that the first reagent composition contains a coupler and a peroxidase.

[0054] Specific examples of the hydrogen donor include aniline derivatives such as N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N-(3-sulfopropyl)aniline (HALPS), N-(3-sulfopropyl)-3-methoxy-5-aniline (HMMPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-4-fluoro-3,5-dimethoxyaniline (FDAOS), and N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine (EMSE). The concentration of the hydrogen donor in the first reagent composition is preferably 1 mM or more, more preferably 1.5 mM or more, from the viewpoint of directing cholesterol in lipoproteins other than sdLDL out of the reaction system, and is preferably 5 mM or less, more preferably 3 mM or less.

[0055] When the hydrogen donor is contained in the second reagent composition described later, the first reagent composition preferably contains a coupler used in the coupling reaction. Examples of couplers that can be used include, but are not limited to, 4-aminoantipyrine (4AA), aminoantipyrine derivatives, vanillin diamine sulfonic acid, methylbenzthiazolinone hydrazone, and sulfonated methylbenzthiazolinone hydrazone. The concentration of the coupler in the first reagent composition is preferably 0.2 mM or more, more preferably 0.3 mM or more, and is preferably 5.0 mM or less, more preferably 3.3 mM or less, from the viewpoint of stably acting on sdLDL with respect to the entire composition of the reaction solution after the addition of the first reagent composition.

[0056] In this embodiment, the first reagent composition has one or more activities selected from the group consisting of cholesterol esterase activity, cholesterol oxidase activity, and sphingomyelinase activity, and contains polyoxyethylene monostyrenated phenyl ether, so that it acts effectively on LDL other than sdLDL and inhibits its action on sdLDL, and has excellent selectivity for LDL other than sdLDL, and can be suitably used for quantifying sdLDL-C. Therefore, by using the first reagent composition in this embodiment, the accuracy of the measurement of sdLDL-C can be improved. In this embodiment, the first reagent composition is used for quantifying sdLDL-C, for example, in combination with the second reagent composition described below.

[0057] (kit) In this embodiment, the kit is used for quantifying sdLDL-C in a sample, and includes the above-mentioned first reagent composition and a second reagent composition for quantifying sdLDL-C. The kit is specifically used in a method for quantifying sdLDL-C comprising two or more steps, in which the first and second reagent compositions are used in different steps, preferably in the order of the first and second reagent compositions. The composition of the second reagent composition will be described in more detail below.

[0058] (Second Reagent Composition) The second reagent composition is specifically a reagent composition for quantifying sdLDL-C. The components of the second reagent composition vary depending on the configuration of the first reagent composition, but may be any composition that allows sdLDL-C to be quantified, and known substances may be used.

[0059] Specific examples of components contained in the second reagent composition include an enzyme, a buffer solution, a salt, a surfactant, a protein having no enzymatic activity, a preservative, and either a hydrogen donor or a coupler. In a preferred configuration of the kit, the first reagent composition contains either a hydrogen donor or a coupler but not the other, and the second reagent composition contains either a hydrogen donor or a coupler but not the other.

[0060] An example of the enzyme is peroxidase. The second reagent composition has, for example, peroxidase activity. From the viewpoint of accurately measuring sdLDL-C in the second step, the peroxidase activity of the second reagent composition is preferably 500 U / L or more, more preferably 1000 U / L or more, and preferably 10000 U / L or less. However, when the first reagent composition contains peroxidase activity, the peroxidase activity is carried over to the second step, so the concentration of peroxidase in the second reagent can be reduced or eliminated.

[0061] The types of buffer and salt can be appropriately selected depending on, for example, the type of enzyme contained in the second reagent composition. Specific examples of the buffer include those described above for the first reagent composition.

[0062] The pH of the second reagent composition is preferably 6.0 or more, more preferably 6.5 or more, from the viewpoint of maintaining the enzyme activity in the composition and improving the storage stability of the reagent, and is preferably 8.0 or less, more preferably 7.5 or less.

[0063] The surfactant may be, for example, a surfactant that acts on sdLDL. From the viewpoint of stably quantifying sdLDL-C, the second reagent composition preferably contains a surfactant that acts on sdLDL. The surfactant acting on sdLDL may be a surfactant that selectively acts on sdLDL, such as a surfactant that acts only on sdLDL, or it may be a surfactant that also acts on lipoproteins other than sdLDL, or a surfactant that acts on all lipoproteins.

