Biochemical testing reagents, methods for producing the same, and biochemical testing methods

Abstract

The present invention aims to provide biochemical testing reagents and biochemical testing methods that suppress turbidity, which is a problem when measuring a target substance in a sample using various measurement methods such as oxidation-reduction reactions, enzyme reactions, and antigen-antibody reactions, and that can measure the target substance without affecting the reaction system and / or with a high reaction rate. [Solution] A mixture is obtained by adding an alkyl ether type nonionic surfactant with a cloud point of 33-40°C to the sample, and the target substance contained in the mixture is measured by a biochemical method.

Description

Technical Field

[0001] The present invention relates to a biochemical test reagent and a biochemical test method capable of suppressing turbidity derived from a biological sample and measuring a measurement target substance in a biochemical test for measuring a measurement target substance contained in a sample derived from a living body without affecting the main reaction for measuring the measurement target substance.

Background Art

[0002] Biochemical tests are tests for measuring a measurement target substance contained in a specimen such as whole blood, serum, plasma, body fluid, or urine derived from a patient by using various biochemical reactions such as enzyme reactions and antigen-antibody reactions. However, due to turbidity derived from the specimen, the influence of various reaction interfering substances and non-specific reaction causative substances, it may not be possible to accurately measure the measurement target substance. In order to solve such problems, various studies have been made. Most simply, methods such as measuring a blank value, measurement by diluting the specimen, physical separation or decomposition of causative substances, and addition of additives have been widely studied.

[0003] Particularly, as additives, various additives such as ionic substances and surfactants that change the pH and polarity of the reagent are used. Surfactants are given various functions by varying their molecular structure, and many surfactants are used in this technical field. For example, it is known that by adding a surfactant having a high turbidity-reducing effect, a low cloud point, and high lipophilicity, and a substance that raises the cloud point, the turbidity of the highly lipophilic surfactant itself is eliminated, and while maintaining the original turbidity-removing effect of the surfactant, sample measurement can be performed without affecting the binding reaction between CRP and anti-CRP antibody (Patent Document 1).

[0004] On the other hand, it is known that by using a combination of two types of polyoxyethylene (hereinafter referred to as POE) - polyoxypropylene (hereinafter referred to as POP) alkyl ethers having different properties, turbidity caused by lipids in a specimen can be suppressed (Patent Document 2).

Prior Art Documents

Patent Documents

[0005] [Patent Document 1] Japanese Patent Publication No. 2001-188065 [Patent Document 2] Japanese Patent Publication No. 2017-198669 [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention provides a biochemical testing reagent, a method for producing the same, and a biochemical testing method using the reagent, which suppress turbidity, a problematic issue when measuring a target substance in a sample using a measurement method that utilizes various reaction principles such as oxidation-reduction reactions, enzyme reactions, and antigen-antibody reactions, and which can measure the target substance without affecting the reaction system and / or increasing the reaction rate of the main reaction. [Means for solving the problem]

[0007] Typically, oxidation-reduction reactions, enzymatic reactions, and antigen-antibody reactions involving specific binding with the target substance are used to biochemically measure the target substance contained in a sample. However, it is known that nonionic surfactants added to the reaction solution for purposes such as sample solubilization, turbidity suppression, and suppression of nonspecific reactions can affect the main reaction related to the measurement of the target substance.

[0008] Furthermore, when an aqueous solution of a nonionic surfactant is heated and the temperature is gradually increased, the surfactant cannot be solubilized at the cloud point temperature, resulting in turbidity. For this reason, there has been a need for a measurement reagent and measurement method using a nonionic surfactant with an appropriate cloud point that suppresses turbidity of the sample reaction solution and does not affect the main reaction. As a result of diligent research, the inventors have found that by using an alkyl ether type nonionic surfactant with a cloud point of 33-40°C, it is possible to suppress turbidity of the sample reaction solution and measure the target substance without inhibiting the main reaction that measures the target substance, or by increasing the reaction rate of the main reaction.

