Method for stabilizing biochemical diagnostic measuring reagent in which catalase and chromogen coexist

Abstract

To provide a biochemical diagnostic measuring reagent in which catalase and a chromogen coexist, capable of suppressing a decrease in catalase activity caused by light exposure and exhibiting stable reagent activity, and a method for stabilizing the biochemical diagnostic measuring reagent.SOLUTION: By causing alcohols to coexist in a biochemical diagnostic measuring reagent in which catalase and a chromogen coexist, a decrease in activity of catalase due to light exposure is suppressed.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a biochemical diagnostic reagent that has stable reagent activity by suppressing the decrease in catalase activity due to exposure to light by adding alcohols to an aqueous solution containing catalase and a chromogen. The present invention is useful in life science fields such as clinical testing, immunology, and medicine, and analytical fields such as analytical chemistry. [Background technology]

[0002] Reactions using oxidoreductases are widely used in biochemical diagnostic assay reagents. A widely used method involves generating hydrogen peroxide from the analyte present in body fluids such as blood (serum, plasma) and urine, then reacting the hydrogen peroxide with a coloring agent such as a chromogen in the reagent to convert the coloring agent into a pigment. The concentration of the analyte is then determined by colorimetric analysis based on the change in absorbance of the pigment. Catalase decomposes hydrogen peroxide to produce water and oxygen. In biochemical diagnostic reagents, one method involves eliminating hydrogen peroxide derived from substances other than the analyte (interfering substances) present in body fluids before detecting the analyte. To eliminate the hydrogen peroxide derived from these interfering substances, a widely used method involves adding catalase to the reagent.

[0003] For example, in the case of a creatinine measurement reagent, the first reagent decomposes interfering substances such as creatine and sarcosine present in body fluids, and then the hydrogen peroxide generated by the decomposition is eliminated by catalase. Subsequently, in the second reagent, hydrogen peroxide is generated from the analyte creatinine, and in the presence of peroxidase, the hydrogen peroxide undergoes oxidative condensation with a coupler and a chromogen (a hydrogen donor) to form a pigment. Thus, in the field of clinical testing, catalase is often used together with peroxidase, a coupler, and a chromogen (a hydrogen donor).

[0004] In the case of a reagent composition containing a first reagent and a second reagent as a creatinine measurement reagent, when catalase is formulated for the above-mentioned purpose, catalase is formulated in the first reagent to eliminate hydrogen peroxide generated from interfering substances in the first reagent, and a method is known in which a chromogen is also formulated in the first reagent at the same time (Patent Document 1).

[0005] Catalase is unstable in aqueous solutions, and known methods for stabilizing it include adding methanol (Patent Document 2), adding 15-20% glycerin, 4-6% sodium citrate, 3-8% ethanol, and 5-10% salt (Patent Document 3), and dissolving it in an ethanol and sugar alcohol solution (Patent Document 4).

[0006] However, all of these methods show the stability of an aqueous solution containing catalase when stored at a given temperature for a certain period of time, and do not describe the effect of exposing an aqueous solution containing catalase or catalase and a chromogen to light. Liquid biochemical diagnostic reagents are typically packaged in translucent bottles or boxes and are generally provided in a state that protects them from light to some extent. However, these bottles are not completely light-blocking, and it is difficult to completely avoid light exposure from the reagent preparation process to use, such as during the reagent manufacturing and filling processes, and after opening packaging materials such as box or bottle lids. Therefore, biochemical diagnostic reagents must be robust against light exposure in order to calculate accurate measurement results. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. WO2023-190087 [Patent Document 2] Japanese Patent Application Laid-Open No. 2002-223795 [Patent Document 3] Japanese Patent Application Publication No. 53-024093 [Patent Document 4] Japanese Patent Publication No. 139384 / 1984 Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors have prepared a biochemical diagnostic reagent containing a chromogen and catalase, and when using it to measure biological components, they have found that the activity of catalase decreases when the reagent is exposed to light for a long period of time. After detailed investigation, they have found that an aqueous solution containing only catalase is not affected by light exposure, but that a decrease in catalase activity occurs only in an aqueous solution containing both a chromogen and catalase.

[0009] As mentioned above, catalase and chromogens are often used together in biochemical diagnostic assay reagents. However, when catalase and chromogens coexist, the activity of catalase decreases upon exposure to light, which may result in inaccurate measurements of biological components.

