Method for suppressing inter-agent differences in measurement reagents when measuring cholesterol in high-density lipoproteins in a sample.

Abstract

This invention provides a method for suppressing inter-agent differences in measurement reagents when measuring cholesterol in high-density lipoproteins in a sample. [Solution] A cholesterol measurement reagent for high-density lipoproteins, consisting of a first reagent and a second reagent, is used. A dye is generated from a chromogen using hydrogen peroxide produced by an oxidase, and the absorbance based on this dye is optically measured. Based on this, the cholesterol value in the high-density lipoprotein in the sample is calculated and measured, characterized in that the cholesterol value in the high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 250 seconds after dispensing the second reagent.

Description

Technical Field

[0001] The present invention relates to measurement of cholesterol levels in high-density lipoprotein (High Density Lipoprotein; HDL). More specifically, the present invention relates to a method for suppressing inter-reagent differences in measurement reagents for measured values of cholesterol in high-density lipoprotein in a sample. The present invention is useful in fields of life science such as clinical testing, clinical pathology and medicine, as well as in chemical fields such as analytical chemistry, and the like.

Background Art

[0002] Hypo-high-density lipoprotein cholesterolemia is regarded as important as an independent risk factor for arteriosclerotic diseases (see Non-Patent Document 1). A method is widely used, which uses a reagent for measuring cholesterol in high-density lipoprotein comprising a first reagent and a second reagent, wherein a surfactant is contained in the first reagent and / or the second reagent, a pigment is generated from a chromogen by hydrogen peroxide generated by an oxidase, the absorbance based on the pigment is optically measured, and the cholesterol level in high-density lipoprotein in a sample is calculated and measured based on the absorbance (see Patent Document 1, Patent Document 2 and Patent Document 3).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0004]

Non-Patent Document 1

[0005] The inventors noticed a difference between the two reagents when measuring cholesterol levels in high-density lipoproteins in a sample using a reagent consisting of a first reagent and a second reagent. In other words, we realized that we were not accurately measuring the cholesterol levels in high-density lipoproteins in the samples. Furthermore, the difference between measuring reagents refers to the difference in measured values ​​between measuring reagents when the sample being measured is the same and the formulation of the measuring reagent used for the measurement is the same.

[0006] Therefore, the object of the present invention is to provide a method for suppressing inter-agent differences in measurement reagents when measuring cholesterol in high-density lipoprotein in a sample. [Means for solving the problem]

[0007] The inventors of the present invention have conducted extensive research on suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample. They have found that the above problem can be solved by measuring the cholesterol level in high-density lipoprotein in the sample based on the absorbance measured within 250 seconds after dispensing the second reagent, and have completed the present invention.

[0008] In other words, the present invention consists of the following inventions. (1) A method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample, characterized in that when using a cholesterol measurement reagent consisting of a first reagent and a second reagent, a dye is generated from a chromogen by hydrogen peroxide produced by an oxidase, the absorbance based on the dye is optically measured, and the cholesterol value in high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 250 seconds after dispensing the second reagent. (2) A method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample as described in (1), characterized by calculating and measuring the cholesterol level in high-density lipoprotein in the sample based on the absorbance measured within 237.0 seconds after dispensing the second reagent. (3) A method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample as described in (1), characterized by calculating and measuring the cholesterol level in high-density lipoprotein in the sample based on the absorbance measured within 219.2 seconds after dispensing the second reagent. (4) A method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample as described in (1), characterized by calculating and measuring the cholesterol level in high-density lipoprotein in the sample based on the absorbance measured within 201.4 seconds after dispensing the second reagent. (5) A method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample as described in (1), characterized by calculating and measuring the cholesterol level in high-density lipoprotein in the sample based on the absorbance measured within 183.5 seconds after dispensing the second reagent. [Effects of the Invention]

[0009] The present invention provides a method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoproteins in a sample, thereby enabling accurate measurement of cholesterol levels in high-density lipoproteins. In other words, when measuring cholesterol levels in high-density lipoprotein using a measurement reagent consisting of a first reagent and a second reagent, it is possible to avoid the measurement being affected by differences between the reagents and prevent errors in the measurement results (measured values).

[0010] Therefore, the method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to the present invention makes it possible to obtain accurate measurement results (measured values) that are not affected by inter-agent differences in the measurement reagents.

[0011] As a result, the method of suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to the present invention can prevent misdiagnosis, for example, in a medical setting. [Modes for carrying out the invention]

[0012] 1. General Overview The present invention provides a method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample, which involves using a cholesterol measurement reagent for high-density lipoprotein consisting of a first reagent and a second reagent, generating a dye from a chromogen using hydrogen peroxide produced by an oxidase, optically measuring the absorbance based on the dye, and calculating and measuring the cholesterol value in high-density lipoprotein in the sample based on this, characterized in that the cholesterol value in high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 250 seconds after dispensing the second reagent.

[0013] Furthermore, the present invention provides a method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoproteins in a sample, thereby suppressing inter-agent differences in the measurement of cholesterol in high-density lipoproteins in a sample and enabling accurate measurement of cholesterol levels in high-density lipoproteins.

[0014] 2. Method for measuring cholesterol levels in high-density lipoprotein in a sample The present invention provides a method for measuring the cholesterol level in high-density lipoprotein in a sample, which can be any method that uses a high-density lipoprotein cholesterol measurement reagent consisting of a first reagent and a second reagent, generates a dye from a chromogen using hydrogen peroxide produced by an oxidase, optically measures the absorbance based on the dye, and calculates and measures the cholesterol level in the high-density lipoprotein in the sample based on this measurement. There are no particular limitations on this method.

[0015] This method involves generating a pigment from a chromogen using hydrogen peroxide produced by an oxidase, optically measuring the absorbance based on the pigment, and calculating the cholesterol level in the high-density lipoprotein in the sample based on this. For example, one method involves using an oxidase to generate hydrogen peroxide directly or indirectly from the substance to be measured, mixing and contacting it with peroxidase and a chromogen, leading to a color development system, and optically measuring the pigment produced from the chromogen by the oxidation reaction to measure the generated hydrogen peroxide, thereby calculating and measuring the cholesterol level in the high-density lipoprotein in the sample.

[0016] More specifically, one possible method involves using an oxidase to generate hydrogen peroxide directly or indirectly from the substance to be measured, mixing and contacting it with peroxidase, a chromogen (a hydrogen donor), and a coupler (a condensation target for the chromogen) to create a color development system, and then optically measuring the pigment produced from the chromogen by the oxidation-condensation reaction to measure the amount of hydrogen peroxide generated. This allows for the calculation and measurement of the cholesterol level in the high-density lipoprotein in the sample.

[0017] Further, for example, using an oxidase, hydrogen peroxide is generated directly or indirectly from the target substance to be measured, which is mixed and brought into contact with peroxidase and a chromogen to form a color development system without using the aforementioned coupler, and the pigment generated from the chromogen by the oxidation reaction is optically measured, whereby the generated hydrogen peroxide is measured, and thereby the method for calculating and measuring the cholesterol level in high-density lipoprotein in a sample can also be mentioned.

[0018] 3. Reagent for measuring cholesterol in high-density lipoprotein (1) General overview The reagent for measuring cholesterol in high-density lipoprotein according to the present invention consists of a first reagent and a second reagent.

[0019] Examples of the composition of the first reagent include those containing a buffer, a coupler, a surfactant or modified silicone oil, an oxidase, peroxidase and the like.

[0020] Examples of the composition of the second reagent include those containing a buffer, a chromogen, a surfactant or modified silicone oil, cholesterol esterase and the like.

[0021] Note that flufenamic acid may be contained in the first reagent and / or the second reagent.

[0022] (2) Oxidase In the present invention, the oxidase refers to a substance that catalyzes the reaction of generating hydrogen peroxide, which is an oxidizing substance, directly or indirectly from cholesterol in high-density lipoprotein in a sample, which is the target substance to be measured contained in the sample, and any such substance can be used without particular limitation.

[0023] That is, in the present invention, examples of the oxidase include cholesterol oxidase and the like.

[0024] In the present invention, oxidases of any origin can be used, including, for example, those derived from animals such as humans or cattle, those derived from plants, or those derived from microorganisms such as bacteria or fungi.

[0025] Furthermore, this oxidase may be one prepared by genetic engineering, which involves incorporating the gene for the oxidase from a microorganism into a microorganism such as E. coli, or an oxidase prepared from a microorganism whose properties have been improved through genetic modification or other means.

[0026] In the measurement reaction for cholesterol levels in high-density lipoprotein in a sample according to the present invention, the activity value of the oxidase is not particularly limited, but it is preferably 100 units / L (Unit / L) or more during the measurement reaction.

[0027] Furthermore, the lower limit of the activity value of this oxidase is more preferably 1,000 units / L or more, and particularly preferably 3,000 units / L or more, at the time of the measurement reaction.

[0028] While there is no particular upper limit to the activity level of this oxidase during the measurement reaction, considering costs and other factors, up to 100,000 units / L is sufficient.

[0029] Furthermore, the upper limit of the activity value of this oxidase is more preferably 80,000 units / L or less, and particularly preferably 50,000 units / L or less, at the time of the measurement reaction.

[0030] Furthermore, it is preferable that the cholesterol measurement reagent in high-density lipoprotein in the present invention contains an oxidase with an activity value such that the activity value described above is obtained during the measurement reaction.