[0009] The present invention includes the following embodiments. [1] A biochemical test reagent used for measuring target substances, containing an alkyl ether type nonionic surfactant with a cloud point of 33-40°C. [2] The biochemical test reagent according to [1], wherein the surfactant is a surfactant whose molecular structure contains a polymer [POE,(-C2H4O-); degree of polymerization: 5~20] made of oxyethylene (EO) and a C10~C20 alkyl group. [3] The biochemical test reagent according to [1] or [2], wherein the surfactant is a surfactant whose molecular structure contains a polymer made of oxyethylene (EO) [POE, (-C2H4O-); degree of polymerization: 5~20], a polymer made of oxypropylene (PO) [POP, (-C3H6O-)] and an alkyl group. [4] The biochemical test reagent according to [3], wherein the PO / EO ratio contained in the surfactant is 0.2 to 0.8. [5] A biochemical test reagent according to any one of [1] to [4], wherein the concentration of the surfactant is 0.01% (w / v) to 2% (w / v). [6] The biochemical test reagent according to any one of items [1] to [5], wherein the measurement system for the substance to be measured is a homogeneous or heterogeneous measurement system. [7] A biochemical test reagent according to any one of items [1] to [6], wherein the substance to be measured is selected from the following group. Group of substances measured: glucose, glycated albumin, glycated hemoglobin (HbA1c), 1,5-anhydroglucitol (1,5-AG), pyruvate, lactic acid, urea, uric acid (UA), creatinine (CRE), polyamines, free fatty acids (FA), triglycerides (TG), cholesterol (CHO), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), bile acids, bilirubin, phospholipids, total protein (TP), albumin, hemoglobin, immunoglobulin, aminotransferase aspartate AST, alanine aminotransferase (ALT), creatine kinase (CK), creatine kinase isoenzyme MB (CK-MB), alkaline phosphatase (ALP), lactate dehydrogenase (LHD), gamma-glutamyltransferase (γGT), leucine aminopeptidase (LAP), cholinesterase (ChE), amylase (AMY), amylase (AMY) isoenzyme, human brain natriuretic peptide (BNP), insulin, C-reactive peptide (CRP), procalcitonin (PC) T), soluble interleukin-2 receptor (sIL-2R), pulmonary surfactant protein D (SP-D), thymic and activating regulatory chemokines (TARC / CCL-17), fibrin and fibrinogen degradation products (FDP), D-dimer (DD), soluble fibrin (SF), lipoprotein (Lp(a)), matrix metalloproteinase-3 (MMP-3), prostate-specific antigen (PSA), IgG, IgA, IgM, IgE, IgD, anti-streptolysin O antibody, rheumatoid factor (RF), transferrin, ha Putoglobin, α1-antitrypsin, α1-acid glycoprotein, α2-macroglobulin, hemopexin, antithrombin-III, α-fetoprotein (AFP), oncogenic antigen (CEA), ferritin, hepatitis B enucleated antigen (HBs-Ag), anti-hepatitis B enucleated antibody (Anti-HBs), hepatitis B e antigen (anti-HBeAg), anti-hepatitis B e antibody (Anti-HBe), anti-hepatitis B core antibody (Anti-HBc), SRAS-CoV-2, MRAS virus, RSV, and influenza virus. [8] A method for producing a biochemical test reagent, comprising using an alkyl ether type nonionic surfactant having a cloud point of 33 to 40°C to produce a biochemical test reagent according to any one of items [1] to [7]. [9] A biochemical test method comprising the steps of: obtaining a mixed solution by adding an alkyl ether type nonionic surfactant having a cloud point of 33 to 40°C to a sample; and measuring a target substance contained in the mixed solution by a biochemical method.

[10] The biochemical testing method according to [9], wherein the surfactant is a surfactant whose molecular structure contains a polymer made of oxyethylene (EO) [POE,(-C2H4O-); degree of polymerization: 5~20] and a C10~C20 alkyl group.

[11] The biochemical testing method according to [9] or

[10] , wherein the surfactant is a surfactant whose molecular structure contains a polymer made of oxyethylene (EO) [POE,(-C2H4O-); degree of polymerization: 5~20], a polymer made of oxypropylene (PO) [POP,(-C3H6O-)] and an alkyl group.

[12] The biochemical testing method according to

[11] , wherein the PO / EO ratio contained in the surfactant is 0.2 to 0.8.

[13] The biochemical testing method according to any one of [9] to

[12] , wherein the concentration of the surfactant contained in the mixture is 0.01% (w / v) to 2% (w / v).

[14] The biochemical testing method according to any one of [9] to

[13] , wherein the measurement system for the substance to be measured is a homogeneous or heterogeneous measurement system.