[0010] Therefore, an object of the present invention is to provide a reagent composition for biochemical diagnostic assay reagents containing both a chromogen and catalase, which can maintain catalase activity even when exposed to light for a long period of time. [Means for solving the problem]

[0011] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors have discovered that by adding alcohols to a biochemical diagnostic assay reagent containing catalase and a chromogen, the decrease in catalase activity can be suppressed even when the biochemical diagnostic assay reagent is exposed to light for a long period of time, thereby producing a stable biochemical diagnostic assay reagent.

[0012] That is, the present invention provides the following inventions: (1) A biochemical diagnostic reagent comprising an aqueous solution containing catalase and a chromogen, to which an alcohol is added. (2) The biochemical diagnostic reagent according to (1) above, wherein the biochemical diagnostic reagent is a triglyceride measuring reagent or a creatinine measuring reagent. (3) The biochemical diagnostic measuring reagent according to (1) or (2), wherein the alcohol is ethanol or methanol. (4) A method for stabilizing a biochemical diagnostic reagent, which comprises adding an alcohol to an aqueous solution containing catalase and a chromogen. (5) The method for stabilizing a biochemical diagnostic reagent according to (4) above, wherein the biochemical diagnostic reagent is a triglyceride measuring reagent or a creatinine measuring reagent. (6) The method for stabilizing a biochemical diagnostic measuring reagent according to (4) or (5) above, wherein the alcohol is ethanol or methanol. (7) A biochemical diagnostic reagent, characterized in that catalase and a chromogen coexist in an aqueous solution in which the cumulative illuminance from production to use is 200 Lx·Day or more, and alcohols are added. (8) A method for stabilizing a biochemical diagnostic measurement reagent, comprising adding alcohols to an aqueous solution in which catalase and a chromogen coexist and the accumulated illuminance from production to use is 200 Lx·Day or more. [Effects of the Invention]

[0013] The present invention relates to a biochemical diagnostic assay reagent containing catalase and a chromogen, and by adding alcohols, the decrease in catalase activity can be suppressed even when exposed to light, thereby making it possible to obtain a biochemical diagnostic assay reagent that is stable even when exposed to light. DETAILED DESCRIPTION OF THE INVENTION

[0014] As a means for carrying out the present invention, for example, measurement using a general-purpose automatic analyzer is possible. It is particularly preferable to carry out the measurement using a liquid biochemical measurement reagent composed of a first reagent and a second reagent adapted for an automatic analyzer, but a manual reagent can also be used as long as it is a liquid biochemical diagnostic measurement reagent in which catalase and a chromogen coexist.

[0015] In liquid biochemical assay reagents consisting of a first reagent and a second reagent, catalase is often formulated in the first reagent for the purpose of eliminating hydrogen peroxide generated from interfering substances. Therefore, it is desirable to practice the present invention using the first reagent, but the present invention can be used in either the first or second reagent as long as the aqueous solution contains both catalase and a chromogen.

[0016] The chromogen in the present invention is a substance that, when mixed with and brought into contact with hydrogen peroxide, peroxidase, and a coupler such as 4-aminoantipyrine, undergoes oxidative condensation with the coupler to produce a dye, and is a substance that serves as a hydrogen donor during the oxidative condensation reaction, and examples of such a substance include aniline derivatives and salts thereof. Specifically, for example, 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-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS), N-ethyl-N-(3-sulfopropyl)aniline (ALPS), N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS), N-ethyl-N N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS), N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline (DAPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-4-fluoroaniline (FDAOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N,N-bis(4-sulfobutyl)-3,5-dimethylaniline (MADB), N,N-bis(4-sulfobutyl)-3-methylaniline (TODB), N-ethyl-N-2-hydroxyethyl-m-toluidine (CEMB), or salts thereof.

[0017] Examples of alcohols in the present invention include ethanol, methanol, 1-propanol, etc. Ethanol and methanol are particularly preferred. The concentration of the alcohols to be added in the aqueous solution containing the catalase and chromogen is preferably 0.0009% or more, more preferably 0.009% or more, even more preferably 0.045% or more, and particularly preferably 0.09% or more. There is no upper limit, but considering costs, etc., up to 10% is sufficient.

[0018] The catalases of the present invention include any catalases derived from animals, plants, and microorganisms.

[0019] In the present invention, light exposure refers to light irradiation to which a biochemical diagnostic assay reagent is subjected in each step from production to use, such as the step of producing the biochemical diagnostic assay reagent, the step of storing the reagent, and the step of using the reagent. The type of light to be irradiated is not particularly limited, and examples include light sources present in spaces where typical biochemical diagnostic measurement reagents are manufactured, transported, stored, and used, such as sunlight, LED, incandescent lamp, and fluorescent lamp.