[0031] (3) Cholesterol esterase In the present invention, it is preferable that the cholesterol measurement reagent in high-density lipoprotein contains cholesterol esterase.

[0032] This cholesterol esterase refers to a substance that catalyzes the hydrolysis reaction of cholesterol esters into cholesterol and fatty acids, and any such substance can be used without any particular limitations.

[0033] In the present invention, cholesterol esterase can be of any origin, including, for example, those derived from animals such as humans or pigs, those derived from plants, or those derived from microorganisms such as bacteria or fungi.

[0034] Furthermore, this cholesterol esterase may be prepared by genetic engineering technology, which involves incorporating the gene for the oxidase in question into microorganisms such as E. coli, or by preparing cholesterol esterase from microorganisms whose properties have been improved through genetic modification or other means.

[0035] In addition, during the measurement reaction of cholesterol levels in high-density lipoproteins in the sample of the present invention, the activity value of cholesterol esterase is not particularly limited, but it is preferable that it is 100 units / L (Unit / L) or more during the measurement reaction.

[0036] Furthermore, the lower limit of the activity value of this cholesterol esterase is more preferably 1,000 units / L or more, and particularly preferably 2,500 units / L or more, at the time of the measurement reaction.

[0037] While there is no particular upper limit to the activity level of cholesterol esterase during this measurement reaction, considering costs and other factors, up to 100,000 units / L is sufficient.

[0038] Furthermore, the upper limit of the activity value of this cholesterol esterase is more preferably 80,000 units / L or less, and particularly preferably 50,000 units / L or less, at the time of the measurement reaction.

[0039] Furthermore, it is preferable that the cholesterol measurement reagent in high-density lipoprotein in the present invention contains cholesterol esterase with an activity value such that the activity value described above is obtained during the measurement reaction.

[0040] (4) Peroxidase In the present invention, peroxidase (POD) can be used together with a chromogen, or together with a chromogen and a coupler, to generate a pigment using hydrogen peroxide produced by an oxidase.

[0041] The peroxidase used is not particularly limited as long as it can catalyze a reaction in which a chromogen is oxidized by hydrogen peroxide to produce a pigment, or a reaction in which a chromogen and a coupler are oxidized and condensed by hydrogen peroxide to produce a pigment.

[0042] The peroxidase can be of any origin, including, for example, those derived from animals such as humans or cattle, those derived from plants such as horseradish, or those derived from microorganisms such as bacteria or fungi.

[0043] Furthermore, this peroxidase may be prepared by genetic engineering technology, which involves incorporating the peroxidase gene of a microorganism into a microorganism such as E. coli, or it may be a peroxidase prepared from a microorganism whose properties have been improved by genetic modification or the like.

[0044] In addition, during the measurement reaction of cholesterol levels in high-density lipoprotein in the sample of the present invention, the activity level of peroxidase is not particularly limited, but it is preferably 10 units / L (Unit / L) or higher during the measurement reaction.

[0045] Furthermore, the lower limit of the activity value of this peroxidase is more preferably 50 units / L or more, and particularly preferably 100 units / L or more, at the time of the measurement reaction.

[0046] Furthermore, there is no particular upper limit to the activity level of this peroxidase during the measurement reaction, but considering costs and other factors, up to 500,000 units / L is sufficient.

[0047] Furthermore, the upper limit of the activity value of this peroxidase is more preferably 100,000 units / L or less, and particularly preferably 50,000 units / L or less, at the time of the measurement reaction.

[0048] Furthermore, it is preferable that the cholesterol measurement reagent in high-density lipoprotein in the present invention contains peroxidase with an activity value such that the activity value described above is obtained during the measurement reaction.

[0049] (5) Chromogen In this invention, a chromogen is a substance that can be used to calculate and measure the cholesterol level in high-density lipoprotein in a sample by optically measuring hydrogen peroxide produced by an oxidase using the chromogen. Any such substance can be used without any particular limitations.

[0050] In the measurement reaction for cholesterol levels in high-density lipoproteins in the sample of the present invention, the chromogen may be, for example, a substance that, when mixed and brought into contact with hydrogen peroxide, peroxidase, and a coupler produced by an oxidase, undergoes oxidative condensation with the coupler to produce a pigment. In this case, the colorant acts as a hydrogen donor, releasing hydrogen during the aforementioned oxidation-condensation reaction.

[0051] In this case, examples of colorants include phenol, phenol derivatives, or aniline derivatives.

[0052] Examples of derivatives of this phenol include 4-chlorophenol, 2,4-dichlorophenol, 2,4-dibromophenol, or 2,4,6-trichlorophenol, or salts thereof.

[0053] Furthermore, examples of aniline derivatives include N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N-sulfopropyl-3,5-dimethoxyaniline (HDAPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS), N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline (DAPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-4-fluoroaniline (FDAOS), N-ethyl-N-sulfopropyl-3,5-dimethoxy-4-fluoroaniline (FDAPS), N-(2-carboxyethyl)-N-ethyl-3,5-dimethoxyaniline (CEDB), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS), and N-ethyl-N-(3-sulfopropyl Examples include N-(2-hydroxy-3-sulfopropyl)aniline (ADPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS), N-ethyl-N-(3-sulfopropyl)aniline (ALPS), N-(3-sulfopropyl)aniline (HALPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (TOOS), N-(2-carboxyethyl)-N-ethyl-m-toluidine (CEMB), N-(2-carboxyethyl)-N-ethyl-3-methoxyaniline (CEMO), or N-(3-sulfopropyl)-3-methoxy-5-methylaniline (HMMPS), or salts thereof.

[0054] In addition, during the measurement reaction of cholesterol levels in high-density lipoprotein in the sample of the present invention, the concentration of the chromogen is not particularly limited, but it is preferably 0.005 mM or higher during the measurement reaction.

[0055] Furthermore, the lower limit of the concentration of the colorant is more preferably 0.05 mM or higher, and particularly preferably 0.5 mM or higher, at the time of the measurement reaction.

[0056] Furthermore, there is no particular upper limit to the concentration of the aforementioned colorant during the measurement reaction, but considering costs and other factors, up to 50 mM is sufficient.

[0057] Furthermore, the upper limit of the concentration of the colorant is more preferably 20 mM or less, and particularly preferably 10 mM or less, at the time of the measurement reaction.

[0058] Furthermore, in the present invention, it is preferable that the reagent for measuring cholesterol in high-density lipoprotein in a sample contains the aforementioned chromogen at a concentration such that the concentration is as described above during the measurement reaction.

[0059] Furthermore, in the measurement reaction of cholesterol levels in high-density lipoprotein in the sample of the present invention, the chromogen may be a substance that produces a pigment through an oxidation reaction, for example, by mixing and contacting it with hydrogen peroxide produced by an oxidase and peroxidase (i.e., without mixing and contacting it with a coupler).

[0060] In this case, examples of the colorant include bis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine (BCMA) or salts thereof.

[0061] Furthermore, in the present invention, it is preferable that the reagent for measuring cholesterol in high-density lipoprotein in a sample contains the aforementioned chromogen at a concentration such that the concentration is as described above during the measurement reaction.

[0062] (6) Coupler In the present invention, the coupler is a substance that can produce a pigment by mixing and contacting hydrogen peroxide produced by an oxidase with the coupler, peroxidase, and the aforementioned chromogen (phenol, phenol derivative, or aniline derivative, etc.) as a hydrogen donor, thereby undergoing oxidative condensation with the chromogen. Any such substance can be used without particular limitation.

[0063] Examples of such couplers include 4-aminoantipyrine or its derivatives, phenylenediamine, or 3-methyl-2-benzothiazolinone hydrazone (MBTH). In this invention, it is preferable that the coupler is 4-aminoantipyrine.

[0064] In addition, during the measurement reaction of cholesterol levels in high-density lipoprotein in the sample of the present invention, the concentration of the coupler is not particularly limited, but it is preferably 0.005 mM or higher during the measurement reaction.

[0065] Furthermore, the lower limit of the concentration of the coupler is more preferably 0.05 mM or higher, and particularly preferably 0.5 mM or higher, at the time of the measurement reaction.

[0066] Furthermore, there is no particular upper limit to the concentration of the coupler during the measurement reaction, but considering costs and other factors, up to 50 mM is sufficient.

[0067] Furthermore, the upper limit of the concentration of the coupler is more preferably 20 mM or less, and particularly preferably 10 mM or less, at the time of the measurement reaction.

[0068] Furthermore, in the present invention, it is preferable that the reagent for measuring cholesterol in high-density lipoprotein in a sample contains the aforementioned coupler at a concentration such that the concentration is as described above during the measurement reaction.

[0069] (7) Surfactants In the present invention, it is preferable to include a surfactant in the reagent for measuring cholesterol in high-density lipoprotein in a sample.

[0070] This surfactant may be included in the first reagent of the reagent for measuring cholesterol in high-density lipoprotein in a sample according to the present invention, or in the second reagent, or in both the first reagent and the second reagent.

[0071] Examples of surfactants in the present invention include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants.

[0072] Specific examples of the aforementioned surfactants include, for example, nonionic surfactants such as polyoxyethylene tribenzylphenyl ether, sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil, or polyoxyethylene lanolin; amphoteric surfactants such as betaine acetate; anionic surfactants such as polyoxyethylene alkyl ether sulfate or polyoxyethylene alkyl ether acetate; or cationic surfactants such as cetylpyridinium chloride.

[0073] The surfactant to be included in the reagent for measuring cholesterol in high-density lipoproteins in a sample in the present invention is preferably one that has the effect of selectively measuring cholesterol in high-density lipoproteins in a sample.

