Method of dissolving organic acids

Abstract

This invention provides a method for dissolving organic acids that are poorly soluble in water in a biological sample measurement reagent, and a biological sample measurement reagent utilizing this method. [Solution] After mixing and dissolving the organic acid and surfactant, the surfactant containing the dissolved organic acid is added to the biological sample measurement reagent. In a biological sample measurement reagent that includes this step, the precipitation of the organic acid can be suppressed.

Description

Technical Field

[0001] The present invention relates to a method for dissolving an organic acid poorly soluble in water in an aqueous solution. Further, the present invention relates to a method for preparing a biological sample measurement reagent characterized by containing an organic acid poorly soluble in water. Further, the present invention relates to a biological sample measurement reagent characterized by containing an organic acid poorly soluble in water. The present invention is particularly useful in the fields of chemistry, life science, analytical science, clinical examination, and the like.

Background Art

[0002] > Various lipids such as cholesterol are present in the living body, and each exhibits diverse functions. Therefore, measuring the lipid concentration in a biological sample and observing its fluctuations are essential for disease diagnosis, treatment, early detection, and prevention, and are widely carried out in hospitals, inspection facilities, and the like. Cholesterol and the like exist in the blood as lipoproteins. In order of their low specific gravity, they are called chylomicron, very low density lipoprotein (hereinafter sometimes abbreviated as VLDL), intermediate density lipoprotein (hereinafter sometimes abbreviated as IDL), low density lipoprotein (hereinafter sometimes abbreviated as LDL), and high density lipoprotein (hereinafter sometimes abbreviated as HDL). Cholesterol in LDL is sometimes called LDL-cholesterol (hereinafter sometimes abbreviated as LDL-C), and cholesterol in HDL is sometimes called HDL-cholesterol (hereinafter sometimes abbreviated as HDL-C). In a blood test, it is known that an increase in the LDL-C concentration is a risk factor for atherosclerotic diseases, and conversely, a decrease in the HDL-C concentration is a risk factor for atherosclerotic diseases. Thus, the significance of cholesterol measurement varies depending on the type of lipoprotein, and it is necessary to accurately separate each lipoprotein. [[ID=]14]

[0003] While methods using ultracentrifugation and electrophoresis are known for measuring LDL-C and HDL-C, these methods are cumbersome and time-consuming to measure many samples. Therefore, measurements using biological sample measurement reagents, which can be used with general-purpose automated analyzers, are widely used in hospital laboratories. There are various measurement principles for biological sample measurement reagents, but for example, by reacting lipoproteins contained in the sample with cholesterol esterase and cholesterol oxidase in the biological sample measurement reagent to generate hydrogen peroxide, and then reacting this hydrogen peroxide with the chromogen, coupler, and peroxidase in the biological sample measurement reagent to form a dye, the concentration of LDL-C and HDL-C can be measured by measuring the absorbance derived from this dye.

[0004] Regarding methods for measuring only cholesterol in specific lipoproteins, such as LDL-C and HDL-C, methods involving the addition of certain surfactants, reaction accelerators, etc., are known. For example, methods involving the addition of surfactants (see Patent Document 1) and methods involving the addition of reaction accelerators (see Patent Document 2) are known. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 09-000299 [Patent Document 2] Japanese Patent Publication No. 2012-191953 [Overview of the project] [Problems that the invention aims to solve]

[0006] While conducting research on biological sample measurement reagents for measuring HDL-C, the inventors noticed that reaction accelerators, which are organic acids, sometimes do not dissolve when added to the biological sample measurement reagents and instead precipitate. In such biological sample measurement reagents, the reaction accelerator does not function properly, making it impossible to measure only HDL-C, which presents problems such as the inability to obtain accurate HDL-C measurements or the possibility that measurement may not be possible at all.

[0007] The inability to obtain accurate measurements, or even the inability to perform measurements at all, is a serious problem that can lead to misdiagnosis or delays in disease diagnosis. In the field of biological sample measurement reagents, there is a need for reagents that can provide accurate measurements. Therefore, there is a demand for biological sample measurement reagents that do not cause organic acid precipitation or any other phenomenon that prevents such precipitation. [Means for solving the problem]

[0008] As a result of diligent research into the above-mentioned problems and their solutions, the inventors of the present invention discovered that the precipitation of organic acids can be suppressed by mixing and dissolving a certain type of surfactant with an organic acid and then adding it to a biological sample measurement reagent, thus completing the present invention.