[15] The biochemical testing method according to any one of items [9] to

[14] , wherein the substance to be measured is selected from any one of the following groups. Group of substances measured: glucose, glycated albumin, glycated hemoglobin (HbA1c), 1,5-anhydroglucitol (1,5-AG), pyruvate, lactic acid, urea, uric acid (UA), creatinine (CRE), polyamines, free fatty acids (FA), triglycerides (TG), cholesterol (CHO), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), bile acids, bilirubin, phospholipids, total protein (TP), albumin, hemoglobin, immunoglobulin, aminotransferase aspartate AST, alanine aminotransferase (ALT), creatine kinase (CK), creatine kinase isoenzyme MB (CK-MB), alkaline phosphatase (ALP), lactate dehydrogenase (LHD), gamma-glutamyltransferase (γGT), leucine aminopeptidase (LAP), cholinesterase (ChE), amylase (AMY), amylase (AMY) isoenzyme, human brain natriuretic peptide (BNP), insulin, C-reactive peptide (CRP), procalcitonin (PC) T), soluble interleukin-2 receptor (sIL-2R), pulmonary surfactant protein D (SP-D), thymic and activating regulatory chemokines (TARC / CCL-17), fibrin and fibrinogen degradation products (FDP), D-dimer (DD), soluble fibrin (SF), lipoprotein (Lp(a)), matrix metalloproteinase-3 (MMP-3), prostate-specific antigen (PSA), IgG, IgA, IgM, IgE, IgD, anti-streptolysin O antibody, rheumatoid factor (RF), transferrin, ha Putoglobin, α1-antitrypsin, α1-acid glycoprotein, α2-macroglobulin, hemopexin, antithrombin-III, α-fetoprotein (AFP), oncogenic antigen (CEA), ferritin, hepatitis B enucleated antigen (HBs-Ag), anti-hepatitis B enucleated antibody (Anti-HBs), hepatitis B e antigen (anti-HBeAg), anti-hepatitis B e antibody (Anti-HBe), anti-hepatitis B core antibody (Anti-HBc), SRAS-CoV-2, MRAS virus, RSV, and influenza virus. [Effects of the Invention]

[0010] The present invention provides a biochemical test reagent that suppresses turbidity in a sample reaction solution using an alkyl ether type nonionic surfactant having a cloud point of 33-40°C, and measures a target substance without affecting the main reaction that measures the target substance, as well as a method for producing the same and a biochemical test method using the reagent. [Modes for carrying out the invention]

[0011] [Biochemical test reagents] A first aspect of the present invention is a biochemical test reagent containing an alkyl ether type nonionic surfactant, used for measuring a target substance. By using this reagent, it is possible to qualitatively or quantitatively determine whether or not a target substance is present in a sample.

[0012] In the present invention, any alkyl ether type nonionic surfactant can be used as long as it suppresses turbidity originating from the sample and enables the measurement of the target substance without affecting the main reaction that measures the target substance contained in the sample. However, an alkyl ether type nonionic surfactant having a cloud point in the range of 33°C to 40°C is preferred. Preferably, a surfactant has a cloud point of 33°C to 39°C, more preferably a surfactant has a cloud point of 34°C to 39°C, a surfactant has a cloud point of 34°C to 38°C, or a surfactant has a cloud point of 35°C to 38°C, and most preferably a surfactant has a cloud point of 36°C to 38°C.

[0013] In this invention, the cloud point refers to the temperature at which, when an aqueous solution (0.5-5 w / v%) of an alkyl ether type nonionic surfactant is heated, turbidity occurs as the water molecules bound to the surfactant molecules gradually dissociate. The cloud point can be measured using conventional methods. Specifically, the sample is heated, and the change in the transparency of the sample is observed while gradually increasing the temperature. After the sample becomes cloudy, it is allowed to cool naturally, and the temperature at which the sample becomes transparent again is defined as the cloud point. Alternatively, the viscosity can be continuously measured during the heating process, and the cloud point of the surfactant can be determined from the rapid change in viscosity.

[0014] Alkyl ether type nonionic surfactants preferably include polyoxyethylene alkyl ether type nonionic surfactants (hereinafter simply abbreviated as POE alkyl ethers) containing polyoxyethylene (polyoxyethylene: POE) obtained by polymerizing a plurality of oxyethylene groups (EO, -CH2CH2O-) in their molecular structures. For example, surfactants composed of polymers obtained by adding POE (degree of polymerization of EO: 5 to 20) [(-C2H4O-); degree of polymerization: 5 to 20] to an alkyl group [carbon number 10 to 20 (C10 to C20)] can be mentioned.

[0015] The average number of molecules of EO contained in one molecule of the POE alkyl ether polymer (also referred to as the degree of polymerization or the number of repetitions) is preferably 5 to 20, more preferably 6 to 15, and most preferably 7 to 12. The number of carbon (C) atoms constituting the alkyl group is preferably 10 to 20, more preferably 11 to 15, and most preferably 12 to 14. The alkyl group may be linear or branched, but a linear chain is preferred. The numbers of EO and C can be appropriately adjusted in the production of a surfactant having a cloud point of 33°C to 40°C.