[0074] Furthermore, nonionic surfactants are preferred as surfactants that have such effects.

[0075] Furthermore, polyoxyethylene tribenzylphenyl ether is more preferred as a surfactant having such effects.

[0076] As a surfactant having such an effect, Emulgen (registered trademark; hereinafter the same) B-66 [Kao Corporation] is particularly preferred.

[0077] In addition, during the measurement reaction of cholesterol levels in high-density lipoprotein in the sample of the present invention, the concentration of the surfactant is not particularly limited, but it is preferably 0.001% (w / v) or higher during the measurement reaction.

[0078] Furthermore, the lower limit of the surfactant concentration is more preferably 0.01% (w / v) or higher, and particularly preferably 0.1% (w / v) or higher, at the time of the measurement reaction.

[0079] Furthermore, there is no particular upper limit to the concentration of the surfactant mentioned above, but considering costs and other factors, a concentration of up to 10% (w / v) is sufficient during the measurement reaction.

[0080] Furthermore, the upper limit of the surfactant concentration is more preferably 8.0% (w / v) or less, and particularly preferably 6.0% (w / v) or less, at the time of the measurement reaction.

[0081] Furthermore, in the present invention, it is preferable to include in the measurement reagent for cholesterol in high-density lipoprotein in a sample the aforementioned surfactant at a concentration such that the concentration is as described above during the measurement reaction.

[0082] (8) Modified silicone oil In the present invention, it is preferable that the reagent for measuring cholesterol in high-density lipoprotein in a sample contains denatured silicone oil.

[0083] This modified silicone oil may be included in the first reagent of the reagent for measuring cholesterol in high-density lipoprotein in a sample according to the present invention, or in the second reagent, or in both the first reagent and the second reagent.

[0084] Examples of modified silicone oils in the present invention include side-chain type / polyether modified silicone oil (non-reactive silicone oil).

[0085] The modified silicone oil to be included in the reagent for measuring cholesterol in high-density lipoproteins in a sample in the present invention is preferably one that has the effect of selectively measuring cholesterol in high-density lipoproteins in a sample.

[0086] Furthermore, as a modified silicone oil having such effects, a side-chain type / polyether modified silicone oil (non-reactive silicone oil) is preferred.

[0087] Furthermore, KF-354L [Shin-Etsu Chemical Co., Ltd.] is more preferred as a modified silicone oil having such effects.

[0088] In addition, during the measurement reaction of cholesterol levels in high-density lipoprotein in the sample of the present invention, the concentration of the modified silicone oil is not particularly limited, but it is preferably 0.001% (w / v) or higher during the measurement reaction.

[0089] Furthermore, the lower limit of the concentration of the modified silicone oil is more preferably 0.01% (w / v) or higher, and particularly preferably 0.1% (w / v) or higher, at the time of the measurement reaction.

[0090] Furthermore, there is no particular upper limit to the concentration of the modified silicone oil mentioned above, but considering costs and other factors, a concentration of up to 10% (w / v) is sufficient during the measurement reaction.

[0091] Furthermore, the upper limit of the concentration of the modified silicone oil is more preferably 5% (w / v) or less, and particularly preferably 2.5% (w / v) or less, at the time of the measurement reaction.

[0092] Furthermore, in the present invention, it is preferable to include in the measurement reagent for cholesterol in high-density lipoprotein in a sample the modified silicone oil at a concentration such that the concentration is as described above during the measurement reaction.

[0093] (9) Flufenamic acid In the present invention, it is preferable that the cholesterol measurement reagent in high-density lipoprotein contains flufenamic acid.

[0094] In addition, during the measurement reaction of cholesterol levels in high-density lipoprotein in the sample of the present invention, the concentration of flufenamic acid is not particularly limited, but it is preferably 0.01 mM or higher during the measurement reaction.

[0095] Furthermore, the lower limit of the concentration of flufenamic acid is more preferably 0.05 mM or higher, and particularly preferably 0.1 mM or higher, at the time of the measurement reaction.

[0096] While there is no particular upper limit to the concentration of flufenamic acid used in this measurement reaction, considering costs and other factors, 10 mM is sufficient.

[0097] Furthermore, the upper limit of the concentration of flufenamic acid is more preferably 5 mM or less, and particularly preferably 1 mM or less, at the time of the measurement reaction.

[0098] Furthermore, it is preferable that the cholesterol measurement reagent in high-density lipoprotein in the present invention contains flufenamic acid at a concentration such that the aforementioned concentration is achieved during the measurement reaction.

[0099] (10) pH In the measurement reaction for cholesterol levels in high-density lipoprotein in the sample of the present invention, the pH is not particularly limited, but the pH during the measurement reaction should be within the pH range that allows the measurement reaction to proceed. Furthermore, it is preferable that the pH during the measurement reaction is within or near the optimal pH range for the measurement reaction.

[0100] For example, the pH during the measurement reaction is preferably pH 5.0 (at 20°C) or higher.

[0101] Furthermore, the lower limit of the pH during this measurement reaction is more preferably pH 5.5 (20°C) or higher, and particularly preferably pH 6.0 (20°C) or higher.

[0102] Furthermore, the pH during this measurement reaction is preferably 8.0 (at 20°C) or lower.

[0103] Furthermore, the upper limit of the pH during this measurement reaction is more preferably pH 7.5 (20°C) or lower, and particularly preferably pH 7.0 (20°C) or lower.

[0104] Furthermore, in the present invention, it is preferable to set the pH of the measurement reagent so that the pH of the measurement reagent falls within the aforementioned pH range during the measurement reaction.

[0105] (11) Buffer In the present invention, a buffer can be appropriately and as needed during the measurement reaction of cholesterol levels in high-density lipoproteins in a sample.

[0106] For example, a buffering agent having buffering capacity in the pH range described in "(10) pH" above can be present at a concentration that has buffering capacity during the measurement reaction of cholesterol levels in high-density lipoprotein in a sample.

[0107] Examples of such buffering agents include MES [2-morpholinoethanesulfonic acid], Bis-Tris [2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol], Bis-Trispropane [1,3-bis[tris(hydroxymethyl)methylamino]propane], ADA [N-(carbamoylmethyl)iminodiacetic acid], PIPES [piperazine-1,4-bis( 2-Ethanesulfonic acid), ACES [N-(carbamoylmethyl)-2-aminoethanesulfonic acid], MOPSO [2-hydroxy-3-morpholinopropanesulfonic acid], MOPS [3-morpholinopropane-1-sulfonic acid], MES [2-morpholinoethanesulfonic acid], BES [2-[N,N-bis(2-hydroxyethyl)amino]-1-ethanesulfonic acid], TES [N-tris(hydroxymethyl) )methyl-2-aminoethanesulfonic acid], HEPES [2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid], DIPSO [3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid], TAPSO [3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid], POPSO [piperazine-1,4-bis(2-hydroxypropanesulfonic acid)], HEPPSO [4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropane-3-sulfonic acid)], HEPPS [4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid], EPPS [4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid], Tricine [N-[tris(hydroxymethyl)methyl]glycine], Bicine [N,Examples include N-bis(2-hydroxyethyl)glycine, TAPS (N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), CHES (2-cyclohexylaminoethanesulfonic acid), CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), CAPS (3-cyclohexylaminopropanesulfonic acid), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane, phosphoric acid, phosphates, boric acid, borates, glycine, glycylglycine, imidazole, or tris(hydroxymethyl)aminomethane (Tris), or salts or hydrates thereof.

[0108] Furthermore, in the present invention, it is preferable to set the pH of the measurement reagent so that the pH of the measurement reagent is within the pH range described in "(10) pH" during the measurement reaction. Therefore, it is preferable to include a buffering agent having buffering capacity in the pH range described in "(10) pH" above in the measurement reagent at a concentration that has buffering capacity at the time of the measurement reaction.

[0109] (12) Other ingredients In the present invention, during the measurement reaction of cholesterol levels in high-density lipoprotein in a sample, enzyme substrates, conjugated enzymes, coenzymes, metal ions or metal salts containing them, chelating agents, stabilizers such as proteins like albumin, sugars or polymer compounds, preservatives such as sodium azide or antibiotics, scavenging agents or inhibitors of interfering substances in the sample (for example, potassium ferrocyanide for bilirubin, or ascorbate oxidase for ascorbic acid), or activators may be present as appropriate and as needed.

[0110] While there are no particular limitations on the concentration of these substances present, it is preferable that the concentration be 0.001% (w / v) or higher during the measurement reaction.

[0111] Furthermore, the lower limit of the concentration when these are present is more preferably 0.01% (w / v) or higher, and particularly preferably 0.1% (w / v) or higher, at the time of the measurement reaction.

[0112] While there is no particular upper limit to the concentration of these substances present, considering costs and other factors, a concentration of up to 10% (w / v) is preferable during the measurement reaction.

[0113] Furthermore, the upper limit of the concentration of these substances present is more preferably 5% (w / v) or less, and particularly preferably 2.5% (w / v) or less, during the measurement reaction.

[0114] Furthermore, it is preferable that the reagent for measuring cholesterol in high-density lipoprotein in a sample in the present invention contains the aforementioned component at a concentration such that the concentration is reached during the measurement reaction.