[0064] The surfactant in the second reagent composition may be, for example, a polyoxyethylene derivative, or a surfactant used in a commercially available reagent for measuring total cholesterol, etc. Examples of such surfactants include polyoxyethylene alkylphenyl ethers such as polyoxyethylene octylphenyl ether (e.g., Emulgen 909 (manufactured by Kao Corporation), Triton X-100), and polyoxyethylene alkyl ethers (e.g., Emulgen 707, Emulgen 709 (both manufactured by Kao Corporation)).

[0065] The concentration of the surfactant in the second reagent composition is preferably 0.05% (w / v) or more, more preferably 0.1% (w / v) or more, and even more preferably 0.5% (w / v) or more, from the viewpoint of allowing the surfactant to act stably on sdLDL in the mixed solution after addition of the second reagent composition. From the same viewpoint, the concentration of the surfactant in the second reagent composition is preferably 8.0% (w / v) or less, and more preferably 5.0% (w / v) or less.

[0066] When the second reagent composition contains a coupler, the coupler is preferably one or more compounds selected from the group consisting of 4-aminoantipyrine (4AA), aminoantipyrine derivatives, vanillin diamine sulfonic acid, methyl benzthiazolinone hydrazone, and sulfonated methyl benzthiazolinone hydrazone. The concentration of the coupler in the second reagent composition is preferably 0.5 mM or more, more preferably 1.0 mM or more, and is preferably 15 mM or less, more preferably 10 mM or less, from the viewpoint of allowing the coupler to act stably on sdLDL, relative to the total composition of the reaction solution after the addition of the second reagent composition.

[0067] On the other hand, when the coupler is contained in the first reagent composition, the hydrogen donor is preferably contained in the second reagent composition. In this case, the concentration of the hydrogen donor in the reagent is preferably 3 mM or more, more preferably 4.5 mM or more, and preferably 15 mM or less, more preferably 12 mM or less, based on the total composition of the reaction solution after the addition of the second reagent composition, from the viewpoint of stably acting on sdLDL.

[0068] Specific examples of proteins without enzymatic activity include those mentioned above for the first reagent composition.

[0069] (method) The method of this embodiment is a method for quantifying sdLDL-C in a sample using the above-mentioned first and second reagent compositions. The quantitative method of this embodiment includes the following first and second steps. (First step) A step of applying the first reagent composition to a sample. (Second step) After the first step, the second reagent composition is applied to quantify the amount of cholesterol in the remaining lipoprotein. The first and second reagent compositions are as described above. In this method, by using the first reagent composition of this embodiment, sdLDL-C can be left in the reaction solution with high selectivity in the first step, and therefore sdLDL-C can be measured with high accuracy. Therefore, for example, sdLDL-C can be stably quantified with high accuracy.

[0070] Furthermore, the quantification of sdLDL-C may be carried out, for example, by adding the first reagent composition to a sample (subject sample) and allowing it to react, then adding the second reagent composition and allowing it to react, and measuring the absorbance. The test sample is a blood-derived sample such as serum or plasma, preferably serum. The first and second steps are usually carried out in an automatic analyzer. The amount of the sample and the amount of each reagent composition can be appropriately determined in consideration of, for example, the concentration of the reagent in each reagent composition, but should be within the range applicable to the automatic analyzer. For example, 1 to 10 μL of the specimen sample, 50 to 300 μL of the first reagent, and 25 to 200 μL of the second reagent may be used. Each step will now be described in more detail.

[0071] (1st step) In the first step, the first reagent composition is applied to the sample, thereby eliminating lipoproteins other than sdLDL, and liberating cholesterol from lipoproteins other than sdLDL, which is then led out of the reaction system. More specifically, in the first step, a surfactant that acts on lipoproteins other than sdLDL is applied to the sample in the presence of cholesterol esterase. Then, cholesterol released from lipoproteins is reacted with an enzyme that reacts with cholesterol, such as cholesterol oxidase, and led out of the reaction system. In the first step, known techniques can be used, such as eliminating cholesterol in lipoproteins other than sdLDL and leading it out of the reaction system, aggregating cholesterol in lipoproteins other than sdLDL, or inhibiting it from reacting in a subsequent step.