[0020] Colored or translucent containers are often used as primary containers for biochemical diagnostic reagents. While primary containers may have some light-blocking properties, it is difficult to completely block light. Possible situations where the reagent is exposed to light include during the reagent manufacturing process, lighting inside the refrigerator in which the reagent is stored, when light from indoor lighting penetrates into the refrigerator in which the reagent is stored, and exposure to indoor lighting during use. There are many situations in which the reagent is exposed to light from manufacturing to use, and it is difficult to completely prevent such light exposure. Therefore, regardless of the form of the primary container, the practice of the present invention can suppress the decrease in catalase activity due to light exposure, thereby achieving the effect of stabilizing the biochemical diagnostic measurement reagent.

[0021] For the same reason, even when paper or cardboard containers are used as secondary packaging, the present invention can suppress the decrease in catalase activity due to exposure to light regardless of the shape of the secondary container, thereby achieving the effect of stabilizing the biochemical diagnostic measurement reagent.

[0022] The light exposure time for a reagent embodying the present invention is not particularly limited, but the effect is observed for reagents with an integrated illuminance from production to use of 200 Lx·Day or more, a significant effect for reagents with an integrated illuminance of 300 Lx·Day or more, an even more significant effect for reagents with an integrated illuminance of 400 Lx·Day or more, an even more significant effect for reagents with an integrated illuminance of 800 Lx·Day or more, and an especially significant effect for reagents with an integrated illuminance of 3000 Lx·Day. The cumulative illuminance is calculated by multiplying the illuminance by the number of days. For example, if an object is exposed to light with an illuminance of 1000 Lx for one day (24 hours), the cumulative illuminance is expressed as 1000 Lx x 1 Day = 1000 Lx·Day.

[0023] In the present invention, the term "integrated illuminance" refers to the integrated amount of light received by an aqueous solution containing catalase, a chromogen, and alcohols, or a biochemical diagnostic test reagent, from production to use. The "production" stage, which is the starting point for the integration in the present invention, refers to the preparation of an aqueous solution containing catalase, a chromogen, and alcohols during the preparation process of a biochemical diagnostic test reagent. This aqueous solution only needs to contain catalase, a chromogen, and alcohols, and any other ingredients necessary for preparing the biochemical diagnostic test reagent can be added before or after the preparation. After the preparation of the biochemical diagnostic test reagent, the test reagent can be further processed, such as dispensing into bottles, packaging, and testing. In the present invention, "use," which is the point at which the accumulation of the integrated illuminance is completed, refers to the measurement of an object using a biochemical diagnostic measurement reagent, such as when a sample is measured using an automatic analyzer in a hospital laboratory.

[0024] According to the Japanese Industrial Standard JIS Z9110:2010, the recommended illuminance for "ordinary visual work in general manufacturing plants, such as assembly c, inspection c, testing c, sorting c, and packaging a" is 500Lx, the recommended illuminance for factory "warehouses" is 100Lx, the recommended illuminance for factory "warehouses where work is performed" is 200Lx, and the recommended illuminance for "general examination rooms (for testing blood, urine, etc.) and measurement rooms" in health care facilities is 500Lx.

[0025] Assuming that the illuminance is set in accordance with the above JIS standard, an accumulated illuminance of 200 Lx·Day in a 500 Lx manufacturing plant is equivalent to 9.6 hours of work in an unshaded environment in the manufacturing plant.

[0026] Assuming that the illuminance is set in accordance with the above JIS standard, an accumulated illuminance of 200 Lx·Day in a general testing room (for testing blood, urine, etc.) at a health care facility with a 500 Lx illuminance is equivalent to reagents being exposed to light for 9.6 hours in the general testing room (for testing blood, urine, etc.) at a health care facility. The effects of the present invention are considered in terms of the cumulative illuminance from production to use. For example, if a reagent is exposed to light for 3 hours in a general testing room (for testing blood, urine, etc.) at a health care facility at 500 Lx after 7 hours of work in a non-light-shielded environment at a manufacturing plant in 500 Lx, the cumulative illuminance from production to use is equivalent to 500 Lx x 7 hours + 500 Lx x 3 hours = 208.3 Lx·Day.

[0027] The temperature at which the present invention is carried out is not particularly limited, but is preferably 1 to 37°C, which corresponds to the temperature during the manufacturing, transportation, and use processes of general biochemical diagnostic assay reagents, and more preferably 2 to 8°C, which corresponds to the storage temperature of general biochemical assay reagents.