[0016] As another embodiment of the present invention, the alkyl ether type nonionic surfactant can use a polyoxyethylene-polyoxypropylene alkyl ether type nonionic surfactant (hereinafter simply abbreviated as POE-POP alkyl ether) containing polyoxypropylene (polyoxypropylene: POP) obtained by polymerizing an oxypropylene group (PO, -CH2CH2CH2O-) in its molecular structure in addition to the aforementioned POE. Examples of the block copolymer structure of the POE-POP alkyl ether include the POE-POP-POE type. The EO and PO constituting the POE-POP alkyl ether may be either block polymerization or random polymerization. In addition, even when there is one PO constituting POP, it is called POP for the sake of simplifying the name.

[0017] The average number of PO molecules (also referred to as the degree of polymerization or the number of repetitions) contained in one molecule of the POE-POP alkyl ether polymer is preferably 1 to 16, more preferably 1 to 12, and most preferably 1.5 to 6. In the POE-POP alkyl ether, it is preferable that EO is more than PO, and the PO / EO ratio is preferably 1 or less. Specifically, the PO / EO ratio is preferably 0.2 to 0.8, more preferably 0.25 to 0.6, and most preferably 0.3 to 0.6.

[0018] Specific examples of the alkyl ether type nonionic surfactant used in the present invention include commercially available surfactants such as Emulgen 108 (manufactured by KAO Corporation), Emulgen 707 (manufactured by KAO Corporation), Emulgen LS-106 (manufactured by KAO Corporation), Lionol L950 (manufactured by LION Corporation), Lionol TD-730 (manufactured by LION Corporation), and ADEKA TOL LB-53B (manufactured by ADEKA Corporation). In the biochemical test reagent and the biochemical test method of the present invention, at least one of the above-mentioned alkyl ether type nonionic surfactants may be contained, and two or more may be contained, and they can be appropriately adjusted and used in combination with the auxiliary components described later.

[0019] The concentration of the alkyl ether type nonionic surfactant used in the biochemical test method and the biochemical test reagent of the present invention can be appropriately adjusted according to the reaction conditions. The concentration can be preferably in the range of 0.01% to 2% (w / v), more preferably 0.05% to 1.5% (w / v), and even more preferably 0.1% to 1.3% (w / v) with respect to the total volume of the biochemical test reagent or the total volume of the reaction solution in the biochemical test method.

[0020] The biochemical test reagent and the biochemical test method of the present invention can analyze or measure various measurement target substances contained in a biological sample using the biological sample as a specimen. The biological sample is, for example, tissue cells, blood, serum, plasma, blood cells, lymph fluid, cerebrospinal fluid, urine, feces, tears, nasal discharge, saliva, gastric juice, serous fluid, pancreatic juice, mucus of each tissue, other body fluids or various secretions collected from a living body.

[0021] The substances to be measured may include any of the following: sugars, proteins, lipids, nucleic acids, glycoproteins, glycolipid components and their complexes, and metabolites contained in the sample.Specifically, glucose, glycated albumin, glycated hemoglobin (HbA1c), 1,5-anhydroglucitol (1,5-AG), pyruvate, lactic acid, urea, uric acid (UA), creatinine (CRE), polyamines, free fatty acids (FA), triglycerides (TG), cholesterol (CHO), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), bile acids, bilirubin, phospholipids, total protein (TP), albumin, hemoglobin, immunoglobulin, aspartate aminotransferase (A) ST), alanine aminotransferase (ALT), creatine kinase (CK), creatine kinase isoenzyme MB (CK-MB), alkaline phosphatase (ALP), lactate dehydrogenase (LHD), gamma-glutamyltransferase (γGT), leucine aminopeptidase (LAP), cholinesterase (ChE), amylase (AMY), amylase (AMY) isoenzyme, human brain natriuretic peptide (BNP), insulin, C-reactive peptide (CRP), procalcitonin (PCT), soluble Sexual interleukin-2 receptor (sIL-2R), pulmonary surfactant protein D (SP-D), thymic and activation-regulated chemokines (TARC / CCL-17), fibrin and fibrinogen degradation products (FDP), D-dimer (DD), soluble fibrin (SF), lipoprotein (Lp(a)), matrix metalloproteinase-3 (MMP-3), prostate-specific antigen (PSA), IgG, IgA, IgM, IgE, IgD, anti-streptolysin O antibody, rheumatoid factor (RF), transferrin, haptoglobin Examples include α1-antitrypsin, α1-acid glycoprotein, α2-macroglobulin, hemopexin, antithrombin-III, α-fetoprotein (AFP), oncogenic antigen (CEA), ferritin, hepatitis B outer antigen (HBs-Ag), anti-hepatitis B outer antibody (Anti-HBs), hepatitis B e antigen (anti-HBeAg), anti-hepatitis B e antibody (Anti-HBe), anti-hepatitis B core antibody (Anti-HBc), SRAS-CoV-2, MRAS virus, RSV, and influenza virus.