[0115] 4. Method for calculating and measuring cholesterol levels in high-density lipoproteins in a sample. (1) General Overview In the present invention's method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample, a cholesterol measurement reagent for high-density lipoprotein consisting of a first reagent and a second reagent is used. A dye is generated from a chromogen using hydrogen peroxide produced by an oxidase, and the absorbance based on the dye is optically measured. Based on this, the cholesterol value in the high-density lipoprotein in the sample is calculated and measured. In this method, the cholesterol value in the high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 250 seconds after dispensing the second reagent.

[0116] When measuring cholesterol levels in high-density lipoproteins in a sample according to the present invention, the measurement can be performed by appropriately selecting a method such as the endpoint method or the rate method. Specifically, examples of endpoint methods include the two-point end method.

[0117] Furthermore, in the present invention, a two-step method (two-reagent method) performed in two steps is preferred for measuring the cholesterol level in high-density lipoprotein in the sample.

[0118] Furthermore, in the measurement described in this invention, the measurement wavelength used when optically measuring the generated dye can be appropriately selected from a suitable wavelength in the visible region. In this case, either a single-wavelength method or a two-wavelength method may be used.

[0119] Furthermore, in the measurement described in the present invention, the temperature during the measurement reaction should be set to a temperature within a range such as 30°C to 37°C, in which case the measurement reaction can proceed without the enzymes or other reaction components involved in the measurement reaction being inactivated, denatured, or altered by heat.

[0120] Furthermore, in the measurement described in the present invention, the method for initiating the measurement reaction may be any of the following: adding a substrate to the sample, or adding the sample to the substrate.

[0121] Furthermore, in the measurement described in the present invention, the measurement may be performed by manual methods or by using an automated analyzer or other device.

[0122] (2) Sample The sample in this invention is a sample that may contain cholesterol in high-density lipoprotein, and the purpose is to measure the cholesterol level in this high-density lipoprotein.

[0123] As long as the sample meets the above criteria, there are no particular limitations, but examples of samples include biological samples.

[0124] For example, biological samples can include human or animal blood, serum, plasma, urine, feces, cerebrospinal fluid, saliva, sweat, tears, ascites, amniotic fluid, organs such as the brain, and tissues and cells such as hair, skin, nails, muscles, or nerves.

[0125] In this invention, the sample is preferably a liquid. If the sample is not in liquid form, pretreatment such as extraction or solubilization may be performed according to known methods to incorporate the cholesterol in the high-density lipoprotein into a liquid.

[0126] (3) Measurement of absorbance In the present invention's method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample, a cholesterol measurement reagent for high-density lipoprotein consisting of a first reagent and a second reagent is used. A dye is generated from a chromogen using hydrogen peroxide produced by an oxidase, and the absorbance based on the dye is optically measured. Based on this, the cholesterol value in the high-density lipoprotein in the sample is calculated and measured. In this method, the cholesterol value in the high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 250 seconds after dispensing the second reagent.

[0127] Furthermore, in the method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to the present invention, it is preferable that the cholesterol value in high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 237.0 seconds after dispensing the second reagent.

[0128] Furthermore, in the method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to the present invention, it is preferable that the cholesterol value in high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 219.2 seconds after dispensing the second reagent.

[0129] Furthermore, in the method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to the present invention, it is preferable that the cholesterol value in high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 201.4 seconds after dispensing the second reagent.

[0130] Furthermore, in the method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to the present invention, it is preferable that the cholesterol value in high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 183.5 seconds after dispensing the second reagent.

[0131] Furthermore, in the method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to the present invention, it is preferable that the cholesterol value in high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 166.6 seconds after dispensing the second reagent.

[0132] Furthermore, in the method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to the present invention, it is preferable that the cholesterol value in high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 150.7 seconds after dispensing the second reagent.

[0133] Furthermore, in the method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to the present invention, it is preferable that the cholesterol value in high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 132.9 seconds after dispensing the second reagent.

[0134] Furthermore, in the method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to the present invention, it is preferable that the cholesterol value in high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 115.0 seconds after dispensing the second reagent.

[0135] Furthermore, in the method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to the present invention, it is preferable that the cholesterol value in high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 97.2 seconds after dispensing the second reagent.

[0136] Furthermore, in the method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to the present invention, it is preferable that the cholesterol value in high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 79.4 seconds after dispensing the second reagent.

[0137] Furthermore, in the method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to the present invention, it is preferable that the cholesterol value in high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 61.5 seconds after dispensing the second reagent.

[0138] (4) Specific examples of methods for suppressing inter-agent differences in measurement reagents when measuring cholesterol in high-density lipoprotein in a sample. A specific example of a method for suppressing inter-agent differences in measurement reagents when measuring cholesterol in high-density lipoprotein in a sample according to the present invention is described below.

[0139] (a) Preparation of measurement reagents By dissolving a fixed amount of each of the components listed below in pure water and adjusting the pH to a constant level, the first and second reagents for measuring cholesterol in high-density lipoproteins in a sample were prepared, respectively.

[0140] (i) Reagent 1 Cholesterol oxidase (oxidase) peroxidase N-(2-carboxyethyl)-N-ethyl-m-toluidine hydrochloride [CEMB] (chromogen <hydrogen donor>) Modified silicone oil Flufenamic acid cushioning agent

[0141] (ii) Second reagent Cholesterol esterase 4-aminoantipyrine (coupler) surfactant cushioning agent

[0142] (b) Sample Human serum was used as a sample.

[0143] (c) Quantitative measurement (i) Stage 1 The sample and the first reagent are mixed to prepare a mixture.

[0144] The amounts of the sample and the first reagent to be mixed should be determined appropriately according to the amount of the second reagent, the concentration of the target substance (cholesterol in high-density lipoprotein) contained in the sample, the absorption coefficient of the dye derived from the chromogen and coupler, the measurement wavelength, the specifications of the analytical instrument used, and other conditions. Generally, it is preferable that the sample volume be in the range of 0.5 to 100 μL, and the volume of the first reagent be in the range of 20 to 1,000 μL.

[0145] After preparing this mixture, incubate it. There are no particular restrictions on the incubation time, but it is usually preferable to keep it under 20 minutes, more preferably under 10 minutes, and especially preferable under 5 minutes.

[0146] Furthermore, the incubation temperature should be above the freezing point of the aforementioned mixture. Generally speaking, a higher temperature during the measurement reaction is preferable because it results in a higher reaction rate. However, if the temperature is too high, the enzymes involved in the reaction will denature and become inactive. Therefore, the incubation temperature must be below the temperature at which the enzymes involved in the measured reaction denature and become inactive.

[0147] The incubation temperature is usually 2 to 50°C, but 20 to 37°C is preferred, and 30 to 37°C is more preferred. Furthermore, if the enzyme involved in the measurement reaction is a heat-resistant enzyme, even higher temperatures may be used.

[0148] (ii) Second stage The second reagent is dispensed into the "mixture of sample and first reagent" prepared in the first step described above, and mixed. This is the final reaction solution.

[0149] The amount of the second reagent to be dispensed and mixed should be determined appropriately according to the amount of sample, the amount of the first reagent, the concentration of the target substance (cholesterol in high-density lipoprotein) contained in the sample, the extinction coefficient of the dye derived from the chromogen and coupler, the measurement wavelength, the specifications of the analytical instrument used, and other conditions. Generally, it is preferable that the amount of the second reagent be in the range of 10 to 1,000 μL.

[0150] After preparing this final reaction solution, incubation is performed. There are no particular restrictions on the incubation time, but it is usually preferable to keep it under 20 minutes, more preferably under 10 minutes, and especially preferable under 5 minutes.

[0151] Furthermore, the incubation temperature should be above the temperature at which the final reaction solution freezes. Generally speaking, a higher temperature during the measurement reaction is preferable because it results in a higher reaction rate. However, if the temperature is too high, the enzymes involved in the measurement reaction will denature and become inactive. Therefore, the incubation temperature must be below the temperature at which the enzymes involved in the measurement reaction denature and become inactive. The incubation temperature is usually 2 to 50°C, but 20 to 37°C is preferred, and 30 to 37°C is more preferred. Furthermore, if the enzyme involved in the measurement reaction is a heat-resistant enzyme, even higher temperatures are acceptable.

[0152] The preparation and incubation of this final reaction solution allows the measurement reaction of cholesterol levels in the high-density lipoprotein contained in the sample to proceed.

[0153] Specifically, first, cholesterol esters in high-density lipoproteins are hydrolyzed by cholesterol esterase into cholesterol and fatty acids.

[0154] Next, cholesterol oxidase (an oxidizing enzyme) produces hydrogen peroxide from this cholesterol and oxygen.

[0155] Next, these two molecules of hydrogen peroxide, along with the chromophorus (CEMB) and coupler (4-aminoantipyrine), are converted into a pigment and four molecules of water by peroxidase. [The chromophorus and coupler undergo oxidative condensation to produce the pigment.]

[0156] Next, the absorbance of the final reaction solution derived from the generated dye is measured. In this invention, the absorbance measurement is performed within 250 seconds after dispensing the second reagent.

[0157] Furthermore, this absorbance measurement may be performed using the single-wavelength method, or the two-wavelength method, where measurements are performed at two different wavelengths; the appropriate method can be selected as needed. Furthermore, the wavelength used for measuring absorbance should be appropriately selected according to the absorption wavelength of the dye.

[0158] The absorbance can then be measured using an appropriate method, such as the endpoint method or the rate method.