[0028] The biochemical diagnostic assay reagent of the present invention is not particularly limited as long as it is a liquid reagent containing both catalase and a chromogen, and examples thereof include a triglyceride assay reagent, a creatinine assay reagent, an HDL cholesterol assay reagent, an LDL cholesterol assay reagent, a total cholesterol assay reagent, etc. Furthermore, there is no limitation on the purpose of use of the biochemical diagnostic assay reagent of the present invention, and it can be used, for example, as an in vitro diagnostic drug, a reagent for research purposes, etc.

[0029] In addition to catalase and chromogen, the biochemical diagnostic measurement reagent of the present invention may contain, as appropriate, buffers, conjugated enzymes, coenzymes, enzyme substrates, metal ions or metal salts containing these, chelating agents, proteins such as albumin, stabilizers such as sugars or polymeric compounds, preservatives such as sodium azide or antibiotics, agents for eliminating or suppressing the effects of measurement-interfering substances contained in the sample, surfactants, excipients, or activators, etc., as needed.

[0030] When the present invention is applied to a reagent for measuring triglycerides, a reagent having the following composition can be prepared, for example. 1) First Reagent: Adenosine-5'-triphosphate disodium, glycerol kinase, glycerol-3-phosphate oxidase, N-(2-carboxyethyl)-N-ethyl-m-toluidine (CEMB), catalase, ethanol 2) Second Reagent: Lipoprotein lipase, 4-aminoantipyrine, peroxidase, sodium azide

[0031] First, when the sample and the first reagent are mixed, free glycerol, an interfering substance other than triglycerides in the sample, reacts with the enzyme in the reagent to produce hydrogen peroxide. This hydrogen peroxide is eliminated by catalase, also contained in the first reagent, so there is no change in absorbance. Furthermore, the interfering free glycerol is converted into another substance, leaving only the triglycerides in the sample.

[0032] Next, when the second reagent is added to the liquid mixture of the sample and the first reagent, the triglycerides react with the enzymes in the sample, producing hydrogen peroxide. The catalase contained in the first reagent is inhibited by the sodium azide contained in the second reagent, so after mixing with the second reagent, the catalase does not eliminate the hydrogen peroxide. Instead, the peroxidase, 4-aminoantipyrine, and CEMB contained in the second reagent react with the hydrogen peroxide, producing a pigment color. This color change causes a change in absorbance, allowing the triglyceride concentration to be determined colorimetrically.

[0033] By using the method described above, a triglyceride measurement reagent can be obtained in which the decrease in catalase activity due to light is suppressed by adding alcohols to an aqueous solution containing catalase and a chromogen. However, the formulation of the triglyceride measurement reagent is not limited to this.

[0034] When the present invention is applied to a reagent for measuring creatinine, a reagent having the following composition can be prepared, for example. 1) First Reagent: Creatinase, sarcosine oxidase, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS), catalase, ethanol 2) Second reagent: Creatininase, 4-aminoantipyrine, peroxidase, sodium azide

[0035] First, when the sample and the first reagent are mixed, the enzymes in the reagent react with sarcosine and creatine, which are interfering substances other than creatinine in the sample, to produce hydrogen peroxide. This hydrogen peroxide is eliminated by catalase, which is also contained in the first reagent, so there is no change in absorbance. Furthermore, the interfering substances sarcosine and creatine are converted into other substances, leaving only creatinine remaining in the sample.

[0036] Next, when the second reagent is added to the liquid mixture of the sample and the first reagent, creatinine reacts with the enzymes in the reagent, producing hydrogen peroxide. The catalase contained in the first reagent is inhibited by the sodium azide contained in the second reagent, so after mixing with the second reagent, the catalase does not eliminate the hydrogen peroxide. Instead, the peroxidase, 4-aminoantipyrine, and ADOS contained in the second reagent react with the hydrogen peroxide, producing a pigment color. This color change causes a change in absorbance, allowing the creatinine concentration to be determined by colorimetry.