[0009] In other words, the present invention is as follows: (1) A biological sample measurement reagent characterized by comprising the step of mixing an organic acid and a surfactant and dissolving them, and then adding the surfactant containing the dissolved organic acid to a biological sample measurement reagent in the process of preparation and dissolving it. (2) A biological sample measurement reagent as described in (1) above, wherein the surfactant is at least one of the following: a silicone-based surfactant or a nonionic surfactant. (3) A biological sample measurement reagent as described in (1) above, wherein the surfactant is at least one of the following: side-chain type / polyether-modified modified silicone oil, polyoxyethylene oleyl ether, polyoxyethylene octylphenyl ether, or polyoxyethylene alkyl (12-14) ether. (4) A biological sample measurement reagent according to any one of items (1) to (3) above, wherein the organic acid is at least one of flufenamic acid, fusidic acid, piroxicam, or salts thereof. (5) A biological sample measurement reagent as described in any one of items (1) to (3) above, wherein the biological sample measurement reagent is a biochemical test reagent. (6) A biological sample measurement reagent as described in any one of items (1) to (3) above, wherein the biological sample measurement reagent is an HDL-C measurement reagent. (7) The biological sample measurement reagent described in (4) above, wherein the biological sample measurement reagent is an HDL-C measurement reagent. (8) A method for dissolving an organic acid, characterized in that an organic acid and a surfactant are mixed and dissolved, and then the surfactant containing the dissolved organic acid is added to a biological sample measurement reagent in the process of preparation and dissolved to prepare the biological sample measurement reagent. (9) A method for dissolving an organic acid as described in (8) above, wherein the surfactant is at least one of the following: a silicone-based surfactant or a nonionic surfactant. (10) A method for dissolving an organic acid as described in (8) above, wherein the surfactant is at least one of the following: side-chain type / polyether-modified modified silicone oil, polyoxyethylene oleyl ether, polyoxyethylene octylphenyl ether, and polyoxyethylene alkyl (12-14) ether. (11) A method for dissolving an organic acid according to any one of items (8) to (10) above, wherein the organic acid is at least one of flufenamic acid, fusidic acid, piroxicam, or salts thereof. (12) A method for dissolving an organic acid as described in any one of items (8) to (10) above, wherein the biological sample measurement reagent is a biochemical test reagent. (13) A method for dissolving an organic acid as described in any one of items (8) to (10) above, wherein the biological sample measurement reagent is an HDL-C measurement reagent. (14) A method for dissolving an organic acid as described in (11) above, wherein the biological sample measurement reagent is an HDL-C measurement reagent. [Effects of the Invention]

[0010] This invention allows organic acids to be dissolved in biological sample measurement reagents. Therefore, accurate measurements of HDL-C can be obtained. [Modes for carrying out the invention]

[0011] The present invention will now be described in detail. The following embodiments are illustrative for illustrating the present invention and are not intended to limit the present invention to these embodiments only. The present invention can be implemented in various forms without departing from its spirit.

[0012] 1. Overview For methods to measure only cholesterol in specific lipoproteins, such as LDL-C and HDL-C, methods involving the addition of certain surfactants or reaction accelerators are known. While conducting research on biological sample measurement reagents for measuring HDL-C, the inventors noticed that reaction accelerators, which are organic acids, sometimes do not dissolve when added to the biological sample measurement reagents and instead precipitate. In such biological sample measurement reagents, the reaction accelerator does not function properly, making it impossible to measure only HDL-C, which presents problems such as the inability to obtain accurate HDL-C measurements or the possibility that measurement may not be possible at all. The inability to obtain accurate measurements, or even the inability to perform measurements at all, is a serious problem that can lead to misdiagnosis or delays in disease diagnosis. In the field of biological sample measurement reagents, there is a need for reagents that can provide accurate measurements. Therefore, there is a demand for biological sample measurement reagents that do not cause organic acid precipitation or any other phenomenon that prevents such precipitation. As a result of diligent research into the above-mentioned problems and their solutions, the inventors have discovered that by mixing and dissolving a certain type of surfactant with an organic acid and then adding it to a biological sample measurement reagent, the precipitation of the organic acid can be suppressed. This invention was made based on these findings.

[0013] 2.Dissolution Dissolution in the present invention means that a substance dissolves in a solvent. When a substance is completely dissolved, precipitates, suspended substances, deposited substances, etc. cannot be visually confirmed. That is, when precipitates, suspended substances, deposited substances, etc. can be visually confirmed in the solvent, it can be said that the substance is not completely dissolved. When water, a polar molecule, is the solvent, substances that are also polar molecules are likely to dissolve, but substances that are non-polar molecules are known to be difficult to dissolve. Non-polar molecules include organic compounds and the like. In a biological sample measurement reagent where water is the solvent, organic compounds may be difficult to dissolve, and organic acids, which are a type of organic compound, may also be similarly difficult to dissolve.