[0159] In this invention, it is preferable to measure the absorbance using the two-endpoint method, which is a type of endpoint method. This two-point end method involves first measuring the absorbance (absorbance A1) 1 second after dispensing the first reagent [and before dispensing the second reagent] [measurement of the absorbance of the sample blank], then measuring the absorbance (absorbance A2) 2 seconds after dispensing the second reagent [measurement of the absorbance corresponding to the concentration of the target substance contained in the sample], and finally calculating and measuring the cholesterol level in high-density lipoprotein in the sample based on the absorbance difference obtained by subtracting the absorbance A1 measured 1 second after dispensing the first reagent from the absorbance A2 measured 2 seconds after dispensing the second reagent.

[0160] Furthermore, calculating and measuring the cholesterol level in high-density lipoprotein in a sample from the measured absorbance, absorbance difference, or change in absorbance can be done by appropriately selecting a method such as calculating it from the absorbance difference measured based on the molar extinction coefficient of the dye, or by comparing it with the absorbance difference of a target substance (standard substance) whose concentration or activity value is known.

[0161] Furthermore, in the present invention, it is preferable to calculate and measure the cholesterol level in high-density lipoprotein in the sample by subtracting the reagent blind (reagent blank) from the absorbance difference obtained by measuring the sample.

[0162] The measurement of cholesterol levels in high-density lipoproteins in a sample may be performed manually by the person performing the measurement, or it may be performed using an automated analyzer or other device. [Examples]

[0163] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0164] [Example 1] (Confirmation of the effect of the method for suppressing inter-agent differences in measurement reagents in cholesterol measurement in high-density lipoprotein in a sample according to the present invention) The effectiveness of the method for suppressing inter-agent differences in measurement reagents in cholesterol measurement in high-density lipoprotein samples according to the present invention was confirmed.

[0165] 1. Reagent for measuring cholesterol in high-density lipoprotein (1) Reagent 1

[0166] The following reagent components were dissolved in pure water to the concentrations indicated, and the pH was adjusted to pH 6.0 (20°C) to prepare the first reagent for measuring cholesterol in high-density lipoprotein. (Hereafter, this first reagent for measuring cholesterol in high-density lipoprotein may be referred to as "R1".)

[0167] Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane [Actec Co., Ltd.] 25mM N-(2-carboxyethyl)-N-ethyl-m-toluidine hydrochloride [Actec Co., Ltd.] 1 mM Flufenamic acid [Fujifilm Wako Pure Chemical Industries] 0.4 mM KF-354L [Shin-Etsu Chemical Co., Ltd.] 0.3% (w / v) Cholesterol oxidase [Toyobo Co., Ltd.] 3K Unit / L Peroxidase [Toyobo Co., Ltd.] 0.5K Unit / L

[0168] (2) Second reagent

[0169] The following reagent components were dissolved in pure water to the concentrations indicated, and the pH was adjusted to pH 6.0 (20°C) to prepare the second reagent for measuring cholesterol in high-density lipoprotein. (Hereinafter, this second reagent for measuring cholesterol in high-density lipoprotein may be referred to as "R2.") [Hereinafter, the combination of these first and second reagents for measuring cholesterol in high-density lipoprotein may be referred to as "measuring reagent-1."]

[0170] 2-Morpholinoethanesulfonic acid monohydrate [Actec Co., Ltd.] 40 mM 4-Aminoantipyrine [Actec Co., Ltd.] 1 mM Emulgen B-66<Lot.2562> [Kao Corporation] 3% (w / v) Cholesterol esterase [Asahi Kasei Pharma Corporation] 2.5K Unit / L

[0171] 2. Sample

[0172] (1) Human serum sample-1 A human serum sample (sample number 11) was used as human serum sample-1.

[0173] (2) Human serum sample-2 Human serum sample (sample number 14) was used as human serum sample-2.

[0174] 3. Measurement of human serum sample-1 (1) When measuring absorbance at 19 photometric points (43.7 seconds after dispensing the second reagent) The measurement of human serum sample-1 in 2(1) above using the cholesterol measurement reagent in high-density lipoprotein described in 1 above was performed using a Hitachi 7180 automatic analyzer [Hitachi High-Technologies Corporation] by the two-point end method.

[0175] (a) In the human serum sample-1 of 2(1) above, 200 μL of the first reagent of the cholesterol measurement reagent in high-density lipoprotein of 1(1) above was dispensed into 2.0 μL of the sample and reacted at 37°C.

[0176] (b) Next, the absorbance at 16 points (270.1 seconds after the addition of the first reagent) was measured at a primary wavelength of 600 nm and a secondary wavelength of 700 nm ["Photometry 1"]. (Measurement of absorbance of sample blank)

[0177] (c) Next, at point 17 (287.0 seconds after the addition of the first reagent), 67 μL of the second reagent from 1(2) above was dispensed and reacted at 37°C.

[0178] (d) Next, the absorbance at 19 points (43.7 seconds after the addition of the second reagent) was measured at a primary wavelength of 600 nm and a secondary wavelength of 700 nm ["Photometric 2"]. (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)

[0179] (e) The operations described in (a) to (d) above were repeated a total of three times.

[0180] (f) Next, the cholesterol levels in the high-density lipoprotein contained in the sample were measured as follows: In other words, for each of the operations described in (a) to (e) above, the absorbance difference was determined by subtracting the absorbance value measured in (b) ("Photometric 1") from the absorbance value measured in (d) ("Photometric 2"). Next, the average value of the absorbance difference was calculated by taking the average value from the absorbance difference values ​​obtained for each of the three operations.

[0181] (g) Next, a standard sample [Oriental Yeast Co., Ltd.; OYCcal] whose cholesterol concentration in high-density lipoprotein is known was measured as described in (a) to (f) above. The average absorbance difference obtained by subtracting the average absorbance value measured in (b) ("Photometric 1") from the absorbance value measured in (d) ("Photometric 2") was compared with the average absorbance difference for human serum sample-1 of 2(1) obtained by the operation of (a) to (f) above, and a proportional calculation was performed to obtain the measured value of cholesterol in high-density lipoprotein when human serum sample-1 of 2(1) was measured with the cholesterol measurement reagent for high-density lipoprotein, derived from the absorbance measurement value at 19 points (43.7 seconds after dispensing the second reagent). These values ​​are shown in Table 1.

[0182] (h) Next, the emulsion B-66 (surfactant) of 1(2) above, if the lot is<Lot.2562> From those that say,<Lot.2568> Except for changing to the above, the procedure was carried out as described in (a) to (g) above, and the measured value of cholesterol in high-density lipoprotein was obtained when the human serum sample-1 of 2(1) above, derived from the absorbance measurement at 19 points (43.7 seconds after dispensing the second reagent), was measured with the high-density lipoprotein cholesterol measurement reagent. (Hereafter, the combination of the first and second reagents of the high-density lipoprotein cholesterol measurement reagent at this time may be referred to as "measurement reagent-2".) These values ​​are shown in Table 1.

[0183] (i) Next, the emulsion B-66 (surfactant) of 1(2) above, if the lot is<Lot.2562> From those that say,<Lot.2569> Except for changing to the above, the procedure was carried out as described in (a) to (g) above, and the measured value of cholesterol in high-density lipoprotein was obtained when the human serum sample-1 of 2(1) above, derived from the absorbance measurement at 19 points (43.7 seconds after dispensing the second reagent), was measured with the high-density lipoprotein cholesterol measurement reagent. (Hereafter, the combination of the first and second reagents of the high-density lipoprotein cholesterol measurement reagent at this time may be referred to as "measurement reagent-3".) These values ​​are shown in Table 1. Furthermore, the value obtained by subtracting the minimum value from the maximum value of the cholesterol measurement in high-density lipoprotein obtained when using the aforementioned measurement reagent-1, measurement reagent-2, and measurement reagent-3 was defined as the "inter-reagent difference between measurement reagents." The inter-agent differences in these measurement reagents are also shown in Table 1.

[0184] (2) When measuring absorbance at 20 photometric points (61.5 seconds after adding the second reagent) Except for changing "Measured absorbance at 19 points (43.7 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)" in (d) of (1) above to "Measured absorbance at 20 points (61.5 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)", the procedure was carried out as described in (a) to (i) of (1) above, and the measured value of cholesterol in high-density lipoprotein was obtained when serum sample-1 of 2 (1) above, derived from the measured absorbance at 20 points (61.5 seconds after dispensing the second reagent), was measured with the cholesterol measurement reagent for high-density lipoprotein. [Measurement reagent-1, Measurement reagent-2, and Measurement reagent-3] These values ​​are shown in Table 1. Furthermore, the value obtained by subtracting the minimum value from the maximum value of the cholesterol measurement in high-density lipoprotein obtained when using the aforementioned measurement reagent-1, measurement reagent-2, and measurement reagent-3 was defined as the "inter-reagent difference between measurement reagents." The inter-agent differences in these measurement reagents are also shown in Table 1.

[0185] (3) When measuring absorbance at 21 photometric points (79.4 seconds after dispensing the second reagent) Except for changing "Measured absorbance at 19 points (43.7 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)" in (d) of (1) above to "Measured absorbance at 21 points (79.4 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)", the procedure was carried out as described in (a) to (i) of (1) above, and the measured value of cholesterol in high-density lipoprotein was obtained when the human serum sample-1 of 2 (1) above, derived from the measured absorbance at 21 points (79.4 seconds after dispensing the second reagent), was measured with the cholesterol measurement reagent for high-density lipoprotein. [Measurement reagent-1, Measurement reagent-2, and Measurement reagent-3] These values ​​are shown in Table 1. Furthermore, the value obtained by subtracting the minimum value from the maximum value of the cholesterol measurement in high-density lipoprotein obtained when using the aforementioned measurement reagent-1, measurement reagent-2, and measurement reagent-3 was defined as the "inter-reagent difference between measurement reagents." The inter-agent differences in these measurement reagents are also shown in Table 1.