[0022] In the present invention, "chylous leukemia" refers to a state in which a sample containing excess lipid components such as triglycerides (TG), cholesterol (CHO), or lipoproteins exhibits a suspension-like condition, similar to hyperlipidemia (Nikolac et al, Biochemia Medica, 2014: 24, 57-67, Toshima et al, J. Biol., Macromol., 2013: 13, 21-32). In the measurement of target substances in a sample, chylous leukemia is an interfering factor in the measurement of various target substances, and a reagent with high chylous elimination ability is required to quickly resolve chylous leukemia in order to efficiently process a large number of samples. Chylous leukemia can be measured as a formazine suspension unit. The biochemical testing reagent and biochemical testing method of the present invention have high chylous elimination ability and can measure target substances without affecting the main reaction to measure them later, and / or with a high reaction rate for the main reaction.

[0023] In this invention, the chyle scavenging ability can be measured by the following method. By adding a reagent to a high-lipid serum or sample prepared by adding approximately 0.1-10% of the commercially available lipid component Intralipos (manufactured by Otsuka Pharmaceutical Co., Ltd.) to serum or a buffer solution, and then measuring the absorbance over time using an appropriate wavelength, such as 800 nm / 600 nm (secondary / dominant wavelength), the effect of reducing turbidity due to chyle can be measured. For chyle scavenging ability, it is desirable that the absorbance decreases to a level equivalent to the baseline within approximately 3 minutes after the addition of the reagent.

[0024] The present invention demonstrates that in the main reaction (or this reaction) for measuring the target substance, the biochemical reaction can be maintained to a measurable extent. That is, it indicates a situation in which the reaction system necessary for measuring the target substance, such as oxidation-reduction reactions, enzymatic reactions, and antigen-antibody reactions, reaches a steady state within a certain period of time. Specifically, it is desirable that the biochemical reaction reaches a steady state within about 5 minutes after adding the reagent that causes this reaction.

[0025] An example of the biochemical testing reagent of the present invention includes various reagent components depending on the test item (substance to be measured) of the biochemical test. Specifically, the above example of reagent includes an alkyl ether type nonionic surfactant with a cloud point of 33°C to 40°C and other arbitrary reagent components. Reagent components refer to components that are involved in the reaction system (e.g., enzymes, substrates, antibodies, labeling substances, colorants, etc.) and / or components that are not involved in the reaction system (e.g., stabilizers, preservatives, emulsifiers, solvents, buffers, excipients, etc.).

[0026] In the following, components involved in the reaction system will also be referred to as "main components," and components not involved in the reaction system will also be referred to as "auxiliary components." For example, in vitro diagnostic medical devices, which are a type of biochemical test reagent commonly sold, include products composed of a main component and auxiliary components, and products composed of only a main component or only auxiliary components. In this specification, "reagent components" (i.e., main components and / or auxiliary components) shall not contain alkyl ether-type nonionic surfactants with a cloud point of 33-40°C.

[0027] In the biochemical testing reagent or biochemical testing method of the present invention, if the biochemical testing reagent is composed of one or more reagents, the added alkyl ether type nonionic surfactant may be contained in only one reagent or in each of the two or more reagents. That is, it is sufficient that a surfactant of an appropriate concentration can coexist with the sample and / or reagent components so as to eliminate turbidity originating from the sample when the biochemical testing reagent is added to the sample and so as not to affect the reaction. The biochemical testing reagent of the present invention may be composed of at least one reagent and can be used by adding the reagent to a sample containing the substance to be measured.

[0028] For example, in the case of a biochemical test reagent consisting of only one reagent, it is sufficient that the alkyl ether type nonionic surfactant is contained in that reagent alone at a predetermined concentration. Furthermore, in the case of a biochemical test reagent consisting of two reagents, the alkyl ether type nonionic surfactant may be contained only in the first reagent (R1), which is responsible for sample pretreatment or the primary reaction, or it may be contained in both the first reagent and the second reagent (R2), which is responsible for the main reaction or the secondary reaction. The reagent composition differs depending on which reaction system the first and second reagents are responsible for, depending on the substance being measured. However, this allows for the suppression of sample turbidity, without affecting the main reaction, and with a high reaction rate, enabling the measurement of the target substance.

[0029] Other reagent components used in the biochemical testing reagent or biochemical testing method of the present invention may include auxiliary components other than the main component involved in this reaction, such as other surfactants, buffers, salts, chelating agents, and preservatives.