[0072] When the first reagent composition contains an electron donor, in the first step, the step of eliminating cholesterol produced from lipoproteins other than sdLDL and directing it out of the reaction system may include, for example, forming a colorless quinone in the presence of hydrogen peroxide produced by cholesterol esterase activity and cholesterol oxidase activity in the first reagent composition, and an electron donor.

[0073] In the first step, at least one of monovalent cations and divalent cations or salts thereof can be further added to the reaction solution as an ionic strength adjuster. By adding the ionic strength adjuster, it becomes easier to differentiate sdLDL from L LDL.

[0074] (2nd process) In the second step, the amount of sdLDL-C remaining after the first step is quantified. In the second step, a conventional method for quantifying LDL can be used. For example, there is a method for quantifying the content of LDL-specific aggregates formed by adding an LDL agglutinant by turbidimetric measurement, a method for using an antigen-antibody reaction by an LDL-specific antibody, a method for quantifying decomposition products using an enzyme, etc. The quantification method is selected depending on, for example, the components and composition contained in the second reagent composition.

[0075] When the second reagent composition contains an enzyme, the method can be such that the enzyme is used to quantify the decomposition product. Specifically, the second reagent composition containing one or more cholesterol measuring enzymes selected from the group consisting of cholesterol esterase, cholesterol oxidase, cholesterol dehydrogenase and peroxidase is added to the reaction solution after the first step to liberate and decompose sdLDL-C, and the reaction product is quantified. In this case, the second reagent composition preferably contains the above-mentioned surfactant.

[0076] In this embodiment, the reaction temperature in each step is preferably 2°C to 45°C, and more preferably 25°C to 40°C. The reaction time for each step is preferably 1 to 10 minutes, more preferably 3 to 7 minutes.

[0077] Examples of automatic analyzers used for quantifying sdLDL-C include TBA-120FR, TBA-200FR (all manufactured by Toshiba Corporation), JCA-BM1250, JCA-BM1650, JCA-BM2250 (all manufactured by JEOL Ltd.), HITACHI7180, HITACHI7170 ​​(all manufactured by Hitachi Corporation), AU2700, AU5800, AU680 (all manufactured by OLYMPUS Corporation), cobas c501, cobas c701 (all manufactured by Roche), etc.

[0078] Quantitation of sdLDL-C is carried out, for example, by measuring absorbance in the wavelength region of 580 to 720 nm, preferably 600 to 700 nm.

[0079] Although the embodiments of the present invention have been described above, these are merely examples of the present invention, and various configurations other than those described above can also be adopted. Below, examples of reference forms are given. 1. A step of applying a first reagent composition to a sample; After the step of applying the first reagent composition to the sample, a step of applying a second reagent composition for quantifying small dense LDL cholesterol (sdLDL-C) to quantify cholesterol in the remaining lipoproteins; A reagent composition used as the first reagent composition in the method for quantifying sdLDL-C in the sample, comprising: having one or more activities selected from the group consisting of cholesterol esterase activity, cholesterol oxidase activity and sphingomyelinase activity; A reagent composition comprising polyoxyethylene monostyrenated phenyl ether. 2. The reagent composition described in 1., wherein the degree of polymerization n of polyoxyethylene in the polyoxyethylene monostyrenated phenyl ether is 5 or more and 80 or less. 3. The reagent composition according to 1 or 2, wherein the content of the polyoxyethylene monostyrenated phenyl ether in the reagent composition is 0.05% (w / v) or more and 0.6% (w / v) or less based on the total amount of the reagent composition. 4. A reagent composition described in any one of 1. to 3., wherein the reagent composition further has at least one activity selected from the group consisting of peroxidase activity and catalase activity. 5. A reagent composition according to any one of 1. to 4., wherein the reagent composition contains either a hydrogen donor or a coupler. 6. A first reagent composition comprising the reagent composition according to any one of 1. to 5.; A second reagent composition for quantifying sdLDL-C; A kit for use in quantifying the sdLDL-C in the sample, comprising: 7. The kit described in 6., wherein the second reagent composition has peroxidase activity. 8. The first reagent composition contains either a hydrogen donor or a coupler but not the other; The kit according to 6. or 7., wherein the second reagent composition does not contain one of the hydrogen donor and the coupler but contains the other. EXAMPLES

[0080] In the following, the "%" of the POE monostyrenated phenyl ether concentration is specifically "% (w / v)".

