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 causing the first reagent composition to act on the sample; and a step for, after the step for causing the first reagent composition to act on the sample, causing a second reagent composition for quantifying the sdLDL-C to act on the sample and thereby quantifying cholesterol in a remaining lipoprotein. This reagent composition contains a nonionic surfactant and has cholesterol esterase activity, cholesterol oxidase activity, and sphingomyelinase activity, wherein the contact angle between the reagent composition and a polyethylene terephthalate (PET) substrate is 63.0-67.0º.

Description

[Technical Field]

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

[0002] One technique for measuring LDL cholesterol is described in Patent Document 1 (Japanese Patent Publication No. 2000-325097). This 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 HDL cholesterol and / or LDL cholesterol by measuring the compounds consumed or produced by the reaction with the enzyme in the first or second step. This method does not require a lipoprotein flocculant that increases the turbidity of the reaction solution, there are no restrictions on the enzyme used, and there is no need to add another enzyme in the step of reacting LDL cholesterol. It is a simple and inexpensive way to quantify LDL cholesterol, and if necessary, it can accurately and inexpensively measure HDL cholesterol without forming lipoprotein aggregates that interfere with optical measurement. Therefore, it is considered particularly useful in the field of clinical testing for arteriosclerosis and other conditions. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2000-325097 [Overview of the project] [Problems that the invention aims to solve]

[0004] The inventors investigated the measurement of small dense LDL cholesterol (sdLDL-C) within LDL cholesterol. They found that the technique described in Patent Document 1 has room for improvement in terms of reaction specificity (specifically, correlation with the reference method) and accuracy after sample dilution.

[0005] This invention provides a measurement technique for sdLDL-C that exhibits excellent accuracy and specificity after sample dilution. [Means for solving the problem]

[0006] The inventors investigated how to improve the reaction specificity (specifically, correlation with the reference method) and accuracy after sample dilution for the quantification of sdLDL-C. As a result, they discovered that by setting the contact angle of the reagent composition used for the quantification of sdLDL-C within a specific range, it becomes possible to quantify sdLDL-C with good correlation to the reference ultracentrifugation method and excellent accuracy after sample dilution, thus completing the present invention.

[0007] According to the present invention, the following reagent compositions and kits are provided. [1] A step of reacting the sample with the first reagent composition, After the step of reacting the sample with the first reagent composition, the process involves reacting the sample with a second reagent composition for quantifying small dense LDL cholesterol (sdLDL-C) to quantify the cholesterol remaining in the lipoprotein, A reagent composition used as the first reagent composition for a method of quantifying the sdLDL-C in the sample, comprising: Contains nonionic surfactants, It possesses cholesterol esterase activity, cholesterol oxidase activity, and sphingomyelinase activity, A reagent composition in which the contact angle between the reagent composition and a polyethylene terephthalate (PET) substrate, as measured by the following method 1, is 63.0° or more and 67.0° or less. (Method 1) (1) PET substrate: PET (amorphous polyester) resin sheet, transparent, 2 mm thick (2) Pretreatment: Wipe the surface of an unused PET substrate with a 70% ethanol aqueous solution. Perform the measurement within 5 minutes after wiping. (3) Measurement method and conditions: Droplet method, θ / 2 method, temperature: 15~25℃, syringe: Teflon (registered trademark, same hereinafter) coated 18G, dropping method, dropping volume: 2μL, measurement is taken 1 second after dropping. (4) Calculation of contact angle: Measure five times and calculate the average of the five measurements. [2] The reagent composition according to [1], wherein the viscosity of the reagent composition at 5°C, as measured by the following method 2-1, is 2.5 mPa·s or less. (Method 2-1) (1) Equipment: EMS viscometer (Electro Magnetically Spinning Viscometer) (2) Measurement method and conditions: Measurement method: Electromagnetic spinning method, Motor rotation speed: 1000 rpm, Holding time: 600 seconds, Measurement temperature: 5°C, Sequence loop: 1 time, Sample: 500 μL (3) Calculation of viscosity: Five measurements are taken and the average value of the five measurements is calculated. [3] The reagent composition according to [1] or [2], wherein the viscosity of the reagent composition at 37°C, as measured by the following method 2-2, is 1.05 mPa·s or less. (Method 2-2) (1) Equipment: EMS viscometer (Electro Magnetically Spinning Viscometer) (2) Measurement method and conditions: Measurement method: Electromagnetic spinning method, Motor rotation speed: 1000 rpm, Holding time: 600 seconds, Measurement temperature: 37°C, Sequence loop: 1 time, Sample: 500 μL (3) Calculation of viscosity: Five measurements are taken and the average value of the five measurements is calculated. [4] The reagent composition according to any one of [1] to [3], wherein the nonionic surfactant comprises polyoxyethylene monostyrene-derived phenyl ether. [5] The reagent composition according to [4], wherein the degree of polymerization n of polyoxyethylene in the polyoxyethylene monostyrenated phenyl ether is 5 or more and 80 or less. [6] The reagent composition according to [4] or [5], 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 whole reagent composition. [7] The reagent composition according to any one of [1] to [6], wherein the reagent composition contains either a hydrogen donor or a coupler. [8] The reagent composition according to any one of [1] to [7], wherein the reagent composition further has ascorbic acid oxidase activity. [9] The reagent composition according to any one of [1] to [8], wherein the reagent composition further has at least one activity selected from the group consisting of peroxidase activity and catalase activity.

[10] A first reagent composition comprising the reagent composition according to any one of [1] to [9], A second reagent composition for quantifying the sdLDL-C, and a kit for quantifying the sdLDL-C in the sample, comprising the above.

[11] The kit according to

[10] , wherein the second reagent composition has peroxidase activity.

Effects of the Invention

[0008] According to the present invention, it is possible to provide a measurement technique for sdLDL-C that is excellent in reaction specificity (specifically, correlation with a reference method) and accuracy after dilution of a specimen.

Brief Description of the Drawings

[0009] [Figure 1] It is a figure showing the evaluation result of the accuracy after dilution of the measured value of sdLDL-C of a specimen. [Figure 2] It is a figure showing the evaluation result of the reaction specificity (correlation with a reference method) of the measured value of sdLDL-C. [Figure 3]It is a diagram showing the relationship between the reaction specificity (correlation coefficient in the correlation with the reference method) of the measured value of sdLDL-C and the contact angle. [Figure 4] It is a diagram showing the evaluation results of the dilution linearity of the measured value of sdLDL-C. [Figure 5] It is a diagram showing the evaluation results of the dilution linearity of the measured value of sdLDL-C.

Mode for Carrying Out the Invention

[0010] Hereinafter, embodiments will be described. In this embodiment, the composition such as the measurement reagent can contain each component alone or in combination of two or more. Also, in this specification, "x to y" of the numerical range represents "x or more and y or less", and both the lower limit value x and the upper limit value y are included.

[0011] First, lipoproteins will be described. Lipoproteins are roughly classified into fractions of Very Low Density Lipoprotein (VLDL), Low Density Lipoprotein (LDL), and High Density Lipoprotein (HDL). LDL is further divided into small dense LDL (sdLDL) and other subfractions. sdLDL may also be called small particle LDL, small LDL (SLDL), dense LDL, small, dense LDL, and other LDL may also be called large LDL (L LDL), Light LDL.

[0012] These lipoprotein fractions and subfractions can be distinguished by particle size or specific gravity. Regarding the diameter of the particle size of lipoproteins, it varies depending on the reporter. 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. Regarding the specific gravity of lipoproteins, for example, VLDL is 1.006 or less, LDL is 1.019 to 1.063, and HDL is 1.063 to 1.21.

[0013] Among lipoproteins, the LDL particle diameter can be measured, for example, by gradient gel electrophoresis (GGE) (JAMA, 260, pp. 1917-21, 1988) or NMR (HANDBOOK OF LIPOPROTEIN TESTING 2nd Edition, edited by Nader Rifai et al., pp. 609-623; AACC PRESS: The Fats of Life Summer 2002, LVDD 15 YEAR ANNIVERSARY ISSUE, Volume AVI No. 3, pp. 15-16). Furthermore, specific gravity can be determined, for example, based on analysis by ultracentrifugation (Atherosclerosis, 106, pp. 241-253, 1994; Atherosclerosis, 83, pp. 59, 1990).