[0014] An example of a method for dissolving an organic acid in the present invention is as follows. (1) Add an organic acid to a surfactant and dissolve it. (2) Add (1) to a biological sample measurement reagent and dissolve it. In this method, temperature changes such as heating or cooling may be made. Also, a biological sample measurement reagent may contain raw materials such as enzymes and buffers in addition to the organic acid. These raw materials may be added before the operation of (2), or may be added after the operation of (2). Only some of the raw materials may be added before the operation of (2). For dissolution, any stirring device such as a vibration stirring device, a shaker, or a magnetic stirrer can be used. When it is desired to dissolve two or more types of organic acids, the above operations may be carried out for each type of organic acid, or two or more types of organic acids may be simultaneously added to the surfactant and dissolved. Also, instead of dissolving the organic acid at once, it may be divided into several portions and the above operations may be carried out multiple times for dissolution. Although an example of a method of adding an organic acid to a surfactant has been shown, a method of adding a surfactant to an organic acid can also be used.

[0015] 3. Organic Acid The organic acid in the present invention refers to an organic compound exhibiting acidity, and those having a hydroxy group, carboxy group, sulfo group, thiol group, enol, etc. are applicable. Since organic acids may be used as reaction accelerators for promoting chemical reactions, one or more organic acids may be contained in a biological sample measurement reagent. In addition, organic acids may be contained for purposes other than the above.

[0016] Regarding the organic acid in the present invention, its type is not particularly limited. For example, examples include flufenamic acid, fusidic acid, piroxicam, mefenamic acid, tiglic acid, betamethasone acetate, monensin, mevinolin, or salts thereof. Note that the present invention can be used not only for dissolving organic acids but also for dissolving other organic compounds. For example, it may be used for dissolving terpyridine.

[0017] 4. Surfactant Regarding the surfactant for dissolving the organic acid in the present invention, it is not particularly limited as long as it has the action of dissolving the organic acid. For example, silicone-based surfactants, nonionic surfactants, etc. can be mentioned. Regarding nonionic surfactants, ether-type nonionic surfactants are preferable. Among them, in particular, side-chain type / polyether-modified modified silicone oil, polyoxyethylene oleyl ether, polyoxyethylene octylphenyl ether, polyoxyethylene alkyl (12 - 14) ether are preferable. Note that surfactants may be contained for purposes other than obtaining the effects of the above actions. [[ID=II]]

[0018] At least one type of surfactant for dissolving the organic acid may be prepared and used, or two or more types may be prepared and mixed for use.

[0019] The concentration of the surfactant used to dissolve organic acids is not particularly limited, as long as it is a concentration that effectively dissolves the organic acids. Undiluted surfactants, such as those with water, or surfactants diluted with water, may be used. When expressing the concentration of the surfactant used to dissolve organic acids in weight percentage, examples include concentrations within the following ranges: 1-80%, 5-80%, 10-80%, 20-80%, 40-80%, 60-80%, 1-90%, 5-90%, 10-90%, 20-90%, 40-90%, 60-90%, 80-90%, 1-100%, 5-100%, 10-100%, 20-100%, 40-100%, 60-100%, 80-100%, or 90-100%.

[0020] 5. Reagents for measuring biological samples In the present invention, a biological sample measurement reagent is a reagent capable of measuring enzyme activity, substance concentration, etc., in a biological sample, and is not particularly limited as long as it is such a reagent. Examples of biological sample measurement reagents include biochemical biological sample measurement reagents that utilize biochemical reactions, immunological biological sample measurement reagents that utilize antigen-antibody reactions, and genetic biological sample measurement reagents that utilize gene analysis technology, but in the present invention, a biochemical biological sample measurement reagent is preferred. Examples of biochemical biological measurement reagents include HDL-C measurement reagents.

[0021] The biological sample measurement reagent of the present invention may be used for measurement by the endpoint method or by the rate method, and the method may be selected as appropriate.

[0022] The biological sample measurement reagent of the present invention may be a one-reagent method in which the biological sample is mixed with one reagent for measurement, a two-reagent method in which the biological sample is mixed with two reagents simultaneously or in an appropriate order for measurement, or a multi-reagent method in which the biological sample is mixed with three or more reagents simultaneously or in an appropriate order for measurement, and can be selected as appropriate.

[0023] In the biological measurement reagent of the present invention, the measurement may be performed by a manual method, or it may be performed using a device such as a general-purpose automated analyzer.

[0024] The biological sample measurement reagent of the present invention may contain all or part of its constituent reagents as liquid reagents.

[0025] The biological sample measurement reagent of the present invention can be sold on its own or used for measuring target substances in biological samples.

[0026] The biological sample measurement reagent of the present invention can also be sold or used for measuring target substances in biological samples in combination with other reagents other than the biological sample measurement reagent described above. Examples of other reagents other than the biological sample measurement reagent described above include buffer solutions, sample diluents, reagent diluents, reagents containing substances for calibration, or reagents containing substances for quality control.