[0037] By using the method described above, a creatinine measurement reagent can be obtained in which the decrease in catalase activity due to light is suppressed by adding alcohols to an aqueous solution containing catalase and a chromogen. However, the formulation of the creatinine measurement reagent is not limited to this. [Example]

[0038] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0039] Example 1 (Stability of catalase and effect of ethanol when an aqueous solution containing catalase and a chromogen is exposed to light) The following reagent components were dissolved in pure water to the concentrations shown below to prepare aqueous solutions. Aqueous solution A: Catalase (Kikkoman Biochemifa) 220kU / L Aqueous solution B (comparative example): N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS) [Dojin Chemicals] 2.2 mM Catalase (Kikkoman Biochemifa) 220kU / L Aqueous solution C (present invention): N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS) [Dojin Chemicals] 2.2 mM Catalase (Kikkoman Biochemifa) 220kU / L Ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) 0.09% Aqueous solutions A to C were exposed to 1000 Lx of light for three days (cumulative illuminance 3000 Lx·day) at a storage temperature of 5°C, and the catalase activity after exposure was measured. The results are shown in Table 1. The catalase activity when stored completely shielded from light using aluminum foil was set at 100%, and the residual catalase activity under each condition is shown. In all examples of the present invention, light exposure tests were carried out by placing the solution in an orange reagent container, covering it, and exposing it to light. In all examples of the present invention, catalase activity was evaluated by calculating the amount of hydrogen peroxide lost when hydrogen peroxide and catalase were reacted at 25°C for 5 minutes. The amount of hydrogen peroxide was quantified by colorimetry using a titanium-containing coloring solution. [Table 1]

[0040] The results in Table 1 show that, compared to an aqueous solution containing only catalase (aqueous solution A), the residual catalase activity in the aqueous solution containing both catalase and the chromogen ADOS (aqueous solution B, comparative example) was significantly reduced to 14%. In contrast, the residual catalase activity in the aqueous solution containing both catalase and ADOS to which ethanol was added (aqueous solution C, the present invention) was 103%, demonstrating that the reduction in catalase activity due to light exposure can be suppressed.

[0041] [Example 2] (Study of ethanol concentration) To determine the alcohol concentration to be added to an aqueous solution containing catalase and a chromogen, the following reagent components were dissolved in pure water to the indicated concentrations to prepare aqueous solutions. Note that the ethanol concentrations were adjusted to 0%, 0.0002%, 0.0009%, 0.009%, 0.045%, 0.09%, 0.1%, and 1%, respectively. N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS) [Dojin Chemicals] 2.2 mM Catalase (Kikkoman Biochemifa) 220kU / L Ethanol [Fujifilm Wako Pure Chemicals] 0%, 0.0002%, 0.0009%, 0.009%, 0.045%, 0.09%, 0.1%, 1% The above aqueous solution was exposed to 1000 Lx of light for 3 days (cumulative illuminance 3000 Lx·day) at a storage temperature of 5°C, and the catalase activity after exposure was measured. The results are shown in Table 2. Note that the catalase activity value when stored completely shielded from light using aluminum foil was set at 100%, and the remaining catalase activity value under each condition is shown. [Table 2]

[0042] The results in Table 2 show that when the formulation concentration of ethanol was 0.0009% or higher, an inhibitory effect on the decrease in catalase activity due to exposure to light was observed, with a better effect observed at 0.009% or higher, an even better effect observed at 0.045% or higher, and a particularly good effect observed at 0.09% or higher.

[0043] [Example 3] (Effect of adding alcohols other than ethanol) The following reagent components were dissolved in pure water to the concentrations shown below to prepare aqueous solutions. Aqueous solution D: N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS) [Dojin Chemicals] 2.2 mM Catalase (Kikkoman Biochemifa) 220kU / L Methanol (Kanto Chemical) 0.01% Aqueous solution E: N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS) [Dojin Chemicals] 2.2 mM Catalase (Kikkoman Biochemifa) 220kU / L 1-Propanol (Domestic Chemicals) 1% Aqueous solutions D and E, and aqueous solutions B and C prepared in Example 1 were irradiated with 1000 Lx of light for 3 days (cumulative illuminance: 3000 Lx·Day) at a storage temperature of 5°C, and the catalase activity after exposure to light was measured. The results are shown in Table 3. The catalase activity value when stored completely shielded from light using aluminum foil was set at 100%, and the residual catalase activity value under each condition is shown. [Table 3]

[0044] The results in Table 3 show that, like ethanol, methanol and 1-propanol also have the effect of suppressing the decrease in catalase activity due to exposure to light.