[0014] In this embodiment, the sdLDL to be measured generally refers to a subfraction of the LDL fraction with a diameter of approximately 22.0 to approximately 25.5 nm, or a subfraction with a specific gravity of 1.040 to 1.063. LDL is divided into subfractions based on size because smaller LDL particles are more likely to induce arteriosclerosis and are considered more malignant than other types of LDL, making it necessary to separately measure the smaller particles. The diameter and specific gravity distributions are continuous within LDL, and it is not possible to clearly distinguish which specific gravity levels indicate particularly high malignancy. Therefore, the specific gravity values ​​of 1.040 to 1.063 mentioned above are not established characteristics of sdLDL, but rather represent the higher specific gravity values ​​when the widely used and established LDL specific gravity range of 1.019 to 1.063 is divided at the midpoint. For example, another report fractionates the specific gravity of sdLDL into 1.044 to 1.060 (Atherosclerosis: 106 241-253 1994). While there are slight differences among reporters regarding the specific gravity range for sdLDL, in all cases, the presence of sdLDL within that range is associated with clinical malignancy.

[0015] In this specification, sdLDL specifically refers to LDL with a high specific gravity that is clinically more atherosclerotic inducing than other types of LDL. Furthermore, sdLDL preferably refers to LDL with a specific gravity higher than the median, and more preferably LDL with a specific gravity in the range of 1.044 to 1.063. Additionally, "lipoproteins other than LDL" refers to VLDL or HDL, and may also include chylomicrons and IDL (intermediate density lipoprotein).

[0016] The inventors investigated how to improve the accuracy of quantifying sdLDL-C in a sample. As a result, they found that when quantifying sdLDL-C by a method comprising the steps of reacting a sample with a first reagent composition (first step) and then reacting it with a second reagent composition for quantifying sdLDL cholesterol (sdLDL-C) to quantify the cholesterol in the remaining lipoprotein (second step), 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 also easily acts on the sdLDL being measured, there is a concern that the accuracy of quantifying sdLDL-C in the sample in the second step will decrease. Therefore, the inventors further investigated how to improve the selectivity of the first reagent composition and found that by configuring the first reagent composition to have specific enzyme activity and a contact angle within a specific range, a first reagent composition can be stably obtained that acts with high selectivity on LDL other than sdLDL, enabling the quantitative determination of sdLDL-C with excellent reaction specificity (correlation with the reference method) and accuracy after sample dilution.

[0017] In this embodiment, the first reagent composition contains specific components and has a contact angle within a specific range, making it suitable for quantitative analysis of sdLDL-C and enabling excellent accuracy in the measurement of sdLDL-C. For example, the first reagent composition is used in combination with the second reagent composition, described later, for the quantitative determination of sdLDL-C. The following provides a more detailed description of each reagent composition.

[0018] (Reagent composition (First reagent composition)) In this embodiment, the reagent composition is used as a first reagent composition for a method of quantifying sdLDL-C in a sample, comprising the steps of: reacting a sample with a first reagent composition; and after reacting the sample with the first reagent composition, reacting it with a second reagent composition for quantifying small dense LDL cholesterol (sdLDL-C) to quantify the cholesterol in the remaining lipoprotein. Hereinafter, the reagent composition used as the first reagent composition will also simply be referred to as the "first reagent composition." The first reagent composition contains a nonionic surfactant and has cholesterol esterase activity, cholesterol oxidase activity, and sphingomyelinase activity. The contact angle between the first reagent composition and the polyethylene terephthalate (PET) substrate, as measured by the following method 1, is 63.0° or more and 67.0° or less.

[0019] (Method 1) (1) PET substrate: PET (amorphous polyester) resin sheet, transparent, 2 mm thick (2) Pretreatment: Wipe the surface of an unused PET substrate with a 70% ethanol aqueous solution. Perform the measurement within 5 minutes after wiping. (3) Measurement method and conditions: Droplet method, θ / 2 method, temperature: 15~25℃, syringe: Teflon coated 18G, dropping method, dropping volume: 2μL, measurement is taken 1 second after dropping. (4) Calculation of contact angle: Measure five times and calculate the average of the five measurements. In (2) above, dust and other debris on the PET substrate are wiped off before measurement. Since static electricity affects the contact angle, wipe it off while minimizing friction, and then perform the measurement within 5 minutes. In this embodiment, the contact angle is specifically the static contact angle measured using, for example, DMo-901, 701, 601, 501, DMC-MC3, etc., manufactured by Kyowa Interface Chemical Co., Ltd.

[0020] The inventors have newly discovered that by setting the contact angle between the first reagent composition and the PET substrate within a specific range, it is possible to quantify sdLDL-C with superior reaction specificity (correlation with the reference method) and accuracy after sample dilution. The reason for this is not entirely clear, but it is thought to be as follows: In the quantification of sdLDL-C, for example, the first reagent composition eliminates cholesterol in lipoproteins other than sdLDL in the first step, and the contact angle may be related to the accuracy after sample dilution. The reason is not clear, but when the sample is diluted, the protein components and the target of measurement (sdLDL) contained in the sample are diluted, and when mixed with the first reagent product, this may cause sdLDL to react incorrectly, and sdLDL-C may be eliminated. By controlling the contact angle, this incorrect reaction is suppressed, and it is thought that sdLDL-C can be predicted more accurately. Furthermore, if the contact angle falls outside a specific range, it is expected that even in undiluted samples, the reaction between the lipoproteins contained in the sample and the nonionic surfactant or specific enzymes in the first reagent composition will change when mixed with the first reagent composition in the first step. This erroneous reaction makes accurate measurement of sdLDL-C difficult and is thought to reduce the correlation with the reference method for reaction specificity. Here, the reference method is specifically the ultracentrifugation method.

[0021] The contact angle between the reagent composition and the PET substrate is 63.0° or higher, preferably 63.8° or higher, from the viewpoint of improving the reaction specificity (correlation with the reference method) of sdLDL-C measurement and the accuracy after sample dilution. Furthermore, from the viewpoint of further improving the reaction specificity (correlation with the reference method) of sdLDL-C measurement, the contact angle between the reagent composition and the PET substrate is 67.0° or less, preferably 66.5° or less, and more preferably 66.0° or less.

[0022] In this embodiment, the contact angle with the PET substrate can be controlled by appropriately selecting, for example, the type, combination, and amount of each component contained in the first reagent composition. Among these, adjusting the type and amount of surfactant, and the type and amount of enzymes and proteins are examples of factors that can bring the contact angle with the PET substrate into a desired numerical range.

[0023] (viscosity) The first reagent composition is, specifically, a liquid. From the viewpoint of improving measurement accuracy, the viscosity of the first reagent composition at 5°C may be, for example, 2.5 mPa·s or less, preferably 2.2 mPa·s or less, and more preferably 2.0 mPa·s or less. Furthermore, the viscosity of the first reagent composition at 5°C may be, for example, 1.6 mPa·s or higher, or for example, 1.8 mPa·s or higher.

[0024] The viscosity of the first reagent composition at 37°C is preferably 1.05 mPa·s or less, more preferably 1.00 mPa·s or less, and even more preferably 0.95 mPa·s or less, from the viewpoint of improving measurement accuracy. Furthermore, the viscosity of the first reagent composition at 37°C may be, for example, 0.80 mPa·s or higher, or for example, 0.90 mPa·s or higher.

[0025] Here, the viscosity of the first reagent composition at each temperature is measured by the following Method 2. In Method 2, Method 2-1 is when the measurement temperature is 5°C, and Method 2-2 is when it is 37°C. (Method 2) (1) Equipment: EMS viscometer (Electro Magnetically Spinning Viscometer) (2) Measurement method and conditions: Measurement method: Electromagnetic spinning method, Motor speed: 1000 rpm, Holding time: 600 seconds, Measurement temperature: 5°C (Method 2-1) and 37°C (Method 2-2), Sequence loop: 1 time, Sample: 500 μL (3) Calculation of viscosity: Five measurements are taken and the average value of the five measurements is calculated. In this embodiment, viscosity is measured using, for example, an EMS-1000 manufactured by Kyoto Electronics Manufacturing Co., Ltd.

[0026] The inventors have also newly discovered that by setting the viscosity of the first reagent composition within the sdLDL-C measurement reagent to a specific range, it is possible to quantify sdLDL-C with even greater accuracy. The reason for this is not entirely clear, but it is thought to be as follows: In the quantification of sdLDL-C, for example, during measurement, a reagent probe draws in a certain amount of the first reagent composition and discharges it into the reaction cell. If the viscosity of the first reagent composition used in the step of scavenging cholesterol in lipoproteins other than sdLDL is too high, the discharge amount may change due to the viscosity and the amount scavenged may fluctuate. However, by accurately controlling the viscosity, the release of the reagent from the liquid can be appropriately controlled, and as a result, it is thought that sdLDL-C can be measured more accurately.