[0027] The biological sample measurement reagent of the present invention may be a biological sample measurement reagent kit consisting of a first reagent, a second reagent, or a plurality of other reagents.

[0028] The pH of the biological sample measurement reagent of the present invention can be appropriately selected considering the stability of the contained components during storage, the reaction rate during biological sample measurement, etc. Furthermore, if the biological sample measurement reagent is composed of multiple reagents, the pH of each reagent constituting the biological sample measurement reagent can be appropriately selected considering the stability of the contained components during storage, the reaction rate during sample measurement, etc.

[0029] For example, in the case of HDL-C measuring reagents, the lower limit of the pH when measuring the HDL-C concentration in the sample is preferably 4.0, and particularly preferably 5.0. Furthermore, the upper limit is preferably 8.0, and particularly preferably 7.0. Regarding the pH of this HDL-C measuring reagent, for example, if the lower limit is 4.0, then a range of 4.0 to 7.0 or 4.0 to 8.0 can be given, and if the lower limit is 5.0, then a range of 5.0 to 7.0 or 5.0 to 8.0 can be given.

[0030] The biological sample measurement reagent of the present invention may contain, as necessary, known surfactants, buffers, enzymes, coenzymes, reaction substrates, pH adjusters, preservatives, etc., in addition to organic acids and surfactants for dissolving organic acids. The concentration of each of these components can be appropriately selected considering the stability of the contained components during storage and the reaction rate during biological sample measurement. Furthermore, when the biological sample measurement reagent is composed of multiple reagents, the concentration of each of these components in each reagent constituting the biological sample measurement reagent can be appropriately selected considering the stability of the contained components during storage and the reaction rate during biological sample measurement.The buffering agent is not particularly limited, but examples include acetic acid, citric acid, tartaric acid, carbonic acid, boric acid, phosphoric acid, trishydroxymethylaminomethane, imidazole, glycylglycine, bistris, tricine, bicine, bistrispropane (hereinafter sometimes abbreviated as BTP), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (hereinafter sometimes abbreviated as TES), 2-morpholinoethanesulfonic acid (hereinafter sometimes abbreviated as MES), N-(2-acetamide)iminodiacetic acid (hereinafter sometimes abbreviated as ADA), N-(2-acetamide)-2- Aminoethanesulfonic acid (hereinafter sometimes abbreviated as ACES), piperazine-1,4-bis(2-ethanesulfonic acid) (hereinafter sometimes abbreviated as PIPES), 2-hydroxy-3-morpholinopropanesulfonic acid (hereinafter sometimes abbreviated as MOPSO), 3-morpholinopropane-1-sulfonic acid (hereinafter sometimes abbreviated as MOPS), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (hereinafter sometimes abbreviated as BES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (hereinafter sometimes abbreviated as HEPES) (Also available), 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (hereinafter sometimes abbreviated as DIPSO), 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (hereinafter sometimes abbreviated as TAPSO), piperazine-1,4-bis(2-hydroxypropanesulfonic acid) (hereinafter sometimes abbreviated as POPSO), N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid) (hereinafter sometimes abbreviated as HEPPS), 4-(2-hydroxyethyl)piperazine Examples include -1-(2-hydroxypropane-3-sulfonic acid) (sometimes abbreviated as HEPPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (sometimes abbreviated as TAPS), 2-cyclohexylaminoethanesulfonic acid (sometimes abbreviated as CHES), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (sometimes abbreviated as CAPSO), or N-cyclohexyl-3-aminopropanesulfonic acid (sometimes abbreviated as CAPS), or salts thereof.

[0031] 6. Biological samples In the present invention, a sample refers to an object that is the subject of measurement, such as enzyme activity or substance concentration, and a biological sample refers to an object contained in a living body that is the subject of measurement, such as enzyme activity or substance concentration. The biological sample is not particularly limited as long as it is one of these types of objects. Examples of such biological samples include human or animal blood, serum, plasma, urine, breast milk, feces, semen, cerebrospinal fluid, saliva, sweat, tears, ascites, amniotic fluid, organs such as the brain, and tissues and cells such as hair, skin, nails, muscles, or nerves.

[0032] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0033] [Example 1] (Confirmation of dissolution of organic acids by surfactants) In the preparation of the HDL-C measurement reagent, the dissolution of organic acids by surfactants was confirmed.