[0045] Example 4 (Stability of catalase with chromogens other than ADOS and effect of ethanol) The following reagent components were dissolved in pure water to the concentrations indicated to prepare aqueous solutions. The chromogens were N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS). )-3-methylaniline (TOOS), N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N,N-bis(4-sulfobutyl)-3,5-dimethylaniline (MADB), N,N-bis(4-sulfobutyl)-3-methylaniline (TODB), or N-ethyl-N-2-hydroxyethylene-m-toluidine (CEMB) were prepared in ethanol-free and ethanol-added reagents, with the chromogen concentrations of each being 2.2 mM. Ethanol-free reagent: Catalase (Kikkoman Biochemifa) 220kU / L Chromogens: N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS) [Dojin Chemicals], N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS) [Dojin Chemicals], N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS) [Dojin Chemicals], N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS) [Dojin Chemicals], N-ethyl-N- (2-Hydroxy-3-sulfopropyl)-3-methylaniline (TOOS) [Dojindo Chemical Industries, Ltd.], N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS) [Dojindo Chemical Industries, Ltd.], N,N-bis(4-sulfobutyl)-3,5-dimethylaniline (MADB) [Dojindo Chemical Industries, Ltd.], N,N-bis(4-sulfobutyl)-3-methylaniline (TODB) [Dojindo Chemical Industries, Ltd.], or N-ethyl-N-2-hydroxyethylene-m-toluidine (CEMB) [Dojindo Chemical Industries, Ltd.] Ethanol-added reagent: Catalase (Kikkoman Biochemifa) 220kU / L Ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) 0.09% Chromogens: N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS) [Dojin Chemicals], N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS) [Dojin Chemicals], N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS) [Dojin Chemicals], N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS) [Dojin Chemicals], N-ethyl-N- (2-Hydroxy-3-sulfopropyl)-3-methylaniline (TOOS) [Dojindo Chemical Industries, Ltd.], N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS) [Dojindo Chemical Industries, Ltd.], N,N-bis(4-sulfobutyl)-3,5-dimethylaniline (MADB) [Dojindo Chemical Industries, Ltd.], N,N-bis(4-sulfobutyl)-3-methylaniline (TODB) [Dojindo Chemical Industries, Ltd.], or N-ethyl-N-2-hydroxyethylene-m-toluidine (CEMB) [Dojindo Chemical Industries, Ltd.] The above ethanol-free and ethanol-added reagents were exposed to 1000 Lx of light for 3 days (cumulative illuminance 3000 Lx·Day) at a storage temperature of 5°C, and the catalase activity after exposure was measured. The results are shown in Table 4. Note that the catalase activity value when stored completely shielded from light using aluminum foil was set at 100%, and the remaining catalase activity value under each condition is shown. [Table 4]

[0046] The results in Table 4 show that when chromogens other than ADOS were coexisted with catalase in aqueous solutions, the activity of catalase decreased due to exposure to light. Furthermore, it was found that the addition of ethanol to aqueous solutions in which catalase and various chromogens were coexisted suppressed the decrease in catalase activity due to exposure to light. These results demonstrate that when catalase is coexisted with chromogens, which are hydrogen donors, the activity of catalase decreases due to exposure to light, and that the addition of alcohols suppresses the decrease in catalase activity.

[0047] Example 5 (Application to a triglyceride measurement reagent) The following reagent components were dissolved in pure water to the concentrations shown below, and the pH was adjusted to 7.0 (20°C) to prepare the first reagent for measuring triglycerides. First Reagent (TG)-1 (Comparative Example) PIPES·Na (Oriental Yeast) 53mM Magnesium chloride hexahydrate (Kanto Chemical) 10mM Glycerol kinase (Asahi Kasei Pharma) 0.75 kU / L Glycerol-3-phosphate oxidase (Asahi Kasei Pharma) 4.5kU / L Adenosine triphosphate sodium salt (Oriental Yeast) 1.5mM Catalase (Toyobo) 320kU / L N-Ethyl-N-2-hydroxyethylene-m-toluidine (CEMB) [Actec] 2.1 mM First Reagent (TG)-2 (present invention) PIPES·Na (Oriental Yeast) 53mM Magnesium chloride hexahydrate (Kanto Chemical) 10mM Glycerol kinase (Asahi Kasei Pharma) 0.75 kU / L Glycerol-3-phosphate oxidase (Asahi Kasei Pharma) 4.5kU / L Adenosine triphosphate sodium salt (Oriental Yeast) 1.5mM Catalase (Toyobo) 320kU / L N-Ethyl-N-2-hydroxyethylene-m-toluidine (CEMB) [Actec] 2.1 mM Ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) 0.045% The first reagent (TG)-1 and first reagent (TG)-2 were exposed to 1000 Lx of light for 2, 4, or 6 days at a storage temperature of 5°C (cumulative illuminance of 2000 Lx·Day, 4000 Lx·Day, or 6000 Lx·Day), and the residual catalase activity of the reagents after light exposure was measured. The results are shown in Table 5. The catalase activity value under the condition of complete light shielding with aluminum foil and light exposure was set to 100%, and the residual catalase activity value under each condition is shown. [Table 5]

[0048] The results in Table 5 confirm that the triglyceride measurement reagent of the present invention retains high catalase activity even after exposure to light, demonstrating that the use of the present invention makes it possible to obtain a light-resistant triglyceride reagent.