[0186] (4) When measuring absorbance at 22 photometric points (97.2 seconds after dispensing the second reagent) Except for changing "Measured absorbance at 19 points (43.7 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)" in (d) of (1) above to "Measured absorbance at 22 points (97.2 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)", the procedure was carried out as described in (a) to (i) of (1) above, and the measured value of cholesterol in high-density lipoprotein was obtained when the human serum sample-1 of 2 (1) above, derived from the measured absorbance at 22 points (97.2 seconds after dispensing the second reagent), was measured with the cholesterol measurement reagent for high-density lipoprotein. [Measurement reagent-1, Measurement reagent-2, and Measurement reagent-3] These values ​​are shown in Table 1. Furthermore, the value obtained by subtracting the minimum value from the maximum value of the cholesterol measurement in high-density lipoprotein obtained when using the aforementioned measurement reagent-1, measurement reagent-2, and measurement reagent-3 was defined as the "inter-reagent difference between measurement reagents." The inter-agent differences in these measurement reagents are also shown in Table 1.

[0187] (5) When measuring absorbance at 23 photometric points (115.0 seconds after dispensing the second reagent) Except for changing "Measured absorbance at 19 points (43.7 seconds after dispensing the second reagent) ["Photometric 2"]. (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)" in (d) of (1) above to "Measured absorbance at 23 points (115.0 seconds after dispensing the second reagent) ["Photometric 2"]. (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)", the procedure was carried out as described in (a) to (i) of (1) above, and the measured value of cholesterol in high-density lipoprotein was obtained when human serum sample-1 of 2 (1) above, derived from the measured absorbance at 23 points (115.0 seconds after dispensing the second reagent), was measured with the cholesterol measurement reagent for high-density lipoprotein. [Distribution of "Measurement Reagent-1", "Measurement Reagent-2", and "Measurement Reagent-3"] These values ​​are shown in Table 1. Furthermore, the value obtained by subtracting the minimum value from the maximum value of the cholesterol measurement in high-density lipoprotein obtained when using the aforementioned measurement reagent-1, measurement reagent-2, and measurement reagent-3 was defined as the "inter-reagent difference between measurement reagents." The inter-agent differences in these measurement reagents are also shown in Table 1.

[0188] (6) When measuring absorbance at 24 photometric points (132.9 seconds after dispensing the second reagent) Except for changing "Measured absorbance at 19 points (43.7 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)" in (d) of (1) above to "Measured absorbance at 24 points (132.9 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)", the procedure was carried out as described in (a) to (i) of (1) above, and the measured value of cholesterol in high-density lipoprotein was obtained when human serum sample-1 of 2 (1) above, derived from the measured absorbance at 24 points (132.9 seconds after dispensing the second reagent), was measured with the cholesterol measurement reagent for high-density lipoprotein. [Measurement reagent-1, Measurement reagent-2, and Measurement reagent-3] These values ​​are shown in Table 1. Furthermore, the value obtained by subtracting the minimum value from the maximum value of the cholesterol measurement in high-density lipoprotein obtained when using the aforementioned measurement reagent-1, measurement reagent-2, and measurement reagent-3 was defined as the "inter-reagent difference between measurement reagents." The inter-agent differences in these measurement reagents are also shown in Table 1.

[0189] (7) When measuring absorbance at 25 photometric points (150.7 seconds after dispensing the second reagent) Except for changing "Measured absorbance at 19 points (43.7 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)" in (d) of (1) above to "Measured absorbance at 25 points (150.7 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)", the procedure was carried out as described in (a) to (i) of (1) above, and the measured value of cholesterol in high-density lipoprotein was obtained when the human serum sample-1 of 2 (1) above, derived from the measured absorbance at 25 points (150.7 seconds after dispensing the second reagent), was measured with the cholesterol measurement reagent for high-density lipoprotein. [Measurement reagent-1, Measurement reagent-2, and Measurement reagent-3] These values ​​are shown in Table 1. Furthermore, the value obtained by subtracting the minimum value from the maximum value of the cholesterol measurement in high-density lipoprotein obtained when using the aforementioned measurement reagent-1, measurement reagent-2, and measurement reagent-3 was defined as the "inter-reagent difference between measurement reagents." The inter-agent differences in these measurement reagents are also shown in Table 1.

[0190] (8) When measuring absorbance at 26 photometric points (166.6 seconds after dispensing the second reagent) Except for changing "Measured absorbance at 19 points (43.7 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)" in (d) of (1) above to "Measured absorbance at 26 points (166.6 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)", the procedure was carried out as described in (a) to (i) of (1) above, and the measured value of cholesterol in high-density lipoprotein was obtained when human serum sample-1 of 2 (1) above, derived from the measured absorbance at 26 points (166.6 seconds after dispensing the second reagent), was measured with the cholesterol measurement reagent for high-density lipoprotein. [Measurement reagent-1, Measurement reagent-2, and Measurement reagent-3] These values ​​are shown in Table 1. Furthermore, the value obtained by subtracting the minimum value from the maximum value of the cholesterol measurement in high-density lipoprotein obtained when using the aforementioned measurement reagent-1, measurement reagent-2, and measurement reagent-3 was defined as the "inter-reagent difference between measurement reagents." The inter-agent differences in these measurement reagents are also shown in Table 1.

[0191] (9) When measuring absorbance at 27 photometric points (183.5 seconds after dispensing the second reagent) Except for changing "Measured absorbance at 19 points (43.7 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)" in (d) of (1) above to "Measured absorbance at 27 points (183.5 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)", the procedure was carried out as described in (a) to (i) of (1) above, and the measured value of cholesterol in high-density lipoprotein was obtained when human serum sample-1 of 2 (1) above, derived from the measured absorbance at 27 points (183.5 seconds after dispensing the second reagent), was measured with the cholesterol measurement reagent for high-density lipoprotein. [Measurement reagent-1, Measurement reagent-2, and Measurement reagent-3] These values ​​are shown in Table 1. Furthermore, the value obtained by subtracting the minimum value from the maximum value of the cholesterol measurement in high-density lipoprotein obtained when using the aforementioned measurement reagent-1, measurement reagent-2, and measurement reagent-3 was defined as the "inter-reagent difference between measurement reagents." The inter-agent differences in these measurement reagents are also shown in Table 1.

[0192] (10) When measuring absorbance at 28 photometric points (201.4 seconds after dispensing the second reagent) Except for changing "Measured absorbance at 19 points (43.7 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)" in (d) of (1) above to "Measured absorbance at 28 points (201.4 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)", the procedure was carried out as described in (a) to (i) of (1) above, and the measured value of cholesterol in high-density lipoprotein was obtained when the human serum sample-1 of 2 (1) above, derived from the measured absorbance at 28 points (201.4 seconds after dispensing the second reagent), was measured with the cholesterol measurement reagent for high-density lipoprotein. [Measurement reagent-1, Measurement reagent-2, and Measurement reagent-3] These values ​​are shown in Table 1. Furthermore, the value obtained by subtracting the minimum value from the maximum value of the cholesterol measurement in high-density lipoprotein obtained when using the aforementioned measurement reagent-1, measurement reagent-2, and measurement reagent-3 was defined as the "inter-reagent difference between measurement reagents." The inter-agent differences in these measurement reagents are also shown in Table 1.

[0193] (11) When measuring absorbance at 29 photometric points (219.2 seconds after dispensing the second reagent) Except for changing "Measured absorbance at 19 points (43.7 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)" in (d) of (1) above to "Measured absorbance at 29 points (219.2 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)", the procedure was carried out as described in (a) to (i) of (1) above, and the measured value of cholesterol in high-density lipoprotein was obtained when the human serum sample-1 of 2 (1) above, derived from the measured absorbance at 29 points (219.2 seconds after dispensing the second reagent), was measured with the cholesterol measurement reagent for high-density lipoprotein. [Measurement reagent-1, Measurement reagent-2, and Measurement reagent-3] These values ​​are shown in Table 1. Furthermore, the value obtained by subtracting the minimum value from the maximum value of the cholesterol measurement in high-density lipoprotein obtained when using the aforementioned measurement reagent-1, measurement reagent-2, and measurement reagent-3 was defined as the "inter-reagent difference between measurement reagents." The inter-agent differences in these measurement reagents are also shown in Table 1.

[0194] (12) When measuring absorbance at 30 photometric points (237.0 seconds after dispensing the second reagent) Except for changing "Measured absorbance at 19 points (43.7 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)" in (d) of (1) above to "Measured absorbance at 30 points (237.0 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)", the procedure was carried out as described in (a) to (i) of (1) above, and the measured value of cholesterol in high-density lipoprotein was obtained when human serum sample-1 of 2 (1) above, derived from the measured absorbance at 30 points (237.0 seconds after dispensing the second reagent), was measured with the cholesterol measurement reagent for high-density lipoprotein. [Measurement reagent-1, Measurement reagent-2, and Measurement reagent-3] These values ​​are shown in Table 1. Furthermore, the value obtained by subtracting the minimum value from the maximum value of the cholesterol measurement in high-density lipoprotein obtained when using the aforementioned measurement reagent-1, measurement reagent-2, and measurement reagent-3 was defined as the "inter-reagent difference between measurement reagents." The inter-agent differences in these measurement reagents are also shown in Table 1.