[0034] 1. Preparation of reagents

[0035] (1) Preparation of HDL-C measurement reagent, first reagent, control X-1 The following components were added to pure water in the order listed, from top to bottom, to achieve the concentrations indicated, while adjusting the pH to 6.0 (at 20°C) to prepare control X-1, the first reagent of the HDL-C measurement reagent. N-(2-carboxyethyl)-N-ethyl-m-toluidine hydrochloride (hereinafter sometimes abbreviated as CEMB) 10mM (Dojin Chemical Co., Ltd.) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec) KF-354L 1g / L (Shin-Etsu Chemical Co., Ltd.) Flufenamic acid 0.1 mM (LKT Labs) (2) Preparation of HDL-C measurement reagent, first reagent, control X-2 The following components were added to pure water in the order listed, from top to bottom, to achieve the concentrations indicated, while adjusting the pH to 6.0 (20°C) to prepare the control X-2 of the first reagent for HDL-C measurement. CEMB 10mM (Dojindo Chemical) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec) KF-354L 3g / L (Shin-Etsu Chemical Co., Ltd.) Flufenamic acid 0.1 mM (LKT Labs)

[0036] (3) Preparation of HDL-C measurement reagent, Reagent A-1 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the total amount of flufenamic acid was 280 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was then dissolved in pure water to a concentration of 1 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent A-1, the first reagent for HDL-C measurement. In this reagent, the concentration is 1 g / L of KF-354L and 0.1 mM of flufenamic acid. CEMB 10mM (Dojindo Chemical) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec) (4) HDL-C measurement reagent First reagent Preparation of reagent A-2 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the amount of flufenamic acid was 93.3 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent A-2, the first reagent of the HDL-C measurement reagent. In this reagent, the KF-354L concentration is 3 g / L and the flufenamic acid concentration is 0.1 mM. CEMB 10mM (Dojindo Chemical) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec) (5) HDL-C measurement reagent First reagent Preparation of reagent A-3 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the concentration was 28 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was dissolved in pure water to a concentration of 10 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent A-3, the first reagent of the HDL-C measurement reagent. In this reagent, the concentration is 10 g / L of KF-354L and 0.1 mM of flufenamic acid. CEMB 10mM (Dojindo Chemical) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec) (6) HDL-C measurement reagent First reagent Preparation of reagent A4 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the amount of flufenamic acid was 5.6 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was dissolved in pure water to a concentration of 50 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent A-4, the first reagent of the HDL-C measurement reagent. In this reagent, the concentration is 50 g / L of KF-354L and 0.1 mM of flufenamic acid. CEMB 10mM (Dojindo Chemical) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec) (7) HDL-C measurement reagent First reagent Preparation of reagent A-5 Flufenamic acid (LKT Labs) was added to KF-355A (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the amount of flufenamic acid was 93.3 mg per 10 g of KF-355A. This solution of KF-355A with dissolved flufenamic acid was dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent A-5, the first reagent of the HDL-C measurement reagent. In this reagent, the concentration is 3 g / L of KF-355A and 0.1 mM of flufenamic acid. CEMB 10mM (Dojindo Chemical) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec) (8) HDL-C measurement reagent First reagent Preparation of reagent A-6 Flufenamic acid (LKT Labs) was added to BO-10V (Nikko Chemicals) and dissolved so that the amount of flufenamic acid was 93.3 mg per 10 g of BO-10V. This BO-10V with dissolved flufenamic acid was dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent A-6, the first reagent of the HDL-C measurement reagent. In this reagent, the BO-10V concentration is 3 g / L and the flufenamic acid concentration is 0.1 mM. CEMB 10mM (Dojindo Chemical) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec) (9) HDL-C measurement reagent First reagent Preparation of reagent A-7 Flufenamic acid (LKT Labs) was added to Triton X-100 (Nacalai Tesque) and dissolved so that the amount of flufenamic acid was 93.3 mg per 10 g of Triton X-100. This Triton X-100 with dissolved flufenamic acid was dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent A-7, the first reagent of the HDL-C measurement reagent. In this reagent, the concentration is 3 g / L of Triton X-100 and 0.1 mM of flufenamic acid. CEMB 10mM (Dojindo Chemical) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec) (10) Preparation of Reagent A-8, the first reagent for HDL-C measurement. Flufenamic acid (LKT Labs) was added to BT-9 (Nikko Chemicals) and dissolved so that the total amount of flufenamic acid was 93.3 mg per 10 g of BT-9. This BT-9 with dissolved flufenamic acid was then dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent A-8, the first reagent of the HDL-C measurement reagent. In this reagent, the BT-9 concentration is 3 g / L and the flufenamic acid concentration is 0.1 mM. CEMB 10mM (Dojindo Chemical) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec) (11) Preparation of Reagent A-9, the first reagent for HDL-C measurement. Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the amount of flufenamic acid was 466.5 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was then dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent A-9, the first reagent of the HDL-C measurement reagent. In this reagent, the KF-354L concentration is 3 g / L and the flufenamic acid concentration is 0.5 mM. CEMB 10mM (Dojindo Chemical) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec) (12) HDL-C measurement reagent, first reagent, preparation of reagent A-10 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the concentration was 28 mg per 10 g of KF-354L. This KF-354L with dissolved flufenamic acid was dissolved in pure water to a concentration of 50 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent A-10, the first reagent of the HDL-C measurement reagent. In this reagent, the concentration is 50 g / L of KF-354L and 0.5 mM of flufenamic acid. CEMB 10mM (Dojindo Chemical) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec) (13) Preparation of HDL-C measurement reagent, first reagent, reagent A-11 Flufenamic acid (LKT Labs) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the total amount of flufenamic acid per 10g of KF-354L was 4665mg. This KF-354L with dissolved flufenamic acid was then dissolved in pure water to a concentration of 50g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20℃) to prepare reagent A-11, the first reagent of the HDL-C measurement reagent. In this reagent, the concentrations are 50g / L of KF-354L and 5mM of flufenamic acid. CEMB 10mM (Dojindo Chemical) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec)