[0027] The first reagent composition contains a nonionic surfactant and has specific enzymatic activity. Therefore, when the first reagent composition is added to a sample, it can, for example, act on and eliminate lipoproteins other than sdLDL in the sample. Furthermore, it can stably remove cholesterol from lipoproteins other than sdLDL, especially cholesterol in lb LDL, from the reaction system.

[0028] Here, "surfactant acts (reacts)" means that the surfactant breaks down lipoproteins, releasing cholesterol from them. For example, when we say "surfactant that acts (reacts) with lipoproteins other than sdLDL," it is not required that the surfactant has no effect on sdLDL at all; it is sufficient that it mainly acts on lipoproteins other than sdLDL. Similarly, when we say "surfactant that acts (reacts) with LDL other than sdLDL," it is sufficient that the surfactant mainly acts on LDL other than sdLDL. "Eradication" means breaking down a substance in the sample so that its breakdown products are not detected in subsequent steps. That is, "eradicating cholesterol from lipoproteins other than sdLDL" means breaking down lipoproteins other than sdLDL in the sample so that the cholesterol in these lipoproteins, which are the breakdown products, is not detected in subsequent steps. Similarly, "eradicating cholesterol from LDL other than sdLDL" means that the cholesterol in LDL other than sdLDL in the sample is not detected in subsequent steps.

[0029] "Leaving the reaction system" specifically refers to eliminating, agglutinating, or inhibiting the cholesterol contained in HDL, VLDL, L LDL, etc., so that it does not affect the quantification of sdLDL-C. The components contained in the first reagent composition will be described in more detail below.

[0030] (Surfactants) The first reagent composition contains at least a nonionic surfactant. Specifically, this surfactant is a surfactant that acts on lipoproteins other than sdLDL, and more preferably a surfactant that acts on lipoproteins other than sdLDL but does not act on sdLDL.

[0031] From the viewpoint of further improving the accuracy and specificity of sdLDL-C measurement after sample dilution, the nonionic surfactant is preferably a polyoxyethylene derivative, more preferably a polyoxyethylene polycyclic phenyl ether derivative, even more preferably one or two selected from the group consisting of polyoxyethylene benzyl phenyl ether derivatives and polyoxyethylene styrene-phenyl ether derivatives, and more preferably includes polyoxyethylene monostyrene-phenyl ether (POE monostyrene-phenyl ether). From a similar viewpoint, it is also preferable that the nonionic surfactant includes polyoxyethylene benzyl phenyl ether derivatives and polyoxyethylene styrene-phenyl ether derivatives.

[0032] The POE monostyrene-modified phenyl ether may be composed of multiple compounds with different degrees of polymerization of the POE portion. In this case, the average degree of polymerization of oxyethylene is, for example, 1 or more, preferably 5 or more, more preferably 10 or more, and also, for example, 100 or less, preferably 80 or less, more preferably 50 or less, even more preferably 40 or less, and also, for example, 30 or less, from the viewpoint of improving the accuracy of quantification of sdLDL-C. Here, the average degree of polymerization is determined from the intensity of the oxyethylene signal of the POE monostylenide phenyl ether measured by NMR. For example, in the NMR spectrum of a TMS derivative of POE monostylenide phenyl ether, it can be calculated by the ratio of the signal intensity of the methylene protons (4) of the POE portion (3.5 ppm) to the intensity of the protons (9) of the TMS group (0.08 ppm) as a reference.

[0033] Furthermore, from the viewpoint of more stably improving the accuracy of sdLDL-C quantification, it is preferable to include one or more compounds selected from the group consisting of compounds in which the average degree of polymerization n of oxyethylene in POE monostyrene-phenyl ether is n=10 to 20. Here, the degree of polymerization of oxyethylene, a component of POE monostyrene-containing phenyl ether, is determined by structural analysis using liquid chromatography-mass spectrometry. For example, after separating the POE monostyrene-containing phenyl ether from the first reagent composition using liquid chromatography (LC), the resulting mass spectrum consists of peaks spaced 44 amu apart, due to differences 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 of monostyrene-containing phenol (MW198) from the molecular weight estimated from the m / z of each peak and dividing by 44. Furthermore, the chemical composition of monostyrene-containing phenol (C) can be calculated from the compositional formula obtained from the precise mass of each peak. 14 H 14 The degree of polymerization of oxyethylene in each component can be analyzed by subtracting O) or by performing a product ion scan and conducting a detailed structural analysis.

[0034] From the viewpoint of improving the accuracy of quantification of sdLDL-C, the content of POE monostyrene-modified 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, relative to the entire first reagent composition. Furthermore, from a similar viewpoint, the content of POE monostyrene-modified 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, relative to the entire first reagent composition.

[0035] Here, POE monostyrene-modified phenyl ether is available as an industrial raw material or research reagent, and commercially available products can be used, for example. Alternatively, for example, POE monostyrene-modified phenyl ether may be produced and used. Specifically, it can be produced by the following method and conditions. In other words, POE monostyrene-modified phenyl ether is obtained by adding a predetermined amount of ethylene oxide to monostyrene-modified phenol in the presence of a basic catalyst. Examples of basic catalysts include sodium hydroxide, potassium hydroxide, and alkali metal alkoxides. The addition reaction temperature is preferably 120 to 200°C. On the other hand, styrene-containing phenols are obtained by alkylating phenol and styrene under acid catalyst at a temperature of 70°C to 200°C. As acid catalysts, 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 complexes can be used. In this reaction, by controlling conditions such as the equivalent ratio of phenol to styrene, the type of acid catalyst, and the reaction temperature, monostyrene-containing phenols, in which one styrene molecule is bonded to the benzene ring, can be obtained. Monostyrene-modified phenol can be synthesized, for example, by adding styrene dropwise to phenol under a phosphoric acid catalyst to carry out an alkyl reaction, and then adding a sulfuric acid or magnesium sulfate catalyst to remove unreacted phenol and styrene and complete the alkyl reaction. In this case, the amount of phosphoric acid catalyst used is preferably 0.001 to 0.01 equivalents relative to the phenol. The amount of sulfuric acid or magnesium sulfate catalyst used is preferably 2% 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 as the equivalent ratio of styrene to phenol. Subsequently, after the alkylation reaction is complete, the alkylation product is neutralized by adding aqueous solutions of sodium carbonate, potassium carbonate, sodium hydroxide, or potassium hydroxide, and the resulting neutralized salt is removed by filtration to recover the monostyrene-conjugated phenol.

[0036] Then, in a pressure vessel such as an autoclave, the obtained monostylenide phenol and ethylene oxide can be subjected to an addition reaction using potassium hydroxide as a catalyst under heating and pressure to obtain POE monostylenide phenyl ether. The heating and pressurizing conditions are, for example, a pressure of 1.5 kg / cm². 3The temperature should be approximately 130°C. Furthermore, by adjusting the molar ratio of ethylene oxide to 1 mole of monostyrene-modified phenol, the number of repeating units of the oxyethylene unit, i.e., the degree of polymerization of oxyethylene, can be adjusted.

[0037] Furthermore, in the above method, a mixture of monostyrene-modified phenol, distyrene-modified phenol (with two styrene units attached), and tristyrene-modified phenol (with three styrene units attached) can be obtained as the styrene-modified phenol, and the monostyrene-modified phenol can be separated and purified from the mixture for use.

[0038] The POE monostyrene-containing phenyl ether in the first reagent composition can be identified by methods such as a combination of IR, NMR, and LC-MS analysis. Methods for determining the structure of the POE monostyrene-containing phenyl ether in the first reagent composition include analysis using LC / MS / MS and NMR. Furthermore, the concentration of POE monostyrene-containing phenyl ether in the first reagent composition can be calculated, for example, by HPLC or LC / MS measurement. Alternatively, the concentration of POE monostyrene-containing phenyl ether in the first reagent composition can be obtained by NMR measurement.

[0039] The concentration of the nonionic surfactant in the first reagent composition is preferably 0.01% (w / v) or more, more preferably 0.025% (w / v) or more, even more preferably 0.03% (w / v) or more, even more preferably 0.05% (w / v) or more, even more preferably 0.075% (w / v) or more, even more preferably 0.1% (w / v) or more, even more preferably 0.125% (w / v) or more, and even more preferably 0.25% (w / v) or more, relative to the total composition of the first reagent composition, from the viewpoint of allowing the nonionic surfactant to act stably on lipoproteins other than sdLDL and further improving dilution linearity and accuracy after sample dilution. Furthermore, the concentration of the nonionic surfactant in the first reagent composition is preferably 1.5% (w / v) or less, more preferably 1.0% (w / v) or less, even more preferably 0.875% (w / v) or less, even more preferably 0.75% (w / v) or less, even more preferably 0.6% (w / v) or less, and even more preferably 0.3% (w / v) or less, relative to the total composition of the first reagent composition.