[0030] Other surfactants are surfactants other than alkyl ether-type nonionic surfactants with a cloud point of 33-40°C, and can be used for various purposes, such as adjusting the cloud point of the entire reagent reaction system, promoting the solubilization of lipids and proteins, and suppressing the influence of other reaction inhibitors. Specific examples of other surfactants include nonionic surfactants, which are preferred, such as polyoxyethylene alkyl ether-type surfactants with a cloud point of 33-40°C or higher, polyoxyethylene-polyoxypropylene alkyl ether-type surfactants with a cloud point of 33-40°C or higher, and polyethylene glycol-alkylphenyl ether-type surfactants.

[0031] Examples of buffers used in the biochemical testing reagent or biochemical testing method of the present invention include phosphate buffers, citric acid, acetic acid, maleic acid, N-(2-acetamide)iminodiacetic acid (ADA), 2-morpholinoethanesulfonic acid (MES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-(2-acetamide)-2-aminoethanesulfonic acid (ACES), and N,N-bis(2-hydroxyethyl) Roxyethyl)-2-aminoethanesulfonic acid (BES), 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris), bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (Bis-Tris), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), 3-morpholinopropane-1-sulfonic acid (MOPS), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), 3-[N,N-bis(2-hydroxyethyl] [Tyl)amino]-2-hydroxypropanesulfonic acid (BIPSO), N-[tris(hydroxymethyl)methyl]-2-hydroxy-3-aminopropanesulfonic acid (TAPSO), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]-2-hydroxypropanesulfonic acid (HEPPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid [(H)EPPS], N-[T Examples include solutions containing at least one buffering agent such as tris(hydroxymethyl)methyl]glycine, N,N-bis(2-hydroxyethyl)glycine, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3-amino-2-hydroxypropanesulfonic acid (CAPSO), or N-cyclohexyl-3-aminopropanesulfonic acid (CAPS).

[0032] The concentration of the buffering agent is not particularly limited as long as it is suitable for measurement, but it is preferably between 0.01 mM and 100 mM, and more preferably between 0.1 mM and 100 mM. The pH of the buffer solution is preferably between 5.0 and 8.0. Note that the pH values ​​used herein are those measured at 25°C ± 5°C. In the biochemical testing reagents and biochemical testing methods of the present invention, other surfactants, chelating agents, preservatives, nonspecific reaction inhibitors, inorganic / organic salts, and other excipients may be added depending on the substance to be measured.

[0033] Examples of detection methods used in the biochemical testing reagent or biochemical testing method of the present invention include methods that use metabolites or hydrogen peroxide produced by enzymatic reactions, oxidation-reduction reactions, etc., of the target substance as indicators to induce a colorimetric quantitative reaction, or methods that measure the substance via a labeled substance that utilizes an antigen-antibody reaction with an antibody or antigen that specifically binds to the target substance. Any of these detection methods may be adopted.

[0034] [Method for manufacturing biochemical test reagents] The biochemical testing reagent of the present invention can be manufactured as a reagent containing, as a component, an alkyl ether type nonionic surfactant having a cloud point of 33-40°C, and reagent components capable of measuring each target substance. As described above, if the biochemical testing reagent consists of one or more reagents, the alkyl ether type nonionic surfactant may be added to only one reagent, or it may be added to each of the two or more reagents. The details of each reagent component are as described above.

[0035] [Biochemical testing methods (methods for measuring target substances)] A preferred embodiment of the biochemical testing method of the present invention is a method for measuring a target substance that suppresses turbidity caused by the sample and does not affect the main reaction of the target substance, and includes the following steps (1) to (3). (1) A step to obtain a mixed solution by adding an alkyl ether type nonionic surfactant having a cloud point of 33-40°C to a sample containing the substance to be measured, thereby suppressing the turbidity of the sample (i.e., the turbidity of the mixed solution). (2) A step of adding a reagent to the sample (i.e., the aforementioned mixture) for measuring the substance to be measured by a biochemical method, and carrying out the main reaction for measurement, (3) A step of measuring the substance to be measured using biochemical methods.

[0036] The biochemical testing method of the present invention can be performed using the biochemical testing reagent of the present invention. As described above, each reagent component is as described above. However, the alkyl ether-type nonionic surfactant with a cloud point of 33-40°C used in this invention may be included in one of the reagents constituting the biochemical test reagent, or in two or more reagents. It may also be added separately as a surfactant solution from the reagents constituting the biochemical test reagent.

[0037] For example, when measuring triglycerides in a sample, steps (1) to (3) described above must be performed in order to eliminate both lipemia and free glycerol from the sample. Similarly, when measuring HbA1c in a sample, the same steps are required because hemolysis and peroxide removal must be performed in addition to lipemia removal from the sample.