[0037] (14) Preparation of HDL-C measurement reagent, first reagent, control Y The following components were added to pure water in the order listed, from top to bottom, to achieve the concentrations indicated for each component. The pH was adjusted to 6.0 (at 20°C) to prepare control Y, the first reagent of the HDL-C measurement reagent. CEMB 10mM (Dojindo Chemical) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec) KF-354L 3g / L (Shin-Etsu Chemical Co., Ltd.) Fusidic acid 0.1 mM (Tokyo Chemical Industries)

[0038] (15) HDL-C measurement reagent, first reagent, preparation of reagent B Fusidic acid (Tokyo Chemical Industry Co., Ltd.) was added to KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the amount of fusidic acid was 172.2 mg per 10 g of KF-354L. This KF-354L with dissolved fusidic acid was dissolved in pure water to a concentration of 3 g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20°C) to prepare reagent B, the first reagent of the HDL-C measurement reagent. In this reagent, the KF-354L concentration is 3 g / L and the fusidic acid concentration is 0.1 mM. CEMB 10mM (Dojindo Chemical) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec)

[0039] (16) Preparation of HDL-C measurement reagent, first reagent, control Z The following components were added to pure water in the order listed, from top to bottom, to achieve the concentrations indicated, while adjusting the pH to 6.0 (at 20°C) to prepare control Z for the first reagent of the HDL-C measurement reagent. CEMB 10mM (Dojindo Chemical) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec) KF-354L 3g / L (Shin-Etsu Chemical Co., Ltd.) Piroxicam 0.1 mM (Fujifilm Wako Pure Chemical Industries)

[0040] (17) HDL-C measurement reagents: First reagent, preparation of reagent C Piroxicam (Fujifilm Wako Pure Chemical Industries) was added to 10g of KF-354L (Shin-Etsu Chemical Co., Ltd.) and dissolved so that the total amount of piroxicam was 110.5mg per 10g of KF-354L. This KF-354L with dissolved piroxicam was then dissolved in pure water to a concentration of 3g / L, and each of the components listed below was dissolved to the concentrations indicated, adjusting the pH to 6.0 (20℃) to prepare reagent C, the first reagent of the HDL-C measurement reagent. In this reagent, the concentrations are 3g / L of KF-354L and 0.1mM of piroxicam. CEMB 10mM (Dojindo Chemical) Peroxidase 1 kU / L (Toyobo) Cholesterol oxidase 1 kU / L (Toyobo) MES monohydrate 50mM (Actec)

[0041] 2. Observation of the reagents

[0042] The reagents (1) to (17) prepared in step 1 were observed and visually checked for the presence of precipitates. The results are shown in Table 1.

[0043] [Table 1]

[0044] 3. Summary

[0045] Precipitates were observed in controls X-1~2, Y, and Z, but not in reagents A-1~11, B, and C.

[0046] Regarding precipitates in the HDL-C measurement reagents, precipitates were observed in controls X-1~2, Y, and Z, which were prepared without dissolving organic acids in a surfactant. However, no precipitates were observed in reagents A-1~11, B, and C, which were prepared after dissolving organic acids in a surfactant. This indicates that dissolving organic acids in a surfactant suppresses the precipitation of organic acids.

[0047] This confirms that the process of dissolving organic acids in surfactants has the effect of suppressing the precipitation of organic acids.

[0048] [Example 2] (Measurement of sample using HDL-C measuring reagent) The HDL-C measurement reagent using the present invention was used to measure a sample.