[0040] The nonionic surfactant in the first reagent composition can be identified by methods such as combining IR, NMR, and LC-MS analysis. Methods for determining the structure of the nonionic surfactant in the first reagent composition include analysis using LC-MS and NMR.

[0041] (enzyme) The first reagent composition possesses cholesterol esterase (CHE) activity, cholesterol oxidase (COO) activity, and sphingomyelinase (SMase) activity. This allows the first reagent composition to act stably with lipoproteins other than sdLDL, thereby diverting the cholesterol out of the reaction system. Furthermore, the first reagent composition specifically includes an enzyme having cholesterol esterase activity, an enzyme having cholesterol oxidase activity, and an enzyme having sphingomyelinase activity. More specifically, the first reagent composition includes cholesterol esterase, cholesterol oxidase, and sphingomyelinase. Cholesterol esterase, cholesterol oxidase, peroxidase, and catalase can be derived from, for example, bacteria or fungi, or from plants.

[0042] Here, "possessing cholesterol esterase activity" specifically means that cholesterol esterase is present and that reactions catalyzed by cholesterol esterase can occur. The same applies to the activity of other enzymes, such as cholesterol oxidase activity.

[0043] The cholesterol esterase activity of the first reagent composition is preferably 50 U / L or more, more preferably 100 U / L or more, preferably 3000 U / L or less, more preferably 2500 U / L or less, even more preferably 2000 U / L or less, and even more preferably 1000 U / L or less, and may also be, for example, 1800 U / L or less, or for example, 1500 U / L or less.

[0044] From a similar viewpoint, the cholesterol oxidase activity of the first reagent composition is preferably 100 U / L or more, more preferably 150 U / L or more, 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.

[0045] Since the first reagent composition has sphingomyelinase activity, it can enhance the reactivity to lipoproteins other than sdLDL, especially L LDL, in the step of releasing cholesterol from lipoproteins other than sdLDL and diverting it out of the reaction system (the first step described later). The sphingomyelinase activity of the first reagent composition is preferably 100 U / L or more, more preferably 200 U / L or more, and preferably 3000 U / L or less, and more preferably 2800 U / L or less, from the viewpoint of more stably guiding cholesterol in lipoproteins other than sdLDL out of the reaction system.

[0046] The first reagent composition preferably further has at least one activity selected from the group consisting of peroxidase (POD) activity and catalase activity, from the viewpoint of more stably guiding cholesterol in lipoproteins other than sdLDL out of the reaction system. The first reagent composition more preferably further contains at least one enzyme selected from the group consisting of an enzyme having peroxidase activity and an enzyme having catalase activity, more preferably further contains at least one of peroxidase and catalase, and even more preferably further contains catalase.

[0047] The peroxidase activity of the first reagent composition is preferably 200 U / L or more, more preferably 300 U / L or more, more preferably 3000 U / L or less, more preferably 2500 U / L or less, and also preferably 2000 U / L or less, from the viewpoint of more stably guiding cholesterol in lipoproteins other than sdLDL out of the reaction system. Furthermore, 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.

[0048] The catalase activity of the first reagent composition is preferably 100 KU / L or higher, more preferably 200 KU / L or higher, more preferably 2000 KU / L or lower, and more preferably 1500 KU / L or lower, from the viewpoint of stably removing hydrogen peroxide generated when the first reagent composition is applied to the sample, and from the viewpoint of more stably leading cholesterol in lipoproteins other than sdLDL in the sample out of the reaction system.

[0049] The cholesterol oxidase activity, cholesterol esterase activity, sphingomyelinase activity, peroxidase activity, and calatase activity of the first reagent composition can each be measured, for example, by the following method.

[0050] For measuring cholesterol oxidase activity, a 6 mM cholesterol solution (dissolved in isopropanol) is used as the substrate solution. A diluent (0.1 M phosphate buffer, Triton X100, pH 7.0) is added to the sample to a concentration of 2-4 U / mL. After warming 3 mL of the diluted solution at 37°C for 5 minutes, 0.05 mL of the substrate solution is added. The mixture is then reacted at 37°C, and the change in absorbance at a wavelength of 240 nm is measured. After the reaction at 37°C, the change in absorbance from 2 to 7 minutes is measured, and the cholesterol oxidase activity is calculated. For example, if the change in absorbance is 3 U / L or more, the sample can be said to have cholesterol oxidase activity, and more specifically, it can be said that cholesterol oxidase is present.

[0051] For measuring cholesterol esterase activity, the following are used: substrate (0.04% cholesterol linolenate, 1% Triton X100, 0.6% sodium cholate solution), 300 U / mL cholesterol oxidase solution, enzyme diluent (20 mM phosphate buffer, 0.5 mM EDTA·2Na, 2 mM MgCl2, 0.2% bovine serum albumin (BSA), pH 7.5), and reaction solution (0.06% 4-aminoantipyrine, 0.4% phenol, 7.5 KU / L peroxidase (POD)). After mixing 1.75 mL of reaction solution and 1.0 mL of substrate solution, the mixture is heated at 37°C for 5 minutes, and 0.1 mL of cholesterol oxidase solution is added. After heating at 37°C for 2 minutes, 0.1 mL of the sample to be measured, diluted with the diluent, is added, and the mixture is reacted at 37°C, and the change in absorbance at a wavelength of 500 nm is measured. After the reaction at 37°C, the change in absorbance from 0 to 3.5 minutes is measured to calculate the cholesterol esterase activity. For example, if the change in absorbance is 8 U / L or more, it can be said that the sample has cholesterol esterase activity, and more specifically, that it contains cholesterol esterase.

[0052] The sphingomyelinase activity of the first reagent composition is measured, for example, by the following method: 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 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 sample to be measured, diluted with the diluent, are mixed and heated at 37°C for 5 minutes, after which 0.16 mL of the stop solution is added. After stopping the reaction, the change in absorbance at the primary wavelength of 546 nm and the secondary wavelength of 700 nm is measured, and the sphingomyelinase activity is calculated. For example, if the absorbance change is 2 U / L or more, it can be said that the sample being measured has sphingomyelinase activity, and more specifically, that it contains sphingomyelinase.

[0053] The peroxidase activity of the first reagent composition is measured, for example, by the following method: 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 is mixed with 0.08 mL of the sample to be measured diluted with the diluent, and the mixture is heated at 37°C for 5 minutes. Then, 0.1 mL of reaction solution 2 is added, and the mixture is reacted at 37°C, measuring the change in absorbance at the dominant wavelength of 600 nm and the secondary wavelength of 700 nm. After the reaction at 37°C, the change in absorbance from 2 to 5 minutes is measured, and the peroxidase activity is calculated. For example, if the change in absorbance is 10 U / L or more, it can be said that the sample being measured has peroxidase activity, and more specifically, that it contains peroxidase.

[0054] The catalase activity of the first reagent composition is measured, for example, by the following method. Specifically, a substrate (0.06% hydrogen peroxide, 50 mM phosphate buffer, pH 7.0) is used for catalase activity measurement. After preheating 2.0 mL of the substrate solution at 25°C, it is mixed with 0.1 mL of the sample to be measured, and the change in absorbance at 240 nm is measured. For example, after the reaction at 25°C, the change in absorbance from 0 to 3 minutes is measured and the catalase activity is calculated. For example, if the change in absorbance is 50 U / L or more, it can be said that the sample has catalase activity, and more specifically, that it contains catalase.

[0055] The first reagent composition may further have other enzyme activities, specifically, 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 one or more enzymes having enzymatic activity selected from the group consisting of, for example, enzymes having ascorbic acid oxidase activity and enzymes having lipoprotein lipase activity, and more specifically, it may contain one or more enzymes selected from the group consisting of ascorbic acid oxidase and lipoprotein lipase.

[0056] From the viewpoint of avoiding the influence of coexisting ascorbic acid in the sample on the accuracy of the measurement, the first reagent composition preferably further has ascorbic acid oxidase activity, and more preferably has ascorbic acid oxidase activity. From a similar viewpoint, the first reagent composition preferably comprises an enzyme having ascorbic acid oxidase activity, and more preferably comprises ascorbic acid oxidase.

[0057] The ascorbic acid oxidase activity of the first reagent composition is preferably 0.1 U / mL or higher, more preferably 0.2 U / mL or higher, and preferably 15 U / mL or lower, and more preferably 10 U / mL or lower, from the viewpoint of avoiding the influence of coexisting ascorbic acid in the sample on the accuracy of the measurement.