[0081] (Experimental Example 1) (Examples 1 to 3 and Comparative Example 1) In order to evaluate the reactivity of each surfactant in an sdLDL-C reagent, the following reagents were prepared and measurements were performed.

[0082] (Preparation of Reagent Composition) The first reagent composition of each example was prepared by mixing the following components at the following concentrations and changing the type of surfactant. The mixing composition of the first reagent composition is shown below. (First Reagent Composition) PIPES buffer, pH7.0 50mM Cholesterol esterase 900U / L Cholesterol oxidase 450U / L Sphingomyelinase 525U / L Peroxidase 1250U / L Bovine serum albumin 0.75% (w / v) TOOS 1.5mM 4AA 1mM Surfactant *1

[0083] *1 Surfactant Example 1: POE monostyrenated phenyl ether, POE polymerization degree 11-33 Example 2: POE monostyrenated phenyl ether, POE polymerization degree 13-37 Example 3: POE monostyrenated phenyl ether, POE polymerization degree 50-80 Comparative Example 1: Mixture of POE distyrenated phenyl ether, POE polymerization degree 2-32 and POE tristyrenated phenyl ether, POE polymerization degree 5-30

[0084] (Evaluation of lipoprotein selectivity) In order to evaluate the lipoprotein selectivity in the reaction with the first reagent composition, the reactivities of the above reagent composition with sdLDL and lbLDL were measured. Specifically, sdLDL and lbLDL separated from human serum by ultracentrifugation were used as specimens. In order to confirm the reactivity of the surfactant to the specimen, the above-mentioned first reagent composition was prepared, containing cholesterol esterase, cholesterol oxidase, which are components involved in the color reaction, as well as peroxidase, a hydrogen donor, and a coupler. 3 μL of the specimen was mixed with 150 μL of the above-mentioned first reagent composition, and the difference in absorbance at 600 nm (main wavelength) and 700 nm (secondary wavelength) after 5 minutes at 37° C. (referred to as "absorbance wavelength 600 nm / 700 nm" in Table 1 and Table 2 described below) [mAbs] was measured, and the ratio of the absorbance of lbLDL to the absorbance of sdLDL was calculated. The measurement results are shown in Table 1.

[0085] [Table 1]

[0086] Table 1 shows that in each Example containing POE monostyrenated phenyl ether, the absorbance ratio of lbLDL / sdLDL is larger than that of the Comparative Examples containing POE distyrenated phenyl ether or POE tristyrenated phenyl ether and not containing POE monostyrenated phenyl ether, and that the first reagent composition has excellent selectivity for lbLDL. Therefore, by using the first reagent composition of each example containing POE monostyrenated phenyl ether as a surfactant, sdLDL in a sample can be quantified with higher accuracy.

[0087] (Experimental Example 2) Except for varying the concentration of POE monostyrenated phenyl ether in Example 1, the absorbance ratio of lbLDL / sdLDL was determined in the same manner as in Experimental Example 1. The concentration at which the lbLDL / sdLDL ratio was 1.2 or more was defined as the permissible concentration. The measurement results are shown in Table 2.

[0088] [Table 2]

[0089] As can be seen from Table 2, the first reagent composition had excellent selectivity for lbLDL at each concentration.

[0090] (Experimental Example 3) The first reagent composition was prepared by varying the concentration of the POE monostyrenated phenyl ether used in Example 1 as a surfactant. The accuracy of the measurement of sdLDL-C in a sample with a known concentration was evaluated by a comparative control method when this was combined with the second reagent composition. Specifically, the following first reagent composition and the following second reagent composition were prepared, each having a different concentration of the surfactant used in Example 1. 75 μL of the first reagent composition was added to 2 μL of a serum sample, and the mixture was reacted at 37° C. for 5 minutes. Then, 75 μL of the second reagent composition was added and reacted for 5 minutes. The absorbance at a main wavelength of 600 nm and a sub-wavelength of 700 nm was measured, and the sdLDL-C concentration was calculated from a calibration curve prepared separately. The formulations of the first reagent composition and the second reagent composition are shown below. (First Reagent Composition) PIPES buffer, pH7.0 50mM Cholesterol esterase 300U / L Cholesterol oxidase 600U / L Sphingomyelinase 2700U / L Catalase 1200KU / L Ascorbic acid oxidase 3000U / L Bovine serum albumin 10g / L TOOS 2.0mM Surfactant *1 0.05%, 0.07%, 0.09%, 0.11%, 0.13%, 0.16%, 0.18%, 0.20% or 0.30%