[0049] Example 6 (Application to creatinine measurement reagent) The following reagent components were dissolved in pure water to the concentrations indicated, and the pH was adjusted to 7.35 (20°C) for the first reagent and 7.8 (20°C) for the second reagent to prepare reagents for biochemical measurement diagnosis. First Reagent (CRE)-1 (Comparative Example) TES buffer solution (Kanto Chemical) 20mM N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS) [Dojin Chemicals] 2.2 mM Nymeen S-220 (NOF) 0.15% Sarcosine oxidase (Toyobo) 13kU / L Creatinase (Toyobo) 40kU / L Catalase (Kikkoman Biochemifa) 220kU / L First Reagent (CRE)-2 (present invention) TES buffer solution (Kanto Chemical) 20mM N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS) [Dojin Chemicals] 2.2 mM Nymeen S-220 (NOF) 0.15% Sarcosine oxidase (Toyobo) 13kU / L Creatinase (Toyobo) 40kU / L Catalase (Kikkoman Biochemifa) 220kU / L Ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) 0.045% Second Reagent (CRE) TES buffer solution (Actec) 20mM Creatininase (Toyobo) 2.3mM 4-Aminoantipyrine (Actec) 2mM Peroxidase (Toyobo) 0.37mM Sodium azide (Kanto Chemical) 23mM Potassium ferrocyanide (trihydrate) [Kanto Chemical] 0.23mM The first reagent (CRE)-1 and first reagent (CRE)-2 were exposed to 1000 Lx of light for 3 days or 10 days at a storage temperature of 5°C (cumulative illuminance of 3000 Lx·day or 10000 Lx·day), and the residual catalase activity of the reagents after light exposure was measured. The results are shown in Table 6. The catalase activity value under the condition of complete light shielding with aluminum foil and light exposure was set to 100%, and the residual catalase activity value under each condition is shown. [Table 6]

[0050] The creatinine concentrations measured using the first reagent (CRE)-1 and the second reagent (CRE), and the first reagent (CRE)-2 and the second reagent (CRE) after 10 days of light exposure (cumulative illuminance 10,000 Lx·Day) are shown in Table 7. Note that the creatinine measurement value measured using the reagent that was completely shielded from light with aluminum foil and exposed to light is set to 100%, and the creatinine measurement value for each condition is shown. Creatinine concentration was measured using a Hitachi Model 7180 automatic analyzer. 150 μL of the first reagent was added to 6 μL of sample and incubated at 37°C for 5 minutes. 50 μL of the second reagent was added and incubated at 37°C for 5 minutes. The absorbance during the reaction was measured at a dominant wavelength of 600 nm and a secondary wavelength of 750 nm. The absorbance at the secondary wavelength was subtracted from the dominant wavelength to calculate the concentration. The creatinine concentration was calculated from the difference in absorbance between 34 points (587.426 seconds after adding the first reagent) and 16 points (270.093 seconds after adding the first reagent). The samples were prepared by adding creatine to pooled human serum to the following concentrations: Sample 1 Creatine 0mg / dL Sample 2 Creatine 10mg / dL Sample 3 Creatine 50mg / dL [Table 7]

[0051] The results in Table 6 confirm that the creatinine measurement reagent of the present invention retains high catalase activity even after exposure to light. Furthermore, Table 7 shows that the catalase activity in the creatinine measurement reagent CRE-1 (Comparative Example) is reduced, so the creatine contained in the sample cannot be completely eliminated, resulting in a falsely high creatinine measurement value. However, the creatinine measurement reagent of the present invention retains the catalase activity necessary for eliminating creatine from the sample even after exposure to light, confirming that an accurate creatinine measurement value can be obtained. This demonstrates that a light-resistant creatinine reagent can be obtained by using the present invention.