[0058] The ascorbate oxidase activity of the first reagent composition is measured, for example, by the following method. Specifically, a substrate (10 mM ascorbic acid solution - 0.13 mM EDTA diluted 20-fold with 90 mM KH2PO4 - 5 mM NaHPO4) is used for measuring ascorbate oxidase activity. 1 mL of the substrate is preheated at 30°C, and after 5 minutes, 0.1 mL of the sample to be measured, diluted with 90 mM NaHPO4 solution containing 0.05% BSA, is added and mixed to start the reaction. After 5 minutes, 3.0 mL of reaction stop solution (0.2 N HCl) is added to stop the reaction, and the absorbance at 245 nm is measured to calculate the ascorbate oxidase activity. For example, if the activity level is 10 U / L or higher, the sample to be measured can be said to have ascorbate oxidase activity, and more specifically, it can be said that it contains "ascorbate oxidase".

[0059] The lipoprotein lipase activity of the first reagent composition is preferably 2 U / mL or more, more preferably 50 U / mL or more, and more preferably 300 U / mL or less, and more preferably 200 U / mL or less, from the viewpoint of favorably adjusting its effect on various lipoproteins.

[0060] The lipoprotein lipase activity of the first reagent composition is measured, for example, by the following method. Specifically, for measuring lipoprotein lipase activity, an olive oil emulsion solution (5 g olive oil, 2 mL ethanol, 5 mL 5.0% Triton X100 added, sonicated for 20 minutes, then 25 mL 4% BSA and 15 mL 0.1 M phosphate buffer pH 7.0 added, stirred at room temperature for 1 to 2 hours) is used as the substrate solution. Diluent (20 mM phosphate buffer, 2 mM MgCl2, 0.5 mM EDTA-2Na, pH 7.5) is added to the substrate solution preheated at 37°C for 5 minutes to a concentration of 0.9 to 1.6 U / mL, 0.2 mL of the diluted sample is added, and after heating at 37°C for 15 minutes, 2.0 mL of stop solution (0.2 M trichloroacetic acid) is added. Then, filtration (Toyo filter paper No. 131 or Whatman No. 42) is performed, and the filtrate is collected. Add 3.0 mL of chromogenic reagent (200 mL of 50 mM MES-NaOH buffer, 4 mL of 5% Triton X100, 40 μL of N,N-dimethyl-m-toluidine, 4 mg of 4-aminoantipyrine, 24.2 mg of ATP·2Na·3H2O, 40.7 mg of MgCl2·6H2O, 200 U of glycerol kinase, 500 U of Lα-glycerophosphate oxidase, and 300 U of peroxidase) to 0.05 mL of the filtrate. React the mixture at 37°C for 15 minutes, measure the absorbance at a wavelength of 545 nm, and calculate the lipoprotein lipase activity. For example, if the change in absorbance is 3 U / L or more, it can be said that the sample has lipoprotein lipase activity, and more specifically, that it contains lipoprotein lipase. The lipoprotein lipase is not limited to any enzyme that has the ability to break down lipoproteins; for example, lipoprotein lipases derived from animals or microorganisms can be used.

[0061] Furthermore, 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. First, a sample containing the target enzyme is degraded with trypsin to obtain a fragment peptide, which is then detected using a hybrid mass spectrometer. The protein can be identified by searching a database (e.g., Mascot search) using the mass of the peptide obtained by the mass spectrometer and the spectrum (MS / MS data) of the fragment ions obtained by colliding them with argon gas in the mass spectrometer. If the sequence of the fragment peptide derived from the amino acid sequence in the reagent composition matches a unique sequence with an amino acid sequence registered in the database, it can be considered to contain the target enzyme.

[0062] Furthermore, the enzymes in the first reagent composition and the second reagent composition described later can also be identified by the following quantification method. Specifically, 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 from among the fragment peptides obtained by degrading the target enzyme with trypsin. Unlabeled peptides and peptides labeled with a stable isotope as an internal standard are prepared by chemical synthesis. The sample containing the target enzyme is completely digested with trypsin, a known amount of the stable isotope-labeled peptide is added, and the sample is measured in MRM mode (multiple reaction monitoring mode) using a triple quadrupole mass spectrometer (LC-MS / MS) connected to an HPLC. The mixture of the unlabeled peptide and the known amount of the stable isotope-labeled peptide is similarly measured to create a calibration curve of the internal standard concentration ratio and peak area ratio, and the target enzyme can be quantified by calculating the absolute amount of the peptide to be quantified in the sample.

[0063] (Other ingredients) The first reagent composition may contain components other than those described above. Examples of other components include buffers, salts, proteins that do not have enzymatic activity, preservatives such as sodium azide, hydrogen donors, and couplers.

[0064] The type of buffer can be selected as appropriate. Specific examples of buffers include MOPS (3-morpholinopropanesulfonic acid) buffer, phosphate buffer, Tris buffer, PIPES (piperazine-1,4-bis(2-ethanesulfonic acid)) buffer, and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer. The concentration of the buffer solution is preferably 1 mM or higher, more preferably 5 mM or higher, even more preferably 10 mM or higher, and also preferably 300 mM or lower, preferably 200 mM or lower, even more preferably 150 mM or lower, and even more preferably 100 mM or lower, from the viewpoint of maintaining enzyme activity in the composition and improving the storage stability of the reagent.

[0065] Specifically, salts are added as pH adjusters or ionic strength adjusters. Specific examples of salts include 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 basic substances such as ammonium salts such as ammonium sulfate and ammonium chloride. Furthermore, the inclusion of at least one monovalent cation and a divalent cation, or a salt thereof, in the first reagent composition further facilitates the separation of sdLDL and L LDL. From the viewpoint of enabling the first reagent composition to act more stably as a pH adjuster or 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 more preferably 50 mmol / L or less, and more preferably 30 mmol / L or less, relative to the total composition of the first reagent composition.

[0066] The pH of the first reagent composition is preferably 6.0 or higher, more preferably 6.5 or higher, and preferably 8.0 or lower, and more preferably 7.5 or lower, from the viewpoint of maintaining enzyme activity in the composition and improving the storage stability of the reagent.

[0067] A specific example of a protein that does not possess enzymatic activity is albumin, such as bovine serum albumin (BSA). The albumin concentration of BSA or the like in the first reagent composition is preferably 1 g / L or more, more preferably 2 g / L or more, more preferably 20 g / L or less, and more preferably 10 g / L or less, relative to 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.

[0068] The first reagent composition preferably comprises at least one of a hydrogen donor and a coupler, and more preferably one of either a hydrogen donor or a coupler. In this case, either the hydrogen donor or the coupler is used to remove cholesterol from lipoproteins other than sdLDL from the reaction system in the first step. More specifically, either the hydrogen donor or the coupler is used to react cholesterol in lipoproteins other than sdLDL with cholesterol esterase or cholesterol oxidase, and to convert the generated hydrogen peroxide into a colorless quinone in the presence of peroxidase.

[0069] Specific examples of hydrogen donors 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, and preferably 5 mM or less, and more preferably 3 mM or less, from the viewpoint of guiding cholesterol in lipoproteins other than sdLDL out of the reaction system.

[0070] Furthermore, when the hydrogen donor is used in the second reagent composition described later, it is preferable that the coupler used in the coupling reaction is included in the first reagent composition. 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 higher, more preferably 0.3 mM or higher, and preferably 5.0 mM or lower, and more preferably 3.3 mM or lower, relative to the total composition of the reaction solution after the addition of the first reagent composition, from the viewpoint of stable action on sdLDL.

[0071] (kit) In this embodiment, the kit is used for the quantitative determination of sdLDL-C in a sample and includes the first reagent composition described above and a second reagent composition for the quantitative determination of sdLDL-C. Furthermore, the kit is used specifically in a method for quantifying sdLDL-C that includes two or more steps. In this case, 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.

[0072] (Second reagent composition) The second reagent composition is specifically a reagent composition for quantifying sdLDL-C. The components of the second reagent composition differ from those of the first reagent composition, but any composition that can quantify sdLDL-C is acceptable, and known substances can be used.

[0073] Specific examples of components included in the second reagent composition include enzymes, buffers, salts, surfactants, proteins that do not exhibit enzymatic activity, preservatives, and either a hydrogen donor or a coupler. Furthermore, in a preferred configuration of the kit, the first reagent composition includes either a hydrogen donor or a coupler but not the other, and the second reagent composition includes either a hydrogen donor or a coupler but not the other.

[0074] An example of an enzyme is peroxidase. The second reagent composition also 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, if the first reagent composition contains peroxidase activity, the peroxidase activity will be carried over to the second step, so the concentration in the second reagent can be reduced or omitted.

[0075] The type 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 buffers include those mentioned above for the first reagent composition.