[0195] (13) When measuring absorbance at 31 photometric points (254.9 seconds after dispensing the second reagent) Except for changing "Measured absorbance at 19 points (43.7 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)" in (d) of (1) above to "Measured absorbance at 31 points (254.9 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)", the procedure was carried out as described in (a) to (i) of (1) above, and the measured value of cholesterol in high-density lipoprotein was obtained when human serum sample-1 of 2 (1) above, derived from the measured absorbance at 31 points (254.9 seconds after dispensing the second reagent), was measured with the cholesterol measurement reagent for high-density lipoprotein. [Measurement reagent-1, Measurement reagent-2, and Measurement reagent-3] These values ​​are shown in Table 1. Furthermore, the value obtained by subtracting the minimum value from the maximum value of the cholesterol measurement in high-density lipoprotein obtained when using the aforementioned measurement reagent-1, measurement reagent-2, and measurement reagent-3 was defined as the "inter-reagent difference between measurement reagents." The inter-agent differences in these measurement reagents are also shown in Table 1.

[0196] (14) When measuring absorbance at 32 photometric points (272.7 seconds after dispensing the second reagent) Except for changing "Measured absorbance at 19 points (43.7 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)" in (d) of (1) above to "Measured absorbance at 32 points (272.7 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)", the procedure was carried out as described in (a) to (i) of (1) above, and the measured value of cholesterol in high-density lipoprotein was obtained when human serum sample-1 of 2 (1) above, derived from the measured absorbance at 32 points (272.7 seconds after dispensing the second reagent), was measured with the cholesterol measurement reagent for high-density lipoprotein. [Measurement reagent-1, Measurement reagent-2, and Measurement reagent-3] These values ​​are shown in Table 1. Furthermore, the value obtained by subtracting the minimum value from the maximum value of the cholesterol measurement in high-density lipoprotein obtained when using the aforementioned measurement reagent-1, measurement reagent-2, and measurement reagent-3 was defined as the "inter-reagent difference between measurement reagents." The inter-agent differences in these measurement reagents are also shown in Table 1.

[0197] (15) When measuring absorbance at 33 photometric points (290.6 seconds after dispensing the second reagent) Except for changing "Measured absorbance at 19 points (43.7 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)" in (d) of (1) above to "Measured absorbance at 33 points (290.6 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)", the procedure was carried out as described in (a) to (i) of (1) above, and the measured value of cholesterol in high-density lipoprotein was obtained when human serum sample-1 of 2 (1) above, derived from the measured absorbance at 33 points (290.6 seconds after dispensing the second reagent), was measured with the cholesterol measurement reagent for high-density lipoprotein. [Measurement reagent-1, Measurement reagent-2, and Measurement reagent-3] These values ​​are shown in Table 1. Furthermore, the value obtained by subtracting the minimum value from the maximum value of the cholesterol measurement in high-density lipoprotein obtained when using the aforementioned measurement reagent-1, measurement reagent-2, and measurement reagent-3 was defined as the "inter-reagent difference between measurement reagents." The inter-agent differences in these measurement reagents are also shown in Table 1.

[0198] (16) When measuring absorbance at 34 photometric points (308.4 seconds after dispensing the second reagent) Except for changing "Measured absorbance at 19 points (43.7 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)" in (d) of (1) above to "Measured absorbance at 34 points (308.4 seconds after dispensing the second reagent) [Photometric 2] (Measurement of absorbance corresponding to the concentration of cholesterol in the high-density lipoprotein contained in the sample)", the procedure was carried out as described in (a) to (i) of (1) above, and the measured value of cholesterol in high-density lipoprotein was obtained when human serum sample-1 of 2 (1) above, derived from the measured absorbance at 34 points (308.4 seconds after dispensing the second reagent), was measured with the cholesterol measurement reagent for high-density lipoprotein. [Measurement reagent-1, Measurement reagent-2, and Measurement reagent-3] These values ​​are shown in Table 1. Furthermore, the value obtained by subtracting the minimum value from the maximum value of the cholesterol measurement in high-density lipoprotein obtained when using the aforementioned measurement reagent-1, measurement reagent-2, and measurement reagent-3 was defined as the "inter-reagent difference between measurement reagents." The inter-agent differences in these measurement reagents are also shown in Table 1.

[0199] [Table 1]

[0200] 4. Measurement of human serum sample-2 Except for changing "human serum sample-1" (the one in 2(1) above) used as the sample for measurement in 3 above to "human serum sample-2" (the one in 2(2) above), the procedure was carried out as described in 3 above. Then, in the case of measuring absorbance at 19 photometric points (43.7 seconds after dispensing the second reagent), in the case of measuring absorbance at 20 photometric points (61.5 seconds after dispensing the second reagent), in the case of measuring absorbance at 21 photometric points (79.4 seconds after dispensing the second reagent), in the case of measuring absorbance at 22 photometric points (97.2 seconds after dispensing the second reagent), in the case of measuring absorbance at 23 photometric points (second reagent) In the case of measuring absorbance at 115.0 seconds after dispensing, in the case of measuring absorbance at 24 photometric points (132.9 seconds after dispensing the second reagent), in the case of measuring absorbance at 25 photometric points (150.7 seconds after dispensing the second reagent), in the case of measuring absorbance at 26 photometric points (166.6 seconds after dispensing the second reagent), and in the case of measuring absorbance at 27 photometric points (183.5 seconds after dispensing the second reagent), In each of the following cases, the measured value of cholesterol in high-density lipoprotein was obtained when human serum sample-2 was measured using a high-density lipoprotein cholesterol measurement reagent: absorbance measurement at 28 photometric points (201.4 seconds after dispensing the second reagent), absorbance measurement at 29 photometric points (219.2 seconds after dispensing the second reagent), absorbance measurement at 30 photometric points (237.0 seconds after dispensing the second reagent), absorbance measurement at 31 photometric points (254.9 seconds after dispensing the second reagent), absorbance measurement at 32 photometric points (272.7 seconds after dispensing the second reagent), absorbance measurement at 33 photometric points (290.6 seconds after dispensing the second reagent), and absorbance measurement at 34 photometric points (308.4 seconds after dispensing the second reagent). [Divided into "Measuring Reagent-1", "Measuring Reagent-2", and "Measuring Reagent-3"] These values ​​are shown in Table 2. Furthermore, in the case of measuring absorbance at each of the aforementioned photometric points, the difference between the reagents was obtained by subtracting the minimum value from the maximum value of the cholesterol measurement value in high-density lipoprotein obtained when using the aforementioned reagent-1, reagent-2, and reagent-3, respectively. The inter-agent differences between these measurement reagents are also shown in Table 2.

[0201] [Table 2]

[0202] 5. Discussion [I] Regarding the above [Table 1] (1) From the above [Table 1], the following can be seen: (a) Based on the absorbance measured 308.4 seconds (34 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-1 was measured with the high-density lipoprotein cholesterol measurement reagent was 3.1 mg / dL.

[0203] (b) Based on the absorbance measured 290.6 seconds (33 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-1 was measured with the high-density lipoprotein cholesterol measurement reagent was 3.0 mg / dL.

[0204] (c) Based on the absorbance measured 272.7 seconds (32 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-1 was measured with the high-density lipoprotein cholesterol measurement reagent was 2.7 mg / dL.

[0205] (d) Based on the absorbance measured 254.9 seconds (31 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-1 was measured with the high-density lipoprotein cholesterol measurement reagent was 2.3 mg / dL.

[0206] (2) In relation to the above, the following can be seen from [Table 1] above. (a) Based on the absorbance measured 237.0 seconds (30 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-1 was measured with the high-density lipoprotein cholesterol measurement reagent was 2.2 mg / dL.

[0207] (b) Based on the absorbance measured 219.2 seconds (29 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-1 was measured with the high-density lipoprotein cholesterol measurement reagent was 1.9 mg / dL.

[0208] (c) Based on the absorbance measured 201.4 seconds (28 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-1 was measured with the high-density lipoprotein cholesterol measurement reagent was 1.7 mg / dL.

[0209] (d) Based on the absorbance measured 183.5 seconds (27 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-1 was measured with the high-density lipoprotein cholesterol measurement reagent was 1.4 mg / dL.

[0210] (e) Based on the absorbance measured 166.6 seconds (26 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-1 was measured with the high-density lipoprotein cholesterol measurement reagent was 1.4 mg / dL.

[0211] (f) Based on the absorbance measured 150.7 seconds (25 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-1 was measured with the high-density lipoprotein cholesterol measurement reagent was 1.4 mg / dL.

[0212] (g) Based on the absorbance measured 132.9 seconds (24 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-1 was measured with the high-density lipoprotein cholesterol measurement reagent was 1.0 mg / dL.

[0213] (h) Based on the absorbance measured 115.0 seconds (23 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-1 was measured with the high-density lipoprotein cholesterol measurement reagent was 0.7 mg / dL.

[0214] (i) Based on the absorbance measured 97.2 seconds (22 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-1 was measured with the high-density lipoprotein cholesterol measurement reagent was 0.5 mg / dL.

[0215] (j) Based on the absorbance measured 79.4 seconds (21 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-1 was measured with the high-density lipoprotein cholesterol measurement reagent was 0.5 mg / dL.

[0216] (k) Based on the absorbance measured 61.5 seconds (20 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-1 was measured with the high-density lipoprotein cholesterol measurement reagent was 0.9 mg / dL.