[0038] On the other hand, when measuring the target substance in a sample using a single reaction system, the sample, reagent components, and surfactant are mixed simultaneously or in a single reaction solution. That is, steps (1) to (3) described above can be performed simultaneously. Specifically, in homogeneous measurement systems utilizing antigen-antibody reactions, the measurement is performed using the turbidimetric / motile method based on agglutination, so the processes are performed simultaneously.

[0039] The biochemical testing methods of the present invention are broadly classified into homogeneous methods and heterogeneous methods. In the homogeneous method, the substance to be measured, the enzyme and / or substrate necessary for the reaction to measure the substance, or the antigen or antibody, are ultimately present in the same reaction system, and the substance to be measured is measured without separating the reactants and unreacted products of the redox reaction, enzymatic reaction, or antigen-antibody reaction. For example, in the triglyceride measurement performed in the following example, the turbidity of the sample is suppressed with the first reagent and free glycerol is eliminated by oxidation reaction. Then, the second reagent involved in the reaction system is added to measure the glycerol derived from the triglycerides. After lipase treatment, the glycerol is oxidized, and the chromogenic substrate is measured in the same reaction system, thereby enabling triglyceride measurement.

[0040] Furthermore, in measurement systems for target substances that utilize antigen-antibody reactions, this method measures the target substance without performing a washing process (sometimes called B / F separation) to separate the antigen-antibody complex of the antibody / antigen bound to the target substance from the antibody / antigen not bound to the target substance. Homogeneous methods are measurement methods that are susceptible to the influence of nonspecific binding. On the other hand, heterogeneous methods perform a final separation process for the reactants of the target substance, making them less susceptible to the influence of nonspecific reactions.

[0041] The present invention will be described in detail below with examples illustrating methods for measuring triglycerides (TG) or cholesterol (CHO) in chyle serum, but the present invention is not limited to these examples. [Examples]

[0042] 1) Sample Samples were prepared as follows to measure the chyle elimination rate and reaction rate. A 0.5% Intralipos concentration sample (hereinafter referred to as the Intralipos sample) was prepared by diluting Intralipos (10%, Otsuka Pharmaceutical) with physiological saline (Otsuka Pharmaceutical) to make a 5% solution and mixing it with pooled serum (TG concentration approximately 95 mg / dL, CHO concentration approximately 160 mg / dL) in a 1:9 ratio.

[0043] 2) Reagents for measuring triglycerides (TG) 2-1) Standard prescription

[0044] [Table 1]

[0045] 3) Reagents for measuring cholesterol (CHO) 3-1) Standard prescription

[0046] [Table 2]

[0047] 4) Test reagents Test reagents were prepared by adding the surfactants shown in Table 3 below to the first reagent (R1) for TG measurement at a concentration of 1.0% (w / v) and the second reagent (R2) at a concentration of 0.5% (w / v), and to the first reagent (R1) for CHO measurement at a concentration of 0.8% (w / v) and the second reagent (R2) at a concentration of 0.25% (w / v).

[0048] [Table 3] CAS No.: Chemical Abstract No. C: Average number of carbon atoms in alkyl groups contained in surfactant polymer molecules EO: Average number of ethylene oxide molecules per surfactant polymer molecule PO / EO: Average number of propylene oxide molecules per average number of ethylene oxide molecules

[0049] 5) Measurement Measurements were performed using a Hitachi 7180 automatic analyzer, and the intralipos samples were measured using the following parameters. [Parameters for TG measurement] • Analysis conditions: 2-point end ·Measurement wavelength (secondary / main): 800nm / 600nm • Metering points: 16-34 points • Reaction time: 10 minutes • Sample volume / First reagent volume / Second reagent volume: 2.1 μL / 210 μL / 70 μL

[0050] [Parameters for CHO measurement] • Analysis conditions: 2-point end ·Measurement wavelength (secondary / main): 800nm / 600nm • Metering points: 16-34 points • Reaction time: 10 minutes • Sample volume / First reagent volume / Second reagent volume: 2.7 μL / 200 μL / 67 μL

[0051] 6) Triglyceride measurement results For TG, the change in absorbance after adding reagent R1 to the Intralipos sample was measured to determine the chyle elimination ability, and the change in absorbance after adding reagent R2 was measured to determine the reaction rate. The results of measuring the sample with the test reagent are shown in Table 4. In the reagents to which the nonionic surfactants Emulgen 120, Emulgen 709, Emulgen LS-110, or ADEKA TOL TN-100 shown in the comparative examples were added, the chyle elimination rate after adding reagent R1 was slow, the time until elimination was long, or chyle elimination did not occur.

[0052] On the other hand, the reaction rate for TG was fast only with the reagent containing ADEKA TOL TN-100, while the reaction rate was slow with the reagents containing the surfactants of the other comparative examples. Therefore, the surfactants of the comparative examples not only had low or no chyle scavenging ability, but also a low reaction rate for TG, making it impossible to measure TG.