[0052] Example 7 (Comparison of the effects of ethanol on catalases of different origins) The effect of ethanol was examined when catalase of different origins was substituted for the creatinine measurement reagents First Reagent (CRE)-1 (Comparative Example) and First Reagent (CRE)-2 (Invention) prepared in Example 6. The catalase origins and dosages used were Bovine liver (Kikkoman Biochemifa) 220 kU / L, Corynebacterium glutamicum (Merck) 220 kU / L, Aspergillus niger (Merck) 220 kU / L, or Micrococcus lysodeikticus (Merck) 440 kU / L. The prepared reagent was exposed to 1000 Lx of light for 18 days (cumulative illuminance: 18000 Lx·Day) at a storage temperature of 5°C, and the catalase activity after exposure was measured. The results are shown in Table 8. The catalase activity after exposure to light while completely shielded from light with aluminum foil was set at 100%, and the remaining catalase activity under each condition is shown. [Table 8]

[0053] The results in Table 8 show that in creatinine measurement reagents, which are aqueous solutions containing catalase and a chromogen, the addition of alcohols suppresses the decrease in catalase activity due to light exposure, regardless of the origin of the catalase. In particular, bovine liver-derived catalase showed a particularly good effect in suppressing the decrease in activity.

[0054] Example 8 (Effect of ethanol under low light intensity) The following reagent components were dissolved in pure water to the concentrations shown below, and the pH was adjusted to 7.35 (20°C) to prepare a first reagent for measuring creatinine. First Reagent (CRE)-3 (Comparative Example) TES buffer solution (Kanto Chemical) 20mM N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS) [Dojin Chemicals] 2.2 mM Nymeen S-220 (NOF) 0.15% Sarcosine oxidase (Toyobo) 13kU / L Creatinase (Toyobo) 40kU / L Catalase (Kikkoman Biochemifa) 220kU / L First Reagent (CRE)-4 (present invention) TES buffer solution (Kanto Chemical) 20mM N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS) [Dojin Chemicals] 2.2 mM Nymeen S-220 (NOF) 0.15% Sarcosine Oxidase (Toyobo) 13kU / L Creatinase (Toyobo) 40kU / L Catalase (Kikkoman Biochemifa) 220kU / L Ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) 0.01% The above reagent was exposed to light at 200 Lx, 300 Lx, 400 Lx, 800 Lx, or 3000 Lx for one day at a storage temperature of 5°C (cumulative illuminance: 200 Lx·Day, 300 Lx·Day, 400 Lx·Day, 800 Lx·Day, or 3000 Lx·Day), and the catalase activity after exposure was measured. The results are shown in Table 9. The catalase activity value after exposure to light while completely shielded from light with aluminum foil was set to 100%, and the residual catalase activity value for each condition is shown. [Table 9]

[0055] The results in Table 9 show that in creatinine measurement reagents, which are aqueous solutions containing catalase and a chromogen, the presence of ethanol suppresses the decrease in catalase activity due to exposure to light, and has a stabilizing effect on the biochemical diagnostic measurement reagent. At the very least, the effect was observed when the cumulative illuminance of exposure to light was 200 Lx·Day or more, with a notable effect at 300 Lx·Day or more, a more significant effect at 400 Lx·Day or more, an even more significant effect at 800 Lx·Day or more, and an especially significant effect at 3000 Lx·Day or more.

[0056] From these results, it was confirmed that in an aqueous solution in which catalase and a chromogen coexist, the biochemical diagnostic assay reagent to which alcohols have been added according to the present invention suppresses the decrease in catalase activity due to exposure to light, stabilizes the biochemical diagnostic assay reagent, and enables accurate measurements to be performed using the biochemical diagnostic assay reagent.

Claims

1. A biochemical diagnostic reagent comprising an aqueous solution containing catalase and a chromogen, to which an alcohol is added.

2. 2. The biochemical diagnostic reagent according to claim 1, wherein the biochemical diagnostic reagent is a triglyceride measuring reagent or a creatinine measuring reagent.

3. 3. The biochemical diagnostic reagent according to claim 1, wherein the alcohol is ethanol or methanol.

4. A method for stabilizing a biochemical diagnostic reagent, comprising adding an alcohol to an aqueous solution containing catalase and a chromogen.

5. 5. The method for stabilizing a biochemical diagnostic reagent according to claim 4, wherein the biochemical diagnostic reagent is a triglyceride measuring reagent or a creatinine measuring reagent.

6. 6. The method for stabilizing a biochemical diagnostic reagent according to claim 4, wherein the alcohol is ethanol or methanol.

7. A biochemical diagnostic reagent comprising an aqueous solution containing catalase and a chromogen, and wherein the aqueous solution has an integrated illuminance of 200 Lx·Day or more from the time of production to the time of use, and to which an alcohol is added.

8. A method for stabilizing a biochemical diagnostic reagent, comprising adding an alcohol to an aqueous solution in which catalase and a chromogen coexist and the accumulated illuminance from production to use is 200 Lx·Day or more.

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