[0049] 1. Preparation of reagents

[0050] (18) Preparation of the first reagent for HDL-C measurement Similar to [Example 1], (2) control X-2 of the first reagent of the HDL-C measurement reagent, (3) reagent A-1 of the first reagent of the HDL-C measurement reagent, (4) reagent A-2 of the first reagent of the HDL-C measurement reagent, and (11) reagent A-9 of the first reagent of the HDL-C measurement reagent were prepared. (19) Preparation of the second reagent for HDL-C measurement The following components were added to pure water to the concentrations indicated, and the pH was adjusted to 6.0 (at 20°C) to prepare the second reagent for HDL-C measurement. MES monohydrate 50mM (Actec) 4-aminoantipyrine 30mM (Actec) Emulgen (registered trademark) B-66 10g / L (Kao Corporation) Cholesterol esterase 1 kU / L (Asahi Kasei Pharma) (20) Preparation of HDL-C measurement reagents I prepared the first and second reagents for Quick Auto Neo HDL-C (Sinotest).

[0051] 2. Sample preparation

[0052] HDL Concentrate Lyophilized (TRINA BIOREACTIVES) was dissolved in 3 mL of pure water, and then diluted with physiological saline to prepare an HDL-C standard solution with an HDL-C concentration of 42 mg / dL. Additionally, LDL Concentrate Lyophilized (TRINA BIOREACTIVES) was dissolved in 3 mL of pure water, and then diluted with physiological saline to prepare an LDL-C sample with an LDL-C concentration of 100 mg / dL.

[0053] 3. Measurement of the sample

[0054] Using reagent 1, the HDL-C concentration was measured using LDL-C sample 2 as the measurement sample. The values ​​are shown in Table 2. The HDL-C concentration was measured as follows. a. Measurement method for HDL-C A Hitachi High-Tech 7180 automated analyzer was used for the measurements. 2.0 μL of the sample was mixed with 200 μL of the first reagent of (2) HDL-C measurement reagent (control X-2), and reacted at 37°C for 5 minutes. Then, 67 μL of the second reagent of (19) HDL-C measurement reagent was added and reacted at 37°C for another 5 minutes. The difference in absorbance at the dominant wavelength of 600 nm and the secondary wavelength of 700 nm at 4 minutes and 30 seconds after the addition of the first reagent (point 16) and 3 minutes and 20 seconds after the addition of the second reagent (point 28) was calculated proportionally to the difference in absorbance measured when HDL-C standard solution 2 was used instead of the sample, thereby determining the HDL-C concentration (mg / dL) of the sample. Similar measurements were also performed using (3) reagent A-1, (4) reagent A-2, and (11) reagent A-9 instead of (2) control X-2. Furthermore, similar measurements were performed using (20) Quick Auto Neo HDL-C instead of the combination of (2) control X-2 and (19), and using lipid standard serum (Sinotest) with an HDL-C concentration of 55.2 mg / dL instead of the HDL-C standard solution of (2).

[0055] [Table 2]

[0056] 4. Summary

[0057] When LDL-C samples were measured using the HDL-C measurement reagent with control X-2, the measured HDL-C concentration was 11 mg / dL, compared to 7 mg / dL with the HDL-C measurement reagent using reagent A-1, 9 mg / dL with the HDL-C measurement reagent using reagent A-2, 6 mg / dL with the HDL-C measurement reagent using reagent A-9, and 7 mg / dL with Quick Auto Neo HDL-C.

[0058] The HDL-C measurement reagents using reagents A-1, A-2, and A-9, which utilize the present invention, yielded lower HDL-C concentrations when measuring LDL-C samples compared to the HDL-C measurement reagent using control X-2, which does not utilize the present invention. Furthermore, the measurement values ​​using reagents A-1, A-2, and A-9 were almost the same as those using Quick Auto Neo HDL-C. This indicates that in reagents A-1, A-2, and A-9, which utilize the present invention, the organic acids dissolve without precipitation, thus acting as reaction accelerators and suppressing the measurement of LDL-C.

[0059] This indicates that dissolving organic acids in surfactants has the effect of suppressing the precipitation of organic acids, and that in reagents containing dissolved organic acids, the organic acids act as reaction accelerators, allowing for accurate measurements.

[0060] [Example 3] (Measurement of sample using HDL-C measuring reagent) The HDL-C measurement reagent using the present invention was used to measure a sample.

[0061] 1. Preparation of reagents

[0062] (21) Preparation of the first reagent for HDL-C measurement Reagent A-9, the first reagent of the HDL-C measurement reagent, was prepared in the same manner as in [Example 1]. (22) Preparation of the second reagent for HDL-C measurement A second reagent for (19) HDL-C measurement was prepared in the same manner as in [Example 2]. (23) Preparation of HDL-C measurement reagents I prepared the first and second reagents for Quick Auto Neo HDL-C (Sinotest).