[0076] The pH of the second reagent composition is preferably 6.0 or higher, more preferably 6.5 or higher, and preferably 8.0 or lower, and more preferably 7.5 or lower, from the viewpoint of maintaining enzyme activity in the composition and improving the storage stability of the reagent.

[0077] Examples of surfactants include surfactants that act on sdLDL. Furthermore, the second reagent composition preferably contains a surfactant that acts on sdLDL, from the viewpoint of stably quantifying sdLDL-C. The surfactant acting on sdLDL may be a surfactant that acts selectively on sdLDL, such as a surfactant that acts only on sdLDL, or it may be a surfactant that acts on lipoproteins other than sdLDL, or a surfactant that acts on all lipoproteins.

[0078] Examples of surfactants in the second reagent composition include polyoxyethylene derivatives, and surfactants used in commercially available reagents for total cholesterol measurement can also be used. 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)).

[0079] 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 ensuring stable action on sdLDL in the mixed solution after the addition of the second reagent composition. Furthermore, from a similar 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.

[0080] When the second reagent composition contains a coupler, the coupler is preferably one or more compounds selected from the group consisting of 4-aminoantipyrine (4-AA), aminoantipyrine derivatives, vanillin diaminesulfonic acid, methylbenzthiazolinone hydrazone, and sulfonated methylbenzthiazolinone hydrazone. The concentration of the coupler in the second reagent composition is preferably 0.5 mM or higher, more preferably 1.0 mM or higher, and preferably 15 mM or lower, and more preferably 10 mM or lower, relative to the total composition of the reaction solution after the addition of the second reagent composition, from the viewpoint of stable action on sdLDL.

[0081] On the other hand, if the coupler is included in the first reagent composition, the hydrogen donor is preferably included in the second reagent composition. Specific examples of hydrogen donors include those mentioned above for the first 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, and more preferably 12 mM or less, relative to the total composition of the reaction solution after the addition of the second reagent composition, from the viewpoint of stable action on sdLDL.

[0082] Specific examples of proteins and preservatives that do not possess enzymatic activity include those mentioned above for the first reagent composition.

[0083] (method) The method in this embodiment is a method for quantifying sdLDL-C in a sample using the first and second reagent compositions described above. The quantification method in this embodiment includes the following first and second steps. (Step 1) Step of reacting the sample with the aforementioned first reagent composition. (Second step) After the first step, the cholesterol in the remaining lipoprotein is quantified by reacting it with the second reagent composition described above. The compositions of the first and second reagent compositions are as described above. In this method, by using the first reagent composition in this embodiment, measurements with excellent accuracy and specificity after sample dilution can be performed. For example, it becomes possible to stably quantify sdLDL-C as a reagent with good dilution linearity and high correlation with the reference method.

[0084] Furthermore, the quantitative determination of sdLDL-C can be performed, for example, by adding the first reagent composition to the sample (subject sample) and allowing it to react, then adding the second reagent composition and allowing it to react, and finally measuring the absorbance. The sample to be examined is, for example, a blood-derived sample such as serum or plasma, and is preferably serum. The first and second steps are typically performed in an automated analyzer. The amount of sample and the amount of each reagent composition can be appropriately determined by considering, for example, the concentration of the reagents in each reagent composition, but should be within a range applicable to the automated analyzer. For example, 1 to 10 μL of the sample, 50 to 300 μL of the first reagent, and 25 to 200 μL of the second reagent may be used. The following provides a more detailed explanation of each step.

[0085] (1st step) In the first step, the sample is reacted with the first reagent composition. This eliminates lipoproteins other than sdLDL and releases cholesterol from the lipoproteins other than sdLDL, which is then removed from the reaction system. More specifically, in the first step, a surfactant that preferably acts on lipoproteins other than sdLDL is reacted with the sample in the presence of sphingomyelinase and cholesterol esterase. Then, the cholesterol released from the lipoprotein is reacted with an enzyme that reacts with cholesterol, such as cholesterol oxidase, and removed from the reaction system. In the first step, known techniques can be used, such as eliminating cholesterol in lipoproteins other than sdLDL and removing it from the reaction system, or agglutinating cholesterol in lipoproteins other than sdLDL or inhibiting it from reacting in later steps.

[0086] When the first reagent composition contains an electron donor, the first step of eliminating cholesterol generated from lipoproteins other than sdLDL and removing it from the reaction system may include, for example, forming a colorless quinone in the presence of hydrogen peroxide generated by the sphingomyelinase, cholesterol esterase activity and cholesterol oxidase activity of the first reagent composition, as well as the electron donor.

[0087] Furthermore, in the first step, at least one of a monovalent cation and a divalent cation, or a salt thereof, can be added to the reaction solution as an ionic strength modifier. Adding an ionic strength modifier makes it easier to differentiate between sdLDL and L LDL.

[0088] (2nd process) In the second step, the sdLDL-C remaining after the first step is quantified. Conventional methods for quantifying LDL can be used in the second step. For example, methods include quantifying the content of LDL-specific aggregates formed by adding an LDL coagulant using turbidimetry, using an antigen-antibody reaction with an LDL-specific antibody, or quantifying degradation products using an enzyme. The quantification method is selected, for example, depending on the components and composition of the second reagent composition.

[0089] When the second reagent composition contains an enzyme, the method can be used to quantify the degradation product using the enzyme. Specifically, to the reaction solution after the first step, a second reagent composition containing one or more cholesterol-measuring enzymes selected from the group consisting of, for example, cholesterol esterase, cholesterol oxidase, cholesterol dehydrogenase, and peroxidase is added to release and degrade sdLDL-C, and the reaction product is quantified.

[0090] 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, and more preferably 3 to 7 minutes.

[0091] Examples of automated analyzers used for the quantitative determination of sdLDL-C include the TBA-120FR and TBA-200FR (both manufactured by Toshiba), JCA-BM1250, JCA-BM1650, and JCA-BM2250 (all manufactured by JEOL Ltd.), HITACHI7180 and HITACHI7170 ​​(both manufactured by Hitachi), AU2700, AU5800, and AU680 (all manufactured by OLYMPUS), and cobas c501 and cobas c701 (both manufactured by Roche).

[0092] The quantitative determination of sdLDL-C is performed, for example, by absorbance measurement in the wavelength range of 580 to 720 nm, preferably in the wavelength range of 600 to 700 nm.

[0093] The embodiments of the present invention have been described above, but these are merely examples, and various other configurations can also be adopted. Examples of reference formats are provided below. 1. A step of reacting the sample with the first reagent composition, After the step of reacting the sample with the first reagent composition, the process involves reacting the sample with a second reagent composition for quantifying small dense LDL cholesterol (sdLDL-C) to quantify the cholesterol remaining in the lipoprotein, A reagent composition used as the first reagent composition for a method of quantifying the sdLDL-C in the sample, comprising: Contains nonionic surfactants, It possesses cholesterol esterase activity, cholesterol oxidase activity, and sphingomyelinase activity, A reagent composition in which the contact angle between the reagent composition and a polyethylene terephthalate (PET) substrate, as measured by the following method 1, is 63.0° or more and 67.0° or less. (Method 1) (1) PET substrate: PET (amorphous polyester) resin sheet, transparent, 2 mm thick (2) Pretreatment: Wipe the surface of an unused PET substrate with a 70% ethanol aqueous solution. Perform the measurement within 5 minutes after wiping. (3) Measurement method and conditions: Droplet method, θ / 2 method, temperature: 15~25℃, syringe: Teflon coated 18G, dropping method, dropping volume: 2μL, measurement is taken 1 second after dropping. (4) Calculation of contact angle: Measure five times and calculate the average of the five measurements. 2. The reagent composition according to 1, wherein the viscosity of the reagent composition at 5°C, as measured by the following method 2-1, is 2.5 mPa·s or less. (Method 2-1) (1) Equipment: EMS viscometer (Electro Magnetically Spinning Viscometer) (2) Measurement method and conditions: Measurement method: Electromagnetic spinning method, Motor rotation speed: 1000 rpm, Holding time: 600 seconds, Measurement temperature: 5°C, Sequence loop: 1 time, Sample: 500 μL (3) Calculation of viscosity: Five measurements are taken and the average value of the five measurements is calculated. 3. The reagent composition according to 1. or 2., wherein the viscosity of the reagent composition at 37°C, as measured by the following method 2-2, is 1.05 mPa·s or less. (Method 2-2) (1) Equipment: EMS viscometer (Electro Magnetically Spinning Viscometer) (2) Measurement method and conditions: Measurement method: Electromagnetic spinning method, Motor rotation speed: 1000 rpm, Holding time: 600 seconds, Measurement temperature: 37°C, Sequence loop: 1 time, Sample: 500 μL (3) Calculation of viscosity: Five measurements are taken and the average value of the five measurements is calculated. 4. The reagent composition according to 1. or 2., wherein the nonionic surfactant comprises polyoxyethylene monostyrenated phenyl ether. 5. The reagent composition according to 4, wherein the degree of polymerization n of polyoxyethylene in the polyoxyethylene monostyrenated phenyl ether is 5 or more and 80 or less. 6. The reagent composition according to 4, wherein the content of polyoxyethylene monostyrenated phenyl ether in the reagent composition is 0.05% (w / v) or more and 0.6% (w / v) or less relative to the entire reagent composition. 7. The reagent composition according to 1. or 2., wherein the reagent composition comprises either a hydrogen donor or a coupler. 8. The reagent composition according to 1. or 2., wherein the reagent composition further has ascorbic acid oxidase activity. 9. The reagent composition according to 1. or 2., wherein the reagent composition further has at least one activity selected from the group consisting of peroxidase activity and catalase activity. 10. A first reagent composition comprising the reagent composition described in 1. or 2., A second reagent composition for quantifying the aforementioned sdLDL-C, A kit used for quantifying the sdLDL-C in the sample, comprising the above-mentioned components. 11. The kit according to 10, wherein the second reagent composition has peroxidase activity. [Examples]