[0091] (Second Reagent Composition) PIPES buffer, pH7.0 50mM 4-Aminoantipyrine 4.0mM Peroxidase 5000U / L Sodium azide 0.05% (w / v) Polyoxyethylene alkyl ether 1% (w / v)

[0092] As a comparative control method, sdLDL-C values ​​obtained under the following conditions using ultracentrifugation, specifically, a method in which sdLDL is fractionated by ultracentrifugation and cholesterol is measured, were used to calculate the correlation coefficient with the sdLDL-C concentration calculated from the calibration curve described above. In ultracentrifugation, the density of sdLDL is 1.044-1.063 g / cm 3 The fraction was separated using the method described in Clinical Chemistry, 57, p.57-65, 2011, and the concentration of sdLDL-C was measured. The evaluation results are shown in Figures 1(a) to 1(c), 2(d) to 2(f), and 3(g) to 3(i). These figures show the evaluation results of the accuracy of the measurement values ​​of sdLDL-C concentration, with the horizontal axis representing the measurement values ​​obtained by ultracentrifugation (UCF) and the vertical axis representing the measurement values ​​calculated from the above-mentioned calibration curve. In addition, the POE monostyrenated phenyl ether concentrations and correlation coefficients in Figures 1(a) to 1(c), Figures 2(d) to 2(f), and Figures 3(g) to 3(i) are shown below. Concentration Correlation coefficient r Figure 1(a) 0.05% 0.9085 Figure 1(b) 0.07% 0.9152 Figure 1(c) 0.09% 0.9198 Figure 2(d) 0.11% 0.9263 Figure 2(e) 0.13% 0.9304 Figure 2(f) 0.16% 0.9295 Figure 3(g) 0.18% 0.9296 Figure 3(h) 0.20% 0.9252 Figure 3(i) 0.30% 0.9240

[0093] From Figures 1(c), 2(d) to 2(f), and 3(g) to 3(i) and the above correlation coefficients, it is clear that at POE monostyrenated phenyl ether concentrations of 0.05 to 0.30% in Figures 1(c), 2(d) to 2(f), and 3(g) to 3(i), r is greater than 0.90 in all cases, enabling accurate quantification of sdLDL-C. In particular, when the POE monostyrenated phenyl ether concentration was 0.11 to 0.30%, r>0.92 was obtained, enabling more accurate quantification of sdLDL-C.

[0094] This application claims priority based on Japanese Patent Application No. 2021-070615, filed on April 19, 2021, the disclosure of which is incorporated herein in its entirety.

Claims

1. applying a first reagent composition to a sample; After the step of applying the first reagent composition to the sample, a step of applying a second reagent composition for quantifying small dense LDL cholesterol (sdLDL-C) to quantify cholesterol in the remaining lipoproteins; A reagent composition used as the first reagent composition in the method for quantifying sdLDL-C in the sample, comprising: having cholesterol esterase activity, cholesterol oxidase activity and sphingomyelinase activity; A reagent composition comprising polyoxyethylene monostyrenated phenyl ether.

2. 2. The reagent composition according to claim 1, wherein the degree of polymerization n of polyoxyethylene in the polyoxyethylene monostyrenated phenyl ether is 5 or more and 80 or less.

3. The reagent composition according to claim 1 or 2, wherein the content of the polyoxyethylene monostyrenated phenyl ether in the reagent composition is 0.05% (w / v) or more and 0.6% (w / v) or less based on the total amount of the reagent composition.

4. 3. The reagent composition according to claim 1, further comprising at least one activity selected from the group consisting of peroxidase activity and catalase activity.

5. 3. The reagent composition according to claim 1, wherein the reagent composition comprises one of a hydrogen donor and a coupler.

6. A first reagent composition comprising the reagent composition according to claim 1 or 2; A second reagent composition for quantifying sdLDL-C; A kit for use in quantifying the sdLDL-C in the sample, comprising:

7. The kit of claim 6 , wherein the second reagent composition has peroxidase activity.

8. the first reagent composition comprises either a hydrogen donor or a coupler but not the other; 7. The kit of claim 6, wherein the second reagent composition is free of the one of the hydrogen donor and the coupler and includes the other.

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