[0217] (l) Based on the absorbance measured 43.7 seconds (19 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-1 was measured with the high-density lipoprotein cholesterol measurement reagent was 1.1 mg / dL.

[0218] (3) Summary (a) Based on (1) and (2) above, when using a cholesterol measurement reagent in high-density lipoprotein consisting of a first reagent and a second reagent, generating a dye from a chromogen with hydrogen peroxide produced by an oxidase, optically measuring the absorbance based on the dye, and calculating and measuring the cholesterol value in high-density lipoprotein in a sample based on this, it has been confirmed that by calculating and measuring the cholesterol value in high-density lipoprotein in a sample based on the absorbance measured within 250 seconds after dispensing the second reagent, inter-agent differences in the measurement values ​​of cholesterol in high-density lipoprotein in a sample can be suppressed, and the cholesterol value in high-density lipoprotein in a sample can be accurately measured.

[0219] (b) The effect of suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in the sample is particularly high when the measurement is made using the cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured at 237.0 seconds after dispensing the second reagent, and also particularly high when the measurement is made using the cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured at 219.2 seconds after dispensing the second reagent, and also particularly high when the measurement is made using the cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured at 201.4 seconds after dispensing the second reagent, and also particularly high when the measurement is made using the cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured at 183.5 seconds after dispensing the second reagent, and also particularly high when the measurement is made using the cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured at 166.6 seconds after dispensing the second reagent, and also particularly high when the measurement is made using the cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured at 150.7 seconds after dispensing the second reagent. The effectiveness was particularly high when measured with a cholesterol measurement reagent, and also particularly high when measured with a cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured 132.9 seconds after dispensing the second reagent, and also particularly high when measured with a cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured 115.0 seconds after dispensing the second reagent, and also particularly high when measured with a cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured 97.2 seconds after dispensing the second reagent, and also particularly high when measured with a cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured 79.4 seconds after dispensing the second reagent, and also particularly high when measured with a cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured 61.5 seconds after dispensing the second reagent, or particularly high when measured with a cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured 43.7 seconds after dispensing the second reagent.

[0220] [II] Regarding the above [Table 2] (1) From the above [Table 2], the following can be seen: (a) Based on the absorbance measured 308.4 seconds (34 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-2 was measured with the high-density lipoprotein cholesterol measurement reagent was 2.6 mg / dL.

[0221] (b) Based on the absorbance measured 290.6 seconds (33 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-2 was measured with the high-density lipoprotein cholesterol measurement reagent was 2.4 mg / dL.

[0222] (c) Based on the absorbance measured 272.7 seconds (32 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-2 was measured with the high-density lipoprotein cholesterol measurement reagent was 2.1 mg / dL.

[0223] (d) Based on the absorbance measured 254.9 seconds (31 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-2 was measured with the high-density lipoprotein cholesterol measurement reagent was 1.8 mg / dL.

[0224] (2) In relation to the above, the following can be seen from [Table 2] above. (a) Based on the absorbance measured 237.0 seconds (30 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-2 was measured with the high-density lipoprotein cholesterol measurement reagent was 1.6 mg / dL.

[0225] (b) Based on the absorbance measured 219.2 seconds (29 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-2 was measured with the high-density lipoprotein cholesterol measurement reagent was 1.1 mg / dL.

[0226] (c) Based on the absorbance measured 201.4 seconds (28 points) after dispensing the second reagent, the value of the "inter-reagent difference" of the measured cholesterol in high-density lipoprotein when human serum sample-2 was measured with the high-density lipoprotein cholesterol measurement reagent was 1.2 mg / dL.

[0227] (d) Based on the absorbance measured 183.5 seconds (27 points) after dispensing the second reagent, the value of the "inter-reagent difference" of the measured cholesterol in high-density lipoprotein when human serum sample-2 was measured with the high-density lipoprotein cholesterol measurement reagent was 1.0 mg / dL.

[0228] (e) Based on the absorbance measured 166.6 seconds (26 points) after dispensing the second reagent, the value of the "inter-reagent difference" of the measured cholesterol in high-density lipoprotein when human serum sample-2 was measured with the high-density lipoprotein cholesterol measurement reagent was 0.5 mg / dL.

[0229] (f) Based on the absorbance measured 150.7 seconds (25 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-2 was measured with the high-density lipoprotein cholesterol measurement reagent was 0.6 mg / dL.

[0230] (g) Based on the absorbance measured 132.9 seconds (24 points) after dispensing the second reagent, the "inter-reagent difference" value of the measured cholesterol in high-density lipoprotein when human serum sample-2 was measured with the high-density lipoprotein cholesterol measurement reagent was 0.4 mg / dL.

[0231] (h) Based on the absorbance measured at 115.0 seconds (23 points) after the dispensing of the second reagent, when human serum sample-2 was measured with the reagent for measuring cholesterol in high-density lipoprotein, the value of "inter-reagent difference of the measurement reagent" for the measured value of cholesterol in high-density lipoprotein was 0.2 mg / dL.

[0232] (i) Based on the absorbance measured at 97.2 seconds (22 points) after the dispensing of the second reagent, when human serum sample-2 was measured with the reagent for measuring cholesterol in high-density lipoprotein, the value of "inter-reagent difference of the measurement reagent" for the measured value of cholesterol in high-density lipoprotein was 0.3 mg / dL.

[0233] (j) Based on the absorbance measured at 79.4 seconds (21 points) after the dispensing of the second reagent, when human serum sample-2 was measured with the reagent for measuring cholesterol in high-density lipoprotein, the value of "inter-reagent difference of the measurement reagent" for the measured value of cholesterol in high-density lipoprotein was 0.3 mg / dL.

[0234] (k) Based on the absorbance measured at 61.5 seconds (20 points) after the dispensing of the second reagent, when human serum sample-2 was measured with the reagent for measuring cholesterol in high-density lipoprotein, the value of "inter-reagent difference of the measurement reagent" for the measured value of cholesterol in high-density lipoprotein was 0.4 mg / dL.

[0235] (l) Based on the absorbance measured at 43.7 seconds (19 points) after the dispensing of the second reagent, when human serum sample-2 was measured with the reagent for measuring cholesterol in high-density lipoprotein, the value of "inter-reagent difference of the measurement reagent" for the measured value of cholesterol in high-density lipoprotein was 1.2 mg / dL.

[0236] (3) Summary (a) It is also confirmed from the above (1) and (2) that, when using the reagent for measuring cholesterol in high-density lipoprotein consisting of the first reagent and the second reagent, generating a dye from a chromogen by hydrogen peroxide produced by an oxidase, optically measuring the absorbance based on the dye, and calculating and measuring the measured value of cholesterol in high-density lipoprotein in a sample based thereon, by calculating and measuring the measured value of cholesterol in high-density lipoprotein in the sample based on the absorbance measured within 250 seconds after dispensing the second reagent, the inter-reagent difference of the measurement reagent in the measured value of cholesterol in high-density lipoprotein in the sample can be suppressed, and the cholesterol level in high-density lipoprotein in the sample can be measured accurately.

[0237] (b) The effect of suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in the sample is particularly high when the measurement is made using the cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured at 237.0 seconds after dispensing the second reagent, and also particularly high when the measurement is made using the cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured at 219.2 seconds after dispensing the second reagent, and also particularly high when the measurement is made using the cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured at 201.4 seconds after dispensing the second reagent, and also particularly high when the measurement is made using the cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured at 183.5 seconds after dispensing the second reagent, and also particularly high when the measurement is made using the cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured at 166.6 seconds after dispensing the second reagent, and also particularly high when the measurement is made using the cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured at 150.7 seconds after dispensing the second reagent. The effectiveness was particularly high when measured with a cholesterol measurement reagent, and also particularly high when measured with a cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured 132.9 seconds after dispensing the second reagent, and also particularly high when measured with a cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured 115.0 seconds after dispensing the second reagent, and also particularly high when measured with a cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured 97.2 seconds after dispensing the second reagent, and also particularly high when measured with a cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured 79.4 seconds after dispensing the second reagent, and also particularly high when measured with a cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured 61.5 seconds after dispensing the second reagent, or particularly high when measured with a cholesterol measurement reagent in high-density lipoprotein in the sample based on the absorbance measured 43.7 seconds after dispensing the second reagent.

Claims

1. A method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample, characterized in that when using a cholesterol measurement reagent consisting of a first reagent and a second reagent, a dye is generated from a chromogen by hydrogen peroxide produced by an oxidase, the absorbance based on the dye is optically measured, and the cholesterol value in high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 250 seconds after dispensing the second reagent.

2. A method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to claim 1, characterized by calculating and measuring the cholesterol level in high-density lipoprotein in a sample based on the absorbance measured within 237.0 seconds after dispensing the second reagent.

3. A method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to claim 1, characterized in that the cholesterol level in high-density lipoprotein in a sample is calculated and measured based on the absorbance measured within 219.2 seconds after dispensing the second reagent.

4. A method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to claim 1, characterized by calculating and measuring the cholesterol level in high-density lipoprotein in a sample based on the absorbance measured within 201.4 seconds after dispensing the second reagent.

5. A method for suppressing inter-agent differences in the measurement of cholesterol in high-density lipoprotein in a sample according to claim 1, characterized in that the cholesterol level in high-density lipoprotein in the sample is calculated and measured based on the absorbance measured within 183.5 seconds after dispensing the second reagent.

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