[0094] (Examples 1-7, Comparative Examples 1 and 2) First reagent compositions were prepared for each example with varying contact angles, and sdLDL-C was quantified by combining them with a second reagent composition. The accuracy and specificity (correlation with the reference method) after sample dilution were then evaluated.

[0095] (Preparation of reagent composition) The first and second reagent compositions for each example were prepared by combining the following components at the following concentrations. For the first reagent composition, each example composition was prepared by varying the type and concentration of the nonionic surfactant.

[0096] The formulations of the first and second reagent compositions used in the measurement are shown below. (First reagent composition) PIPES buffer, pH7.0 50mM Cholesterol esterase 300 U / L Cholesterol oxidase 600 U / L Sphingomyelinase 2700 U / L Catalase 1200 KU / L Bovine serum albumin 1.0% (w / v) TOOS 2.0mM Nonionic surfactant *1

[0097] *1 Nonionic surfactants: Polyoxyethylene benzylphenyl ether derivatives and polyoxyethylene styrene-phenyl ether derivatives were used, and the first reagent compositions a to i were prepared by varying the amounts of these added.

[0098] (Second reagent composition) PIPES buffer, pH7.0 50mM 4-aminoantipyrine 4.0 mM Peroxidase 4000 U / L Sodium azide 0.05% (w / v) Polyoxyethylene octylphenyl ether 1% (w / v)

[0099] (Measurement of sdLDL-C) SdLDL-C was measured using the first and second reagent compositions for each example. Specifically, the first and second reagent compositions with different contact angles were prepared for each example. 150 μL of the first reagent composition was added to 3 μL of serum sample and reacted at 37°C for 5 minutes. Then, 50 μL of the second reagent composition was added and reacted for 5 minutes. Absorbance was measured at a primary wavelength of 600 nm and a secondary wavelength of 700 nm, and the sdLDL-C concentration was calculated from the calibration curve.

[0100] (Method for measuring contact angle) A transparent PET (amorphous polyester) resin plate (manufactured by Takiron CI Co., Ltd., PET plate (Petec) product number 6010, 2 mm thick) was used as the PET substrate. The surface of the unused PET substrate was wiped with a 70% ethanol aqueous solution, and measurements were taken within 5 minutes thereafter. Using a contact angle meter (DMo-501, manufactured by Kyowa Interface Science Co., Ltd.), the contact angle was measured 1 second after dropping using the droplet method and the θ / 2 method under the following conditions: temperature: 15-25°C, syringe: Teflon-coated 18G, dropping method, and dropping volume: 2 μL. Each example was measured five times, and the average of the five measurements was calculated. The measurement results are shown in Table 1.

[0101] (Method for measuring viscosity) For the examples and comparative examples, the viscosity of the first reagent composition for each example was measured using an EMS viscometer (EMS-1000, Kyoto Electronics Manufacturing Co., Ltd.) by electromagnetic spinning, with a motor speed of 1000 rpm, a holding time of 600 seconds, measurement temperatures of 5°C and 37°C, a sequence loop of 1, and a sample size of 500 μL. Five measurements were performed at each temperature for each example, and the average value of the five measurements was calculated. The measurement results are shown in Table 1.

[0102] (Accuracy test after sample dilution) Human serum samples of known sdLDL-C concentrations were diluted 2-fold with physiological saline, and the sdLDL-C concentration was measured in each sample before and after dilution. The measured values ​​after dilution were multiplied by 2 and compared with the measured values ​​before dilution, and a linear regression equation was calculated.

[0103] The summary of the measurement results is shown in Table 1. Also, the detailed data for each example are shown in FIGS. 1(a) to 1(i). In FIGS. 1(a) to 1(i), the horizontal axis represents the measured value of the sample before dilution, and the vertical axis represents the measured value of the sample after dilution multiplied by the dilution factor of 2. The evaluation criteria are shown below. A: The slope of the linear regression equation is within 1.00 ± 0.06 (0.94 to 1.06), and the correlation coefficient r of the linear regression equation (the same hereinafter) satisfies r > 0.9 (r 2 > 0.81) B: The slope of the linear regression equation exceeds 1.00 ± 0.06 and is within 1.00 ± 0.10, and r > 0.9 (r 2 > 0.81) C: The slope of the linear regression equation exceeds 1.00 ± 0.10, or r ≤ 0.9 (r 2 ≤ 0.81) From Table 1 and FIGS. 1(a) to 1(i), good accuracy after sample dilution was obtained in each example.

[0104] (Correlation test with the reference method) As a comparative method, the value of ultracentrifugation sdLDL-C (specific gravity 1.044 to 1.063) in each human sample was calculated and compared with the sdLDL-C value of each example.

[0105] The summary of the measurement results is shown in Table 1. Also, the detailed correlation data for each example are shown in FIGS. 2(a) to 2(i). The horizontal axis in FIGS. 2(a) to 2(i) represents the sdLDL-C value calculated by the reference method of ultracentrifugation, and the vertical axis represents the sdLDL-C value in each example. The evaluation criteria are shown below. The evaluations of A and B below were considered qualified. A: The slope is within 1.00 ± 0.11 (0.89 to 1.11), and the correlation coefficient r of the linear regression equation (the same hereinafter) > 0.905 (r 2 > 0.819) B: The slope exceeds 1.00 ± 0.11 and is within 1.00 ± 0.20, and r > 0.900 (r 2 > 0.810) C: The slope exceeds 1.000 ± 0.200, or 0.900 ≤ r (r 2 ≤ 0.81)

[0106] Table 1 and Figures 2(a) to 2(i) show that a good correlation was obtained with the ultracentrifugation method in each embodiment. Figure 3 shows the relationship between the contact angles of the reagent compositions and the correlation coefficient when the reagent compositions are arranged in order from largest to smallest contact angle. The correlation coefficient was higher for the contact angles of reagent compositions b to h.

[0107] (Dilution linearity test) Dilution linearity tests were performed on Comparative Example 1 and Examples 2, 4, and 6 from the examples listed in Table 1. Specifically, high-concentration sdLDL-C samples of known concentration were diluted in physiological saline in 10 steps, and the sdLDL-C concentration of each example in each dilution series was measured. Analysis was performed according to CLSI guideline EP06-A, and the difference plot of nonlinearity was calculated to evaluate the dilution linearity.

[0108] Table 1 summarizes the measurement results, and detailed data is shown in Figures 4(a) to 4(d). In the left figures of Figures 4(a) to 4(d), the horizontal axis (dilute Lv.) shows the dilution series of high-concentration samples, and the vertical axis (Measured value [mg / dL]) shows the sdLDL-C value. In the right figures of Figures 4(a) to 4(d), the horizontal axis (dilute Lv.) shows the dilution series of high-concentration samples, and the vertical axis (Nonlinearity [mg / dL]) shows the nonlinearity of the sdLDL-C value. The evaluation criteria were as follows, in accordance with CLSI guideline EP06-A. Samples with evaluations A and B below were considered acceptable. A: The nonlinearity values ​​(circular plots in Figure 4, right) are within the acceptable range for all points (inside the two solid lines in Figure 4, right: ±3 mg / dL if Linear fit sdLDL-C < 30 mg / dL, ±10% if Linear fit sdLDL-C ≥ 30 mg / dL). C: The Nonlinearity value (circular plot in Figure 4, right) falls outside the acceptable range at least at one point (inside the two solid lines in Figure 4, right: ±3 mg / dL if Linear fit sdLDL-C < 30 mg / dL, ±10% if Linear fit sdLDL-C ≥ 30 mg / dL).