[0063] 2. Measurement of the sample

[0064] The HDL-C concentration was measured using human pooled serum as the sample with reagent 1. The values ​​are shown in Table 3. The HDL-C concentration was measured as follows. a. Measurement method for HDL-C A Hitachi High-Tech 7180 automated analyzer was used for the measurements. 2.0 μL of the sample was mixed with 200 μL of control X-2 of the first reagent of (11) HDL-C measurement reagent, and reacted at 37°C for 5 minutes. Then, 67 μL of the second reagent of (19) HDL-C measurement reagent was added and reacted at 37°C for another 5 minutes. The difference in absorbance at the dominant wavelength of 600 nm and the secondary wavelength of 700 nm at 4 minutes and 30 seconds after the addition of the first reagent (point 16) and 3 minutes and 20 seconds after the addition of the second reagent (point 28) was calculated proportionally to the difference in absorbance measured when using lipid standard serum (Sinotest) with an HDL-C concentration of 55.2 mg / dL instead of the sample, thereby determining the HDL-C concentration (mg / dL) of the sample. In addition, a similar measurement was performed using (23) Quick Auto Neo HDL-C instead of the combination of (11) reagent A-9 and (19).

[0065] [Table 3]

[0066] 3. Summary

[0067] When human pooled serum was measured using the HDL-C measurement reagent A-9, the measured HDL-C concentration was 27 mg / dL, while with QuickAutoNeo HDL-C it was 26 mg / dL.

[0068] Since the HDL-C concentration measured using reagent A-9, which utilizes the present invention, is almost the same as the HDL-C concentration measured using Quick Auto Neo HDL-C, it can be seen that the HDL-C measurement reagent utilizing the present invention accurately measures the HDL-C concentration.

[0069] This indicates that dissolving organic acids in a surfactant has the effect of suppressing the precipitation of organic acids, and in reagents where organic acids are dissolved, the organic acids act as reaction accelerators, allowing for accurate measurement. In the preparation of HDL-C measurement reagents, dissolving organic acids in a surfactant suppresses the precipitation of organic acids and allows them to act as reaction accelerators, thereby enabling accurate measurement of HDL-C concentration.

Claims

1. A biological sample measurement reagent characterized by including a step of mixing and dissolving an organic acid and a surfactant, and then adding the surfactant containing the dissolved organic acid to a biological sample measurement reagent in the process of preparation to dissolve it.

2. The biological sample measurement reagent according to claim 1, wherein the surfactant is at least one of silicone-based surfactants and nonionic surfactants.

3. The biological sample measurement reagent according to claim 1, wherein the surfactant is at least one of the following: side-chain type / polyether-modified modified silicone oil, polyoxyethylene oleyl ether, polyoxyethylene octylphenyl ether, and polyoxyethylene alkyl (12-14) ether.

4. A biological sample measurement reagent according to any one of claims 1 to 3, wherein the organic acid is at least one of flufenamic acid, fusidic acid, piroxicam, or salts thereof.

5. A biological sample measurement reagent according to any one of claims 1 to 3, wherein the biological sample measurement reagent is a biochemical test reagent.

6. A biological sample measurement reagent according to any one of claims 1 to 3, wherein the biological sample measurement reagent is an HDL-C measurement reagent.

7. The biological sample measurement reagent according to claim 4, wherein the biological sample measurement reagent is an HDL-C measurement reagent.

8. A method for dissolving organic acids, characterized by mixing and dissolving the organic acid with a surfactant, and then adding the surfactant containing the dissolved organic acid to a biological sample measurement reagent in the process of preparation to dissolve it further, thereby preparing a biological sample measurement reagent.

9. The method for dissolving an organic acid according to claim 8, wherein the surfactant is at least one of a silicone-based surfactant and a nonionic surfactant.

10. The method for dissolving an organic acid according to claim 8, wherein the surfactant is at least one of the following: side-chain type / polyether-modified modified silicone oil, polyoxyethylene oleyl ether, polyoxyethylene octylphenyl ether, and polyoxyethylene alkyl (12-14) ether.

11. A method for dissolving an organic acid according to any one of claims 8 to 10, wherein the organic acid is at least one of flufenamic acid, fusidic acid, piroxicam, or salts thereof.

12. A method for dissolving an organic acid according to any one of claims 8 to 10, wherein the biological sample measurement reagent is a biochemical test reagent.

13. A method for dissolving an organic acid according to any one of claims 8 to 10, wherein the biological sample measurement reagent is an HDL-C measurement reagent.

14. The method for dissolving an organic acid according to claim 11, wherein the biological sample measurement reagent is an HDL-C measurement reagent.

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