[0053] Compared to the comparative example, the reagents in the examples to which nonionic surfactants with cloud points of 33-40°C, such as Emulgen 108, Emulgen 707, Emulgen LS-106, Lionol L-950, Lionol TD-730, or ADEKA TOL LB-53B, were added showed a significant improvement in the chyle removal rate. The reaction rate was slightly faster or very fast for all reagents except Emulgen 108. Therefore, it was revealed that the reagents in the examples are TG measurement reagents with high chyle removal ability and a high reaction rate that does not affect the TG reaction itself.

[0054] [Table 4] ◎ Chylous elimination or the reaction with TG is very rapid. ○ Chylous elimination or the main reaction with TG is rapid. □ Chylous elimination or the main reaction with TG is slightly faster. △ Chylous elimination or the main reaction with TG is slow. × Chyle does not disappear, or the main reaction with TG is very slow.

[0055] 7) Cholesterol measurement results For CHO, the chyle elimination ability and reaction rate were measured in the same way as for TG, and the results are shown in Table 5. Similar to the TG measurement results, the reagents to which the nonionic surfactants Emulgen 120, Emulgen 709, Emulgen LS-110, or ADEKA TOL TN-100 used in the comparative examples were added showed slow or no lipemia elimination after the addition of reagent R1, indicating low lipemia elimination ability.

[0056] Compared to the comparative examples, the reagents used in the examples, each containing the nonionic surfactant Emulgen 108, Emulgen 707, Emulgen LS-106, Lionol L-950, Lionol TD-730, or ADEKA TOL LB-53B, showed a significantly improved lipemia elimination rate and an increased reaction rate. Therefore, it was revealed that the reagents in the examples are CHO measurement reagents with high lipemia elimination ability and a high reaction rate that does not affect the CHO reaction.

[0057] From these results, it became clear that by using a nonionic surfactant with a cloud point of 33-40°C, it is possible to set conditions that combine both lipemia elimination ability and a good reaction rate. Note that the reagents containing Emulgen 108 and Emulgen 707 used above are for reference only.

[0058] [Table 5] ◎ Chylous elimination or the reaction with CHO is very rapid. ○ Chylous elimination or the main reaction with CHO is rapid. □ Chylous elimination or the main reaction with CHO is slightly faster. △ Chylous elimination or the main reaction with CHO is slow. × Chyle does not disappear, or the reaction with CHO is very slow.

Claims

1. This is a biochemical test reagent used for measuring target substances, containing an alkyl ether-type nonionic surfactant with a cloud point of 33-40°C. A biochemical test reagent wherein the surfactant is a surfactant whose molecular structure contains a polymer made of oxyethylene (EO) [POE, (-C2H4O-); degree of polymerization: 5-20], a polymer made of oxypropylene (PO) [POP, (-C3H6O-)], and an alkyl group.

2. The biochemical test reagent according to claim 1, wherein the alkyl group has 10 to 20 carbon atoms.

3. The biochemical test reagent according to claim 1, wherein the PO / EO ratio contained in the surfactant is 0.2 to 0.

8.

4. The biochemical test reagent according to claim 1, wherein the concentration of the surfactant is 0.01% (w / v) to 2% (w / v).

5. The biochemical test reagent according to claim 1, wherein the measurement system for the substance to be measured is a homogeneous or heterogeneous measurement system.

6. Using an alkyl ether type nonionic surfactant with a cloud point of 33-40°C, A method for producing a biochemical test reagent, comprising producing a biochemical test reagent according to any one of claims 1 to 5.

7. A biochemical testing method comprising the steps of: obtaining a mixed solution by adding an alkyl ether-type nonionic surfactant having a cloud point of 33 to 40°C to a sample; and measuring the target substance contained in the mixed solution by a biochemical method. A biochemical testing method wherein the surfactant is a surfactant whose molecular structure contains a polymer made of oxyethylene (EO) [POE, (-C2H4O-); degree of polymerization: 5-20], a polymer made of oxypropylene (PO) [POP, (-C3H6O-)], and an alkyl group.

8. The biochemical testing method according to claim 7, wherein the alkyl group has 10 to 20 carbon atoms.

9. The biochemical testing method according to claim 7, wherein the PO / EO ratio contained in the surfactant is 0.2 to 0.

8.

10. The biochemical testing method according to claim 7, wherein the concentration of the surfactant contained in the mixture is 0.01% (w / v) to 2% (w / v).

11. The biochemical testing method according to claim 7, wherein the measurement system for the substance to be measured is a homogeneous or heterogeneous measurement system.

Images