[0109] Table 1 and Figures 4(a) to 4(d) show that good dilution linearity was obtained in each example.

[0110] Figures 1(a) to 1(i), 2(a) to 2(i), 3, 4(a) to 4(d), and Table 1 show that in each example, sdLDL-C measurement was possible with excellent dilution linearity, accuracy after sample dilution, and correlation with the reference method.

[0111] [Table 1]

[0112] (Examples 8-10) (Preparation of reagent composition) The following components were combined at the specified concentrations, and the type of surfactant was varied to prepare the first reagent composition for each example. The composition of the first reagent composition is shown below. (First reagent composition) PIPES buffer, pH7.0 50mM Cholesterol esterase 900 U / L Cholesterol oxidase 450 U / L Sphingomyelinase 525 U / L Bovine serum albumin 0.75% (w / v) Catalase 1200 KU / L 4AA 1mM Surfactants *2

[0113] *2 Surfactants Example 8: POE monostyrene-derived phenyl ether, POE degree of polymerization 11-33 Example 9: POE monostyrene-derived phenyl ether, degree of polymerization of POE 13-37 Example 10: Polyoxyethylene benzylphenyl ether derivative

[0114] (Evaluation of lipoprotein selectivity) To evaluate the lipoprotein selectivity in the reaction with the first reagent composition, the reactivity of sdLDL and lbLDL with the reagent composition was measured, respectively. Specifically, sdLDL and lbLDL separated from human serum by ultracentrifugation were used as samples. A first reagent composition was prepared to confirm the reactivity of the surfactant with the samples. To allow for simple evaluation by measuring absorbance after generating a color reaction using only the first reagent composition, a first reagent composition containing 1.5 mM TOOS and 1250 U / L peroxidase was used for evaluation in each example, instead of 1200 KU / L catalase in the above composition.

[0115] 3 μL of the sample was mixed with 150 μL of the first reagent composition for each example. The difference in absorbance at 600 nm (primary wavelength) and 700 nm (secondary wavelength) after 5 minutes at 37°C (indicated as "Absorbance Wavelength 600 nm / 700 nm" in Table 2) [mAbs] was measured, and the ratio of the absorbance of lbLDL to the absorbance of sdLDL was determined. The measurement results are shown in Table 2.

[0116] [Table 2]

[0117] Table 2 shows that by using the first reagent composition of each example, sdLDL in the sample can be quantified more accurately. Furthermore, Table 2 shows that among the examples, the first reagent composition containing POE monostyrene-modified phenyl ether exhibits even better selectivity for lbLDL in the first step, in terms of LDL absorbance ratio.

[0118] (Comparative Example 3) (Preparation of reagent composition) In Examples 1-7 and Comparative Examples 1 and 2, a first reagent composition using polyoxyethylene styrene-phenyl ether sulfate as the anionic surfactant was prepared according to the method described above. The contact angle and viscosity were measured according to the method in the above examples, and the accuracy after sample dilution was evaluated. In measuring the accuracy after sample dilution, in addition to the first reagent composition of Comparative Example 3, a first reagent composition with the same formulation as in Example 4 was prepared as a control and measured. The measurement results are shown in Table 3. Table 3 also shows the measurement results for the contact angle and viscosity of Example 4 (Table 1). Detailed data is shown in Figures 5(a) and 5(b). Figures 5(a) and 5(b) show the measurement results for the first reagent composition with the same formulation as in Example 4 and Comparative Example 3, respectively.

[0119] [Table 3]

[0120] As shown in Figure 5(b), in Comparative Example 3, which used an anionic surfactant, sdLDL could not be accurately quantified after diluting the sample, whereas in the formulation of Example 4 (Figure 5(a)), which used a nonionic surfactant, accurate quantification was possible even after sample dilution. Furthermore, as shown in Figures 5(a) and 1(e), it was confirmed that the selectivity after sample dilution could be evaluated with excellent reproducibility for compositions with the same formulation.

[0121] This application claims priority based on Japanese Patent Application No. 2022-064966, filed on 11 April 2022, and incorporates all of its disclosures herein.

Claims

1. A step of reacting the sample with the first reagent composition, After the step of reacting the sample with the first reagent composition, the process involves reacting the sample with a second reagent composition for quantifying small dense LDL cholesterol (sdLDL-C) to quantify the cholesterol remaining in the lipoprotein, A reagent composition used as the first reagent composition for a method of quantifying the sdLDL-C in the sample, comprising: Contains surfactants, The surfactant is at least one nonionic surfactant selected from the group consisting of polyoxyethylene benzylphenyl ether derivatives and polyoxyethylene styrene-phenyl ether derivatives. It possesses cholesterol esterase activity, cholesterol oxidase activity, and sphingomyelinase activity, A hydrogen donor and a coupler, or a hydrogen donor, but not the other. The hydrogen donor is one or more compounds selected from the group consisting of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline, N-ethyl-N-(3-sulfopropyl)-3-methylaniline, N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-(3-sulfopropyl)aniline, N-(3-sulfopropyl)-3-methoxy-5-aniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-4-fluoro-3,5-dimethoxyaniline, and N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine. The coupler is one or more compounds selected from the group consisting of 4-aminoantipyrine, vanillin diamine sulfonic acid, methylbenzthiazolinone hydrazone, and sulfonated methylbenzthiazolinone hydrazone. The contact angle between the reagent composition and the polyethylene terephthalate (PET) substrate, as measured by the following method 1, is 63.0° or more and 67.0° or less. The second reagent composition comprises a buffer, an enzyme, a preservative, and a surfactant, and contains the hydrogen donor and the other of the coupler, but does not include one of the latter. (Method 1) (1) PET substrate: PET (amorphous polyester) resin sheet, transparent, 2 mm thick (2) Pretreatment: Wipe the surface of the unused PET substrate with a 70% ethanol aqueous solution. Measurements should be taken within 5 minutes after wiping. (3) Measurement method and conditions: Droplet method, θ / 2 method, temperature: 15-25°C, syringe: Teflon coated 18G, dropping method, dropping volume: 2 μL, measurement taken 1 second after dropping. (4) Calculation of contact angle: Measure five times and calculate the average of the five measurements.

2. The reagent composition according to claim 1, wherein the viscosity of the reagent composition at 5°C, as measured by the following method 2-1, is 2.5 mPa·s or less. (Method 2-1) (1) Apparatus: EMS viscometer (Electro Magnetically Spinning Viscometer) (2) Measurement method and conditions: Measurement method: Electromagnetic spinning method, Motor rotation speed: 1000 rpm, Holding time: 600 seconds, Measurement temperature: 5°C, Sequence loop: 1 time, Sample: 500 μL (3) Calculation of viscosity: Five measurements are taken and the average value of the five measurements is calculated.

3. The reagent composition according to claim 1 or 2, wherein the viscosity of the reagent composition at 37°C, as measured by the following method 2-2, is 1.05 mPa·s or less. (Method 2-2) (1) Apparatus: EMS viscometer (Electro Magnetically Spinning Viscometer) (2) Measurement method and conditions: Measurement method: Electromagnetic spinning method, Motor rotation speed: 1000 rpm, Holding time: 600 seconds, Measurement temperature: 37°C, Sequence loop: 1 time, Sample: 500 μL (3) Calculation of viscosity: Five measurements are taken and the average value of the five measurements is calculated.

4. The reagent composition according to claim 1 or 2, wherein the nonionic surfactant comprises polyoxyethylene monostyrene-derived phenyl ether.

5. The reagent composition according to claim 4, wherein the degree of polymerization n of polyoxyethylene in the polyoxyethylene monostyrene-phenyl ether is 5 or more and 80 or less.

6. The reagent composition according to claim 4, wherein the content of polyoxyethylene monostyrenated phenyl ether in the reagent composition is 0.05% (w / v) or more and 0.6% (w / v) or less relative to the entire reagent composition.

7. The reagent composition according to claim 1 or 2, wherein the reagent composition used as the first reagent composition further has ascorbic acid oxidase activity.

8. The reagent composition according to claim 1 or 2, wherein the reagent composition further has at least one activity selected from the group consisting of peroxidase activity and catalase activity.

9. A first reagent composition comprising the reagent composition according to claim 1 or 2, A second reagent composition for quantifying the aforementioned sdLDL-C, A kit used for quantifying the sdLDL-C in the sample, including the above.

10. The kit according to claim 9, wherein the second reagent composition has peroxidase activity.

Images